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Medullary Thyroid CarcinomaVerbeek, Hans

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Medullary Thyroid Carcinoma

From diagnosis to treatment

Hans Verbeek



Verbeek, H.H.G.

Cover: Wouter van de Gronde, www.woutr.nl

Printed by: Ipskamp Drukkers

ISBN: 978-90-367-7392-8 (print)

978-90-367-7391-1 (eBook)

Copyright © 2014 H.H.G. Verbeek, The Netherlands

All rights reserved. No part of this thesis may be reproduced, stored in a retrieval

system, or transmitted in any form or by any means, without prior written

permission of the author.

Financial support for printing of this thesis was kindly provided by: The Endocrinology Fund, as part

of the Ubbo Emmius Fund, University Medical Center Groningen and University of Groningen.



Proefschrift

ter verkrijging van de graad van doctor aan de

Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 7 januari 2015 om 16.15 uur

door

Hans Hendrik Gijsbert Verbeek

geboren op 1 september 1985

te Emmen

Promotores

Prof. dr. T.P. Links

Prof. dr. J.T.M. Plukker

Prof. dr. R.M.W. Hofstra

Beoordelingscommissie

Prof. dr. W.J.G. Oyen

Prof. dr. I.H.M. Borel Rinkes

Prof. dr. E.G.E. de Vries

Contents

Chapter 1 General introduction and aims of the thesis 7

Chapter 2 Medullary thyroid cancer, a tumour with many appearances 17

Chapter 3 Calcitonin testing for detection of medullary thyroid cancer 27

in patients with thyroid nodules

Chapter 4 Fewer cancer reoperations for medullary thyroid cancer 57

after initial surgery according to ATA guidelines

Chapter 5 PET imaging in thyroid carcinoma 73

Chapter 6 Clinical relevance of 18F-FDG PET and 18F-DOPA PET in 91

recurrent medullary thyroid carcinoma

Chapter 7 The effects of four different tyrosine kinase inhibitors on 109

medullary and papillary thyroid cancer cells

Chapter 8 Summary, discussion and future perspectives 125

Nederlandse samenvatting 139

Dankwoord 147

Curriculum vitae 151

Appendices 153

Paranimfen:

B. Kok

C.H. Verbeek

Chapter 1

General introduction and aims of the thesis

Chapter 1

8

General introduction

The thyroid

One of the largest endocrine organs of the human body is the thyroid. This butterfly-shaped

organ is located in the neck, in front of the trachea, directly below the larynx (Figure 1A). The

thyroid is composed of follicles, surrounded by follicular and parafollicular cells (C-cells)

(Figure 1B). Endocrine organs, such as the thyroid, produce hormones; biochemical active

messengers which are released in the blood and regulate body functions. The thyroid produces

thyroxine (T4), tri-iodotyronine (T3) (thyroid hormones) and calcitonin. The thyroid

hormones are involved in the metabolic rate throughout the body, while calcitonin exerts an

effect on calcium levels.

A. B.

Figure 1 Schematic representation of the thyroid gland (A) and (B) a histological section of thyroid tissue

showing the different thyroid cells.

Thyroid hormone production is a complex process and involves selective uptake of iodine

which is bound to thyroglobulin, a large protein synthesized in the follicular thyroid cells to

form eventually thyroxine and tri-iodotyronine. To release these hormones in the

bloodstream, they are cleaved from the thyroglobulin which takes place intracellular. In the

circulation, thyroid hormones are bound for 99% to the thyroxine binding globulin, and only

1% is free available for uptake by tissue cells.

C- cells

Follicular cells

Follicle


9

Thyroid hormones act throughout the whole body and increase metabolism by activating

transcription of many genes. Although thyroxine is much more present in the circulation, tri-

iodotyronine is the active form of thyroid hormone and therefore almost all thyroxine is

diodinated in order to have an effect on gene transcription.

Calcitonin is the other hormone of the thyroid produced in the parafollicular C-cells, which

account for about 0.1% of all thyroid cells. These cells have another embryological origin

than the follicular thyroid cells. Calcitonin is a 32-amino acid peptide. The main effect of

calcitonin is decreasing serum calcium levels, mainly by inhibiting bone resorption.1,2

However, in comparison to the parathyroid hormone (PTH) secreted by the parathyroids, the

effect of calcitonin on calcium metabolism is limited.

Thyroid nodules

Thyroid nodules are common in the general population; they are detected in up to 7% of

patients with neck palpation and on ultrasound even up to 70%.3-6 Functional imaging

methods such as PET imaging also frequently reveal thyroid nodules.7 Thyroid nodules are

more present in women than in men and the incidence of thyroid nodules also increases with

age.3,8,9 Benign nodules can be caused by clonal expansion of follicular cells (hyperplasia),

increase of the colloid follicles (colloid nodules), formation of cysts (cystic nodules) or

inflammation (thyroiditis).10,11 Most nodules are benign; only 5% to 10% of patients with

palpable thyroid nodules have thyroid cancer.

Thyroid cancer

Five major histological types of thyroid cancer are recognized; papillary, follicular,

medullary, poorly differentiated and undifferentiated (anaplastic) cancer. Papillary and

follicular cancer (also known as differentiated thyroid cancer) arise from the follicular

epithelium and account for 80-90% of all thyroid cancers. Medullary thyroid cancer arises

from the parafollicular C-cells and accounts for approximately 5%-10% (Figure 2). Poorly

differentiated thyroid carcinoma also arises from the follicular epithelium but exhibits a more

aggressive growth pattern compared to differentiated thyroid carcinoma, although less

aggressive than undifferentiated thyroid carcinoma. In undifferentiated thyroid carcinoma

undifferentiated cells exhibit features indicative of epithelial differentiation and

immunohistochemical staining is generally negative for thyroglobulin and calcitonin.

Undifferentiated thyroid carcinoma covers the last 5%-10% of the cases.11

Chapter 1

10

Medullary thyroid cancer

MTC was first described in a patient 1906 as a “malignant goiter with amyloid” by Jaquet.12

In 1959, Hazard defined a case of thyroid carcinoma with a solid non follicular structure with

amyloid in the stroma as a MTC.13 MTC can occur sporadically (75%) or as part of a familial

syndrome called Multiple Endocrine Neoplasia type 2 (MEN 2). This syndrome is caused by

a mutation in the ‘REarranged during Transfection’ (RET) gene and a MEN2A and MEN2B

variant are discerned. Other manifestations of the MEN2 syndromes are a pheochromocytoma

and hyperparathyroidism (MEN2A) or neurofibromatosis (MEN2B) (Table 1).

Figure 2 Histological section of medullary thyroid carcinoma

Table 1 Clinical expression of familial MTC-associated syndromes14

FMTC MEN 2A MEN2B

MTC 100% 100% 100%

Pheochromocytoma 0% 10-60% 50%

Hyperparathyroidism 0% 10-25% 0%

Marfanoid habitus 0% 0% 100%

Intestinal ganlioneuromatosis 0% 0% 60-90%

Mucosal neuromas 0% 0% 70-100%

Clinical presentation and diagnosis

Most MTC patients present with an asymptomatic palpable solitary thyroid nodule or lymph

node. Some patients have symptoms such as dyspnea, dysphagia, coughing or hoarseness.

Due to excessive calcitonin production diarrhea or flushing may occur.15,16 In very rare cases

ectopic ACTH production of the neuroendocrine cells can cause Cushing syndrome.17 At


11

presentation, about 50% of the MTC patients have lymph node metastases and distant

metastasis are diagnosed in around 15% of patients.18,19 Fine needle aspiration cytology is

generally the first diagnostic procedure for thyroid nodules. However, the sensitivity of this

procedure, without the use of additional immunohistochemical analysis, for detecting MTC is

limited.20 Since MTC originates from the calcitonin producing C-cells, this hormone can be

used as a sensitive tumour marker. Another tumour marker used in MTC is carcinoembryonic

antigen (CEA), however this marker is less sensitive. Serum calcitonin levels are not only

determined in patients suspected of MTC, but are also used as screening tool for detection of

MTC in patients with thyroid nodules. Although calcitonin testing in patients with thyroid

nodules can detect MTC in an early stage, it also increases the risk of unnecessary surgery as

a proportionate number of patients with thyroid nodules have an elevated basal calcitonin

based on other causes than MTC (e.g. thyroiditis, idiopathic, sepsis and chronic renal

failure).21-24

Treatment

According to the current American Thyroid Association (ATA) guidelines, treatment for

sporadic MTC consists of complete surgical removal of the thyroid (total thyroidectomy) and

the adjacent lymph nodes in the central compartment (central compartment dissection). If the

disease has spread to lateral lymph nodes of the neck, a lateral lymph node dissection is also

indicated.25 However a proportionate number of MTC patients cannot be cured due to the

extensiveness of the disease at presentation.26,27 In contrast to papillary and follicular thyroid

cancer, MTC patients do not benefit from adjuvant radioactive iodine treatment as the C-cells

do not have uptake of iodine.28 Therefore adequate surgery is of crucial importance in MTC,

including meticulous nodal dissection in the central and/or lateral neck. Although current

ATA guidelines provide clear recommendations for the surgical approach, the effect of

adherence to these recommendations on the outcome of MTC patients remains unclear.

Follow-up

Despite extensive surgery, a large proportion of MTC patients cannot be cured.29 Although

many patients with residual disease have a good life expectancy, some will develop

progressive disease.30 Therefore follow-up is important including regular determinations of

calcitonin and CEA. If these tumour markers are elevated or increasing, further diagnostic

work-up is needed using morphological (US/MRI/CT) and functional imaging (positron

Chapter 1

12

emission tomography (PET)). Although calcitonin and CEA doubling times are currently the

most reliable markers for progression, time consuming serial measurements are required for

accurate determination. Early detection of progressive disease is important because

appropriate therapeutic interventions, such as local surgical treatment, may delay

symptomatic deterioration. Therapeutic strategies are based on the outcome of the imaging

procedure and the doubling time of the tumour markers, and covers a wait and see policy with

close monitoring, a surgical intervention or systemic (targeted) therapy, if possible in a

clinical trial.

Systemic treatment

Traditional chemotherapeutic regimens have not been proven to be effective in the palliative

treatment of MTC. Recently developed tyrosine kinase inhibitors have shown improvement of

progression free survival in MTC patients.31,32 However, most studies have used one

particular TK inhibitor without analysis of the mutations present in the tumour. This makes it

difficult to compare these compounds for different patient groups. Most tyrosine kinase

inhibitors target multiple intracellular pathways, which can cause next to the intended effect

also side-effects, including cardiac toxicity and hand-foot syndrome.33,34 Therefore careful

consideration must be given when applying these new therapies.

Aims and outlines of the thesis

This thesis covers problems encountered in the diagnosis and treatment of primary and

recurrent MTC. The aims of the studies in this thesis were to:

1. Address the value of calcitonin testing for detection of MTC in patients with thyroid

nodules.

2. Evaluate the recommendations regarding surgical treatment by the current ATA

guidelines for MTC patients.

3. Detect MTC patients with progressive recurrent disease in an early stage.

4. Optimize treatment with targeted therapy (tyrosine kinase inhibitors) for MTC patients

with RET mutations/translocations with recurrent disease.

Chapter 2 encompasses an introduction to MTC and the difficulties in diagnosis. To illustrate

the different clinical presentation and behaviour of MTC, three patients with different stages


13

of disease are presented. A brief overview of currently used methods for diagnosis, treatment

options and follow-up is provided.

MTC patients detected in an early stage of the disease have a better prognosis. As almost all

MTCs secrete calcitonin, systematic determination of calcitonin might detect these tumours in

patients presenting with a thyroid nodule. However, the role of routine calcitonin testing

remains unclear, and no consensus exists between different guidelines. Chapter 3 focuses on

the value of routine calcitonin testing for detection of MTC in patients with thyroid nodules.

A formal systematic meta-analysis was performed to determine the diagnostic accuracy of the

calcitonin test. Sixteen studies were eventually included in which 72368 patients with nodular

thyroid disease underwent basal calcitonin testing and 187 MTC patients were identified.

Summary estimates of sensitivity and specificity for different cut-off values and subgroups

were determined.

Surgery is the most important therapeutic option for curative treatment of MTC. For MTC

patients presenting with a palpable thyroid nodule without suspected lymph node

involvement, treatment consists of a total thyroidectomy and central compartment dissection.

In many institutes this approach is followed by an unilateral elective nodal dissection of the

lateral neck. When suspected nodal involvement is present, a therapeutic lateral lymph node

dissection, according to the American Thyroid Association (ATA) 2009 guidelines is

performed. As the effect of adherence to these recommendations on the outcome of MTC

patients is unclear, we retrospectively evaluated these guidelines with respect to locoregional

control and clinical outcome. In Chapter 4 we reviewed the surgical and pathology reports of

86 patients treated between 1980 and 2010 in two major tertiary referral centres. We

compared the clinical outcome (reoperations, biochemical cure, survival and complications)

of the patients treated adequately according to ATA guidelines versus patients treated

inadequately. Furthermore, we examined to which extent clinical outcome of patients was

influenced by one-step versus a two-step intended curative surgical procedure, institute of

initial curative surgery (experienced centre versus non-centre hospital). Finally, influence of

patient and tumour characteristics on clinical outcome were evaluated.

After surgery, follow-up is important in patients with MTC, as a large proportion of patients

with biochemical residual disease will develop clinical recurrent disease. Although prognosis

is good in most patients with recurrent disease, some patients develop progressive disease.

Chapter 1

14

Regular determinations of calcitonin and CEA are useful in the early identification of these

patients, because therapeutic interventions, such as surgery for local tumour control, can be of

value in these patients. If tumour markers increase, further diagnostic work-up with

anatomical and/or functional imaging is required. In Chapter 5 we aimed to provide an

overview of the available PET imaging methods used for MTC and other types of thyroid

cancer. In Chapter 6 we investigated the potential of 18F-deoxyglucose (FDG-PET) and 18F-

diphenylalanine (DOPA-PET) to identify progression in MTC patients. PET positivity was

compared with biochemical parameters (calcitonin and CEA serum levels and doubling times)

in 47 patients. In a subgroup of 21 patients whole body metabolic burden (WBMTB) was

assessed with standardized uptake value and the number of lesions, and compared with

biochemical parameters. Furthermore, survival was compared with 18F-DOPA PET or 18F-

FDG PET positivity.

In MTC, but also PTC patients with progressive disease, systemic targeted therapy with

tyrosine kinase inhibitors is currently considered. As activating mutations or rearrangements

in the RET gene can cause MTC and PTC, tyrosine kinase inhibitors that target the RET

receptor might be promising. In Chapter 7 we aimed to determine which inhibitor is the most

effective and if there is rationale for mutation based therapy. We cultured and treated three

cell lines expressing a MEN2A (MTC-TT), a MEN2B (MZ-CRC-1) mutation, and a

RET/PTC (TPC-1) rearrangement. We compared four tyrosine kinase inhibitors (axitinib,

sunitinib, vandetanib and cabozantinib) in vitro. We evaluated the effects on cell proliferation,

RET expression, RET autophosphorylation and on RET downstream pathways (Extracellular

Signal-regulated Kinase (ERK)).

In Chapter 8 a general discussion is provided and future perspectives are addressed. In

Chapter 9 a summary of this thesis in Dutch is given.


15

References

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calcitonin. J Clin Endocrinol Metab 1983;57:819-824.

2. Karsdal MA, Henriksen K, Arnold M, Christiansen C. Calcitonin: a drug of the past or for the future?

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year study of the incidence of thyroid malignancy. Ann Intern Med 1968;69:537-540.

4. Rallison ML, Dobyns BM, Meikle AW, Bishop M, Lyon JL, Stevens W. Natural history of thyroid

abnormalities: prevalence, incidence, and regression of thyroid diseases in adolescents and young

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5. Wiest PW, Hartshorne MF, Inskip PD, et al. Thyroid palpation versus high-resolution thyroid

ultrasonography in the detection of nodules. J Ultrasound Med 1998;17:487-496.

6. Tan GH, Gharib H. Thyroid incidentalomas: management approaches to nonpalpable nodules

discovered incidentally on thyroid imaging. Ann Intern Med 1997;126:226-231.

7. Soelberg KK, Bonnema SJ, Brix TH, Hegedus L. Risk of malignancy in thyroid incidentalomas detected

by 18F-fluorodeoxyglucose positron emission tomography: a systematic review. Thyroid 2012;22:918-

925.

8. Vanderpump MP, Tunbridge WM, French JM, et al. The incidence of thyroid disorders in the

community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 1995;43:55-68.

9. Mazzaferri EL. Management of a solitary thyroid nodule. N Engl J Med 1993;328:553-559.

10. Salabe GB. Pathogenesis of thyroid nodules: histological classification? Biomed Pharmacother

2001;55:39-53.

11. Schmid KW. Molecular pathology of thyroid tumors. Pathologe 2010;31 Suppl 2:229-233.

12. Jaquet AJ. Ein fall von metastasierenden amyloidtumoren (lymphosarcoma). Virchows Archiv

1906:251-267.

13. HAZARD JB, HAWK WA, CRILE G,Jr. Medullary (solid) carcinoma of the thyroid; a clinicopathologic

entity. J Clin Endocrinol Metab 1959;19:152-161.

14. de Groot JW, Links TP, Plukker JT, Lips CJ, Hofstra RM. RET as a diagnostic and therapeutic target in

sporadic and hereditary endocrine tumors. Endocr Rev 2006;27:535-560.

15. Beressi N, Campos JM, Beressi JP, et al. Sporadic medullary microcarcinoma of the thyroid: a

retrospective analysis of eighty cases. Thyroid 1998;8:1039-1044.

16. Mure A, Gicquel C, Abdelmoumene N, et al. Cushing's syndrome in medullary thyroid carcinoma. J

Endocrinol Invest 1995;18:180-185.

17. Hijazi YM, Nieman LK, Medeiros LJ. Medullary carcinoma of the thyroid as a cause of Cushing's

syndrome: a case with ectopic adrenocorticotropin secretion characterized by double enzyme

immunostaining. Hum Pathol 1992;23:592-596.

18. Kebebew E, Ituarte PH, Siperstein AE, Duh QY, Clark OH. Medullary thyroid carcinoma: clinical

characteristics, treatment, prognostic factors, and a comparison of staging systems. Cancer

2000;88:1139-1148.

19. Modigliani E, Cohen R, Campos JM, et al. Prognostic factors for survival and for biochemical cure in

medullary thyroid carcinoma: results in 899 patients. The GETC Study Group. Groupe d'etude des

tumeurs a calcitonine. Clin Endocrinol (Oxf) 1998;48:265-273.

20. Bugalho MJ, Santos JR, Sobrinho L. Preoperative diagnosis of medullary thyroid carcinoma: fine needle

aspiration cytology as compared with serum calcitonin measurement. J Surg Oncol 2005;91:56-60.

21. Lips CJ, Landsvater RM, Hoppener JW, et al. Clinical screening as compared with DNA analysis in

families with multiple endocrine neoplasia type 2A. N Engl J Med 1994;331:828-835.

22. Landsvater RM, Rombouts AG, te Meerman GJ, et al. The clinical implications of a positive calcitonin

test for C-cell hyperplasia in genetically unaffected members of an MEN2A kindred. Am J Hum Genet

1993;52:335-342.

23. Machens A, Haedecke J, Holzhausen HJ, Thomusch O, Schneyer U, Dralle H. Differential diagnosis of

calcitonin-secreting neuroendocrine carcinoma of the foregut by pentagastrin stimulation.

Langenbecks Arch Surg 2000;385:398-401.

Chapter 1

16

24. Niccoli P, Brunet P, Roubicek C, et al. Abnormal calcitonin basal levels and pentagastrin response in

patients with chronic renal failure on maintenance hemodialysis. Eur J Endocrinol 1995;132:75-81.

25. Kloos RT, Eng C, Evans DB, et al. Medullary thyroid cancer: management guidelines of the American

Thyroid Association. Thyroid 2009;19:565-612.

26. Machens A, Gimm O, Ukkat J, Hinze R, Schneyer U, Dralle H. Improved prediction of calcitonin

normalization in medullary thyroid carcinoma patients by quantitative lymph node analysis. Cancer

2000;88:1909-1915.

27. Scollo C, Baudin E, Travagli JP, et al. Rationale for central and bilateral lymph node dissection in

sporadic and hereditary medullary thyroid cancer. J Clin Endocrinol Metab 2003;88:2070-2075.

28. Meijer JA, Bakker L, Valk GD, et al. Radioactive Iodine in the treatment of Medullary Thyroid

Carcinoma: a controlled multicenter study. Eur J Endocrinol 2013.

29. Machens A, Schneyer U, Holzhausen HJ, Dralle H. Prospects of remission in medullary thyroid

carcinoma according to basal calcitonin level. J Clin Endocrinol Metab 2005;90:2029-2034.

30. Rendl G, Manzl M, Hitzl W, Sungler P, Pirich C. Long-term prognosis of medullary thyroid carcinoma.

Clin Endocrinol (Oxf) 2008;69:497-505.

31. Wells SA,Jr, Robinson BG, Gagel RF, et al. Vandetanib in patients with locally advanced or metastatic

medullary thyroid cancer: a randomized, double-blind phase III trial. J Clin Oncol 2012;30:134-141.

32. Elisei R, Schlumberger MJ, Muller SP, et al. Cabozantinib in progressive medullary thyroid cancer. J Clin

Oncol 2013;31:3639-3646.

33. Ye L, Santarpia L, Gagel RF. The evolving field of tyrosine kinase inhibitors in the treatment of

endocrine tumors. Endocr Rev 2010;31:578-599.

34. Kapiteijn E, Schneider TC, Morreau H, Gelderblom H, Nortier JW, Smit JW. New treatment modalities

in advanced thyroid cancer. Ann Oncol 2012;23:10-18.

Chapter 2

Medullary thyroid cancer, a tumour

with many appearances

Hans H.G. Verbeek, Jan Willem B. de Groot, John T.M. Plukker, Robert M.W. Hofstra

Adrienne H. Brouwers, Michiel N. Kerstens, Thera P. Links

Ned Tijdschr Geneesk 2010; 154: A1818 (translated from Dutch)

Chapter 2

18

Abstract

Medullary thyroid cancer (MTC) has a variable clinical presentation. We present 3 patients

with this endocrine tumour. The first patient, a 41-year-old woman complaining of diarrhoea,

a painful abdomen, weight loss and sensibility disorders in both legs, had metastases of MTC

in the spine, with little progression during 2 years of follow-up. The second patient, a 64-year-

old woman suffering from a painful nodule in the neck and a painful shoulder, was diagnosed

with MTC and liver, lung and bone metastases. She died after 14 months due to progressive

disease. The third patient, an 81-year-old woman with hyperparathyroidism, was

coincidentally diagnosed with MTC after goitre surgery at the age of 67. When she was

evaluated for rising calcitonin levels, a pheochromocytoma was found. RET mutation analysis

confirmed a MEN2A syndrome. Current diagnostic procedures of MTC may include positron

emission tomography with 18F-deoxyglucose (18F-FDG PET) and 18F-diphenylalanine (18F-

DOPA PET). MTC is usually treated surgically. Tyrosine kinase inhibitors also appear to

offer potential new therapeutic possibilities.

MTC, a tumour with many appearances

19

Introduction

Medullary thyroid carcinoma (MTC) is a rare endocrine tumour which originates from the

calcitonin producing C-cells in the thyroid. The serum level of calcitonin has therefore a great

diagnostic value as tumour marker. Also the serum level of carcinoembryonic antigen (CEA)

is often elevated, but is less specific for MTC. In the Netherlands, 20-30 MTC patients are

diagnosed yearly. MTC can occur sporadically (75% of cases) or familiarly as part of the

multiple endocrine neoplasia type 2 syndrome (MEN2). Of this syndrome, caused by a

mutation in the ‘REarranged during Transfection (RET)’ gene, a MEN2A and MEN2B variant

are known (Table 1).1 Here we illustrate the broad spectrum of presentation and the clinical

course of MTC with 3 patients. Furthermore the current diagnostic modalities and treatment

options are discussed.

Patient A, a 41 year old woman, presented with a multinodular goitre. Repeated fine needle

aspiration cytology (FNAC) of the thyroid did not reveal malignancy. Four years later she

presented with diarrhoea, a painful lower abdomen and a weight loss of 13 kg. Additional

investigation, which included gastroduodenoscopy, colonoscopy, abdominal and transvaginal

ultrasound (US), did not lead to a diagnosis. She then developed an abnormal walking pattern

with sensibility dysfunction of both legs. Magnetic resonance imaging (MRI) showed a

tumour in the sixth thoracic vertebrae with compression of the myelum. Surgical debulking

took place and the pathologist diagnosed a metastasis of a MTC. The calcitonin and CEA

were highly elevated (respectively 143,150 ng/l (ref 0.3-12 ng/l) and 1400 ug/l (ref 0.5-5

ug/l)). The serum calcium was normal and there were no hints of pheochromocytoma.

Additional RET-mutation analysis did not show a mutation. Further investigations for staging

with fluor-18-deoxyglucose (18F-FDG) positron emission tomography (PET) and fluor 18-

dihydroxyphenylalanine (18F-DOPA) PET showed the primary process in the thyroid with

extensive bone metastasis (Figure 1). A total thyreoidectomy with lymph node dissection of

the central compartment was performed. In this procedure all lymph nodes and fat tissue

between both carotids was removed, from the os hyoideum cranially to the v. brachiocheplica

caudally. After surgery a single dose with 150 mCurie I131 MIBG was given for persisting

diarrhoea, with a subjectively good response. At this moment four years after MIBG therapy

there is slowly progressive disease, which is too slow for inclusion in a clinical trial with a

tyrosine kinase inhibitor.

Chapter 2

20

Table 1 Clinical characteristics of patients with familial medullary thyroid carcinoma,

multiple endocrine neoplasia (MEN) 2A and MEN2B.

Clinical characteristic Prevalence (%)

Familial MTC* MEN2A MEN2B

Medullary thyroid carcinoma 100 100 100

C-cell hyperplasia 100 100 100

Pheochromocytoma 0 10-60 50

Hyperparathyroidism 0 10-25 0

Neurofibromatosis 0 0 60-90

Marfanoid habitus 0 0 100

* Families are described in which only MTC occurs without other endocrine neoplasia’s.

Patient B, a 64–year old female presented with a painful nodule in the neck and a painful

right shoulder in another hospital. FNAC of the nodule showed a MTC. Ultrasound of the

liver, CT-imaging of the abdomen and bone scintigraphy showed metastasis in the liver and

skeleton, upon which referral to our centre was made. Additional imaging with 18F-FDG PET

showed besides liver metastasis also neck and lung metastasis. Calcitonin serum levels were

strongly elevated (650 ng/l); CEA was not determined and no biochemical clues existed for a

pheochromocytoma or hyperparathyroidism. RET-mutation analysis was not performed

because the patient was above 50 years and there was no clinical suspicion of a MEN2

syndrome. For local control of the primary tumour, a total thyroidectomy with a central and

lateral lymph node dissection was performed. External radiotherapy was given

postoperatively in the neck and mediastinum (70 Gy in 35 fractions). There was, however,

biochemical progression and progression on 18F-FDG and 18F-DOPA PET, especially of the

bone metastasis, for which palliative radiotherapy was given. Because of her poor clinical

condition, no systemic therapy was started. The patient deceased fourteen months after initial

presentation.

Patient C, an 81 year old woman, had at the age of 67 undergone in another hospital a

subtotal thyroidectomy and parathyroidectomy for a primary hyperparathyroidism and goitre.

Histopathological investigation revealed a hyperplasia of the parathyroids and a coincidental

MTC; no information on tumour size was available. An additional total thyroidectomy

without lymph node dissection was performed.


21

Figure 1 Positron emission tomography with 18

F-DOPA PET (left) and 18

F-FDG PET (right) of patient A. The 18

F-

DOPA PET shows clear uptake in the primary tumour in the neck. Extensive bone metastasis in the skull, spine,

skull and both femora is present. There is physiological uptake of 18

F-DOPA in the putamen, the caudate

nucleus, kidneys and bladder. The 18

F-FDG PET shows also clear uptake in the primary tumour and focal uptake

in the pelvis and right femur. There is faint uptake in the area of the 6th

thoracic vertebra, because of surgical

debulking 6 weeks before the scan. There is physiological uptake in the brain, kidneys, bladder and colon.

The patient was referred to our centre for further analysis of rising serum calcitonin levels; the

CEA concentration was not elevated. For a period of four years she had paroxysmal occurring

heat sensations without other symptoms. Blood pressure was 170/74 mm Hg, and further

physical examinations revealed no abnormalities. Biochemical investigation showed, besides

a raised serum calcitonin concentration, also increased metanephrines in plasma and urine,

suggestive of a pheochromocytoma. Imaging revealed enlarged lymph nodes in the neck and

an adrenal tumour on the left.

A laparoscopic adrenalectomy was performed with removal of a pheochromocytoma. DNA

analysis was performed because of the clinical presentation, despite the high age. The patient

was carrier of a Cys618Phe mutation of the RET-gene, a single base substitution in codon

618, in exon 10, resulting in the amino acid substitution of cysteine by phenylalanine. This

Chapter 2

22

confirmed the clinical diagnosis MEN2A. Because of the high age of the patient and the lack

of clinical symptoms a ‘wait and see’ policy for the MTC was adopted. In the family of the

patient genetic analysis was performed over four generations. Of the 40 family members

investigated, 19 carried the Cys618Phe mutation.

These patients show the varied clinical course of MTC. The presentation of patients A and B

is characteristic for a sporadic MTC, in which at the time of diagnosis extensive metastasis is

already present. It’s likely that a MTC was already present when patient A presented with

goitre. This shows that patients with metastasized disease can survive for many years. The

progressive and fatal nature of MTC is illustrated by the clinical course of patient B. The

clinical course of patient C shows the sometimes mild course of MTC with a RET-mutation.

Most RET-mutations result in an aggressive biological behaviour, but in some patients the

clinical course is more favourable. The mutation of our patient had great implications for her

family, because carriers of the mutation are candidates for prophylactic thyroidectomy, with

or without central compartment dissection and lifelong follow-up for possible occurrence of a

pheochromocytoma and primary hyperparathyroidism.

Clinical presentation

Patients with MTC most often present with a palpable tumour in the neck. More than 50% of

patients already have lymph node metastases at the time of diagnosis and 15% have distant

metastasis.2 Patient A presented with diarrhoea, a symptom that can occur due to

hypersecretion of calcitonin.1 In patients with long term unexplained diarrhoea, MTC can be

considered in differential diagnosis. Retrospectively, patient C presented with at that time

unrecognised manifestations of MEN2A. Failure to recognise this syndrome can lead to

inadequate diagnosis and therapy. The patient did have a pheochromocytoma and had

therefore an increased risk of a potentially fatal hypertensive crisis.3

Diagnosis

According to current guidelines, an ultrasound guided FNAC in a solitary thyroid nodule is

preferred.4 In 63%-89% of MTC patients this gives the correct diagnosis, and 91%-100% of

MTC patients are operated based on FNAC results.1 When FNAC is inconclusive,

determination of serum calcitonin and CEA can be helpful. Calcitonin is a sensitive tumour

marker (sensitivity 98%), but there is still discussion about the cost-effectiveness because


23

thyroid nodules are common, MTC is relatively rare and false-positive findings occur

frequently.1,5

Morphological imaging with CT or MRI can be used for staging. Before starting treatment

it’s important to know if and where MTC metastases are present. To determine this,

functional imaging with 18F-FDG PET and 18F-DOPA PET can be used, because

morphological imaging is less sensitive. Fluor-18-DOPA is a relatively new tracer for

imaging of neuroendocrine tumours. DOPA is a precursor in the catecholamine synthesis,

which specifically occurs in many of these tumours. 18F-DOPA PET has the highest

sensitivity while 18F-FDG PET is more often positive in patients with a progressive tumour.6

Because of the risk of MEN2, which inherits autosomal dominantly, every patient with

MTC under the age of 50 is a candidate for genetic screening. Pre-operative determination of

serum calcium and metanephrines is indicated in all patients with MTC to rule out

hyperparathyroidism or pheochromocytoma. Manifestations of a MEN2 can then be

diagnosed and treated at an early stage.

When a RET-mutation is established in a patient, genetic screening of family members is

necessary to offer carriers – including children – a prophylactic thyroidectomy.7 The age of

children undergoing such a procedure varies between one and ten year and depends on the

mutation. Early recognition and adequate treatment of MTC in this way can prevent severe

morbidity and mortality.

Treatment

Primary tumour

The treatment of MTC is primary surgical and consists of a total thyroidectomy and possible

additional lymph node dissection.8 The extent of the lymph node dissection depends on the

expansion of the primary tumour and the presence of lymph node or distant metastasis.

Recurrence

The treatment of locoregional recurrent disease is also surgical. If there is curative intent an

extensive systematic central and lateral lymph node dissection is performed. If there are

distant metastases, the procedure is less extensive and more focussed on locoregional control,

and locoregional radiotherapy can be given.9 Iodine-131 can be given if there is proven uptake

of this tracer.1 Another option for therapy is radioactive labelled octreotide.10 However both

Chapter 2

24

therapies have modest results. Currently no effective systemic treatment is available and only

treatment in a clinical trial is advised.2

Follow-up

Measurement of serum calcitonin and CEA levels are important in the follow-up of patients

with MTC. A raised or raising tumour marker indicates local recurrent disease or metastasis.

Additional morphological and functional imaging can determine localisation, after which

possible treatment can be given.

Prognosis

The 10 year survival rate of MTC is around 75%.1 The most important prognostic factors are

the extent of the primary tumour at the time of diagnosis and the presence of lymph node or

distant metastasis. Despite the wide spectrum of available diagnostic modalities, MTC is often

diagnosed in a late stadium and survival has barely increased during the last decades.1

New therapeutic options

A large proportion of sporadic MTC patients has persistent disease activity with locoregional

recurrent disease and distant metastasis. Tyrosine kinase inhibitors might give these patients

new perspectives. These drugs target tyrosine kinase mediated signal transduction in

malignant C-cells. The RET receptor is a tyrosine kinase which is active in a large proportion

of the MTC patients, causing uninhibited proliferation of C-cells.1,7 The RET-receptor might

therefore be a good target for this antiproliferative therapy.

Multikinase inhibitors

Multikinase inhibitors like vandetanib and XL-184, which not only target the RET-receptor

but also other receptors like the vascular endothelial growth receptor (VEGFR) and the

mesenchymal-epithelial-transitionfactor (MET)-receptor are promising, with reported tumour

responses in 20%-33% of patients and stable disease in 25%-53%.11,12 However, in a

proportion of patients no effects have been seen. Therefore development of new drugs or

combination therapy is necessary.1,7


25

Conclusion

MTC is a rare tumour with different presentations. Because an apparent sporadic MTC can be

the first manifestation of a MEN2 syndrome, pheochromocytoma and hyperparathyroidism

have to be ruled out preoperatively through biochemical analysis. Furthermore RET-mutation

analysis is recommended, at least in patients under the age of 50 and in patients with a clinical

suspicion. In this case, family members can also be screened and prophylacticly treated if a

mutation is found. MTC patients are preferably treated in a multidisciplinary centre with

extensive experience in thyroid surgery, endocrinology, genetics and nuclear medicine.

Chapter 2

26

References





2000;88:1139-1148.

3. Milos IN, Frank-Raue K, Wohllk N, et al. Age-related neoplastic risk profiles and penetrance

estimations in multiple endocrine neoplasia type 2A caused by germ line RET Cys634Trp (TGC>TGG)

mutation. Endocr Relat Cancer 2008;15:1035-1041.

4. Links TP, Huysmans DA, Smit JW, et al. Guideline 'Differentiated thyroid carcinoma', including

diagnosis of thyroid nodules. Ned Tijdschr Geneeskd 2007;151:1777-1782.

5. Costante G, Durante C, Francis Z, Schlumberger M, Filetti S. Determination of calcitonin levels in C-cell

disease: clinical interest and potential pitfalls. Nat Clin Pract Endocrinol Metab 2009;5:35-44.

6. Koopmans KP, de Groot JW, Plukker JT, et al. 18F-dihydroxyphenylalanine PET in patients with

biochemical evidence of medullary thyroid cancer: relation to tumor differentiation. J Nucl Med

2008;49:524-531.



8. de Groot JW, Links TP, Sluiter WJ, Wolffenbuttel BH, Wiggers T, Plukker JT. Locoregional control in

patients with palpable medullary thyroid cancer: results of standardized compartment-oriented

surgery. Head Neck 2007;29:857-863.

9. Kebebew E, Kikuchi S, Duh QY, Clark OH. Long-term results of reoperation and localizing studies in

patients with persistent or recurrent medullary thyroid cancer. Arch Surg 2000;135:895-901.

10. Kwekkeboom DJ, de Herder WW, Kam BL, et al. Treatment with the radiolabeled somatostatin analog

[177 Lu-DOTA 0,Tyr3]octreotate: toxicity, efficacy, and survival. J Clin Oncol 2008;26:2124-2130.

11. Wells SA,Jr, Gosnell JE, Gagel RF, et al. Vandetanib for the treatment of patients with locally advanced

or metastatic hereditary medullary thyroid cancer. J Clin Oncol 2010;28:767-772.

12. Kurzrock R, Sherman S, Hong D, et al. A phase 1 study of XL184, a MET, VEGFR2, and RET kinase

inhibitor, administered orally to patients (pts) with advanced malignancies, including a subgroup of pts

with medullary thryoid cancer (MTC). EORTC 2008:119.

Chapter 3

Calcitonin testing for detection of medullary thyroid

cancer in patients with thyroid nodules

Hans H.G. Verbeek, Jan Willem B. de Groot, Wim J. Sluiter, Anneke C. Muller Kobold

Edwin R. van den Heuvel, John T.M. Plukker, Thera P. Links

Protocol published in: Cochrane Database of Systematic Reviews 2012, Issue 10

Review submitted

Chapter 3

28

Abstract

Background Thyroid nodules are very common. Calcitonin is a sensitive tumour marker for

the detection of medullary thyroid carcinoma (MTC). Although the European Thyroid

Association's guideline advocates calcitonin determination in patients with thyroid nodules,

the role of routine calcitonin testing in patients with thyroid nodules is still debatable.

Objectives The objective of this review was to determine the diagnostic accuracy of

calcitonin testing in the detection of MTC in patients with thyroid nodules.

Search methods We searched The Cochrane Library, MEDLINE, EMBASE and Web of

Science from inception to March 2013.

Selection criteria We included all retrospective and prospective cohort studies in which all

patients with thyroid nodules had undergone determination of basal calcitonin levels (and

stimulated calcitonin, if performed).

Data collection and analysis Two review authors independently scanned all retrieved records.

Data was extracted by using a standard data extraction form. We assessed risk of bias and

applicability using the QUADAS-2 (quality assessment of diagnostic accuracy studies) tool.

We obtained summary estimates of the expected operating points (sensitivity and specificity)

for each threshold using the HSROC model.

Main results In 16 studies, 73052 patients with nodular thyroid disease were identified.

Prevalence of MTC was 0.26% (n=187). Summary estimates of sensitivity and specificity for

basal calcitonin testing were 99.2% (95% CI 96.4%-100%) and 98.7% (95% CI 97.5%-100%)

respectively. For stimulated calcitonin testing sensitivity was slightly lower (98.5%; 95% CI

93.9%-100%) while specificity was higher (99.9%; 95% CI 99.7%-100%). The positive

predictive value (PPV) of basal calcitonin testing was 7.5% and for stimulated calcitonin

testing 72%.

Authors' conclusions Both basal and stimulated calcitonin testing have a high sensitivity and

specificity. The value of routine testing in patients with thyroid nodules remains questionable,

due to the low PPV of basal calcitonin testing. Whether routine calcitonin testing improves

prognosis in MTC patients remains unclear.

Calcitonin for detection of MTC

29

Background

Thyroid nodules are very common in the general population, and they can be found in 2.3% to

6.9% of all adults.1-3 Ultrasound detects an even higher frequency of thyroid nodules (17% to

69%).4 Thyroid nodules are more prevalent in women than in men (1.5% to 2% vs. 6.4% to

10%) and the incidence increases with age.1,5,6 Of all patients with thyroid nodules who

undergo fine needle aspiration (FNA), approximately 7.7% to 12% have thyroid cancer and in

3.3% to 3.7% of these patients medullary thyroid cancer (MTC) will be diagnosed.7-11

MTC is a neuro-endocrine tumour originating from the parafollicular C-cells. These C-

cells secrete calcitonin, a 32-amino acid peptide, which can be used as a sensitive tumour

marker. The 10-year survival for MTC is about 75%, but the prognosis depends on the extent

of the primary tumour, the presence of nodal disease and distant metastases.12 The primary

treatment for MTC is surgery, consisting of a total thyroidectomy with central compartment

dissection and even more extensive lymph node dissection depending on the extent of the

disease. Some patients develop recurrent disease, which limits the therapeutic options.

Patients with progressive disease may benefit from newly developed targeted therapies,

although early diagnosis of MTC and adequate surgical treatment remain crucial for a

favourable prognosis.13

Calcitonin is elevated in virtually all MTC patients and therefore a very sensitive tumour

marker, although MTC does not always produce calcitonin.14,15 On the other hand

hypercalcitoninaemia can also be caused by other conditions such as thyroiditis, sepsis,

hypercalcaemia, hypergastrinaemia, other neuroendocrine tumours, chronic renal failure,

chronic pulmonary disease, acute trauma, inhalation injury and pseudohypoparathyroidism.16-

19

In the recent guidelines of the American Thyroid Association (ATA) the diagnostic work-

up of a thyroid nodule consists, after history, physical examination and TSH determination, of

a diagnostic ultrasound and FNA when a nodule is seen on ultrasound. The role of calcitonin

testing in the work-up of thyroid nodules is unclear and there is no clinical consensus on

calcitonin testing. While the ATA's revised evidence-based guidelines for thyroid cancer do

not recommend for or against calcitonin determination, the European Thyroid Association's

consensus-based guideline advocates calcitonin determination in all patients with thyroid

nodules.20-22 Based on these guidelines and several studies, routine calcitonin testing is

practiced in multiple centres, while the use remains disputed.

Chapter 3

30

Despite the high sensitivity and specificity, only a small number of patients with elevated

calcitonin levels have MTC. This is due to the low prevalence of MTC. Accordingly, the

positive predictive value (PPV) in most studies is low, although some studies do report PPVs

of up to 100%.23 Furthermore, the cut-off level of calcitonin has not yet been established and

there are indications that different subgroups of patients need specific cut-off points, since

there are gender specific cut-off levels.24 Perhaps only a subset of patients should undergo

calcitonin testing. It is also unclear whether calcitonin testing can contribute to longer overall

survival or will increase the quality of life of MTC patients. Finally, to determine its role in

the evaluation of thyroid nodules the cost-effectiveness of calcitonin testing is also

important.25,26

Role of calcitonin testing

There are several potential roles for calcitonin testing in the diagnostic work-up of thyroid

nodules (Figure 1). First it can be used as a screening tool. Screening, however, implies that

the entire healthy population will undergo determination of calcitonin, which is currently not

effective or clinically relevant. Therefore we focus only on calcitonin testing in patients with

thyroid nodules, detected through palpation or ultrasound. It can be performed in all patients

with thyroid nodules at an early stage and before FNA (Figure1: I). In this case the supposed

sensitivity is very high but a great number of patients will have false positive results which

might lead to unnecessary surgery (resulting in life-long thyroid hormone supplementation

and risk of recurrent nerve damage and hypoparathyroidism). As FNA is also commonly used

for diagnosing other types of thyroid cancer which do not secrete calcitonin, calcitonin testing

as a replacement for FNA is irrational and clinically not relevant.

Calcitonin testing can be used as an add-on test after FNA in patients with suspicious or

indeterminate cytology (Figure 1: II). In this case the number of false positives will be lower,

but some MTC patients might be missed (when cytology is benign) with the risk that MTC in

these patients will be diagnosed at a later stage or not at all. Calcitonin testing can also be

used as a preoperative test in all patients who will undergo thyroid surgery (Figure 1: III). In

that case not all MTC patients will be detected but the risk of patients who undergo an

operation receiving too restricted surgery decreases. This form of calcitonin testing will not be

included in this review as it is more focused on preoperative assessment of tumour type than

on screening.


31

This review will address the value of calcitonin testing for diagnosing MTC in patients

with thyroid nodules for the triage and add-on roles of the calcitonin test. We want to give

more insight into the different sensitivities and specificities for these different roles. By

providing data on the diagnostic accuracy of the calcitonin test in light of the low prevalence

of MTC in thyroid nodules we want to contribute to the discussion on the role of the

calcitonin test in patients with thyroid nodules.

Figure 1 Possible roles of calcitonin testing

Chapter 3

32

Index tests

The available test for diagnosing MTC in thyroid nodules is the calcitonin assay. The former

radioimmunoassays for calcitonin measurement recognised the monomeric and the dimeric

form of calcitonin, as well as its precursors leading to false-positive results. The more recent

and most commonly used immunometric assays mainly recognise the mature, monomeric

form of calcitonin. They rely on a 'sandwich' formation by two monoclonal or polyclonal

antibodies recognising different epitopes on calcitonin.27 However, limitations still exist in the

calcitonin assays. If a one-step assay is applied, in case of an extremely high calcitonin

concentration, all the antibodies including the signal antibodies are saturated with the antigen,

preventing a sandwich formation. Then, the antigen concentration measured may be falsely

low (also known as the ‘high dose hook’).28 Furthermore, also mainly in one-step assays, the

presence of heterophilic antibodies may give erroneously high results of calcitonin by cross-

linking the antibodies in the absence of calcitonin.29,30 Very rarely ‘blocking’ heterophilic

antibodies are also able to produce false-negative results.31 Alternative methods for

quantification, such as mass spectrometry may circumvent this problem, as was also shown

for thyroglobulin.32 Furthermore, despite the World Health Organization international

reference preparation for human calcitonin, differences exist between the same type of assays

of different manufacturers, making it even more difficult to compare results from different

studies and to establish an optimal cut-off value.27,33,34

To improve the specificity of the calcitonin assay, calcitonin stimulation tests with

pentagastrin or calcium are used.35 These stimulation tests can distinguish calcitonin secreted

by MTC from other sources of calcitonin but there are some limitations.36 Stimulation with

pentagastrin can induce unpleasant side effects, such as nausea, vomiting or skin rash.37

Furthermore, pentagastrin is not available in several countries. Calcium stimulation tests are

better tolerated but are not routinely used although an increasing number of small studies have

advocated the use of calcium.38-40 We planned to perform a heterogeneity analysis on whether

basal calcitonin, stimulated calcitonin, or both, were determined and also the type of

stimulation test used.

Alternative tests

The alternative test for diagnosing MTC in patients with thyroid nodules is fine needle

aspiration cytology (FNAC) with eventually immunohistochemical examination in suspicious

lesions. FNAC is an accurate and cost-effective method for evaluation of thyroid nodules, but


33

the sensitivity for diagnosis of MTC is not optimal, ranging from 63% to 89%.41-43 The

outcome of the FNAC in these studies resulted in surgery in 91% to 100% of patients.

Although a large proportion of the patients received surgery despite incorrect FNAC results,

this might be an inadequate test as MTC requires a different surgical approach than

differentiated thyroid cancer. Other techniques, such as measuring calcitonin levels in

washout fluids of fine needle aspirates may improve accuracy but few studies have reported

on this in limited numbers of patients.44-46

Rationale

A number of studies and reviews on this topic advocate calcitonin testing for detection of

MTC. These studies are hard to compare, however, since they have different inclusion criteria

and different cut-off points for calcitonin levels. Moreover, there is no consensus between the

American and European guidelines on thyroid nodules. Calcitonin testing in patients with

thyroid nodules is associated with a high rate of false-positive results and a low PPV. It has

not been established that calcitonin testing reduces MTC-related mortality in these patients.

Cheung et al. stated that calcitonin testing in the US is cost-effective at the same level as

mammography screening and advocates calcitonin testing in subgroups of patients such as

young men with larger thyroid nodules, but this also remains a matter for debate.26

Objectives

The objective of this review was to determine the diagnostic accuracy of calcitonin testing in

the detection of MTC in patients with thyroid nodules.

Investigation of sources of heterogeneity

We planned to investigate several potential sources of heterogeneity, including differences in

cut-off values, assay types and different verification methods. Possible factors that were

evaluated as source for heterogeneity were;

• Age.

• Gender.

• Nodules detected by palpation or ultrasound.

• Nodule size.

• Number of nodules.

• Sonographic morphology of thyroid nodules.

Chapter 3

34

• FNA procedures performed through ultrasound guidance versus palpation.

• Basal versus stimulated calcitonin testing.

Methods

Criteria for considering studies for this review

Types of studies

We included all retrospective and prospective cohort studies in which all patients with thyroid

nodules had undergone determination of basal calcitonin levels (and stimulated calcitonin, if

performed).

Participants

We included patients with nodular thyroid disease (defined as solitary thyroid disease

(toxic/non-toxic), multinodular thyroid disease (toxic/non-toxic), autonomously functioning

thyroid nodule) found by palpation or on ultrasound in whom calcitonin testing was

performed. We distinguished between studies in which calcitonin testing was performed as a

triage (before FNAC) or as an add-on test (after FNAC). We included patients with coexisting

non-nodular disease such as autoimmune thyroid disease (Graves' disease or Hashimoto's

thyroiditis) and subacute thyroiditis. We excluded patients with only non-nodular thyroid

disease. If studies included both patients with nodular and non-nodular disease, we included

them only if it was possible to separate the calcitonin levels and surgical outcomes of these

patient groups or if fewer than 10% of patients had non-nodular disease. We excluded patients

with known sporadic or familiar MTC (MEN2A/B, FMTC) prior to calcitonin screening. We

also excluded studies that included these patients and did not describe them separately.

Index tests

The index tests for this review included all serum tests used to determine basal and stimulated

serum calcitonin levels.

Target conditions

The target condition was MTC.


35

Reference standards

The optimal clinical reference standard for diagnosis of MTC was considered

histopathological examination of the thyroid after surgery of all patients, even patients

without elevated calcitonin levels. In all of the studies, however, the problem of differential

verification was encountered and only patients with (markedly) elevated calcitonin levels or

patients with suspicious cytology had histological verification (although some patients did

undergo surgery for other reasons e.g. mechanical complaints due to a multinodular goitre).

We planned therefore to make use of other reference standards such as clinical follow-up. A

follow-up of at least three years was considered adequate as most clinically relevant MTCs

will be identified at that time, while longer follow-up carries the risk that MTC patients are

diagnosed while not having the disease at the time of calcitonin testing. To determine whether

standard of verification significantly influences accuracy, we planned to include method of

verification in the heterogeneity analysis.

Search methods for identification of studies

Electronic searches

We used the following sources for the identification of trials.

• The Cochrane Library.

• MEDLINE.

• EMBASE.

• Web of Science.

For detailed search strategies please see Appendix 1. The Editorial Base of the Cochrane

Metabolic and Endocrine Disorders Group provided support for generating the optimal search

strategy. We used PubMed's 'My NCBI' (National Centre for Biotechnology Information)

email alert service for the identification of newly published studies using a basic search

strategy (see Appendix 1). We included studies published in English language.

Searching other resources

We examined the references lists of relevant publications for additional studies. We searched

in Pubmed for related articles of relevant studies.

Chapter 3

36

Data collection and analysis

Selection of studies

To determine the studies to be assessed further, two review authors (HHGV, JWBG)

independently scanned the abstract, title or both sections of every record retrieved. All

potentially relevant articles were investigated as full text. Any disagreements were resolved

by a third reviewer (TPL). A PRISMA (preferred reporting items for systematic reviews and

meta-analyses) flow-chart of study selection was made.47

Data extraction and management

We extracted data on study design and study population using a standard data extraction form

(Appendix 2), in which we included the following items:

• Study design.

• Included number of patients.

• Inclusion and exclusion criteria.

• General patient characteristics.

• Type of calcitonin assay and cut-off values.

• Number of patients with nodular thyroid disease.

• Number of patients with palpable nodules and/or nodules on ultrasound.

• Number of patients who had undergone calcitonin testing and number of positive

patients.

• Number of patients operated and reason for operation.

• Number of patients with known follow-up and outcome of follow-up.

• Histological outcome of patients operated.

• Number of patients with MTC.

Assessment of methodological quality

We assessed risk of bias and applicability using the QUADAS-2 (quality assessment of

diagnostic accuracy studies) tool. We rated each of the four key domains (patient selection,

index test, reference standard, flow and timing) using the signalling questions as developed by

the QUADAS-2 group.48 The criteria for each signalling question are provided in Appendix 3.

We scored all items in the QUADAS-2 tool as ‘yes’, ‘no’ or ‘unclear', and used graphs to

present overall scores of risk of bias and applicability for each domain.


37

Statistical analysis and data synthesis

We incorporated true positives, false positives, true negatives and false negatives of each

study in a 2x2 table and calculated test sensitivity and specificity with corresponding 95%

confidence intervals. For extraction of data, we used pre-specified cut-offs based on previous

literature with different cut-offs for basal and stimulated calcitonin levels. These cut-off

values were 10,15, 20, 30, 50 and 100 pg/ml for basal calcitonin levels and 100 pg/ml and 200

pg/ml for stimulated calcitonin levels. We entered the data into RevMan 5.2.3, to graphically

present coupled forest plots, showing the pairs of sensitivity and specificity of each study, for

each threshold.

Investigations of heterogeneity

We used SAS software for meta-analysis. We obtained summary estimates of the expected

operating points (sensitivity and specificity) for each threshold using the HSROC model.49

Depending on the number of included studies and available data, covariates were added in this

model, for investigation of possible sources of heterogeneity.

Sensitivity analyses

We performed sensitivity analyses on the different domains scored on the QUADAS-2 tool, in

order to explore the influence of the quality of the included studies.

Results

Results of the search

A total of 2947 unique records were identified by our search in January 2012 and updated

searches in June 2012 and March 2013. An additional two records were identified by

examining references list of relevant publications. One other relevant publication was also

included. Screening of all records resulted in 35 publications that were eligible for further

evaluation. After assessment 19 articles were excluded. Eventually 16 studies were included

in this review (Figure 2).

Chapter 3

38

Included studies

Characteristics of the 16 included studies are shown in the table Characteristics of included

studies (Appendix 4).50-65 A total of 73052 patients with nodular thyroid disease were

included in these studies, of which 72368 underwent basal calcitonin testing with or without

stimulated calcitonin testing as shown in Table 1. A total of 187 MTC patients were

identified. Three studies performed only basal calcitonin testing, whereas in thirteen studies

both basal and stimulated calcitonin testing was performed.

Figure 2 Study flow diagram.

Calcitonin assays

Two studies used an radio immunometric assay (RIA) for determination of calcitonin,

including one study which during the study period switched from a RIA assay to an

immunoradiometric assay (IRMA).50,51 Five other studies used also an IRMA assay.52,54,56,60,61


39

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Chapter 3

40

Two of these five studies switched during the study period to a chemiluminescence assay

(ICMA).56,61 The remaining nine studies used an ICMA assay.53,55,57-59,62-65 In conclusion,

thirteen studies used only one calcitonin assay during their study period, while three studies

used two assays. One of these three studies, that switched from an IRMA to an ICMA assay,

used the ICMA assay only in 14 out of 702 patients, and was therefore in further analyses

regarded as using an IRMA assay.61 The other two studies that switched from calcitonin assay

were not included in the covariate analysis regarding assay type.

In total, calcitonin assays of nine different manufacturers were used (Appendix 4;

Characteristics of included studies). Especially in the seven studies using a RIA or IRMA

assay a large heterogeneity in manufacturers was present (n=7); some studies used during the

study period assays from 2 different producers. Within the nine studies using a ICMA assay,

one study did not report the manufacturer,65 while in the other studies an assay was used from

one of two producers.

Verification method

Differential verification was present in all studies; all patients with a (highly) elevated basal

and/or stimulated calcitonin underwent surgery, while only a subset of patients with negative

calcitonin tests had surgery. We considered clinical follow-up of calcitonin negative patients

as an appropriate alternative for detection of missed MTC patients. However, none of the

included studies did report on clinical follow-up of all of their calcitonin negative patients.

Only in the study of Hasselgren follow-up was performed that consisted of cross linkage with

a national thyroid cancer database.61

Calcitonin as triage or add-on test

None of the studies included, provided explicit information on the role of calcitonin testing in

the diagnostic pathway of thyroid nodules. In nine studies FNA was described in the materials

and methods section as part of the diagnostic protocol. Most of these studies stated that

surgery was indicated if basal or stimulated calcitonin was clearly elevated (e.g. >100 pg/ml)

regardless of the results of FNA. In these studies the role of calcitonin testing can be

considered as a triage test in which calcitonin positive patients are subjected to surgery, while

calcitonin negative patients require more diagnostic work-up in the form of FNA. In all

studies in which FNA was not described in the diagnostic protocol, calcitonin testing was

performed in all included patients, independent of another diagnostic procedure, and if


41

markedly elevated an indication to perform surgery. Therefore in these studies calcitonin

testing was also regarded as a triage test.

Patient and study characteristics

Average and/or median age was described in twelve studies, but only one study reported the

results of calcitonin testing specified in different age groups.58 Information on gender of the

included patients was provided in 15 studies, although only in seven studies detailed

information on outcome was given for both sexes. In nine studies information was available

on whether thyroid nodules in the included patients were detected through palpation or US;

four studies included patients with thyroid nodules found by US and five studies included

patients with thyroid nodules detected through US or palpation. With regard to nodule size

only one study provided information on summary measures of size for the included patients,

although no detailed information was provided for patients with elevated calcitonin levels. No

study presented information on number of nodules or US morphology of all patients. In four

studies information was given on whether FNA procedures were performed through palpation

or US; in one study both techniques were performed, in the three others US-guided FNA was

performed.

Excluded studies

In the table Characteristics of excluded studies (Appendix 5) reasons for exclusion for the 19

excluded studies are shown. Of six studies only a meeting abstract was available and no full

text article was published.66-71 Four articles were written in non-English language.72-75 Two

studies used a study population that was also described in a later publication.76,77 Three

studies did not specify the numbers of patients with thyroid nodules.78-80 Three studies

reported on calcitonin testing in pre-operative patients, which is not the topic of this review.81-

83 One study reported on calcitonin testing in isthmic thyroid nodules; because this patient

group evaluated only nodules in a specific part of the thyroid, we excluded this study.84

Methodological quality of included studies

In Figure 3 the overall quality of the 16 included studies is shown, with regard to the risk of

bias and concerns about applicability scored according to the QUADAS 2 domains. In the

domain Patient selection, one study scored high on the risk of bias as patients were included

who showed evidence of growth during follow-up examinations.53 This might have increased

Chapter 3

42

the rate of included patients with a malignancy. In all studies the risk of bias by the

conduction or interpretation of the calcitonin test was scored low. The risk of bias with the

conduct or interpretation of the reference standard was unclear in all studies, for the reference

standard in calcitonin negative patients, was not described. Due to this lack of reference

standard, resulting in a verification bias, the risk of bias with regard to flow and timing was in

all studies expect one regarded as high. In the only study using a cross linkage with a national

thyroid cancer database the risk was scored as unclear.61 No concerns of applicability existed

in all studies. In Figure 4 the individual quality assessment of all studies can be found.

Figure 3 Risk of bias and applicability concerns graph: review authors' judgements about each domain

presented as percentages across included studies

Findings

The sensitivity of the reported basal calcitonin testing cut-off in the included studies ranged

from 83% to 100%, while the specificity ranged from 94% to 100% (Figure 5 and Figure 6).

In the study of Schuetz et al. no MTC patients with nodular thyroid disease were identified so

sensitivity could not be calculated.57 The summary estimates of sensitivity and specificity

were 99.2% and 98.7% respectively (95% Confidence intervals (CI) 96.4%-100% and 97.5%-

100% respectively).

Effect of cut-off value

We extracted data for several cut-off values from the included studies, ranging from 10 pg/ml

to 100 pg/ml. With the cut-off value of 10 pg/ml the sensitivity of the individual studies

ranged from 92% to 100% while specificity ranged from 95% to 99%; summary estimates

were 99.9% (95% CI: 99.1-100%) for sensitivity and 96.8% (95% CI: 95.5%-98.1%) for

specificity (Table 2). With the highest cut-off value of 100 pg/ml sensitivities ranged from

42%-100% and specificity from 95%-100%. Summary estimates of sensitivity and specificity


43

for the different cut-off values of basal calcitonin >10 pg/ml could not be calculated due to

limited number of studies.

Figure 4 Risk of bias and applicability concerns summary: review authors'

judgements about each domain for each included study

Effect of stimulated calcitonin

Thirteen studies were included in the analysis of stimulated calcitonin. Sensitivity of

individual studies ranged between 82% and 100%. Specificity ranged from 99% to 100%

(Figure 7). Summary estimates were 98.5% (95% CI 93.9%-100%) for sensitivity and 99.9%

(95% CI: 99.7%-100%) for specificity, respectively (Summary of findings Table 1, Figure 8).

Chapter 3

44

Due to limited numbers of studies no summary estimates could be calculated for different cut-

off values.

Table 1 Summary of findings

Summary points (95% CI) No. of patients (studies)

Basal calcitonin

Reported cut-off value Sensitivity 99.2% (96.4%-100%)

Specificity 98.7% (97.5%-99.9%)

72369 (16)

10 pg/ml Sensitivity 99.9% (99.1%-100%)

Specificity 96.8% (95.5%-98.1%)

44393 (10)

Basal and stimulated reported calcitonin

Reported cut-off value Sensitivity 98.5% (93.9%-100%)

Specificity 99.9% (99.7%-100%)

69702 (13)

Subgroup analysis

Gender

Female

Male

Sensitivity 95.9% (83.1%-100%)

Specificity 99.0% (97.6%-100%)

Sensitivity 100% (NA-NA*)

Specificity 98.2% (95.9%-100%)

14858 (6)

4339 (6)

Figure 5 Forest plot of basal reported cut-off values.


45

Figure 6 Summary ROC Plot of basal reported cut-off values.

Effect of gender

In Summary of findings Table 1 summary estimates for basal reported calcitonin for

subgroups of females and males are reported. For females the sensitivity ranged between 96%

and 100% and the specificity ranged between 96% and 100%. For males the sensitivity ranged

between 82% and 100% and the specificity ranged and between 90% and 100% (Figure 9).

Summary estimates for females were 95.9 % (95% CI 83.1%-100%) for sensitivity and 99.0%

(95% CI: 97.6%-100%) for specificity. Summary estimates for males were 100% (95% CI

NA) and 98.2% (95% CI: 95.9%-100%) for sensitivity and specificity respectively. Only one

study used gender specific basal calcitonin cut-off values for all included patients.53 Another

study which used two assays during the study period also had a gender specific cut-off for the

second assay, but this concerned only 14 patients.61 The study of Rink et al. used gender

specific stimulated calcitonin cut-off values.60 Due to limited number of studies no summary

estimates could be calculated for different cut-off values.

Chapter 3

46

Figure 7 Forest plot of basal and stimulated reported cut-off values.

Effect of assay type, detection method of thyroid nodules and FNA method

Due to limited number of studies no summary estimates could be calculated for subgroups

with respect to assay type or manufacturer, detection method of thyroid nodules and method

of FNA.

Figure 8 Summary ROC Plot of basal and stimulated reported cut-off values.


47

Sensitivity analysis

We planned to perform a sensitivity analysis with regard to quality items scored with the

QUADAS 2 tool. However, no large differences were seen between studies regarding quality

items. Only two studies had aberrant scores with regard to the risk of bias on the domains of

patient selection and flow and timing (Figure 4).

Figure 9 Forest plot of basal calcitonin reported cut-off values for females and males.

Chapter 3

48

Discussion

Summary of main results

In this review we included 16 studies for determination of the diagnostic accuracy of

calcitonin testing in patients with thyroid nodules. We found high summary estimates of

sensitivity and specificity for the reported basal calcitonin cut-off value of all studies. For

reported basal and stimulated calcitonin sensitivity was slightly lower and specificity slightly

increased. In subgroup analysis, sensitivity in females was lower, while specificity was higher

compared to males.

Strengths and weaknesses of the review

We evaluated the diagnostic accuracy of calcitonin testing for detection of medullary thyroid

cancer in patients with thyroid nodules with a comprehensive search of literature, and

performing a formal diagnostic meta-analysis.

One of the major limitations of this review is the lack of adequate reference standards for

calcitonin negative patients in nearly all included studies. This increases the risk that false

negative patients are missed and the reported sensitivities are overestimated. In two of the

included studies false negative MTC patients were identified, although in the study of

Vierhapper et al. two of the three false negative patients were not operated and a histological

diagnosis was not obtained.56 In studies performing preoperative calcitonin testing, the rate of

false negative MTC patients ranged between 4.3%- 12.5% of all MTC patients identified.18,83

Because the prevalence of MTC is low in patients with thyroid nodules, even a small number

of false negative patients can markedly affect sensitivity. The clinical relevance of these false

negative MTC can be discussed as in most calcitonin testing studies this are patients with a

micro MTC without nodal metastasis. However, reports also exist on MTC patients with more

aggressive disease and undetectable calcitonin levels.85

Another limitation of this study is the small number of studies that could be included in

final analyses. Due to this small number only for a few subgroups summary estimates could

be calculated. Furthermore, no optimal cut point could be assessed due to the heterogeneity of

the used cut-off levels in the different studies. This was further complicated by the large

heterogeneity in assay types and manufacturers.


49

Other reports

Other reviews have been published on the value of calcitonin testing in the detection of

medullary thyroid carcinoma, although no systematic reviews were performed. Daniels

provided an overview of 15 studies but included also patients with preoperative calcitonin

testing and multiple studies of one study group. The author concluded that due to the large

reservoir of undetected of medullary thyroid micro carcinoma's of uncertain malignant

potential, and the unavailability of pentagastrin in the US, calcitonin testing is not indicated in

the United States and Canada.86 Costante et al. evaluated 11 studies in their review and

concluded that the question whether to routinely measure calcitonin remained unsolved

because no evidence exists whether testing actually reduces MTC-related mortality.87

Applicability of findings to the review question

This review provides summary estimates of sensitivity and specificity for basal and stimulated

calcitonin. The role of calcitonin testing in the diagnostic evaluation in thyroid nodules

remains unclear. The final purpose of calcitonin testing is to detect MTC patients in an early

stage, in which the chance of biochemical cure improves and the prognosis of patients. The

findings of this review indicate that calcitonin testing is a very sensitive and specific test, but

this has to be interpreted bearing in mind the low prevalence of MTC. The positive predictive

value of calcitonin testing is therefore low, especially with lower cut-off values. Although

several conditions are known to cause increased calcitonin levels, still in a fairly large

proportion of patients with elevated calcitonin levels MTC cannot be excluded. Repeated

calcitonin testing and follow up in these patients is therefore warranted. A number of these

patients will be operated without histological evidence of a MTC. Some patients will have C-

Cell hyperplasia, but the clinical relevance of this finding and its malignant potential remain

unclear.

Cost effectiveness in health care becomes more and more important. In this review no

formal cost effectiveness analysis is performed, so no validated statements can be made.

However, Cheung et al performed in 2008 a cost effectiveness analysis in which calcitonin

testing was concluded to be cost effective similar to colonoscopy and mammography

screening.26 In their hypothetical model, several parameters had an important influence on

cost-effectiveness, such as specificity of the calcitonin test and prevalence of MTC. Cheung et

al. used a cut-off value of 50 pg/ml with a specificity of 98 % in the base line model, almost

similar to the 98.7% specificity we found of the basal calcitonin test. However, the MTC

Chapter 3

50

prevalence established in our review was 0.26%; one third of the prevalence used by Cheung

et al. With a three times lower prevalence, costs will also increase almost three times. In a

review of autopsy studies the prevalence of occult MTC was estimated to be 0.14%.88

Although it is not known if all MTC's detected at autopsy are clinically irrelevant and will be

detected through calcitonin screening, a proportion of these tumours will be, further lowering

the prevalence of clinically relevant MTC's. Our findings with regard to sensitivity, specificity

and MTC prevalence, applied to the cost-effectiveness model of Cheung et al. imply that

basal calcitonin testing does not seem to be cost effective. The effects on cost-effectiveness of

a combined basal and stimulated calcitonin on cost effectiveness is more difficult to estimate.

In their model Cheung et al. give a sensitivity and specificity for this combined approach of

80% and 98% respectively while our summary estimates show both a higher sensitivity and

specificity. However, it is likely that also this model is influenced by the prevalence of MTC,

decreasing cost-effectiveness with lower prevalence.

Implications for practice

Calcitonin testing can be a sensitive and specific instrument for detecting MTC in thyroid

nodules. However, due to the low prevalence of MTC its role as a screening tool remains

unclear. If we apply our findings from a basal calcitonin test with a cut-off of 10 pg/ml to a

population of 10000 patients with a MTC prevalence of 0.26% (mean prevalence of the

included studies), 26 patients will have MTC. All these 26 patients will have an elevated basal

calcitonin test, while 319 patients without MTC also will have an elevated basal calcitonin.

The positive predictive value of the calcitonin test in this situation is 7.5%. Surely not all

patients with an elevated basal calcitonin will be operated, but even if a cause of elevated

calcitonin can be found in 90% of the false positive patients, more than 50% (n=32) of the

patients will be operated unnecessary. Increasing cut-off values results in a higher positive

predictive value but at the cost of missed MTC patients. An optimal cut-off value for is

therefore difficult to generate. Also the variation between assays used in different studies

makes it hard to establish a common cut-off value. Adding a stimulated calcitonin test

increases specificity with a very little effect on sensitivity. Still all 26 patients with MTC will

be detected and only 10 patients will have a false positive stimulated calcitonin test. In this

scenario, the positive predictive value increases almost 10 times to 72%. However, the most

commonly used stimulative, pentagastrin, is not available in several countries.

The major reason to perform calcitonin testing is to ultimately improve prognosis of MTC

patients. The supposed value of calcitonin testing is the detection of MTC patients in an


51

earlier stage in which biochemical cure is still possible. However, to assess this, one has to

know which of the MTC patients would not have been detected trough regular examinations

(US/FNA/optional calcitonin), and which of these detected patients by calcitonin testing have

or will develop a clinically relevant MTC. Some studies demonstrate that survival was

significantly improved after introduction of routine calcitonin testing in patients with thyroid

nodules.54,89,90 However, these studies have made their comparison with a historical group,

and other factors might also have contributed to improved survival. These factors include, for

instance, improved surgical treatment strategies and use of ultrasound. Furthermore, it is also

interesting to note that MTC was supposedly detected at an earlier stage in these studies, but

the age of the screened MTC patients was not lower compared to the patients that were not

screened.54,89 As MTC is considered to be a slow growing tumour which takes several years to

become clinically evident, the fact that MTC patients in the screened groups are of equal or

even higher age, might indicate that additional MTC patients have been detected who would

have otherwise had an indolent course of their disease. Another indication that otherwise

undetected and possibly indolent MTC patients are identified is the increased number of MTC

patients detected in shorter periods in the calcitonin tested patients compared to the historical

cohorts.54,90

Implications for research

This review shows that the diagnostic accuracy of calcitonin testing in MTC is high.

However, this conclusion is based on studies in which the MTC prevalence in calcitonin

negative patients might have been underestimated. Future studies should therefore report

more accurately on the follow-up of calcitonin negative patients, to ensure that no MTC

patients are missed, and also to provide more information on the clinical behaviour of these

tumours. Furthermore accurate reporting of assay type and manufacturer is crucial for

establishing optimal cut-off points for diagnosis of MTC. Also the role of the calcitonin test,

being a triage or add-on test next to FNA (with or without measurement of calcitonin in

washout fluids) should be further evaluated. Furthermore reporting results for subgroups may

also identify subgroups with a higher MTC prevalence in which calcitonin testing can be

more cost effective.

Chapter 3

52

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carcinoma by calcitonin measurement in fine-needle aspiration biopsy specimens. Thyroid

2007;17:635-638.

47. Liberati A, Altman DG, Tetzlaff J, et al. The PRISMA statement for reporting systematic reviews and

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48. Whiting PF, Rutjes AW, Westwood ME, et al. QUADAS-2: a revised tool for the quality assessment of

diagnostic accuracy studies. Ann Intern Med 2011;155:529-536.

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49. Macaskill P. Empirical Bayes estimates generated in a hierarchical summary ROC analysis agreed

closely with those of a full Bayesian analysis. J Clin Epidemiol 2004;57:925-932.

50. Rieu M, Lame MC, Richard A, et al. Prevalence of sporadic medullary thyroid carcinoma: the

importance of routine measurement of serum calcitonin in the diagnostic evaluation of thyroid

nodules. Clin Endocrinol (Oxf) 1995;42:453-460.

51. Ozgen AG, Hamulu F, Bayraktar F, et al. Evaluation of routine basal serum calcitonin measurement for

early diagnosis of medullary thyroid carcinoma in seven hundred seventy-three patients with nodular

goiter. Thyroid 1999;9:579-582.

52. Hahm JR, Lee MS, Min YK, et al. Routine measurement of serum calcitonin is useful for early detection

of medullary thyroid carcinoma in patients with nodular thyroid diseases. Thyroid 2001;11:73-80.

53. Hatzl-Griesenhofer M, Pichler R, Bogner S, et al. Results of calcitonin screening in a Central Upper

Austrian Region. J Endocr Genet 2002;3:75-85.

54. Elisei R, Bottici V, Luchetti F, et al. Impact of routine measurement of serum calcitonin on the

diagnosis and outcome of medullary thyroid cancer: experience in 10,864 patients with nodular

thyroid disorders. J Clin Endocrinol Metab 2004;89:163-168.

55. Karanikas G, Moameni A, Poetzi C, et al. Frequency and relevance of elevated calcitonin levels in

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56. Vierhapper H, Niederle B, Bieglmayer C, Kaserer K, Baumgartner-Parzer S. Early diagnosis and curative

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thyroid disorders. Thyroid 2005;15:1267-1272.

57. Schuetz M, Beheshti M, Oezer S, et al. Calcitonin measurements for early detection of medullary

thyroid carcinoma or its premalignant conditions in Hashimoto's thyroiditis. Anticancer Res

2006;26:723-727.

58. Papi G, Corsello SM, Cioni K, et al. Value of routine measurement of serum calcitonin concentrations in

patients with nodular thyroid disease: A multicenter study. J Endocrinol Invest 2006;29:427-437.

59. Costante G, Meringolo D, Durante C, et al. Predictive value of serum calcitonin levels for preoperative

diagnosis of medullary thyroid carcinoma in a cohort of 5817 consecutive patients with thyroid

nodules. J Clin Endocrinol Metab 2007;92:450-455.

60. Rink T, Truong PN, Schroth HJ, Diener J, Zimny M, Grunwald F. Calculation and validation of a plasma

calcitonin limit for early detection of medullary thyroid carcinoma in nodular thyroid disease. Thyroid

2009;19:327-332.

61. Hasselgren M, Hegedus L, Godballe C, Bonnema SJ. Benefit of measuring basal serum calcitonin to

detect medullary thyroid carcinoma in a Danish population with a high prevalence of thyroid nodules.

Head Neck 2010;32:612-618.

62. Herrmann BL, Schmid KW, Goerges R, Kemen M, Mann K. Calcitonin screening and pentagastrin

testing: predictive value for the diagnosis of medullary carcinoma in nodular thyroid disease. Eur J

Endocrinol 2010;162:1141-1145.

63. Giovanella L, Verburg FA, Imperiali M, Valabrega S, Trimboli P, Ceriani L. Comparison of serum

calcitonin and procalcitonin in detecting medullary thyroid carcinoma among patients with thyroid

nodules. Clin Chem Lab Med 2012:1-5.

64. Schneider C, Kobe C, Schmidt M, et al. Calcitonin screening in patients with thyroid nodules. Diagnostic

value. Nuklearmedizin 2012;51:228-233.

65. Grani G, Nesca A, Del Sordo M, et al. Interpretation of serum calcitonin in patients with chronic

autoimmune thyroiditis. Endocr Relat Cancer 2012;19:345-349.

66. Lepage M, Bounaud MP, Breton I, et al. Calcitonin Determination in Assessment of Cold Thyroid-

Nodules - Prospective-Study on 195 Patients. Revue De Medecine Interne 1992;13:S364-S364.

67. Mariss P, Kammeier A, Thermann M, Emrich D, Raute-Kreinsen U. Calcitonine screening in patients

with nodular goiter for detection of medullary thyroid cancer. Eur J Nucl Med 2001;28:1043-1043.

68. Mariss P, Kammeier A, Thermann M, Emrich D, Raute-Kreinsen U. Untersuchungen des

serumcalcitonin zur diagnostik medullärer schilddrüsenkarzinome bei patienten mit struma nodosa.

Nuklearmedizin 2001;40:A60-A60.

69. Lipp RW, Grohs S, Mader J, Obermaier-Pitsch B, Pieber T, Piswanger-Soelkner J. Routine calcitonin

screening for medullary thyroid cancer detection in an Austrian population. Eur J Nucl Med Mol

Imaging 2011;38:S100.


55

70. Zaplatnikov K, Soukhov V. Nuclear Medicine methods and prognostic significance of calcitonin and

pentagastrin test at diagnosis of sporadic MTC. European Journal of Nuclear Medicine and Molecular

Imaging 2012;39:S594-S594.

71. Marui S, Danilovic DS, Camargo RY, Lando VS, Knobel M, Batista MC. The utility of serum calcitonin

measurement in thyroid nodule evaluation: Experience of a single university hospital in Sao Paulo,

Brazil. Endocr Rev 2012;33.

72. Henry JF, Denizot A, Niccoli P, Gramatica L, Conte-Devolx B, De Micco C. Preoperative diagnosis of

sporadic medullary thyroid carcinoma. Interest of routine measurement of serum calcitonin. Lyon Chir

1995;91:467-472.

73. Henry JF, Denizot A, Puccini M, Niccoli P, Conte-Devolx B, Fie Micco C. Early diagnosis of sporadic

medullary cancer of the thyroid: Contribution of routine calcitonin assay. Presse Med 1996;25:1583-

1588.

74. Lopez-Guzman A, Escola CA, Andia VM, Arranz A, Garcia B, Del Campo AG. Routine calcitonin

measurement in nodular thyroid disease. Endocrinol Nutr 2002;49:222-226.

75. Shong YK, Choi CS, Park HY, Cho BY. Clinical Significance of Routine Measurement of Serum Calcitonin

in Korean Patients with Thyroid Nodules as a Screening test of Sporadic Thyroid Medullary Carcinoma.

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76. Pacini F, Fontanelli M, Fugazzola L, et al. Routine measurement of serum calcitonin in nodular thyroid

diseases allows the preoperative diagnosis of unsuspected sporadic medullary thyroid carcinoma. J

Clin Endocrinol Metab 1994;78:826-829.

77. Vierhapper H, Raber W, Bieglmayer C, Kaserer K, Weinhausl A, Niederle B. Routine measurement of

plasma calcitonin in nodular thyroid diseases. J Clin Endocrinol Metab 1997;82:1589-1593.

78. Kaserer K, Scheuba C, Neuhold N, et al. C-cell hyperplasia and medullary thyroid carcinoma in patients

routinely screened for serum calcitonin. Am J Surg Pathol 1998;22:722-728.

79. Iacobone M, Niccoli-Sire P, Sebag F, De Micco C, Henry JF. Can sporadic medullary thyroid carcinoma

be biochemically predicted? Prospective analysis of 66 operated patients with elevated serum

calcitonin levels. World J Surg 2002;26:886-890.

80. Mirallie E, Iacobone M, Sebag F, Henry JF. Results of surgical treatment of sporadic medullary thyroid

carcinoma following routine measurement of serum calcitonin. Eur J Surg Oncol 2004;30:790-795.

81. Niccoli P, Wion-Barbot N, Caron P, et al. Interest of routine measurement of serum calcitonin: study in

a large series of thyroidectomized patients. The French Medullary Study Group. J Clin Endocrinol

Metab 1997;82:338-341.

82. Gibelin H, Essique D, Jones C, Levillain P, Marechaud R, Kraimps JL. Increased calcitonin level in thyroid

nodules without medullary carcinoma. Br J Surg 2005;92:574-578.

83. Chambon G, Alovisetti C, Idoux-Louche C, et al. The use of preoperative routine measurement of basal

serum thyrocalcitonin in candidates for thyroidectomy due to nodular thyroid disorders: results from

2733 consecutive patients. J Clin Endocrinol Metab 2011;96:75-81.

84. Papi G, Rossi G, Corsello SM, et al. Nodular disease and parafollicular C-cell distribution: results from a

prospective and retrospective clinico-pathological study on the thyroid isthmus. 2010;162:137-143.

85. Frank-Raue K, Machens A, Leidig-Bruckner G, et al. Prevalence and clinical spectrum of nonsecretory

medullary thyroid carcinoma in a series of 839 patients with sporadic medullary thyroid carcinoma.

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86. Daniels GH. Screening for medullary thyroid carcinoma with serum calcitonin measurements in

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88. Valle LA, Kloos RT. The prevalence of occult medullary thyroid carcinoma at autopsy. J Clin Endocrinol

Metab 2011;96:E109-E113.

89. Karga H, Giagourta I, Papaioannou G, et al. Changes in risk factors and Tumor Node Metastasis stage

of sporadic medullary thyroid carcinoma over 41 years, before and after the routine measurements of

serum calcitonin. Metabolism 2010.

90. Alevizaki M, Saltiki K, Rentziou G, et al. Medullary thyroid carcinoma: the influence of policy changing

in clinical characteristics and disease progression. Eur J Endocrinol 2012;167:799-808.

56

Chapter 4

Fewer cancer reoperations for medullary thyroid

cancer after initial surgery according to ATA guidelines

Hans H.G. Verbeek, Johannes A.A. Meijer, Wouter T. Zandee, Kelvin H. Kramp

Wim J. Sluiter, Johannes W. Smit, Job Kievit, Thera P. Links, John T.M. Plukker

Annals of Surgical Oncology 2014 [Epub ahead of print]

Chapter 4

58

Abstract

Introduction Surgery is still the only curative treatment for medullary thyroid cancer (MTC).

We evaluated clinical outcome in patients with locoregional MTC with regard to adequacy of

treatment following ATA guidelines and number of sessions to first intended curative surgery

in different hospitals.

Methods We reviewed all records of MTC patients (n=184) treated between 1980 and 2010

in two tertiary referral centres in the Netherlands. Symptomatic MTC (palpable tumour or

suspicious lymphadenopathy) patients without distant metastasis were included (n=86).

Patients were compared with regard to adequacy of surgery according to ATA

recommendations, tumour characteristics, number of local cancer reoperations, biochemical

cure, clinical disease free survival (DFS), overall survival (OS) and complications.

Results Adherence to ATA guidelines resulted in fewer cancer-related reoperations (0.24 vs.

0.60; p=0.027) and more biochemical cure (40.9% vs. 20%; p=0.038). Surgery according to

ATA-guidelines on patients treated in referral centres was significantly more often adequate.

(59.2% vs. 26.7%; p=0.026). Tumour size and LN+ were the most important predictors for

clinical recurrence (relative risk: RR 4.1 (size >40 mm); 4.1 (LN+) and death (RR 4.2 (size

>40 mm); 8.1 (LN+).

Conclusions ATA-compliant surgery resulted in fewer local reoperations and more

biochemical cure. Patients in referral centres more often underwent adequate surgery

according to ATA-guidelines. Size and LN+ were the most important predictors for DFS and

OS.

Fewer reoperations after ATA compliant surgery

59

Introduction

At present, surgery offers the only possibility of cure for medullary thyroid carcinoma (MTC).

The American Thyroid Association (ATA) guidelines recommend total thyroidectomy and

bilateral lymph node dissection (LND) of the central compartment (level VI) as initial

treatment.1 In case of regional node involvement, lateral compartment LND (level II–V)

should be performed. Consensus with regard to indication, timing and extent (ipsi- or

bilateral) of the lateral LND is lacking.2-4 Adherence to ATA guidelines may improve disease

specific survival (DSS) in patients with locoregional disease.5 However, other studies reported

that extent of cervical lymphadenectomy, although important for staging and locoregional

control, did not increase biochemical cure or survival.6,7

Besides LND extent, both surgeons’ experience and the volume of the centre are important

factors, as they are associated with complication rates and length of hospital stay.8 However,

many MTC patients are still treated primarily in low-volume hospitals, and are at risk of

receiving inadequate initial surgical management.9,10

Patients initially treated in an inadequate manner are usually considered candidates for

completion surgery. One third of these patients may benefit from meticulous surgical

eradication resulting in long-term disease free survival (DFS).11 However, reoperations are

technically challenging with potentially increased morbidity, including hypoparathyroidism

and recurrent nerve damage.11-13

The first aim of this retrospective cohort study was to evaluate ATA recommendations for

primary surgical treatment by comparing clinical outcomes (i.e. number of reoperations,

biochemical cure, survival and complications) after the first curative intended surgical

resection in patients treated with ATA-compliant surgery versus patients treated with non-

ATA compliant surgery (A). Secondly, we investigated whether clinical outcome was

influenced by a one-step (versus two step) intended curative surgical procedure (B) or by the

location of initial curative surgery (experienced centre versus hospital) (C). Finally, we

evaluated the influence of patient and tumour characteristics on clinical outcome.

Chapter 4

60

Materials and methods

Patients and tumour characteristics

The records of all 184 MTC patients treated between 1980 and 2010 in two major tertiary

referral centres in the Netherlands, the University Medical Centre in Groningen (UMCG) and

the Leiden University Medical Centre (LUMC), were reviewed retrospectively. Inclusion

criteria were: histologically proven symptomatic MTC at presentation (palpable tumour

and/or suspicious lymph nodes), available required data, treatment with curative intent and

clinical absence of distant metastases upon diagnosis and within 6 months after initial

intended curative surgery. Sixty-six patients did not meet the above criteria and were

excluded, as were 32 patients with hereditary MTC who had a preventive thyroidectomy

based only on mutation analysis or increased calcitonin levels. Eventually 86 patients were

included for final analyses (Table 1).

Table 1 Patient and tumour related characteristics

Characteristic N (%)

Sex

Female

Male

41 (47.7)

45 (52.3)

Type

Sporadic

Hereditary

60 (69.8)

26 (30.2)

Age (y)

Median

Range

46.6

9 - 84

Tumour size (cm)

<2

2-4

>4

Unknown

37 (43.0)

29 (33.7)

18 (20.9)

2 (2.3)

Lymph node involvement

N0

N1a

N1b

N1x*

26 (30.2)

13 (15.1)

46 (53.5)

1 (1.2)

Stage (TNM 7)

Stage I

Stage II

Stage III

Stage IVa

Unknown

11 (12.8)

13 (15.1)

15 (17.4)

45 (52.3)

2 (2.4)

Number of patients (N) with percentages in parentheses (%)

unless otherwise indicated. *Not known if N1a or N1b.


61

Patients were categorized as; (A) those in whom initial intended curative surgery was

performed in adherence to current ATA guidelines with adequate (group A1) or inadequate

surgery (group A2); (B) those in whom intended curative surgery was performed in a one-

step (group B1) or two-step (group B2) procedure and (C) those whose surgery was

performed in a centre (UMCG/LUMC; group C1/C3) or a non-centre (low-volume) hospital

(group C2) (Figure 1).

Figure 1 Distribution of patients (total 86) with adequate and inadequate surgery (group A) in surgery with

curative intent in one or two sessions (group B) and surgery in a centre or non-centre hospital (group C).

*According to American Thyroid Association guidelines.

Review of surgery and pathology reports

All surgical and pathology reports were independently reviewed and scored by at least three

of four reviewers. Differences in scoring outcome were solved by consensus. First the type of

thyroidectomy (diagnostic or therapeutic) was scored and whether it was a hemi-, subtotal or

total thyroidectomy. Secondly, the types of LND were categorized as lymphadenectomy in

the central (level VI) or lateral region (levels II-VI), and in a unilateral or bilateral neck

Chapter 4

62

dissection. The LND was scored as complete or partial(including lymph -node picking), using

anatomical structures and borders defined by the American Head and Neck Society and

American Academy of Otolaryngology-Head and Neck Surgery.14,15 We evaluated mean and

total numbers of resected nodal categories and tumour positive lymph nodes (LN+). In all

patients the time and location (centre vs. non-centre hospital) of first surgery with curative

intent was documented, and whether it was performed in one or two sessions.

Using ATA guidelines, the first intended curative surgery was scored as being carried out

adequately or inadequately. Adequate surgery consisted of total thyroidectomy with complete

central compartment dissection (level VI) combined with a uni- or bilateral LND, levels II–V,

when there was a preoperative suspicion of metastatic lateral lymph nodes during clinical or

radiological examination.1

Evaluation of putative selection bias

To exclude putative selection bias of non-referred patients we compared data of one referral

centre (UMCG) with data from the national cancer registry (data available during last

decade). This registry reported 50 MTC patients, which was equal to the number treated in the

centre; this indicated that in a non-centre hospital there was no significant number of MTC

patients treated successfully, without later referral to the tertiary centre.

Follow-up and outcome

Follow-up consisted of regular calcitonin and carcinoembryonic antigen (CEA)

measurements, and when these were markedly elevated further evaluation was carried out

with morphological imaging (US, CT, or MRI), functional imaging (PET) or US guided fine

needle aspiration biopsy (FNAB). Biochemical cure was defined as normalization of

calcitonin and CEA levels after the first surgical procedure with curative intent, and before

any further intervention. Recurrence was defined as any lesion with positive cytology or

suspected lesion upon imaging with significantly increased tumour markers. Indeterminate

lesions appearing in imaging without confirmation by other imaging methods or acquired

cytology and/or histology during follow-up, were considered tumour negative. The time

between the first curative intended surgery and clinical recurrence or death was used to define

disease free survival (DFS), disease specific survival (DSS) and overall survival (OS). All

cancer reoperations and complications after first surgery with curative intent were

documented. Recurrent laryngeal nerve palsy diagnosed by indirect laryngoscopy was


63

considered permanent if persisting >6 months. Hypoparathyroidism was considered

permanent if calcium supplementation was necessary >6 months.

Statistical analysis

For statistical analysis we used IBM SPSS statistics 20. For comparison of continuous

normally distributed variables we used Student’s t test or One way ANOVA and for

continuous variables not normally distributed we used the Mann-Whitney U test or Kruskall

Wallis test. The Chi-square test or Fisher exact test was applied for categorical data. For

survival analysis we used a standardized survival model as described earlier.16 The

significance level was p <0.05, 2-sided.

Results

Distribution of patients and tumour characteristics between different groups

An overview of all 86 included patients according to adequacy of first curative intended

surgery (A), number of required sessions (B) and institute (C) is given in Figure 1. Correlated

patient and tumour characteristics are shown in Table 2.

Analysis A: Adequacy of surgery according to ATA guidelines

Surgical procedures according to ATA guidelines were initially adequate in 46 patients

(Figure 1: group A1) and inadequate in 40 patients (Figure 1; group A2; Supplementary Table

1). Adequately treated patients had significant more LN’s examined (24.7 vs. 13.7; p=0.016),

but the number of tumour positive LN’s was almost equal (9.6 vs. 7.9; Table 2).

Analysis B: Number of performed sessions to obtain intended curative surgery

Intended curative surgery was obtained in one session in 66 patients (Figure 1; group B1),

while in 20 patients a two-step procedure was necessary to obtain intended curative surgery

(Figure 1; group B2). Supplementary Table 2 lists the initial procedures in patients requiring

two-step surgery. Patients treated in one-step had significantly more LN involvement (77.3%

vs. 45%, p=0.006) and stage IVa disease (60% vs. 31.6%, p=0.003) (Table 2).

Chapter 4

64

Analysis C: Institute of first intended curative surgery

Sixty-four patients received all surgical procedures in a tertiary referral centre (Figure 1;

group C1). Fifteen of the other 22 patients were treated with curative intent in a non-centre

hospital (group C2). Seven patients received complementary intended curative surgery in a

centre after undergoing a diagnostic hemithyroidectomy in a non-centre hospital (group C3).

Adequate surgery was more often performed in centres (59.2%; 42/71), as compared with

non-centre hospitals (4/15) (relative risk, RR=2.22; p=0.026). Two-step surgery was

significantly more often needed for patients treated initially in non-centre hospitals (54.5%;

12/22) as compared to patients treated in a centre (8/64; p<0.001) (Table 2).

Clinical outcome with regard to ATA-compliant or ATA-non-compliant surgery

(Group A)

Cancer reoperations

After surgery with curative intent, 32 patients underwent a cancer reoperation; 29 of the latter

had histological confirmed MTC. Local cancer reoperations (n=34) were performed on 25

patients. Three patients had four distant reoperations and one patient had a local and two

distant reoperations. The median interval between initial curative surgery and cancer

reoperation was 33 (range 4–331) months. The mean number of local reoperations was

significantly lower in adequately versus inadequately treated patients (total re-operations 11

vs. 24; average 0.24 vs. 0.60, p=0.027; Table 2).

Biochemical cure

Biochemical cure after first intended curative surgery occurred in 31 patients (36%). At the

last follow-up 26 patients (30.9%) had biochemical cure and in adequately treated patients it

was significantly higher (40.9% vs. 20 %, p=0.038; Table 2).

Clinical recurrent disease and survival

During follow-up, 38 patients developed clinical recurrent disease (16 only local recurrences,

7 only distant metastasis and 15 both local and distant). Adequacy of intended curative

surgery did not predict clinical DFS, OS or DSS (Table 3).


65

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Chapter 4

66

Complications

Postoperative hypoparathyroidism (n=31: 36%), was not significant different between patients

treated adequately or inadequately (41.3% vs. 30%). Also, the postoperative recurrent nerve

palsy rate (n=10: 12.2%) was not significant different between the two groups (9.1% vs.

15.8%).

Table 3 Evaluation of risk factors for clinical disease free (DFS) and overall survival (OS)

Clinical DFS OS

Univariate Bivariate* Univariate Bivariate*

RR

(95% CI) P

RR

(95% CI) P

RR

(95% CI) P

RR

(95% CI) P

Surgery according to

ATA guidelines (yes)

1.06

(0.56 – 2.02)

0.84

0.65

(0.28 – 1.48)

0.30

Centre curative surgery

(non-centre)

1.18

(0.52 – 2.67)

0.69

0.45

(0.11 – 1.84)

0.26

Curative surgery in

one/two sessions (two)

0.82

(0.36 - 1.85)

0.63

0.57

(0.19 - 1.65)

0.30

Sex (female) 0.55

(0.29 – 1.05)

0.07

0.61

(0.32 – 1.17)

0.13

0.24

(0.09 – 0.65)

0.005

0.36

(0.12 – 1.03)

0.06

Type MTC (hereditary) 0.51

(0.25 – 1.04)

0.06

0.65

(0.32 – 1.33)

0.24

0.18

(0.03 – 1.05)

0.06

0.36

(0.05 – 2.43)

0.29

Tumour size (>40 mm) 4.1

(2.23 – 7.65)

7. 10-6

4.20

(1.87 – 9.44)

5. 10-4

LN involvement 4.1

(1.73 – 9.75)

0.001

3.18

(1.30 – 7.74)

0.01

8.08

(1.50 – 43.47)

0.015

4.39

(0.70 – 27.55)

0.11

TNM stage (4a) 2.9

(1.48 – 5.49)

0.002

2.41

(1.24 – 4.69)

0.01

6.80

(1.94 – 23.87)

0.003

4.75

(1.26 – 17.82)

0.02

*Corrected for tumour size. DFS: disease free survival. OS: overall survival. RR: relative risk. ATA: American

Thyroid Association. MTC: medullary thyroid carcinoma. LN: lymph node.

Clinical outcome with respect to number of sessions (Group B)

No difference was found in cancer reoperations (average 0.36 vs. 0.55), biochemical cure at

last follow-up (28.1% vs. 40.0%) and survival between patients treated by one-step versus

two-step intended curative surgery (Table 2 and 3) After a two-step surgery patients had

significantly less postoperative hypoparathyroidism (15 % vs. 42.4%, p=0.025), also after

correction for LN+ and TNM stage. No significant difference existed in recurrent nerve palsy

(15.9% vs. 0%).


67

Clinical outcome with respect to centre of first curative intended surgery (Group C)

Between patients treated in a centre as opposed to a non-centre, the clinical outcome did not

differ. There were no differences in cancer reoperations (mean 0.34 vs. 0.73) and biochemical

cure at last follow-up (29.0% vs. 40.0 %; Table 2), nor on clinical DFS, DSS and OS (Table

3). Hypoparathyroidism and recurrent nerve palsy occurred in 35.2% vs. 40% (p=NS) and

10.5% vs. 20% (p=NS).

Clinical outcome with respect to patient and tumour characteristics

Patient and tumour characteristics did not affect the number of reoperations. Biochemical cure

at last follow-up was significantly lower in patients with LN involvement and stage IVa

disease (19.0% vs. 57.7%, p<0.001 and 16.2% vs. 48.7%, p=0.001). Most tumour-related

factors were significant predictors for clinical recurrence (Table 3). After correction for

tumour size as the most significant predictor, LN involvement and TNM stage IVa remained

significant risk factors for clinically recurrent disease

Favourable significant predictors for OS and DSS were female gender, small tumour size

(<40 mm), no LN involvement and TNM stage I-III. With correction for tumour size, only the

TNM stage remained a significant predictor. After correction for TNM stage, tumour size was

the most significant factor for survival, with a RR of 3.92 (p=0.002).

Patients with familial MTC more often had postoperative hypoparathyroidism (53.8% vs.

28.3%, p=0.024) while recurrent nerve palsy more frequently occurred in sporadic MTC

(17.9% vs 0% in familial MTC, p=0.026) and stage IVa disease (19.5% vs 2.6% in stage III

disease, p=0.029).

Chapter 4

68

Discussion

In this study, we emphasize the importance of adequate initial surgery to optimize cure and

locoregional control of MTC. Patients treated adequately according to current ATA guidelines

had significantly fewer local cancer reoperations and more frequent biochemical cure during

follow-up after primary surgery with curative intent. Despite significantly more advanced

disease in patients treated with one-step surgery with curative intent, no difference was

observed in the number of local cancer reoperations or biochemical cure in these patients as

compared to patients requiring a two-step surgery. Tumour characteristics were the most

important predictors for biochemical cure, clinical DFS, DSS and OS.

Important issues in the surgical treatment of MTC patients are the indication and extent of

lateral LND. Because of lack of evidence, leading experts and guidelines advocate different

surgical approaches.1-4,17 For symptomatic MTC patients with limited local disease the ATA

recommends lateral LND if there is suspicion of lateral LN involvement, while the BTA

guidelines advocate bilateral selective LND in pT2-4 tumours or palpable lymph nodes in

central or lateral compartment.1,17 Although systematic LND has improved biochemical cure,

it is still difficult to obtain cure in patients with extensive lymph node involvement.18-22

Indeed, not the extent of operation but rather the baseline tumour characteristics such as

preoperative calcitonin and TNM stage are independent predictors for biochemical cure.7,21

The survival benefits of extensive LND remains contradictory.18,23 Although we did not

specifically evaluate the extent of LND’s, a significant survival benefit with respect to ATA-

compliant surgery was not observed, whereas the latter resulted more often in biochemical

cure. Panigrahi et al. recorded a significantly shorter DSS in patients with regional nodal

disease not treated according to ATA recommendations, which is in contrast to the results of

our study.5 However, the authors included a relative large proportion of patients (16%) with a

subtotal or no thyroidectomy. Moreover, they categorized patients receiving an incomplete

LND as having been treated according to the guidelines. Although our study is more

representative for current practice because of our strict definition of adequate surgery,

comparison between both studies remains difficult.

While results on cure and survival remain contradictory, extensive LND is advocated for

preventing locoregional recurrences, reoperations and future complications.18,23,24 We found

fewer cancer reoperations and more biochemical cure in patients adequately treated according

to current ATA guidelines, a fact which is beneficial for MTC patients. We showed a higher


69

rate of ATA-compliant surgery in referral centres, underscoring earlier data dealing with high

volume hospitals and surgery for thyroid cancer.10

Preoperative work-up by experienced surgeons is advocated, as preoperative diagnosis is

still difficult to establish. An illustration in this study was the performance of a diagnostic

hemithyroidectomy in a substantial number of patients. Despite less advanced disease in these

patients the number of cancer reoperations was relatively high (after second completion

surgery) and biochemical cure was not more frequent. The avoidance of a diagnostic

hemithyroidectomy might have resulted in a more radical initial procedure, and probably with

fewer reoperations. Therefore an adequate diagnosis before initial surgery is crucial in order

to reach locoregional control.25-27

Based on the retrospective character of this study there is a possibility of selection bias.

Other limitations include the relatively low number of patients and possible understaging, due

to limited sensitivity of imaging techniques in the early study period and in patients

inadequately treated.

Conclusion

This study shows the importance of adequate surgery according to ATA guidelines in order to

obtain biochemical cure and regional disease control, leading to fewer cancer reoperations.

This was achieved even in the presence of advanced disease in which size and LN+

commonly predict outcome. Our results indicate a higher rate of adequate surgery in tertiary

referral centres, and support centralized treatment for all patients with confirmed or suspected

MTC.

Chapter 4

70

References



2. Leboulleux S, Baudin E, Travagli JP, Schlumberger M. Medullary thyroid carcinoma. Clin Endocrinol

(Oxf) 2004;61:299-310.

3. Machens A, Dralle H. Biomarker-based risk stratification for previously untreated medullary thyroid

cancer. J Clin Endocrinol Metab 2010;95:2655-2663.

4. Moley JF. Medullary thyroid carcinoma: management of lymph node metastases. J Natl Compr Canc

Netw 2010;8:549-556.

5. Panigrahi B, Roman SA, Sosa JA. Medullary thyroid cancer: are practice patterns in the United States

discordant from American Thyroid Association guidelines? Ann Surg Oncol 2010;17:1490-1498.

6. Kandil E, Gilson MM, Alabbas HH, Tufaro AP, Dackiw A, Tufano RP. Survival implications of cervical

lymphadenectomy in patients with medullary thyroid cancer. Ann Surg Oncol 2011;18:1028-1034.

7. Yip DT, Hassan M, Pazaitou-Panayiotou K, et al. Preoperative basal calcitonin and tumor stage

correlate with postoperative calcitonin normalization in patients undergoing initial surgical

management of medullary thyroid carcinoma. Surgery 2011;150:1168-1177.

8. Sosa JA, Bowman HM, Tielsch JM, Powe NR, Gordon TA, Udelsman R. The importance of surgeon

experience for clinical and economic outcomes from thyroidectomy. Ann Surg 1998;228:320-330.

9. Kebebew E, Greenspan FS, Clark OH, Woeber KA, Grunwell J. Extent of disease and practice patterns

for medullary thyroid cancer. J Am Coll Surg 2005;200:890-896.

10. Lifante JC, Duclos A, Couray-Targe S, Colin C, Peix JL, Schott AM. Hospital volume influences the choice

of operation for thyroid cancer. Br J Surg 2009;96:1284-1288.

11. Fialkowski E, DeBenedetti M, Moley J. Long-term outcome of reoperations for medullary thyroid

carcinoma. World J Surg 2008;32:754-765.

12. Gimm O, Ukkat J, Dralle H. Determinative factors of biochemical cure after primary and reoperative

surgery for sporadic medullary thyroid carcinoma. World J Surg 1998;22:562-7; discussion 567-8.

13. Dralle H, Sekulla C, Haerting J, et al. Risk factors of paralysis and functional outcome after recurrent

laryngeal nerve monitoring in thyroid surgery. Surgery 2004;136:1310-1322.

14. Robbins KT, Clayman G, Levine PA, et al. Neck dissection classification update: revisions proposed by

the American Head and Neck Society and the American Academy of Otolaryngology-Head and Neck

Surgery. Arch Otolaryngol Head Neck Surg 2002;128:751-758.

15. Robbins KT, Shaha AR, Medina JE, et al. Consensus statement on the classification and terminology of

neck dissection. Arch Otolaryngol Head Neck Surg 2008;134:536-538.



17. British Thyroid Association, Royal College of Physicians. Guidelines for the management of thyroid

cancer. Report of the Thyroid Cancer Guidelines Update Group. London: Royal College of Physicians;

2007.

18. Dralle H, Damm I, Scheumann GF, et al. Compartment-oriented microdissection of regional lymph

nodes in medullary thyroid carcinoma. Surg Today 1994;24:112-121.






2000;88:1139-1148.



2000;88:1909-1915.



23. Pelizzo MR, Boschin IM, Bernante P, et al. Natural history, diagnosis, treatment and outcome of

medullary thyroid cancer: 37 years experience on 157 patients. Eur J Surg Oncol 2007;33:493-497.


71

24. Moley JF, Dilley WG, DeBenedetti MK. Improved results of cervical reoperation for medullary thyroid

carcinoma. Ann Surg 1997;225:734-40; discussion 740-3.



Metab 1997;82:338-341.

26. Elisei R. Routine serum calcitonin measurement in the evaluation of thyroid nodules. Best Pract Res

Clin Endocrinol Metab 2008;22:941-953.




Chapter 4

72

Supplementary data

Supplementary Table 1 First intended curative surgical procedure in

inadequately* treated patients (group A2, N = 40)

Type of treatment N

Incomplete thyroidectomy and no CCD 1

No CCD 8

Incomplete CCD

(only node picking or unilateral CCD) 22

Incomplete CCD and LND

(only node picking or unilateral CCD and incomplete

neck dissection not including all levels II-V)

5

Incomplete LND

(incomplete neck dissection not including all levels

II-V)

4

*According to ATA guidelines. CCD: Central compartment (level VI) dissection,

LND: Lymph node dissection.

Supplementary Table 2 First surgical procedure in pts requiring two sessions

(group B2, N = 20)

Type of surgery N

(Diagnostic) hemithyroidectomy 15

Hemithyroidectomy with unilateral LND 2

Total thyroidectomy 1

Total thyroidectomy with bilateral node picking 1

Total thyroidectomy with unilateral LND 1

LND: Lymph node dissection

Chapter 5

PET Imaging in thyroid carcinoma

Hans H.G. Verbeek, Ha T.H. Phan, Adrienne H. Brouwers

Klaas P. Koopmans, Thera P. Links

Methods of Cancer Diagnosis, Therapy and Prognosis. Vol. 7. Editor: M.A. Hayat. Springer

Sience + Business Media BV 2010.

Chapter 5

74

Introduction

Thyroid cancer is the most common endocrine malignancy. It is divided in several types with

papillary, folliculary, and Hürthecell cancer (also called differentiated thyroid cancer)

originating from the follicular epithelial cells as the most common types (>90%). Other types

are medullary thyroid carcinoma (a neuroendocrine tumour originating from the calcitonin

producing C-cells) (3%-10%) and anaplastic carcinoma (often a dedifferentiated form of the

other types) (2%-10%).1

There is a different treatment for each of these types of thyroid cancer. In differentiated

thyroid cancer the initial therapy is total thyroidectomy with or without lymph node

dissection, followed by adjuvant radioactive iodine therapy. Radioactive iodine therapy with 131I can be used successfully due to the active uptake of iodine in tumour cells of thyroid

origin. However, this property can be lost during dedifferentiation, which limits the use of this

therapy in anaplastic carcinoma. Medullary thyroid tumour cells show no iodine uptake at all

and curative options are therefore mainly limited to surgical resection of primary tumour and

metastases.2

The prognosis for differentiated thyroid cancer is good, with an average 10-years survival

between 80% and 95%. However, these tumours can dedifferentiate, which results in limited

therapeutic options, leading to a much poorer prognosis with a 5-years survival of 30%.

Medullary thyroid carcinoma has a 10-years survival of 20%-70%, and for anaplastic thyroid

carcinoma the median survival is 2-6 months.3-5

Imaging is especially important in determining the right therapeutic approach for patients

with differentiated and medullary thyroid carcinoma. Different imaging techniques are

available such as computed tomography (CT), magnetic resonance imaging (MRI),

conventional nuclear scintigraphy, positron emission tomography (PET), etc.. While MRI and

CT are imaging techniques which show morphologic structures, PET imaging depicts

pathophysiological processes and is described as functional imaging. The value of PET

imaging is emerging mainly in the follow-up of thyroid cancer. Several PET imaging

techniques are available for different types of thyroid cancer and these techniques and their

applications are discussed in this chapter.

PET imaging in thyroid carcinoma

75

Positron Emission Tomography

Positron emission tomography imaging is a technique used in nuclear medicine which is

based on the use of positron emitting isotopes in specific molecules which are relevant for

specific metabolic pathways. The PET technique yields a high resolution and the capability to

quantify the amount of radioactivity measured in a specific region. Positrons, emitted by an unstable atom nucleus, are the antiparticles of electrons and have

the same mass, but an opposite charge. Positrons are not detected by a PET camera, but

photons, which are formed when a positron fuses with an electron, are detected. This means

that the positron binds with an electron to form a positronium which annihilates. In this

annihilation process all the mass is converted into energy by which two photons are formed.

These photons, always carrying an energy of 511 keV, are emitted in opposite directions

under an angle of 180º and are detected by the PET camera.6

Radioisotopes used in PET imaging usually have a short half live, such as carbon-11(11C;

half-life (T½) 20 min), nitrogen-13 (13N; T½ 10 min), oxygen-15(15O; T½ 2 min) and fluorin-

18(18F; T½ 110 min). Positron emitting radionuclides are in most cases produced by

bombarding the target material with highly accelerated particles (deuterons or protons) using

a cyclotron and inducing a nuclear reaction. Centres which use isotopes with a very short half-

life, such as 15O, 13N, and 11C, need to have an on-site cyclotron. Other longer living isotopes

can be made elsewhere and then transported to the PET imaging facility.7

A cyclotron is a type of particle accelerator which accelerates charged particles using a

high-frequency, alternating voltage. A perpendicular magnetic field causes the particles to

assume a circular orbit so that they reencounter the accelerating voltage many times. When

the particles are accelerated fast enough they are bombarded on to specific atoms and

radionuclides are formed. These radionuclides are trapped and transported to the laboratory

where they can be processed for clinical use. The resulting end-products are called

radiotracers. The most commonly used tracers are precursors for metabolic pathways,

although many other tracer types exist. After preparation and careful quality monitoring,

tracers can be injected.

The imaging device used is the PET camera. Most PET cameras consist of a ring of special

detectors which are well suited for the detection of 511 keV gamma rays. Software registers

only simultaneously entering photonpairs on different detectors. This is called coincidence

detection. Coincidence detection removes the need for a lead collimator such that the

Chapter 5

76

sensitivity of the PET imaging system is much higher than for the conventional Anger camera

that is used in planar nuclear imaging or single photon emission computed tomography

(SPECT). Both the specific block detector structure and the absence of a collimator contribute

to a higher resolution as compared with SPECT. Another advantage of PET is that the amount

of radioactivity injected in the body can be quantitatively determined. PET is a non-invasive,

sensitive imaging tool for depicting of molecular and biochemical processes without changing

its physical properties.6

In contrast to radiologic imaging which shows the morphologic structures, nuclear

medicine techniques depict pathophysiological processes and are also described as functional

imaging methods. The imaging with PET is considered to be an useful diagnostic tool for the

detection of cancer, brain diseases, and coronary artery diseases.

Combined PET/CT

The combination of PET and CT scanning is a new promising imaging technique. The

integrated PET/CT scanner allows acquisition of CT and PET images in one session. The

combination of morphologic and functional imaging leads to more precise anatomical

localization of tumour lesions. The localization of tumour foci is important for initiating the

appropriate treatment such as surgery. Especially in patients with a negative radioiodine scan

where surgery is the only therapeutic option, the integrated PET/CT scan can be helpful in

guiding therapeutic management.

18Fluorine-fluorodeoxyglucose (

18F-FDG) PET

Mechanism

Fluorodeoxyglucose (FDG) is a glucose analogue and is used as a precursor for glucose

metabolism. In both benign and malignant tissue it enters the cell by the same glucose

transporters. However, the need for glucose in malignant cells is strongly increased because

these cells have a considerably less efficient energy metabolism.8 For example, the energy

production per molecule of glucose in malignant cells is decreased because anaerobic

glycolysis is strongly increased instead of the much more efficient energy production from the

citric acid cycle. This inefficient use of glucose is the basis for the preferential uptake of

glucose or an offered glucose analogue as FDG in malignant cells.


77

In the cell FDG is phosphorylated by a hexokinase enzyme into FDG-6 phosphate which,

in contrast to glucose-6-phospate, cannot be further metabolized. Therefore, the FDG-6-

phosphate does not leave the cell and becomes trapped intracellulary. The final quantity of

FDG-6-phosphate is proportional to the glycolytic rate of the cell. Besides the increased

glycolysis, it has been demonstrated that in malignant cells levels of transmembrane glucose

transporters (e.g., the GLUT-1 transporter) and possibly some hexokinase isoenzymes are also

increased, also resulting in increased FDG uptake.9

However, in all metabolically active tissues, such as brain cells, active muscles, and

activated macrophages, increased glucose metabolism leads to an increased FDG uptake. 18F-

FDG PET is, therefore, a marker for glucose metabolism in general. In most normal tissues

(e.g., liver, kidney, intestine, muscle and some tumour cells) the level of phosphate activity is

variable; nevertheless, FDG-6-phospate accumulation is lower than in malignant tissues. In

addition, some benign tissues require more glucose.10,11 So, the uptake mechanism of FDG

with irreversible trapping in malignant tissue is ideal for PET imaging and has been applied

widely in oncology. However, it is important to make a correct interpretation of these PET

images, for non-malignant tissue also has FDG uptake.

Scan method

Uptake of 18F-FDG occurs rapidly after administration and the amount taken up increases

with time. The most applied imaging moment is 60-90 min after tracer administration. The

reason is that the excretion of 18F-FDG via the kidneys reduces 18F-FDG in the blood, which

causes clearance of ‘background’ uptake and the decay of fluorin-18 (T½ 110 min). Generally

the patient preparation consists of an 18F-FDG injection in a fasting condition and after oral

prehydration. The injected dose varies between 2-8 MBq/kg.

Clinical application

Thyroid nodules

The value of 18F-FDG PET in the distinction between malignant and benign thyroid nodules

before surgery is unclear. Several studies reported that 18F-FDG PET is useful in the

preoperative evaluation of cytologicaly inconclusive nodules with a high negative predictive

value.12-14 De Geus-Oei et al. observed that the probability for thyroid cancer increased from

14% (pre-PET) to 32% (post-PET) in case the nodule was positive on 18F-FDG PET.12 In this

study 18F-FDG PET could reduce the number of futile hemithyroidectomies by 66%.

Chapter 5

78

A. 18F-FDG PET

B. CT-lung C. 18F-FDG PET/CT

Figure 1 These are the images of a 68-year old male known with follicular thyroid cancer. This patient showed

increased serum Tg level (14 ng/ml) suspected for recurrent or metastatic disease. Blind treatment with 131

I

was given followed by a post-treatment whole body scan (WBS) after 10 days which was negative. 18

F-FDG PET

(A) showed a focal lesion in the lower lobe of the left lung (arrow), confirmed by CT (B, arrow). Picture C

showed the fusion image of 18

F-FDG PET and CT for the lesion in the left lung (arrow).

However, recent studies by Kim et al. and Bogsrud et al., demonstrated that 18F-FDG PET is

not helpful in differentiating between malignant and benign nodules, and therefore has only

limited value in preoperative evaluation of indeterminate thyroid nodules.15,16

So, conflicting results are reported on the usefulness of 18F-FDG PET in the prediction of

malignancy in thyroid nodules in case of inconclusive cytology, and therefore further research

is needed. Meanwhile histopathological examination remains the gold standard.

Differentiated thyroid cancer (DTC): follow-up

More information is available regarding the value of 18F-FDG PET in the follow-up of thyroid

cancer such as the detection of recurrences or metastases, especially in patients with a

negative radioiodine scan or in patients who has lost the ability to accumulate iodine. A


79

complementary uptake of 18F-FDG and radioiodine can be present, which is known as the

‘flip-flop’ phenomenon and was first described by Joensuu and Ahonen.17 This phenomenon

might be explained by the degree of tissue differentiation. Well differentiated thyroid tissue

has the capability to take up iodine but is metabolically inactive while less differentiated

thyroid cancer tissue loses its capability to trap iodine and becomes metabolically more

active. This makes 18F-FDG PET scanning the method of choice for the detection of 131I

negative metastases of differentiated thyroid carcinoma.18

Performance of 18F-FDG PET during thyrotropin (TSH) stimulation improves the results in

comparison to the scanning during the euthyroid state (during thyroxin treatment) as was

shown by van Tol et al..19 In vitro studies have shown a stimulating effect of TSH on Glut 1

expression and glucose transport.20,21 This increase in glucose carriers results in a higher

uptake of glucose and also 18F-FDG in thyroid cancer cells, which improves the result of the

PET-scan. Stimulation with exogenous TSH (recombinant human(rh) TSH) also increases 18F-FDG uptake by differentiated thyroid cancer, and therefore more lesions can be detected

and tumour/background contrast is enhanced.22,23 The influence of rhTSH on the background

is not well-known, but there is evidence that rhTSH increases 18F-FDG uptake in the tumour

lesion itself.

Several studies have been performed to assess the value of 18F-FDG PET imaging in the

follow up of thyroid cancer. Hooft et al. performed a meta-analysis of studies that investigated

the role of 18F-FDG PET in patients with thyroid cancer after negative radioiodine

scintigraphy and elevated serum thyroglobulin.24 The diagnostic accuracy of these studies was

assessed. Observed sensitivity and specificity in these studies were ranging from 70%-95%

and 77%-100%, respectively. Furthermore, they observed that there are methodological

problems in these studies such as small sample size, validity of reference tests, and short

follow-up. Nonetheless, 18F-FDG PET is now considered a valuable diagnostic imaging tool

in the follow-up of 131I-negative patients for the detection of recurrences or metastases.

However, it is not known whether PET is superior to bone scintigraphy in the detection of

bone metastases in thyroid cancer. Comparative studies of bone scans and 18F-FDG PET are

lacking. In a retrospective study, 24 patients had undergone both 18F-FDG PET and bone

scans within six months because of suspected bone metastases.25 This study shows that bone

scintigraphy is still valuable in differentiated thyroid cancer, as it was found that 38% of bone

metastases could be missed on 18F-FDG PET. Further prospective studies in a higher number

of patients are required to define the exact role of bone scan and 18F-FDG PET in the

detection of bone metastases in patients with differentiated thyroid cancer (DTC).

Chapter 5

80

Combined or integrated 18F-FDG PET/CT in patients with negative 131I scans and elevated

thyroglobulin (Tg) showed that the diagnostic accuracy can be improved compared to 18F-

FDG PET or CT alone. The combination of these imaging techniques has also led to a change

in patient management and therapy, e.g., extension of surgery by providing precise anatomical

localization of the recurrent or metastatic disease.26

Medullary thyroid cancer (MTC)

The detection of recurrence or metastases in MTC is difficult and there is no single method

sensitive enough to reveal all MTC recurrences or metastases. In comparison with the

calcitonin tumour marker nearly all imaging modalities (Ultrasonography, CT, MRI, and

scintigraphy) have limited sensitivities. The clinical role of 18F-FDG PET in the diagnosis and

staging of recurrent and metastatic MTC seems promising.27

The sensitivity and specificity of 18F-FDG PET ranges between 73%-88% and 76%-80%,

respectively. In a study by de Groot et al., 18F-FDG PET was performed in patients with

elevated serum tumour markers after total thyroidectomy.28 Compared with 111In-octreotide

imaging(lesion based sensitivity: 41%), 99mTc(V)DMSA scintigraphy (57%) and

morphological imaging (87%), 18F-FDG PET (96%) was superior. However, morphological

imaging will always be needed because 18F-FDG PET only yields functional data and no

morphological information, which is necessary to assess resectability.

The combination of 18F-FDG PET/CT can have a useful role in medullary thyroid cancer.

Because surgery only can cure the disease, precise anatomical localization and the extent of

the recurrent or metastatic disease is mandatory. However, little data and case reports have

shown an increased diagnostic accuracy so further studies are needed.27

18Fluorine-dihydroxyphenylalanine (

18F-DOPA)

Mechanism

The mechanism responsible for uptake of 18Fluorine-dihydroxyphenylalanine (18F-DOPA) in

medullary thyroid carcinoma is probably the strongly upregulated transmembrane transport of

amino acids via the large amino acid transporters in medullary thyroid carcinoma cells. It is

not yet clear whether the increased uptake of 18F-DOPA PET is the result of the increased

transporter capabilities or the increased metabolic activity of the catecholamine pathway.


81

After transmembrane transport, 18F-DOPA is intracytoplasmatically converted into

dopamine by the enzyme aromatic acid decarboxylase (AADC). The formed 18F-dopamine is

transported into secretory vesicles via the vesicular mono aminoacid transporters (VMAT) in

which it can be further metabolized to 18F-noradrenalin and 18F-adrenalin.29 Although the 18F

atom influences the metabolism of 18F-DOPA there is no or little effect on the transport into

the intracellular environment. In the kidneys, 18F-DOPA is rapidly converted into 18F-dopamine

which is than excreted actively in urine. This conversion can be inhibited by oral

administration of carbidopa prior to tracer administration.30

Carbidopa also lowers the physiological uptake of 18F-DOPA in the pancreas, but it is yet

unknown which mechanism is responsible for this decrease in pancreatic uptake. The

reduction in renal and urinary activity leads to a better image quality in the surroundings of

the urinary system. Also, the reduced uptake in the pancreas makes the identification of

lesions in the pancreatic region easier. It can be speculated that by reducing the excretion of 18F-DOPA, more 18F-DOPA is available for neuroendocrine tumour lesions; thereby,

increasing the tumour to background ratio, leading to a better discrimination of

neuroendocrine lesions.

Scan method

In most centres, patients are prepared with oral administration of carbidopa, either in a fixed

dose or in a dose calibrated to body weight. Patients are scanned in a fasting condition for 4-6

h. In most centres a whole body study will be performed ranging from the skull to the upper

femora. The average injected dose is 200 MBq, the radiation burden ~ 4 mSv.30 Attenuation

correction is applied, either by using a CT in a PET-CT machine or by using camera-specific

attenuation protocols.


Medullary thyroid cancer

Although 18F-DOPA PET is not yet in widespread use, it is a promising new functional

imaging procedure for imaging neuroendocrine tumours. More and more centres gain access

to this tracer either via on-site production or production elsewhere. Hoegerle et al. were the

first to describe the use of 18F-DOPA PET in medullary thyroid cancer.31 In this study 18F-

DOPA PET was compared with 18F-FDG PET, SRS, and CT/MRI. A high precision of 18F-

DOPA PET was observed in the diagnosis of lymph node metastases (sensitivity 88%), while

Chapter 5

82

organ metastases were better detected with conventional imaging (sensitivity 13%). In the

recently published study by Koopmans et al. diagnostic accuracy was assessed for 18F-DOPA

PET in patients with carcinoid tumours which are, like medullary thyroid carcinoma,

neuroendocrine tumors.32 Compared to conventional somotostatine receptor scintigraphy

(SRS) they showed improved sensitivity of 18F-DOPA PET in staging and identify carcinoid

tumours.

A. 18F-FDG PET B. 18F-DOPA PET

Figure 2 These are the images of a patient known with medullary thyroid cancer. In this patient 18

F-FDG PET

(A) and 18

F-DOPA PET (B) were performed. The 18

F-DOPA PET (B) showed multiple lesions in the liver and

several lesions in the spinal column(arrows) while the 18

F-FDG PET showed hardly any lesions.

The value of 18F-DOPA PET for the detection of recurrent or residual disease in 21 patients

with postsurgically elevated calcitonin or CEA was assessed by Koopmans et al..33 They

compared 18F-DOPA PET with 18F-FDG PET, 99mT(V)DMSA, and CT/MRI. 18F-DOPA PET

was superior to conventional imaging for the detection of MTC on patient (sensitivity 87%)

and regional (89%) level. On lesional level 18F-DOPA PET (sensitivity 71%) was equal to

morphological imaging (64%) but superior to 18F-FDG PET (30%) and 99mT(V)DMSA (19%).

In the recent study by Beuthien-Baumann et al., 18F-DOPA-PET also seems to be more

specific than 18F-FDG PET for the detection of metastases of MTC.34 Thus, compared with 18F-FDG PET and conventional imaging techniques, 18F-DOPA PET provides better results in

the imaging of medullary thyroid cancer. However, it is still unclear if this improved imaging

results in different therapeutic approaches, and so further research is needed.


83

11C-Methionine (MET) PET

Mechanism

Proteins play an important role in virtually all biological processes. Proteins are built from a

set of 20 amino acids. Amino acid transport across the cell membranes into the cells occurs

primarily via carrier-mediated processes. Amino acid transport is generally increased in

malignant transformation.35-37 This increased protein metabolism in cancer cells is important

for metabolic tumour imaging, for which radiolabeled amino acids can be applied. These

amino acid tracers could help in imaging areas where 18F-FDG is limited such as the

interference of high (physiologic) 18F-FDG uptake in the brain. Another reason is that amino

acid imaging is less influenced by inflammatory disease. The most frequently used

radiolabeled amino acid is L-[methyl-11C]-methionine. Normal biodistribution of radiolabeled

methionine occurs in the pancreas, liver, spleen, kidney, and salivary glands.

Scan method

11C-MET PET scanning can be performed 10 to 20 min after intravenous injection of a fixed

dose or a dose calibrated on body weight (suggested range is 70 MBq to 1100 MBq), in a

fasting condition for 2-6 h. Images are corrected, either by using a CT in a PET/CT machine

or by using camera-specific attenuation protocols.


Differentiated thyroid cancer

The need for new tracers and improvement of diagnostic tools in thyroid cancer is growing.

So far, no data on the application of methionine (MET) PET in thyroid cancer are available.

The general feasibility of amino acid imaging in many tumour types has been sufficiently

shown.37

It is imaginable that thyroid cancer could sufficiently concentrate amino acids due to its

metabolically inert nature and high protein synthesis (e.g., thyroglobulin). In a feasibility

study by Phan et al., 11C-MET PET has been compared with 18F-FDG PET in the detection of

recurrent or metastatic disease in 20 patients with negative 131I scans and elevated Tg.38 Six of

the 20 patients showed uptake on both PET scans, but the abnormalities were more 18F-FDG-

avid and more extensive on the 18F-FDG PET in 3 patients. In four of the 20 patients uptake

Chapter 5

84

was only observed on the 11C-MET PET; however, no anatomical localization could be

confirmed. Presently, the significance of the MET uptake in these four patients is unclear, so

the clinical value of 11C-MET PET in the detection of recurrent DTC disease still has to be

proven in the (long-term) follow-up.

A. 11C-MET PET B. 18F-FDG PET

Figure 3 These are the images of a 68-y old female known with papillary thyroid cancer. This patient had

unreliable Tg due to the presence of Tg antibodies (which were increasing in the course of the follow-up). The

post-treatment 131

I whole body scan (WBS) was negative. Due to suspicion of dedifferentiated, metastatic

disease 11

C-MET PET (A) and 18

F-FDG PET (B) were performed. 11

C-MET PET (A) showed lesions in the

mediastinum with slightly to moderate 11

C-MET uptake. 18

F-FDG PET (B) also showed multiple lesions in the

mediastinum, but the lesions showed clearly higher 18

F-FDG uptake and the abnormalities were more

extensive.

124Iodine-PET

Mechanism

Iodine-124 is a positron emitting isotope, which is suitable for PET imaging, with a half-life

of 4.2 days.39 This isotope has been used for dosimetric purposes or thyroid volume

measurements.40 While the radioisotopes 123I and especially 131I are used on a wide scale in

diagnosis and treatment of many thyroid disorders, 124I has received little attention.

Chemically identical to nonradioactive iodine, this radioisotope allows thyroid cancer imaging

with the high resolution PET technique.39,40


85

Scan method

The 124I -PET scan can be obtained 24 h to 6 days after administration of 74-100 MBq of 124I.

A whole body PET scan (from the upper thigh up until the top of the skull) can be performed

in 2D or 3D mode, using standard energy window setting of 350-650 keV (or energy window

setting 425-650 keV or 460-562 keV in case of the presence of high amounts of 131I.41


Differentiated thyroid cancer

Accumulation of iodine is a highly specific characteristic for differentiated thyroid cancer

(DTC) cells. In patients with increasing or recurrent detectable Tg a blind treatment (meaning

after a negative diagnostic 131I scan) with high dose 131I followed by a post-treatment 131I scan

is used as a diagnostic tool. However, this strategy with (unnecessary) high radiation exposure

must be taken into account in patients without 131I uptake in their metastases. Besides the high

radiation exposure, there is a high TSH level which potentially stimulates thyroid cancer cell

growth.

Based on the higher spatial resolution, 124I-PET is potentially able to detect recurrent

disease in DTC with a higher sensitivity than (diagnostic) 131I scans. With this higher

sensitivity and the possibility to combine the 124I-PET scan with morphologic imaging, such

as CT data, an appropriate therapeutic decision in terms of surgery and/or additional high dose 131I can be made. 124I-PET imaging might, therefore, become the diagnostic tool of choice in

the follow-up of DTC. In the study by Freudenberg et al., 124I-PET (/CT) modalities were

compared with the high dose 131I-WBS in 12 patients with DTC.42 They showed an overall

lesion detectability of 87 %, 83%, and 100% for 124I, 131I-WBS and combined 124I-PET/CT

respectively. So, these 124I-PET (/CT) modalities are promising diagnostic tools and are

suitable alternative to the high dose 131I-WBS in the follow-up of DTC patients.

In a prospective, feasibility study by Phan et al., 20 patients with advanced DTC (T4,

extranodal tumour growth, distant metastasis) underwent a low-dose diagnostic 131I scan, a 124I PET scan, and a high-dose (posttreatment) 131I scan.43 The 131I images were compared to

the 124I-PET images. 124I-PET proved to be a superior diagnostic tool as compared to low dose

diagnostic 131I scans, and showed comparable findings with the post-treatment 131I-WBS

which was in agreement with the study by Freudenberg et al. and Abdul Fatah et al..42,44

Therefore, 124I-PET could be used as a diagnostic tool in the follow-up of patients with DTC

for the favourable radiation exposure burden compared to the high dose diagnostic 131I-WBS

Chapter 5

86

and the superior diagnostic accuracy compared to low dose diagnostic 131I-WBS and the

fusion possibility with CT which improves clinical decision making.

A. Post-treatment 131I-WBS B. 124I-PET

C. 18F-FDG PET

Figure 4 These are the images of a 68-year old male known with follicular thyroid cancer. This patient showed

increased serum Tg level (14 ng/ml) suspected for recurrent or metastatic disease. Blind treatment with 131

I

was given followed by a post-treatment whole body scan (WBS) after 10 days (A), which was negative. The 124

I-

PET (B) was also negative, besides physiological uptake in the salivary glands, oesophagus, gastro-intestinal

tract, kidney and bladder. 18

F-FDG PET (C) showed a focal lesion in the lower lobe of the left lung (arrow),

confirmed by CT. This complementary uptake of radioiodine and 18

F-FDG is known as the flip-flop phenomenon.

Conclusion

This chapter on PET imaging in thyroid cancer gives an overview of the different PET

techniques available in thyroid cancer. PET imaging is based on two principles: the ability of

unstable atom nucleus to emit positrons and the labelling of organic molecules, which are

used in specific metabolic pathways.


87

For differentiated thyroid cancer the most frequently used PET technique is 18F-FDG PET

imaging, which is based on the use of a glucose analogue. Although its application in the

preoperative assessment of thyroid nodules is still unclear, it is considered an useful tool in

the follow up of differentiated thyroid cancer. The use of PET/CT scanning which combines

functional imaging with morphological imaging is promising, providing more accurate

localization of tumour sites, which is important for further treatment.

Other PET radiotracers have been developed: the clinical value of amino acid tumour

imaging with 11C-MET PET is unclear and still has to be proven in the follow-up of

differentiated thyroid cancer. Another relative new PET imaging technique is the iodine

isotope 124I. Compared with the high dose diagnostic 131I whole body scan, 124I-PET showed

similar findings and can therefore be used as a diagnostic tool in the follow-up of

differentiated thyroid cancer.

For the follow-up of medullary thyroid cancer 18F-FDG PET is also the most employed

imaging technique. However, 18F-DOPA PET, which is based on a precursor of dopamine,

seems to be superior compared to 18F-FDG PET in the follow up of medullary thyroid cancer.

A potential new tracer is 11C-5-HTP, which is based on a precursor of serotonin and already

has been applied in neuroendocrine tumours. The value of this technique has to be further

assessed in medullary thyroid cancer.

The need for new tracers and advanced PET imaging to improve the diagnostic

sensitivities and accuracy in detection, staging, and follow-up of thyroid cancer patients is

growing. Knowledge of the pathogenesis, the molecular characteristics and the behaviour of

the tumour cell is crucial for developing of specific tracers and techniques, e.g., radiolabeled

Tg, (rh) TSH. Although new tracers are developed and applied in patients, little data are

available on the changes in therapeutic management these new PET-techniques give. While

PET-imaging is still in development, more research is needed to assess the effects on therapy

of these new developments. In conclusion, PET imaging is a useful diagnostic tool in thyroid

cancer and new promising techniques are developed which could further improve the

diagnostic accuracy and therapeutic approaches.

Chapter 5

88

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18. Schluter B, Bohuslavizki KH, Beyer W, Plotkin M, Buchert R, Clausen M. Impact of FDG PET on patients

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22. Petrich T, Borner AR, Otto D, Hofmann M, Knapp WH. Influence of rhTSH on [(18)F]fluorodeoxyglucose

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23. Chin BB, Patel P, Cohade C, Ewertz M, Wahl R, Ladenson P. Recombinant human thyrotropin

stimulation of fluoro-D-glucose positron emission tomography uptake in well-differentiated thyroid

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24. Hooft L, Hoekstra OS, Deville W, et al. Diagnostic accuracy of 18F-fluorodeoxyglucose positron

emission tomography in the follow-up of papillary or follicular thyroid cancer. J Clin Endocrinol Metab

2001;86:3779-3786.

25. Phan HT, Jager PL, Plukker JT, Wolffenbuttel BH, Dierckx RA, Links TP. Detection of bone metastases in

thyroid cancer patients: bone scintigraphy or 18F-FDG PET? Nucl Med Commun 2007;28:597-602.

26. Shammas A, Degirmenci B, Mountz JM, et al. 18F-FDG PET/CT in patients with suspected recurrent or

metastatic well-differentiated thyroid cancer. J Nucl Med 2007;48:221-226.

27. Nanni C, Rubello D, Fanti S, et al. Role of 18F-FDG-PET and PET/CT imaging in thyroid cancer. Biomed

Pharmacother 2006;60:409-413.

28. de Groot JW, Links TP, Jager PL, Kahraman T, Plukker JT. Impact of 18F-fluoro-2-deoxy-D-glucose

positron emission tomography (FDG-PET) in patients with biochemical evidence of recurrent or

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29. Pearse AG. The APUD cell concept and its implications in pathology. Pathol Annu 1974;9:27-41.

30. Brown WD, Oakes TR, DeJesus OT, et al. Fluorine-18-fluoro-L-DOPA dosimetry with carbidopa

pretreatment. J Nucl Med 1998;39:1884-1891.

31. Hoegerle S, Altehoefer C, Ghanem N, Brink I, Moser E, Nitzsche E. 18F-DOPA positron emission

tomography for tumour detection in patients with medullary thyroid carcinoma and elevated

calcitonin levels. Eur J Nucl Med 2001;28:64-71.

32. Koopmans KP, de Vries EG, Kema IP, et al. Staging of carcinoid tumours with 18F-DOPA PET: a

prospective, diagnostic accuracy study. Lancet Oncol 2006;7:728-734.



2008;49:524-531.

34. Beuthien-Baumann B, Strumpf A, Zessin J, Bredow J, Kotzerke J. Diagnostic impact of PET with 18F-

FDG, 18F-DOPA and 3-O-methyl-6-[18F]fluoro-DOPA in recurrent or metastatic medullary thyroid

carcinoma. Eur J Nucl Med Mol Imaging 2007;34:1604-1609.

35. BUSCH H, DAVIS JR, HONIG GR, ANDERSON DC, NAIR PV, NYHAN WL. The uptake of a variety of amino

acids into nuclear proteins of tumors and other tissues. Cancer Res 1959;19:1030-1039.

36. Isselbacher KJ. Sugar and amino acid transport by cells in culture--differences between normal and

malignant cells. N Engl J Med 1972;286:929-933.

37. Jager PL, Vaalburg W, Pruim J, de Vries EG, Langen KJ, Piers DA. Radiolabeled amino acids: basic

aspects and clinical applications in oncology. J Nucl Med 2001;42:432-445.

38. Phan HT, Jager PL, Plukker JT, Wolffenbuttel BH, Dierckx RA, Links TP. Comparison of 11C-methionine

PET and 18F-fluorodeoxyglucose PET in differentiated thyroid cancer. Nucl Med Commun

2008;29:711-716.

39. Pentlow KS, Graham MC, Lambrecht RM, et al. Quantitative imaging of iodine-124 with PET. J Nucl

Med 1996;37:1557-1562.

40. Eschmann SM, Reischl G, Bilger K, et al. Evaluation of dosimetry of radioiodine therapy in benign and

malignant thyroid disorders by means of iodine-124 and PET. Eur J Nucl Med Mol Imaging

2002;29:760-767.

41. Lubberink M, van Schie A, de Jong HW, van Dongen GA, Teule GJ. Acquisition settings for PET of 124I

administered simultaneously with therapeutic amounts of 131I. J Nucl Med 2006;47:1375-1381.

42. Freudenberg LS, Antoch G, Jentzen W, et al. Value of (124)I-PET/CT in staging of patients with

differentiated thyroid cancer. Eur Radiol 2004;14:2092-2098.

43. Phan HT, Jager PL, Paans AM, et al. The diagnostic value of 124I-PET in patients with differentiated

thyroid cancer. Eur J Nucl Med Mol Imaging 2008;35:958-965.

44. Abdul Fatah S, Brans B, Huijberts M, Sels J, Nieuwenhuijzen Kruseman A, Teule G. Impact of I-124 PET-

CT in the loco-regional staging of differentiated thyroid carcinoma. J NUCL MED MEETING ABSTRACTS

2007;48:127P-c.

90

Chapter 6

Clinical relevance of 18

F-FDG PET and 18

F-DOPA PET in

recurrent medullary thyroid carcinoma

Hans H.G. Verbeek, John T.M. Plukker, Klaas P. Koopmans, Jan Willem B. de Groot

Robert M.W. Hofstra, Anneke C. Muller-Kobold, Anouk N.A. van der Horst-Schrivers

Adrienne H. Brouwers, Thera P. Links

J Nucl Med. 2012; 53: 1863-71

Chapter 6

92

Abstract

Introduction The transition from stable to progressive disease is unpredictable in patients

with biochemical evidence of medullary thyroid carcinoma (MTC). Calcitonin and

carcinoembryonic antigen (CEA) doubling times are currently the most reliable markers for

progression, but for accurate determination serial measurements are required which need time.

We compared 18F-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET)

and 18F-dihydroxyphenylanaline (18F-DOPA) PET with biochemical parameters and survival

to assess whether these imaging modalities could be of value in detecting progressive disease.

Methods We evaluated outcome of 18F-FDG PET and/or 18F-DOPA PET with calcitonin and

CEA doubling times in 47 MTC patients. A subgroup of patients was included in whole

metabolic burden (WBMTB) analysis, with determination of standardized uptake values

(SUV) and number of lesions. WBMTB of 18F-DOPA PET and 18F-FDG PET was compared

with biochemical parameters. Furthermore survival was compared with 18F-DOPA PET

and/or 18F-FDG PET positivity.

Results In 38 out of 40 patients with 18F-FDG PET doubling times were available. There was

a significant correlation with 18F-FDG PET positivity. Doubling times were <24 months in

77% (n=10/13) of 18F-FDG PET positive patients, while 88% (n=22/25) of 18F-FDG PET

negative patients had doubling times >24 months (p<0.001). Between doubling times and 18F-

DOPA PET positivity no significant correlation existed. 18F-DOPA PET detected significantly

more lesions (75%, 56 of 75) compared to 18F-FDG PET (47%, 35 of 75) in the 21 patients

included in WBMTB analysis (p=0.009). Calcitonin and CEA levels correlated significantly

with WBMTB on 18F-DOPA PET but doubling times did not. 18F-FDG PET positivity was a

more important indicator for poor survival in patients with both scans performed.

Conclusion 18F-FDG PET is superior in detecting patients with biochemical progressive

disease and identifying patients with a poor survival. Although 18F-DOPA PET has less

prognostic value it can more accurately assess the extent of the disease in patients with

residual MTC. Hence, both scans are informative regarding tumour localization and

behaviour. Based on these results we designed a clinical flow diagram for the general practice

in detecting recurrent MTC.

18F-FDG PET and

18F-DOPA PET in MTC

93

Introduction

Medullary thyroid carcinoma (MTC) accounts for about 4% of all thyroid cancers. The

overall 10 year survival ranges between 40% and 80% and has not increased substantially in

the past few decades.1-3 Unfortunately, even in MTC that is clinically confined to the neck,

many patients already have metastatic disease and are beyond cure even by surgery.

Furthermore, though the overall survival in patients with only biochemical evidence of

residual MTC is good, a number of patients will develop progressive and symptomatic

disease.4 Early identification of these patients is clinically relevant because appropriate

therapeutic interventions may delay symptomatic deterioration. However, the transition from

a stable status to a progressive disease course is unpredictable and it is hard to identify

patients who may benefit from early intervention.

Calcitonin is a specific tumour marker for MTC, carcinoembryonic antigen (CEA) is less

specific, but can also be useful.5 Currently, short calcitonin and CEA doubling times are

considered the best available indicators to assess progressive disease, MTC recurrence and

cancer mortality.6,7 Calcitonin and CEA levels can fluctuate, however, and determination of

the doubling times needs serial measurement for 12-24 months and is therefore time-

consuming.

Most imaging techniques have a moderate sensitivity in detecting MTC.8 Positron emission

tomography (PET) using the radioactive tracers 18F-fluoro-2-deoxy-D-glucose (18F-FDG) and

more recently 18F-dihydroxyphenylanaline (18F-DOPA) are available for the staging and

follow-up of MTC.9-15 Some studies have suggested that 18F-FDG PET might be more

sensitive in patients with a short calcitonin doubling time.16,17 Furthermore, a higher

metabolic activity, expressed as the maximum standardized uptake value (SUV), on 18F-FDG

PET compared with the maximum SUV on 18F-DOPA PET, might be related to a more

aggressive tumour type.18 PET also enables determination of the total tumour load expressed

as the whole-body metabolic burden (WBMTB), reflecting metabolic tumour activity, as was

shown in a recent study of 18F-DOPA PET in carcinoid patients.19

In this retrospective study of patients with biochemical evidence of MTC, our aim was to

assess the ability of 18F-FDG PET and 18F-DOPA PET to discriminate between patients with

progressive disease and patients with stable disease.

Chapter 6

94

Materials and methods

Patients

We analysed all patients with histologically proven MTC seen at the Department of

Endocrinology for follow-up and who had undergone 18F-FDG PET and/or 18F-DOPA PET

for detection of residual or metastatic MTC between 2002 and 2010. We excluded patients

with undetectable calcitonin levels, patients with concurrent systemic treatment at the time of 18F-FDG PET or 18F-DOPA PET, and patients with less than 2 calcitonin or CEA values at

the time of 18F-FDG PET or 18F-DOPA PET imaging. For WBMTB analysis, we excluded

patients with more than 6 months between 18F-FDG PET and 18F-DOPA PET imaging.

Several patients (n=21) were also described in a previous study assessing the value of 18F-

DOPA PET in patients with MTC.16 That study was approved by the local medical ethics

committee, and the patients gave written informed consent to participate in it. After

completion of that study PET was performed as part of standard patient care; therefore in

concordance with national law no further Institutional Board Review approval was required.

We initially analysed 47 MTC patients (Figure 1). In group A, composed of 40 patients, 18F-FDG PET was performed and we compared outcome with doubling times (n=38) and

survival (n=37). For the 38 patients composing group B, 18F-DOPA PET was performed, and

we compared outcome with biochemical parameters (n=36) and survival (n=34).Thirty one

patients had undergone both scans and in 24 patients these scans were performed within 6

months of each other. We performed WBMTB and survival analysis in respectively, 21 and

22 patients (group C), of which 14 and 15 patients respectively, were also included in the

previous study.16 The number of patients participating in each analysis and reasons for

exclusion are shown in Figure 1. Patient characteristics of the different groups are shown in

Table 1.

18

F-DOPA PET, 18

F-FDG PET and image analysis

18F-FDG and 18F-DOPA were locally produced as described previously.20 All patients were

studied after a 6-h fasting period, were allowed to continue all medication, and were

encouraged to drink water. For 18F-FDG PET, data acquisition started after 60 or 90 min after

injection of 18F-FDG intravenously(5 MBq/kg; range 250-824 MBq). For 18F-DOPA PET,

whole body 2-dimensional-PET images were acquired 60 min after the intravenous

administration of a standard dose of 18F-DOPA (range 70-220 MBq). To reduce tracer

18F-FDG PET and

18F-DOPA PET in MTC

95

decarboxylation and subsequent renal clearance and thereby increase tracer uptake in tumour

cells, patients received carbidopa (2 mg/kg; maximum 150 mg) orally as pre-treatment 1 h

before the 18F-DOPA injection.

Figure 1 Flow diagram for inclusion and analysis of MTC patients.*Insufficient biochemical data for calculation

of doubling times. †Insufficient follow-up data.

‡n = 1 without suitable scan for WBMTB analysis due to

technical problems. pts = patients.

18F-FDG PET and 18F-DOPA PET images were interpreted by two dedicated nuclear

medicine specialists as part of routine patient care and were subsequently independently

reviewed. We calculated the WBMTB, defined as the sum of the metabolic burden of each

tumour lesion in the PET image, for both PET methods. We defined metabolic burden as

mean SUV × volume of tumour lesion obtained from the PET image using a volume of

interest that was enclosed by a 40 % isodensity contour (Figure 2).21,22 We categorized

patients according to differences in WBMTB uptake on paired 18F-FDG and 18F-DOPA PET

scans; more than 10% WBMTB on 18F-FDG PET, more than 10% WBMTB on 18F-DOPA

PET, equal uptake (less than 10% difference) or no uptake on both scans.

Chapter 6

96

Biochemical analysis

Calcitonin was determined using an enzyme-linked immunosorbent assay (Biomerica, Irvine,

Califorina, USA) with a reference value of 0.3-12 ng/L. CEA levels were measured using a

chemiluminescent microparticle immunoassay (Abbott Laboratories, North Chicago, Illinois,

USA) with a reference value of 0.5-5.0 µg/L.

Table 1 Patient characteristics

18

F-FDG PET

analysis

(group A; n = 38)

18F-DOPA PET

analysis

(group B; n = 36)

WBMTB

analysis

(Group C; n = 21)

Sex

Male

Female

19

19

17

19

10

11

Age (y)

Mean

Range

53.2

19-79

52.4

19-79

56.7

19-79

Type

Sporadic

Familial

18

20

18

18

12

9

Calcitonin (ng/L)

Median

Range

346.2

1.8-161,275

825

17.8-240,325

817

17.8-161,275

CEA (µg/L)

Median

Range

10.2

0.5-2620

12.3

0.5-2620

9.7

0.5-2620

Calcitonin doubling time

<24 mo

>24 mo

13 (34%)

25 (66%)

13 (36%)

23 (64%)

9 (43%)

12 (57%)

CEA doubling time

<24 mo

>24 mo

6 (19%)

32 (81%)

5 (14%)

30* (86%)

3 (14%)

18 (86%)

Calcitonin and CEA doubling time

Calcitonin or CEA <24 mo

Calcitonin and CEA >24 mo

13 (34%)

25 (66%)

14 (39%)

22 (61%)

9 (43%)

12 (57%)

PET

Positive

Negative

13 (34%)

25 (66%)

16 (44%)

20 (56%)

10 (48%)

11 (52%)

*Of one patient CEA doubling time could not be calculated. mo = months.

18F-FDG PET and

18F-DOPA PET in MTC

97

Calcitonin and CEA serum levels and doubling times

For calculating the calcitonin and CEA doubling time, we used in principle 4 values (with a

minimum of 2), obtained within a median period of 11 months (range 2-47 months) around 18F-FDG PET and 18F-DOPA PET imaging. We used the average of these values for further

analysis. We calculated exponential growth curves aB, using standard linear regression of the

serum levels on time and doubling times as ln(2)/B. To identify progressive patients we

defined biochemical progressive disease as a calcitonin or CEA doubling time of less than 24

months in concordance with the study of Giraudet et al..6

Figure 2 Determination of volume of interest (VOI) and standardized uptake value (SUV) for calculation of the

whole metabolic burden. On this 18

F-FDG PET scan four lesions (respectively subcarinal, in the lateral

hemithorax, and in the liver region) are enclosed by a 40% iso-contour, after manual designation, with

automatic calculation of SUVmean, SUVmax and lesion volume.

Follow-up

Follow-up was performed according to current guidelines, consisting of regular determination

of calcitonin and CEA.23 If there was an elevation in one of these tumour markers, further

evaluation was performed with morphological or functional imaging. Depending on the

outcome of imaging, the therapeutic strategy was determined.


For statistical analysis we used PASW statistics 18 (SPSS Ltd.). We performed a χ2 test for

comparison of PET outcome and doubling times. Correlation between WBMTB of 18F-FDG

Chapter 6

98

PET and 18F-DOPA PET and calcitonin or CEA levels and doubling times was calculated

with Spearman’s r test. To determine the optimal calcitonin cut-off level for 18F-FDG PET

and 18F-DOPA PET, we calculated the maximum value of sensitivity multiplied by specificity,

as derived from ROC curve analysis. We performed a χ2 test for comparison of uptake and

WBMTB category with doubling times or a Fisher exact test when the frequency of cells with

an expected value of 5 was higher than 20%. For comparison of the number of detected

lesions between 18F-FDG PET and 18F-DOPA PET, a McNemar test was used. For survival

analysis we used the Kaplan Meier method, and the log-rank test for comparison. The

significance level was 0.05, 2-sided.

Results

Patients

18F-FDG PET and biochemical parameters (Group A)

We analysed 38 patients for outcome of 18F-FDG PET and calcitonin or CEA levels and

doubling times. 18F-FDG PET was positive in 13 patients (34%) (Table 2). In 18F-FDG PET-

positive patients, levels of calcitonin and CEA were significantly higher and more patients

had calcitonin and CEA doubling times less than 24 months. Positive and negative predictive

values for biochemical progressive disease were 77% and 88% respectively in 18F-FDG PET-

positive and -negative patients. In ROC curve analysis, we found an optimal calcitonin cut-off

of 874 ng/L for PET positivity, with a sensitivity of 69% and a specificity of 70% for the

detection of tumour lesions.

18

F-DOPA PET and biochemical parameters (Group B)

Of the 36 patients analysed for the outcome of 18F-DOPA PET and biochemical parameters, 18F-DOPA PET was positive in 16 (44%) (Table 3). Calcitonin and CEA levels differed

significantly between 18F-DOPA PET positive and -negative patients, but there was no

significant difference in doubling times. The positive and negative predictive values for

progressive disease were 56% and 75%, respectively, in 18F-DOPA PET-positive and -

negative patients. In ROC curve analysis, we found a calcitonin cut-off of 825 ng/l to be

optimal for PET positivity, with a sensitivity and specificity of 88% and 80%, respectively,

for detection of tumour lesions.

18F-FDG PET and

18F-DOPA PET in MTC

99

Table 2 Biochemical parameters of patients with 18FDG PET (Group A)

18

F-FDG PET

Positive (n = 13)

18F-FDG PET

Negative (n = 25)

P

Calcitonin (ng/L)

Median

Range

2320

(60.4 – 161,275)

246

(1.8 – 18565)

0.040

CEA (ug/L)

Median

Range

32.4

(0.8 -2620)

6.5

(0.5-187)

0.006


< 24 mo

> 24 mo

10 (77%)

3 (23%)

3 (14%)

22 (86%)

< 0.001

CEA doubling time

< 24 mo

> 24 mo

6 (46%)

7 (54%)

0

25 (100%)

0.001


Calcitonin or CEA < 24 mo

Calcitonin and CEA > 24 mo

10 (77%)

3 (23%)

3 (14%)

22 (86%)

< 0.001

Mo = months.

Table 3 Biochemical parameters of patients with 18F-DOPA PET (Group B)

*Of 1 pt CEA level was not available. †Of 1 pt CEA dt could not be calculated. mo = months.

WMBTB results of 18

F-FDG PET and 18

F-DOPA PET (Group C)

For the 21 patients with both 18F-FDG PET and 18F-DOPA PET who were included in

WBMTB analysis, the results for both scans were negative in 11 patients. Of the remaining 10

patients, 4 had higher WBMTB on 18F-FDG PET, another 4 had higher WBMTB on 18F-

DOPA PET, and 2 had equal WBMTBs (Table 4). The total number of lesions found was 75,

and 18F-DOPA PET detected significantly more lesions than 18F-FDG PET (56 vs. 35)

18

F-DOPA PET

Positive (n = 16)

18F-DOPA PET

Negative (n =20)

P

Calcitonin (ng/L)

Median

Range

3626

(88 – 240,325)

287

(17.8 – 2320)

< 0.001

CEA (ug/L)

Median

Range

36.6

(1.2 – 2620)

6.6

(0.5 – 72)

<0.001


< 24 mo

> 24 mo

8 (50%)

8 (50%)

5 (25%)

15 (75%)

NS

CEA doubling time

< 24 mo

> 24 mo

4 (27%)

11 (73%)

1 (5%)

19 (95%)

NS




9 (56%)

7 (44%)

5 (25%)

15 (75%)

NS

Chapter 6

100

(p=0.009). In PET-positive patients, WBMTB on 18F-DOPA PET was significantly correlated

with calcitonin levels (r=0.82) (p=0.013) and CEA levels (r=0.88) (p=0.004) but not with

doubling times. There was no significant correlation between WBMTB of 18F-FDG PET and

calcitonin and CEA levels or doubling times. Between the different WBMTB categories and

calcitonin and CEA doubling times, no significant relation was found.

Table 4 Biochemical parameters and WBMTB in different WBMTB categories (Group C)

WBMTB Category

18F-DOPA >

18F-

FDG (n= 4)

18F-FDG >

18

F-DOPA (n = 4)

18F-DOPA =

18F-FDG (n = 2)

Negative P

Calcitonin (ng/L)

Median

Range

13052

832-161,275

650

89-1,066

14958

6,679-22,236

246

18-1,030

0.015

CEA (µg/L)

Median

Range

727

22-2620

14.2

0.8-29.3

1088

32.4 -2144

3.1

0.5-28.1

0.002




1

3

3

1

2

0

3

8

NS

No. of lesions 18

F-FDG

Mean

Total 18

F-DOPA

Mean

Total

1.3

5

9.5

38

5.3

21

2.5

10

4.5

9

4

8

-

-

WBMTB (cm3)

18F-FDG

Median

Range 18

F-DOPA

Median

Range

55.4

0 – 121

271.6

15.3 – 983

83.3

18.8 - 920

6.1

0 - 465

275

11.5 – 538

271

12.5 - 530

-

-

Treatment based on PET

Eight patients underwent reoperation because of recurrent disease. In 5 patients, PET showed

local disease and contributed to the decision for surgery. 18F-FDG PET was performed in 4

and positive in 2. 18F-DOPA PET was performed in 4 patients and positive in 3. All PET

lesions were confirmed on histological examination. In the other 3 patients, PET was negative

and surgery was performed because of positive conventional imaging or palpable

abnormalities. All patients who underwent reoperation had no clinical progression during

follow-up (range 6.6–106 months). Seven patients received targeted treatment with tyrosine

kinase inhibitors. 18F-FDG PET imaging was performed in 6 patients and all showed

18F-FDG PET and

18F-DOPA PET in MTC

101

metastatic disease, 18F-DOPA PET was performed in 5 and showed metastatic disease in 4.

Three patients developed stable disease. The other 27 patients did not receive surgical or

systemic treatment during follow-up.

Survival and PET outcome

In the 42 patients of whom follow-up data were available, median follow-up was 63.8 months

(range 2.3-114 months). During follow-up 11 patients died: 7 because of progressive MTC, 3

because of other causes (prostate cancer, oesophageal cancer and sepsis due to perforated

appendicitis) and in 1 patient for whom the reason of death was unknown. In 37 patients with 18F-FDG PET imaging and sufficient follow-up, survival was significantly lower in 18F-FDG

PET positive patients than in 18F-FDG PET negative patients (p<0.001) (Figure 3A). The

same was true for 18F-DOPA PET positive compared with -negative patients (n=34)

(p=0.019) (Figure 3B). However, in univariate analysis of patients who had undergone both 18F-FDG PET and 18F-DOPA PET (n=22), the survival in patients with a positive 18F-FDG

PET was lower and independent of 18F-DOPA PET outcome, whereas survival in 18F-DOPA

PET positive patients was dependent of 18F-FDG PET outcome (p=0.018) (Figure 3C). Figure

4 shows a patient with biochemical progressive disease and uptake on both scans.

Figure 3 Kaplan Meier curve of survival (in years) after 18

F-FDG PET (A), 18

F-DOPA PET (B) and both 18

F-FDG

PET and 18

F-DOPA PET(C).

Chapter 6

102

Figure 4 MTC patient with uptake on both 18

F-DOPA PET (left) and 18

F-FDG PET (right). On 18

F-DOPA-PET

lesions are seen in the right supraclavicular region, the right hemithorax and there is slight uptake subcarinal. In

the abdomen there are several lesions with faint uptake. Also on 18

F-FDG-PET uptake is seen in the right

supraclavicular region, right hemithorax and intensive uptake subcarinal. Furthermore several lesions are seen

in the liver region. Calcitonin and CEA levels were highly elevated (23236 ng/L (ref 0.3-12 ng/L) and 2144 ug/L

(ref 0.5-5.0 μg/L)) and calcitonin and CEA doubling times were short; 13 months and 12 months respectively.

The patient died 29 months after scans were performed due to progressive disease.

Discussion

In this study, 18F-FDG PET was superior to 18F-DOPA PET in identifying patients with

progressive disease. Unlike 18F-DOPA PET positivity, 18F-FDG PET positivity correlated

significantly with biochemical progressive disease. Furthermore, we showed that 18F-FDG

PET- and 18F-DOPA PET positive patients, had a significantly decreased survival. However,

univariate analysis in patients for whom both scans were performed showed that 18F-FDG

PET positivity had the most influence on survival. WBMTB analysis showed that metabolic

activity on 18F-DOPA PET correlated significantly with calcitonin and CEA levels.

Differences (>10%) in WBMTB on 18F-FDG PET and 18F-DOPA PET could not distinguish

stable from progressive disease.

In a previous study of our institute focusing on detecting residual disease with both 18F-

FDG PET and 18F-DOPA PET, we already described the superiority of 18F-FDG PET in 2

18F-FDG PET and

18F-DOPA PET in MTC

103

patients with progressive disease.16 This outcome is probably based on the fact that aggressive

(dedifferentiated) disease has a higher glucose metabolism and consequently higher 18F-FDG

uptake. This observation was also made by others but the described series are rather

small.14,15,17,18 Bogsrud et al. showed a higher mortality in 18F-FDG PET positive patients than

in 18F-FDG PET negative patients.24 However, survival data in patients with 18F-DOPA PET

have not been described before. This study shows that progressive patients can be identified

with both PET techniques, taking into account biochemical parameters and survival.

For 18F-FDG PET of patients with progressive MTC, not only have higher sensitivities

been described but also increased tracer intensity. Marzola et al. included only patients with

short doubling times (6-9 months) and showed significantly higher maximum SUV on 18F-

FDG PET versus 18F-DOPA PET, although patient- and lesion-based sensitivity of 18F-DOPA

PET was higher.18 In our WBMTB analysis, we did not find a significant difference in

doubling times between patients with a higher uptake on 18F-FDG PET and patients with a

higher uptake on 18F-DOPA PET. This lack of significance could have been caused by the

small number of patients with positive scan results in WBMTB analysis (n=11) or the

different doubling time cut-offs used for defining progressive disease.

Although the doubling times of calcitonin and CEA have thus far been the most reliable

indicators of recurrence and progressive disease in MTC, cut-off values are still a matter of

discussion. Meijer et al. showed a higher hazard ratio for recurrence for a calcitonin doubling

time cut-off of 12 months (hazard ratio, 5.33) than 24 months (hazard ratio, 2.93), but warned

about interpreting these cut-off values with caution.7 Moreover that study focuses on disease

recurrence and not progression in general. We based our 24 months cut-off for doubling times

on the results of the study by Giraudet et al., who compared doubling times with progression

according to the Response Evaluation Criteria in Solid Tumours (RECIST). They found

progressive disease in 94% of patients with doubling times less than 25 months while 86%

had stable disease when doubling times were more than 24 months.6

Our results show a significant correlation between WBMTB on 18F-DOPA PET and

calcitonin and CEA levels, demonstrating that 18F-DOPA PET might be a good indicator of

tumour load. Although 18F-FDG PET is better in distinguishing progressive disease, 18F-

DOPA PET seems to be more important in assessing the extent of residual disease. In our

WBMTB analysis, 18F-DOPA PET also detected more tumour lesions than did 18F-FDG PET.

On the whole, 18F-DOPA PET is superior to 18F-FDG PET with a higher patient-based

sensitivity (64% vs. 48%, respectively [range, 38%-83% vs. 17%-64%, respectively]) and

lesion-based sensitivity (72% vs 52%, respectively [range 52%-94% vs. 28%-62%]) (Table

Chapter 6

104

5).12-15,17,18 However, in line with the study of Kauhanen et al. and a recent review by Wong et

al., combining both modalities increases sensitivity and is complementary.14,25

Nevertheless, many patients with biochemical recurrent disease do not show lesions on

currently available imaging modalities. Most of these patients have moderately elevated

tumour markers and long doubling times, probably because of the nature of calcitonin-

producing metastases (sclerotic, necrotic or calcified) and their small size.26 A previous study

of our centre showed that MTC lesions are best detected on 18F -DOPA PET above >500 ng/L

and ROC curve analysis in the current study found a cut-off value of 825 ng/l to be optimal in

distinguishing 18F -DOPA PET-positive from -negative patients.16 This cut-off value is also

dependent on the resolution of the PET camera system, which with new developments

becomes increasingly sensitive. Also, the combination of PET with CT increases the yield of

these scans and lowers the threshold for localization of tumour lesions.27

The negative predictive value for biochemical progressive disease in our study was 88%

for 18F-FDG PET and 75% for 18F-DOPA PET. However, there are still patients - both in our

study (n=3) and in other series – who have rapidly increasing tumour markers but do not have

positive functional imaging results.18 In these patients, there is still need for other modalities

for the detection of occult MTC. Yet, the first results of new tracers like 68Ga-somatostatin

analogues or 11C-Methionine are not convincing.15,28,29

Table 5 Patient and lesion based sensitivity of 18F-FDG PET and 18F-DOPA PET. PET patient based sensitivity % (n) PET lesion based sensitivity % (n)

N 18

F-FDG 18

F-DOPA Combined Total no. of

lesions

18F-FDG

18F-DOPA

Hoegerle et al. 2001 11 64% (7) 64% (7) 73% (8) 27 44% (12) 63% (17)

Beuthien-Baumann et

al. 2007 15 47% (7) 47% (7) 60% (9) NA NA NA

Beheshti et al. 2009 26 58% (15) 81% (21) 85% (22) 53 62% (33) 94% (50)

Marzola et al. 2010 18 61% (11) 83% (15) 89% (16) 111 58% (64) 76% (84)

Kauhanen et al. 2011 19 53% (10) 58% (11) 63% (12) 118 47% (55) 52% (61)

Treglia et al. 2012 18 17% (3) 72% (13) 72% (13) 72 28% (20) 85% (61)

This study 21‡ 38% (8) 38% (8) 48% (10) 75 47% (35) 75% (56)

Total 128 48% (61) 64% (82) 70% (90) 456 48% (219) 72% (329)

*Average calcitonin, median not available. †Only 19 pts with data available.

‡Only patients included in WBMTB

analysis.

18F-FDG PET and

18F-DOPA PET in MTC

105

On the basis of the results of this and previous studies, we recommend a combined approach

for patients with recurrent MTC and increasing tumour markers (Figure 5). Conventional

imaging of the neck (ultrasound, MRI or CT) to detect localized disease can be followed by 18F-FDG PET or PET/CT to identify progressive disease. In the case of a negative 18F-FDG

PET result or the presence of only localized resectable disease (head and neck region), an 18F-

DOPA PET or PET/CT scan is recommended, to exclude distant metastasis and support the

decision for local surgery.

Figure 5 Flow-diagram for combined approach of 18

F-FDG PET and 18

F-DOPA PET in patients with recurrent

MTC and increasing tumour markers. If 18

F-FDG PET or 18

F-DOPA PET shows distant metastatic disease, targeted

therapy can be considered. If there is resectable localized disease on 18

F-FDG PET or 18

F-DOPA PET, with an

anatomical substrate, surgery could be considered. If both 18

F-FDG PET and 18

F-DOPA PET are negative, follow-

up would be appropriate.

Chapter 6

106

This study is limited by its retrospective character and the differences in 18F-FDG PET uptake

time, which can result in differences in the mean SUV. Most of our patients who were

included in the WBMTB analysis had an uptake time of 60 min (n=16). Because the WBMTB

for determination of tumour load depends not only on the mean SUV but also on tumour

volume and number of lesions we concluded that a slight difference in mean SUV does not

significantly influence our results. Furthermore, there could be a selection bias in patients

undergoing only 1 type of scan, or both scans. However, no significant difference existed in

patient characteristics (including doubling times) between these 2 groups (data not shown).

Other limitations are the small study size, which is often the case with rare tumours, and the

fact that not all PET lesions were histologically confirmed.

Conclusion

In MTC patients, 18F-FDG PET positivity seems to be associated with biochemical

progressive disease and significantly affects survival. 18F-DOPA PET has a higher sensitivity

than 18F-FDG PET, and WBMTB on 18F-DOPA PET can be related to the tumour load.

Therefore, 18F-DOPA PET seems to be more important in assessing the extent of the disease

in patients with residual disease whereas 18F-FDG PET can more accurately identify patients

with progressive disease. Both scans may be used to guide therapeutic strategies in patients

with recurrent MTC.

18F-FDG PET and

18F-DOPA PET in MTC

107

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Chapter 7

The effects of four different tyrosine kinase inhibitors

on medullary and papillary thyroid cancer cells

Hans H. G. Verbeek*, Maria M. Alves*, Jan Willem B. de Groot, Jan Osinga

John T. M. Plukker, Thera P. Links, Robert M. W. Hofstra

*The first two authors contributed equally to this study

J Clin Endocrinol Metab. 2011; 96: E991-5

Chapter 7

110

Abstract

Context Medullary and papillary thyroid carcinoma (MTC and PTC) are two types of thyroid

cancer that can originate from activating mutations or rearrangements in the RET gene.

Therapeutic options are limited in recurrent disease, but because RET is a tyrosine kinase

(TK) receptor involved in cellular growth and proliferation, treatment with a TK inhibitor

might be promising. Several TK inhibitors have been tested in clinical trials, but it is unknown

which inhibitor is most effective and whether there is any specificity for particular RET

mutations.

Objective We aimed to compare the effect of four TK inhibitors (axitinib, sunitinib,

vandetanib, and XL184 (cabozantinib)) on cell proliferation, RET expression and

autophosphorylation, and ERK activation in cell lines expressing a MEN2A (MTC-TT), a

MEN2B (MZ-CRC-1) mutation, and a RET/PTC (TPC-1) rearrangement.

Design The three cell lines were cultured and treated with the four TK inhibitors. Effects on

cell proliferation and RET and ERK expression and activation were determined.

Results XL184 and vandetanib most effectively inhibited cell proliferation, RET

autophosphorylation in combination with a reduction of RET expression, and ERK

phosphorylation in MTC-TT and MZ-CRC-1, respectively. TPC-1 cells showed a decrease in

RET autophosphorylation after treatment with XL184, but no effect was observed on ERK

activation.

Conclusion There is indeed specificity for different RET mutations, with XL184 being the

most potent inhibitor in MEN2A and PTC and vandetanib the most effective in MEN2B in

vitro. No TK inhibitor was superior for all the cell lines tested, indicating that mutation-

specific therapies could be beneficial in treating MTC and PTC.

Effects of TK inhibitors on MTC and PTC cells

111

Introduction

Medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC) can be caused by

activating mutations or rearrangements of the rearranged during transfection (RET) gene.

MTC originates from the calcitonin-producing C cells of the thyroid and can occur

sporadically (75%) or as part of a familial cancer syndrome (25%). The latter occurs as

multiple endocrine neoplasia (MEN) type 2 syndrome (MEN2A and MEN2B) or as familial

MTC.1-3 PTC is the most common thyroid cancer (80% of all thyroid cancers) and originates

from the follicular epithelial cells of the thyroid. In 2.5%–40% of PTC, a RET rearrangement

is found, although this percentage is higher in patients exposed to radiation.1

Total thyroidectomy and extensive lymph node dissection is the curative treatment for

MTC and PTC, followed by radioiodine ablation in PTC. However, recurrent disease is often

seen in sporadic MTC, and until recently, reoperation was the only therapeutic option. In

iodine-refractory PTC, no effective adjuvant therapy is available as well.4,5 New systemic

therapies are therefore needed for both recurrent MTC and PTC.

With RET being the gene involved in a subset of MTC and PTC, it is logical to consider

the encoded receptor as an important target for systemic therapy. RET is expressed in all

tumour cells and continuous autophosphorylation on its tyrosine kinase (TK) residues caused

by mutations (MEN2) or rearrangements (PTC) on RET results in a constant activation of

downstream signalling pathways that ultimately lead to tumour growth.1 Therefore, inhibition

of RET phosphorylation and its downstream pathways could be of great value.

Small-molecule inhibitors that selectively inhibit TK have been proven to be effective in

the treatment of several neoplastic diseases.6-8 A number of these clinically useful inhibitors

target TK receptors that belong to the same family group of proteins as RET.9 Several TK

inhibitors have already been tested in vitro and evaluated in phase II clinical trials (Table 1).

In a large number of patients (25%–81%), a stable disease state can be established, and some

patients even show a partial response (2%–33%).10-19

Because most studies have focused on one particular TK inhibitor and have not looked for

mutation specificity, it is hard to compare these compounds for the different patient groups.

We therefore set out to compare the efficiency of four recently developed TK inhibitors,

XL184 (cabozantinib), vandetanib, sunitinib, and axitinib, using three cell lines: MTC-TT

reported to be derived from a sporadic MTC expressing a C634W RET mutation, MZ-CRC-1

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112

derived from a patient with metastatic sporadic MTC expressing an M918T RET mutation,

and TPC-1 derived from a patient with PTC expressing a RET/PTC-1 rearrangement.

Table 1 Molecular targets and clinical trials performed with tyrosine kinase inhibitors in

medullary thyroid carcinoma (MTC) patients.

Molecular targets No. of

patients

Tumor response

% (n)

Stable disease

% (n)

Ref.

Imatinib

Bcr-Abl, PDGFR α

and β, c-Fms, c-Kit,

RET

24 0 38 (9) 10,11

Sorafenib

RAF, VEGFR2 and 3,

PDGFR, RET

21 10 (2) 86 (18) 12

Motesanib VEGFR 1, 2, and 3,

PDGFR, c-Kit, RET

91 2 (2) 81 (74)

13

Gefitinib* EGFR 4 0 0 14

Vandetanib

VEGFR, EGFR, RET 30

19

20 (6)

16 (3)

53 (16)

53 (10)

15

16

Axitinib

VEGFR 1, 2, and 3, 11 18 (2) 27 (3) 17

Sunitinib*

RET, VEGFR, PDGFR

6 0 83 (5) 18

XL184#

MET, VEGFR-2, RET 37 (35 with

measurable disease)

29 (10) 41 (15) 19

* Clinical trial performed in advanced thyroid cancer patients, not only MTC patients;

# Phase 1 trial.

Materials and Methods

Cell culture

MZ-CRC-1, MTC-TT, TPC-1, and HEK293 (human embryonic kidney cells) cell lines were

cultured as described in Supplemental Materials and Methods.

Tyrosine kinase inhibitors

XL184 (Exelixis, San Francisco, CA), vandetanib (AstraZeneca, Zoetermeer, The

Netherlands), and sunitinib and axitinib (Pfizer, Capelle a/d IJssel, The Netherlands) were

made up as a stock solution of 10 mM in dimethylsulfoxide (DMSO).

Cell proliferation assays

MTC-TT and MZ-CRC-1cells were plated in 200 µl medium at concentrations of 4 × 104 cells

per well. TPC-1 and HEK293 cells were plated at a density of 2 × 103 cells per well. After

overnight incubation, increasing concentrations of TK inhibitor solutions (0, 0.005, 0.05, 0.1,


113

0.5, and 5 µM) were added. A concentration of 0.1% DMSO was used in all experiments.

Control cells were grown without DMSO or TK inhibitor. Proliferation was measured at 1, 4,

and 7 d using a cell proliferation kit (MTT assay; Roche, Almere, The Netherlands) according

to the manufacturer’s instructions. The concentration that led to 50% growth inhibition (IC50)

was determined using linear interpolation at r=0.5 (Supplemental Table 1). If IC50

concentrations were between 0.5 and 5 µM, additional cell proliferation assays were

performed (Supplemental Figure 1, A–D). All experiments were performed in triplicate.

Cell lysates and Western blot analysis

MZ-CRC-1, MTC-TT, and TPC-1 cells were treated with 0, IC50, and maximal

concentrations of the different TK inhibitors for 0, 2, and 5 d. Cell lysates were prepared as

described in Supplemental Materials and Methods, and supernatants were stored at -80C

before they were further processed for SDS-PAGE followed by Western blot analysis. The

antibodies used are described in Supplemental Materials and Methods. All experiments were

performed in duplicate.

RNA extraction and RT-PCR

MTC-TT and MZ-CRC-1 cells, treated with 0, IC50, and maximal concentrations

(Supplemental Table 1) of XL184 and vandetanib, respectively, were collected after 0, 2, and

5 d. RNA extraction and RT-PCR procedures are described in the Supplemental Materials and

Methods.


The statistical analysis was performed using the software program SPSS version 16.0.

Results

Effect of different TK inhibitors on cell proliferation

A dose-dependent decrease in cell proliferations was seen for all four inhibitors (Figure 1, A–

C, and Supplemental Figure 1, A–D). In contrast, HEK293 cells, who do not endogenously

express RET, showed only minor effects on proliferation at concentrations lower than 0.5 µM

(Supplemental Figure 2A). Based on the IC50 values (Supplemental Figure 2B and

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114

Supplemental Table 1), we observed that XL184 was the most effective inhibitor of MTC-TT

(IC50=0.04 µM) and TPC-1 (IC50=0.06 µM) proliferation, whereas for MZ-CRC-1,

vandetanib inhibited cell proliferation at the lowest concentration (IC50=0.26 µM). DMSO

did not seem to have a significant negative effect on cell growth of any of the cell lines tested

(Supplemental Table 2).

Effect of XL184 and vandetanib on RET autophosphorylation

Inhibition of RET autophosphorylation on tyrosine 1062 was observed for the three cell lines

tested after 2 d of treatment with XL184 (MTC-TT and TPC-1) and vandetanib (MZ-CRC-1).

However, only vandetanib was able to induce this effect with IC50 levels (Figure 1D). For

MTC-TT and TPC-1, 5 d exposure to IC50 concentrations of XL184 were necessary to reduce

RET autophosphorylation levels (Figure 1, E and F). To investigate whether the decrease in

RET autophosphorylation was due to reduced levels of RET expression, the membranes were

stripped and probed for RET. MTC-TT and MZ-CRC-1 cells showed a dose-dependent

response after 5 d incubation with XL184 and vandetanib, respectively. At IC50 values, a

marked decreased in RET expression was observed, and at maximal concentrations, RET was

no longer detectable (Figure 1, D and F). To determine whether this effect was due to

inhibition of RET transcription, RET expression levels were determined. For MTC-TT, no

effect on RET transcription levels was observed. However, for MZ-CRC-1, 5 d exposure to

maximal concentrations of vandetanib led to a decrease in RET expression (Supplemental

Figure 3, A–C). For TPC-1 cells, the total amount of RET remained relatively unchanged

even after 5 d exposure to maximal concentrations of XL184 (Figure 1F).

Effect of XL184 and vandetanib on RET downstream signalling pathways

RET is involved in the activation of several signalling pathways, including the MAPK

pathway.20 Therefore, we evaluated the effect of XL184 and vandetanib in ERK activation.

For MTC-TT and MZ-CRC-1, ERK phosphorylation was markedly reduced with IC50 levels

of XL184 and vandetanib, respectively, and was totally inhibited when maximal

concentrations were used (Figure 1, D and E). However, XL184 was able to exert this effect

after only 2 d exposure. Interestingly, this reduction of ERK activation was related to a

decrease in ERK expression (Figure 1, D and E). For TPC-1, no effect on ERK expression

and activation was observed, even when maximal concentrations of XL184 were used (Figure

1F).


115

Figure 1 Effect of XL184, vandetanib, sunitinib, and axitinib on cell proliferation. A–C, Dose-response curves

of MZ-CRC-1 (A), MTC-TT (B), and TPC-1 (C) cell lines incubated with different concentrations of XL184,

vandetanib, sunitinib, and axitinib; D–F, effect of XL184 and vandetanib on RET expression, RET

autophosphorylation, ERK expression, and ERK phosphorylation: MZ-CRC-1 cells treated with IC50

concentration and 5 μM vandetanib for 2 and 5 d (D), MTC-TT (E), and TPC-1 (F) cells treated with IC50

concentration and 5 μM XL184 for 2 and 5 d. Error bars shown correspond to SD.

Discussion

We compared the effects of four TK inhibitors, XL184, vandetanib, sunitinib, and axitinib, on

cell proliferation and RET inhibition and looked for mutation specificity using cell lines

harbouring a MEN2A mutation (MTC-TT), a MEN2B mutation (MZ-CRC-1), and a

RET/PTC rearrangement (TPC-1). Our results showed that all four TK inhibitors are capable

of reducing cell proliferation to some extent. However, XL184 was found to be the most

efficient inhibitor for MEN2A and PTC-derived cell lines, whereas vandetanib proved to be

the most potent inhibitor for MEN2B.

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116

We also showed that XL184 and vandetanib were able to decrease RET

autophosphorylation and expression levels in MTC-TT and MZ-CRC-1 cells, respectively.

However, only vandetanib exerted this effect by inhibiting RET transcription. It is possible

that for XL184, lysosomal or proteasomal degradation is involved, as was described for other

inhibitors.20 For TPC-1 a marked decrease in RET phosphorylation levels was detected, but

surprisingly, RET/PTC expression levels increased after exposure to XL184. This dual effect

might be the result of a negative feedback mechanism to compensate a reduction in RET

activation. Furthermore, it shows that XL184 exerts its effect in PTC by direct inhibition of

RET autophosphorylation and that lysosomal or proteasomal degradation may not be effective

due to the presence of the fusion protein.

Finally, we explored the effect of these drugs in a downstream signalling pathway directly

activated by RET, the MAPK pathway. For MTC-TT and MZ-CRC-1, exposure to XL184

and vandetanib, respectively, induced a marked decrease in ERK phosphorylation.

Interestingly, a reduction on ERK expression was also observed for these cell lines,

suggesting a possible effect of XL184 and vandetanib on ERK transcription. For the TPC-1

PTC model system, no change in ERK phosphorylation was detected after exposure to

XL184. ERK phosphorylation through other TK receptors and TK effector molecules, e.g.

BRAF, are likely to exert a stronger effect in ERK phosphorylation than RET. It is possible

that for PTC, a combination of different inhibitors targeting RET and, e.g. BRAF

(serine/threonine-protein kinase B-Raf), could circumvent this problem and thus result in an

even more effective treatment for this type of cancer.

Because no TK inhibitor was superior for the cell lines tested, our in vitro results suggest

that mutation-specific therapies could be beneficial for the treatment of MTC and PTC.

However, because only three different mutations were analysed, additional mutational studies

are necessary to confirm this specificity.

To date, no distinction has been made between the different RET-related mutational

subtypes (MEN2A/MEN2B/PTC) in clinical trials, and it is known that aspecific targeting of

TK inhibitors can also contribute to antitumor effects. However, the risk of severe side effects

for the patients in combination with the development of resistance to the TK inhibitors

incorrectly used reinforces the need of mutation specific therapies. Furthermore, the combined

use of different TK inhibitors for multiple targeting will also benefit from this knowledge,

because only then optimal combinations of inhibitors can be chosen.

In conclusion, our results are in general agreement with the outcome of the clinical trials,

supporting the idea that XL184 and vandetanib are two potent inhibitors for tumour response


117

in MTC. We also showed that there is specificity of these inhibitors for the treatment of

different RET mutations, which suggest that mutation-specific therapies will likely improve

the outcome of ongoing studies. Thus, reanalysis of already performed trials based on

mutation status is more than worthwhile.

Acknowledgments

We thank Exelixis, AstraZeneca, and Pfizer for providing the TK inhibitors for investigation

and Jackie Senior for editing the manuscript.

Chapter 7

118

References



2. Hofstra RM, Landsvater RM, Ceccherini I, et al. A mutation in the RET proto-oncogene associated with

multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature

1994;367:375-376.

3. Eng C, Mulligan LM. Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2

syndromes, related sporadic tumours, and hirschsprung disease. Hum Mutat 1997;9:97-109.



5. Cooper DS, Doherty GM, Haugen BR, et al. Management guidelines for patients with thyroid nodules

and differentiated thyroid cancer. Thyroid 2006;16:109-142.

6. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine

kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031-1037.

7. Demetri GD, von Mehren M, Blanke CD, et al. Efficacy and safety of imatinib mesylate in advanced

gastrointestinal stromal tumors. N Engl J Med 2002;347:472-480.

8. McArthur GA, Demetri GD, van Oosterom A, et al. Molecular and clinical analysis of locally advanced

dermatofibrosarcoma protuberans treated with imatinib: Imatinib Target Exploration Consortium

Study B2225. J Clin Oncol 2005;23:866-873.



10. de Groot JW, Zonnenberg BA, van Ufford-Mannesse PQ, et al. A phase II trial of imatinib therapy for

metastatic medullary thyroid carcinoma. J Clin Endocrinol Metab 2007;92:3466-3469.

11. Frank-Raue K, Fabel M, Delorme S, Haberkorn U, Raue F. Efficacy of imatinib mesylate in advanced

medullary thyroid carcinoma. Eur J Endocrinol 2007;157:215-220.

12. Lam ET, Ringel MD, Kloos RT, et al. Phase II clinical trial of sorafenib in metastatic medullary thyroid

cancer. J Clin Oncol 2010;28:2323-2330.

13. Schlumberger MJ, Elisei R, Bastholt L, et al. Phase II study of safety and efficacy of motesanib in

patients with progressive or symptomatic, advanced or metastatic medullary thyroid cancer. J Clin

Oncol 2009;27:3794-3801.

14. Pennell NA, Daniels GH, Haddad RI, et al. A phase II study of gefitinib in patients with advanced thyroid

cancer. Thyroid 2008;18:317-323.

15. Wells SA,Jr, Gosnell JE, Gagel RF, et al. Vandetanib for the treatment of patients with locally advanced

or metastatic hereditary medullary thyroid cancer. J Clin Oncol 2010;28:767-772.

16. Robinson BG, Paz-Ares L, Krebs A, Vasselli J, Haddad R. Vandetanib (100 mg) in patients with locally

advanced or metastatic hereditary medullary thyroid cancer. J Clin Endocrinol Metab 2010;95:2664-

2671.

17. Cohen EE, Rosen LS, Vokes EE, et al. Axitinib is an active treatment for all histologic subtypes of

advanced thyroid cancer: results from a phase II study. J Clin Oncol 2008;26:4708-4713.

18. Cohen EE, Needles BM, Cullen KJ, et al. Phase 2 study of sunitinib in refractory thyroid cancer. Journal

of Clinical Oncology 2008;26.

19. Kurzrock R, Sherman S, Hong D, et al. A phase 1 study of XL184, a MET, VEGFR2, and RET kinase

inhibitor, administered orally to patients (pts) with advanced malignancies, including a subgroup of pts

with medullary thryoid cancer (MTC). EORTC 2008:119.

20. Plaza-Menacho I, Mologni L, Sala E, et al. Sorafenib functions to potently suppress RET tyrosine kinase

activity by direct enzymatic inhibition and promoting RET lysosomal degradation independent of

proteasomal targeting. J Biol Chem 2007;282:29230-29240.


119

Supplementary data

Materials and Methods

Cell culture

MTC-TT and TPC-1 cell lines were cultured in RPMI medium (Gibco, Breda, The

Netherlands) supplemented with 15% or 10% (for TPC-1) fetal bovine serum (FBS,

BioWhittaker, Lonza,Verviers, Belgium), 1% L-glutamine (Gibco) and 1% penicillin/strepto-

mycin (Gibco). MZ-CRC-1 and HEK293 cell lines were cultured in DMEM high medium

containing 4.5 g/L of glucose, L-glutamine and pyruvate (Gibco), supplemented with 15% or

10% (for HEK293) FBS and 1% penicillin/streptomycin. All cells were maintained at 37°C

and 5% CO2 atmosphere.

Cell lysates and Western blot analysis

Cells were washed with ice-cold PBS and incubated with lysis buffer (m-PER, Thermo Fisher

Scientific, Rockford, IL, USA) containing protease (Thermo Fisher Scientific) and

phosphatase (Thermo Fisher Scientific) inhibitors, for 30 min on ice. Cell lysates were

collected by scraping and cleared by centrifugation at 14,000 rpm for 10 min in a pre-cooled

(4°C) centrifuge.

The following antibodies were used for Western blot: RET (H-300), RET (C-19), p-RET

(Y1062) (Santa Cruz Biotechnology, Heidelberg, Germany); ERK, p-ERK (Cell Signaling,

Danvers, MA, USA) and actin (C4, MP Biomedicals, Illkirch, France). Secondary antibodies

used were goat anti-rabbit IgG-HRP (Bio-Rad, Veenendaal, The Netherlands) and goat anti-

mouse IgG-HRP (Bio-Rad). All the experiments were performed in duplicates.

RNA extraction and RT-PCR

Cells were washed with PBS (Gibco) and RNA extraction was performed using the RNeasy

mini kit© (Qiagen, Venlo, The Netherlands). First strand cDNA was originated using the

Ready-To-Go You-Prime First-Strand Beads© kit (GE Healthcare, Zeist, The Netherlands)

according to the manufacturer’s instructions. The following primers were used: RET forward

(5’-CCGTGAAGATGCTGAAAGAG-3’); RET reverse (5’-

AGAGGCCGTCGTCATAAATC-3’); GAPDH forward (5’-

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120

TGAAGGTCGGAGTCAACGGATTTGGT-3’); GAPDH reverse (5’-

GCAGAGATGATGACCCTTTTGGCTC -3’).


121

Supplementary Tables

Supplementary Table 1 IC50 concentrations of the different TK inhibitors

in different cell lines

Cell-line

Compound MTC-TT MZ-CRC TPC-1

Sunitinib

0.78 + 0.19 μM 0.71 + 0.15 μM 0.37 + 0.03 μM

Vandetanib

0.47 + 0.33 μM 0.26 + 0.07 μM 0.41 + 0.09 μM

Axitinib

1.56 + 0.04 μM > 5 μM 1.14 + 0.03 μM

XL184

0.04 + 0.03 μM > 5 μM 0.06 + 0.02 μM

MTC-TT cells express a MEN2A mutation, MZ-CRC-1 cells express a MEN2B mutation,

and TPC-1 cells express a RET/PTC rearrangement.

Supplementary Table 2 Effect of DMSO on cell proliferation of MZ-CRC-1, MTC-TT and TPC-1

Cell proliferation

Sign. Control (SD) 0 concentration (SD)

MZ-CRC-1 104.7% (10.2%) 100% (7.0%) 0.35 (NS)

MTC-TT 94.2% (18.4%) 100% (14.5%) 0.11 (NS)

TPC-1 100.3% (10.4%) 100% (12.1%) 0.93 (NS)

Control cells were grown without DMSO or inhibitors, cells in the “0”condition were grown without inhibitors

but in the presence of 0.1% DMSO.

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122

Supplementary Figures

Supplementary Figure 1 Additional dose response curves for (A) MTC-TT cells treated with sunitinib, (B)

MTC-TT cells treated with axitinib, (C) MZ-CRC-1 cells treated with sunitinib and (D) TPC-1 cells treated with

axitinb.


123

Supplementary Figure 2 (A) Dose response curves for HEK293 cell line incubated with different

concentrations of XL184, vandetanib, sunitinib and axitinib. (B) Overview of the IC50 values (the concentration

that lead to 50% cell death) of the four tyrosine kinase inhibitors for MTC-TT, MZ-CRC-1 and TPC-1 cell lines.

Error bars shown correspond to standard deviation. * Concentrations are above 5 µM.

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Supplementary Figure 3 Effects of XL184 and vandetanib on RET mRNA levels. (A) MTC-TT cells treated

with IC50 concentration and 5 µM of XL184 showed no effect on RET transcription levels. (B) MZ-CRC-1 cells

treated with IC50 concentration and 5 µM of vandetanib showed a decrease in RET transcription levels after

prolonged exposure (5 days) to 5 µM of this inhibitor. (C) Graphical representation of RET bands’ intensity after

correction for GAPDH levels confirmed a reduction of RET mRNA levels for MZ-CRC-1 cells after prolonged

exposure to 5 µM of vandetanib. MTC-TT cells express a MEN2A mutation (RET C634R) and MZ-CRC-1 cells

express a MEN2B mutation (RET M918T). IC50 = concentration that leads to 50% cell death. GAPDH was used

as an internal control.

Chapter 8

Summary, discussion and future perspectives

Chapter 8

126

Summary

Medullary thyroid carcinoma (MTC) arises from the parafollicular C-cells and accounts for

approximately 5% of all thyroid cancers.1 MTC occurs sporadically (75%) or in a familial

form as part of one of the inherited syndromes known as familial MTC (FMTC) and Multiple

Endocrine Neoplasia type 2 (MEN 2). A MEN2A and MEN2B variant of the syndrome are

discerned and mutations in the ‘REarranged during Transfection’ (RET) gene are responsible

for these syndromes. Other manifestations of the MEN2 syndromes include

pheochromocytoma (MEN 2A and 2B) and hyperparathyroidism (MEN2A) or

neurofibromatosis (MEN2B).2

At presentation most MTC patients have an asymptomatic palpable solitary thyroid nodule

or palpable lymph node(s). Fifty percent of the patients have lymph node metastasis and 10%

will have distant metastasis at the time of diagnosis.3,4 Usually, the first diagnostic procedure

for thyroid nodules is fine needle aspiration cytology (FNAC) with ultrasound guidance.

However, without the use of specific immunohistochemical analyses, the sensitivity of this

procedure for detecting MTC is limited.5 As MTC originates from the calcitonin producing C-

cells, this hormone may also be used as a screening tool in patients with thyroid nodules to

detect MTC in an early stage of the disease. This application is a matter of discussion as a

proportionate number of patients has an elevated calcitonin caused by other conditions than

MTC.6,7

The initial treatment of patients with sporadic MTC without identified metastases consists

of a total thyroidectomy with an elective lymphadenectomy of regional lymph nodes in the

central compartment (level VI) (central compartment dissection (CCD)), according to the

current American Thyroid Association (ATA) guidelines. If the disease has already spread to

the lateral lymph nodes of the neck, a modified radical uni- or bilateral dissection of the

lateral lymph nodes (levels II-V) (lateral node dissection (LND)) is also indicated.8 As

surgery offers the only chance on cure, some surgeons not only perform an elective CCD, but

also an elective LND because of a relative risk of nodal (micro) metastases. A considerable

number of MTC patients (>50%) are beyond cure, because the disease has been so broadly

metastasized, even when MTC presented as a locoregional disease.9,10 Unlike other types of

thyroid cancer, MTC patients will not benefit from adjuvant radioactive iodine treatment.11

Therefore, adequate surgery in MTC is of crucial importance for optimal locoregional control

and potential cure.


127

Although more than 50% of MTC patients cannot be cured despite extensive surgery, many

patients with minimal residual disease have a good life expectancy.12,13 To monitor disease

progression, careful follow-up, consisting of regular determinations of the tumour markers

calcitonin and carcinoembryonic antigen (CEA) is important.14 If these tumour markers are

increasing, diagnostic work-up with morphological and functional imaging (including

Positron Emission Tomography (PET)) is advocated. Calcitonin and CEA doubling times are

currently the most reliable markers for progression, but accurate determination requires serial

measurements which are time consuming. Early detection of progression may be important,

because appropriate therapeutic interventions may delay symptomatic deterioration.

Depending on the extent of the disease, based on imaging and the rate of elevation of tumour

markers, several therapeutic strategies can be considered; a ‘wait and see’ approach with close

monitoring, surgical intervention (for locoregional control) or treatment with systemic

(targeted) therapy.

Chemotherapy has not yet been proven to be effective in the palliative treatment of MTC.

Recent preclinical and clinical research involving tyrosine kinase (TK) inhibitors, have shown

an improvement of the progression free survival in MTC patients.15,16 However, it is unknown

whether the effectiveness of these inhibitors is dependent on the somatic or germ line RET

mutation of the tumour or patient. Different RET mutations give rise to different

configurationally changes of the RET protein.17 For instance, the mostly found mutation

found in sporadic MTC, the ’MEN2B mutation’, changes the ATP pocket of the RET protein,

to which these inhibitors bind. We therefore speculate that selecting patients based on specific

RET mutations may increase the effectiveness of targeted therapy. Furthermore, most TK

inhibitors have several targets activating multiple pathways, which can cause, next to tumour

regression, also side-effects such as cardiac toxicity or hand-foot syndrome.18,19 Therefore,

careful selection of patients is important in which mutation specific therapy can be of value.

This thesis covers several important clinical issues in the diagnosis and treatment of primary

and recurrent (inherited) MTC. The aim of this thesis was to evaluate and to improve both

diagnostic and therapeutic modalities in the treatment of patients with MTC by: (1)

Addressing the value of calcitonin testing for detection of MTC in patients with thyroid

nodules; (2) Evaluating the recommendations regarding surgical treatment by the current

ATA guidelines for MTC patients; (3) Investigating the value of 18F-FDG PET and 18F-

DOPA PET for detection progressive recurrent MTC and (4) Evaluating different tyrosine

kinase inhibitors for treatment of MTC.

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In Chapter 1 a general introduction and the aims and outlines of this thesis are presented.

In Chapter 2 three patients with different stages of disease were presented to illustrate the

variety in clinical presentation and behaviour of MTC. Based on these patients a short

overview of the presentation, diagnosis, treatment and follow-up of MTC was provided.

In Chapter 3 the diagnostic accuracy of the calcitonin test for detection of MTC in patients

with thyroid nodules was evaluated. MTC patients detected in an early stage of the disease

have a better prognosis, so early detection of MTC in patients with thyroid nodules can be

beneficial. As almost all MTC’s secrete calcitonin, standard determination of calcitonin may

detect these tumours in patients with thyroid nodules. However, no consensus exists whether

or not to perform routine calcitonin testing in patients with thyroid nodules. A meta-analysis

was performed to evaluate the diagnostic accuracy of the calcitonin test. Sixteen studies were

eventually included. Summary estimates for different cut-off values were determined.

Sensitivity was high for lower basal cut-off values and combined basal and stimulated

calcitonin testing. Specificity did slightly increase with higher cut-off values and stimulated

testing. Overall, the diagnostic accuracy of calcitonin testing was high. However, for the

interpretation of this accuracy, the low prevalence of MTC has also to be taken into account.

The rarity of MTC decreases the positive predictive value and thereby the clinical

applicability of routine calcitonin testing for the detection of MTC. The low positive

predictive value carries the risk of patients being operated unnecessary. Moreover, the cost-

effectiveness of calcitonin screening for the early detection of MTC is still a matter of

discussion following the overall low prevalence of MTC.

In Chapter 4 we investigated, whether the current American Thyroid Association (ATA)

recommendations are of clinical benefit for MTC patients with respect to the surgical

treatment. No evidence exists for the optimal surgical treatment, especially with regard to the

extent of the lateral lymph node dissection (LND). Therefore, different surgical strategies are

advocated by experts and guidelines. The ATA guidelines recommend total thyroidectomy

and LND of the central compartment (level VI) as the initial treatment.8 In case of regional

node involvement, systematic LND of the lateral compartments (level II – V) should be

performed. Retrospectively, we reviewed the surgical and pathology reports of 86 patients and

compared the clinical outcome (reoperations, biochemical cure, survival and complications)

of the patients treated by ATA-compliant surgery versus the patients treated by ATA-non-


129

compliant surgery. Furthermore we examined to which extent clinical outcome of patients

was influenced by (1) one-step versus a two-step intended curative, (2) the location of the

initial curative surgery (experienced referral centre versus non-centre hospital), and (3) patient

and tumour characteristics. Our results indicated that patients treated adequately according to

ATA guidelines had significantly fewer reoperations compared to the inadequately operated

patients. Moreover, these patients remained significantly more often biochemically cured. We

could not demonstrate a significant effect on the clinical outcome of the patients treated in an

experienced referral centre hospital, but these patients received significantly more often

adequate surgery. Tumour size and lymph node involvement showed to be the most important

predictors for clinical disease free (DFS) and overall survival (OS).

In Chapter 5 we discussed the role of Positron Emission Tomography (PET) imaging using

different radiotracers in the staging and follow-up of papillary thyroid cancer (PTC) and

MTC. PET imaging is based on the use of positron emitting isotopes. For the detection of

thyroid cancers, several radiotracers are available. 18Fluorine-Fluorodeoxyglucose (18F-FDG),

a glucose analogue, is frequently used. While its use for discriminating between benign and

malignant thyroid nodules is contradictory, 18F-FDG can be of value in the follow-up of

differentiated thyroid cancer (DTC). Especially in patients with a negative radioiodine scan

but a detectable tumour marker thyroglobulin 18F-FDG can localize disease activity. Other

available tracers for the use during follow-up of DTC are 11C-Methionine PET (11C-MET-

PET) and 124Iodine-PET (124I-PET). While the value of 11C-MET-PET seems limited, 124I-PET

may be a superior diagnostic tool in comparison to the 131Iodine whole body scintigraphy

(WBS). In MTC patients, 18F-FDG also has been used, but 18Flurorine-

dihydroxyphenylalanine (18F-FDOPA) seems to be more sensitive. This radiotracer makes use

of a strongly upregulated transmembrane transport of amino acids via the large amino acid

transporters. In conclusion, PET imaging is a useful diagnostic tool in thyroid cancer although

the optimal radiotracer depends on the type of cancer and the intent of imaging

(staging/follow-up).

In Chapter 6 we evaluated the outcome of both 18F-FDG PET and 18F-DOPA PET with

calcitonin and carcinoembryonic (CEA) doubling times in 47 MTC patients. Early

identification of MTC patients with progressive residual disease is relevant because

appropriate therapeutic interventions may delay symptomatic deterioration. Calcitonin

doubling time is the most reliable marker for progression, but for accurate calculation, serial

Chapter 8

130

measurements over a considerable period are needed. Most morphological imaging techniques

like CT or MRI have moderate sensitivities for detecting recurrent MTC, but in the last

decade 18F-FDG PET and 18F-DOPA PET have become available for staging and follow-up of

MTC. We assessed the ability of 18F-FDG PET and 18F-DOPA PET to discriminate patients

with progressive disease and patients with stable disease. PET positivity was compared with

biochemical parameters (calcitonin and CEA serum levels and doubling times) and survival.

In a subgroup of patients whole body metabolic burden (WBMTB) was assessed with

standardized uptake value and the number of lesions. The WMBTB was compared with

biochemical parameters.

We observed that 18F-FDG-PET positivity was significantly correlated with both calcitonin

and CEA levels and their doubling times. Although 18F-DOPA PET positivity was

significantly correlated with the calcitonin and CEA levels, no significant correlation existed

with doubling times. 18F-DOPA PET detected significantly more lesions compared to 18F-

FDG PET in the 21 patients included in WBMTB analysis. However, 18F-FDG PET positive

was a more important indicator for poor survival. Both scans are therefore important in the

follow-up of patients; while 18F-DOPA PET is better in assessing the extent of the disease, 18F-FDG PET can more accurately identify patients with progressive disease.

In Chapter 7 we described the effects of four different tyrosine kinase inhibitors on MTC and

PTC cell lines. MTC and PTC can be caused by activating or rearrangements in the RE-

arranged during Transfection (RET) gene. This gene encodes for the RET tyrosine kinase

(TK) receptor which is involved in cellular growth and proliferation. Several TK inhibitors

have been tested in clinical trials, but it is unknown if there is specificity for particular RET

mutations and which inhibitor is the most effective. We cultured three cell-lines expressing a

MEN2A (MTC-TT), a MEN2B (MZ-CRC-1) mutation, and a RET/PTC (TPC-1)

rearrangement. We treated these cell lines with four different tyrosine kinase inhibitors

(axitinib, sunitinib, vandetanib and cabozantinib (XL184)) and compared the effect on cell

proliferation, RET expression and autophosphorylation, and RET downstream pathways

(Mitogen-Activated Protein Kinase (MAPK) pathway; involved in cell differentiation,

proliferation and survival).

A dose-dependent decrease in cell proliferation was found in all four tested tyrosine kinase

inhibitors. Cabozantinib was the most effective inhibitor of the MEN2A and RET/PTC cell

line, whereas vandetanib was the most effective inhibitor for the MEN2B cell line. Both

cabozantinib and vandetanib were able to decrease RET autophosphorylation and RET


131

expression levels in MEN2A and MEN2B cells. However, only vandetanib exerted this effect

by inhibiting RET transcription. A marked decrease in RET phosphorylation was detected for

RET/PTC cells, but RET/PTC expression was increased after exposure to cabozantinib. With

regard to downstream targets of RET, the MAPK pathway, and more specifically

Extracellular Signal-regulated Kinase (ERK) phosphorylation and expression, was markedly

decreased in MEN2A and MEN2B cells after exposure to cabozantinib and vandetanib

respectively. In RET/PTC cells no change in ERK phosphorylation and expression was

observed. Our results show that both vandetanib and cabozantinib are potent inhibitors for

tumour progression in MTC. We also found a specificity of the TK inhibitors for different

RET-mutations, suggesting mutation-specific therapies might be of benefit for MTC and PTC

patients.

Discussion and future perspectives

Although several new diagnostic and therapeutic technologies have been developed, the

prognosis of patients with medullary thyroid cancer has not improved in the last decades. The

current diagnostic and therapeutic approaches will be discussed and future perspectives are

provided.

Calcitonin as a routine test in patients with thyroid nodules

Whether or not to perform routine calcitonin testing is still a matter of debate. Although a

clearly elevated calcitonin level is highly suggestive for MTC, moderately elevated levels are

seen in a large number of non-MTC patients. While the European Thyroid Association (ETA)

guidelines recommend routine determination of calcitonin, the ATA guidelines do not

recommend either for or against routine measurement.8,20 Based on current literature, routine

testing is performed in several centres. In our systematic meta-analysis we showed that the

calcitonin test had a high diagnostic accuracy in terms of sensitivity and specificity. However,

the high diagnostic accuracy itself does not advocate routine calcitonin testing. Due to the low

prevalence of MTC in patients with thyroid nodules (range 0.11%-0.85%), the positive

predictive value of the calcitonin test is low.21-24 This is especially true if a low basal

calcitonin cut-off value is used and no additional stimulation tests are performed. With a cut-

off value of 10 pg/ml, only 7.5% of patients with an elevated calcitonin level will have a

histological proven MTC. Another important problem for evaluation of the routine use of the

Chapter 8

132

calcitonin test is the variation between assays, which make comparison between different

study groups difficult.25 Although not all patients with elevated calcitonin levels will be

operated on, a considerable proportion of these patients will be operated unnecessary, if the

supposed diagnosis MTC was not confirmed with immunohistochemical (IHC) based

cytological examination.

Cheung et al. performed a formal cost-effectiveness model on the routine calcitonin test in

thyroid nodules and concluded that calcitonin testing had a comparable cost-effectiveness to

mammography.26 However, if we apply our findings in this model, routine calcitonin testing

is not cost-effective, especially due to the lower prevalence we established in the evaluated

studies. Cost-effectiveness can be improved in several ways, such as applying the calcitonin

test only in subgroups of patients with a higher prevalence of MTC. Further studies should

focus on identification of such subgroups (e.g. young male patients, or patients with large

thyroid nodules). Based on current literature there seems no role for routine calcitonin testing

in all patients with thyroid nodules.

Instead of using the calcitonin test as a triage test next to or before FNA, calcitonin testing

can also be used as an add-on test after FNA. Determining calcitonin levels in patients with

inconclusive FNAC (Bethesda 3) results might be a more cost-effective approach in

comparison to routine calcitonin testing in all thyroid nodule patients. Measuring calcitonin in

FNA aspirates can increase the sensitivity of FNAC for diagnosis of MTC. Recently the use

of FNA-calcitonin measurement has been advocated in addition to routine calcitonin testing

as a possible alternative to stimulated calcitonin testing.27 The (cost-)effectiveness of this

approach is unclear as it still requires routine calcitonin testing and needs further evaluation.

Another application of the calcitonin test may be as a routine pre-operative test. The

reported sensitivity of routine pre-operative testing is lower compared to routine calcitonin

testing in all patients. This lower sensitivity is likely the result from a verification bias; studies

including only pre-operative patients have histological verification in all calcitonin-negative

patients while studies including all patients have only histological confirmation in a limited

number of calcitonin-negative patients (e.g. operated for other causes).28,29 The sensitivity of

pre-operative calcitonin testing is for detection of MTC is higher than FNAC and can increase

the rate of correct pre-operative diagnoses. This can result in more adequate initial surgical

procedures which is of crucial importance in MTC patients. Few studies have reported on the

value of pre-operative calcitonin testing and more research is needed whether this approach

can be cost-effective.


133

Optimal surgical treatment for MTC patients

Although the current ATA guidelines provide specific recommendations for the surgical

approach, the effect of adherence to these recommendations on the outcome of MTC patients

is unclear. We demonstrated that patients who were not treated adequately according to

guidelines had more locoregional reoperations and less biochemical cure at follow-up. We

also observed that patients treated in a non-centre institute had less often adequate surgery

compared to patients treated in a centre. Our findings underscore the importance to organize

the treatment of MTC patients in specialized tertiary referral centres, with maximal surgical

experience but also a multidisciplinary approach including endocrinologists, surgeons,

radiologists, nuclear physicians and pathologists. One of the main problems in the treatment

of MTC is to determine accurately the extent of the disease. Even with state of the art

diagnostic methods it is difficult to determine locally advanced disease and in particular

optimal nodal staging. On the other hand, although accurate imaging is crucial for optimal

locoregional surgery, it remains unclear when the point of cure is passed due to undetectable

distant metastases even with extended (elective) surgical dissections.30

Although total thyroidectomy and central compartment lymph node dissection are accepted

as standard procedure in common practice, it still remains unclear whether or not to perform a

(bi)lateral lymph node dissection of level II-V in MTC patients, especially in the elective

treatment of patients without apparent lymph node metastases in the lateral compartments. A

number of authors recommend an elective lateral lymph node dissection whenever lymph

node metastases are present in the central lymph node compartment (level VI).31 Other

authors suggest a (uni)lateral lymph node dissection when calcitonin levels are elevated >20

pg/ml and a bilateral lymph node dissection with calcitonin >200 pg/ml. Using such a cut-off

level for calcitonin minimizes the risk that MTC patients receive inadequate surgery.

However, as a consequence there is a risk of over treating patients as nearly 90% of MTC

patients with calcitonin between 20-200 pg/ml have no lateral lymph node metastases.32

Long-term results on cure and survival of the different proposed strategies are lacking in

patients without apparent lateral lymph node metastasis, and the current ATA guidelines are

indeterminate. In absence of such data, careful follow-up of treated patients is crucial. Either

way, comprehensive pre-operative evaluation is important and functional imaging techniques

can play an important role pre-operative staging. These techniques can also identify patients

who may not benefit from surgical treatment. The optimal surgical treatment for MTC

patients thus remains difficult; another reason to advocate treatment in an experienced centre.

Chapter 8

134

Functional imaging in the follow-up of MTC

We showed that 18F-FDG PET was superior in detecting patients with progressive disease,

while 18F-DOPA PET was better in assessing the extent of residual disease. We therefore

proposed a flow diagram with a combined approach of 18F-FDG PET and 18F-FDOPA PET in

patients with increasing tumour markers. Using this flow diagram, progressive MTC patients

who do not benefit from (local) surgical treatment but are candidates for systemic treatment

can be identified. A proportion of patients with progressive disease - indicated by rapidly

increasing tumour markers - still have false negative imaging results.33 Other imaging

modalities in these patients are necessary for detection of recurrent tumour lesions to assess

the optimal (therapeutic) approach. However, first results for new tracers such as 68Ga-

somatostatin analogues or 11C-methionine are not convincing. Compared with 18F-FDG PET

and 18F-FDOPA PET, 68Ga-somatostatin analogue did not identify additional lesions or led to

a change in TNM status.34,35 Although 11C-MET PET had a higher sensitivity for detecting

cervical metastasis compared to 18F-FDG PET, it was not superior compared to ultrasound.36

Further applications of nuclear imaging may be valuable in the early evaluation of

therapeutic response of targeted therapy, thereby identifying patients who may benefit from

this treatment. A preclinical study with vandetanib showed for instance that 18F-FDG PET

was able to assess metabolic changes after three days of treatment. A down-regulation of key

genes in the glycolysis pathway was observed resulting in a reduction of uptake of

deoxyglucose both in vitro and in vivo.37 Other improvements in the future may focus on

developing radiotracers which are able to selectively image mutated receptors, as was shown

for a mutant form of EGFR in lung carcinoma.38 As somatic mutations exist in the RET

receptor in a fair proportion of sporadic MTC patients, using selective radiotracers may better

identify these lesions and also serve as a new treatment modality.

New treatment modalities

In the last years, new systemic therapeutic options have become available for the treatment of

MTC patients. We showed in a comparison of four different TK inhibitors targeting RET that

vandetanib and cabozantinib were the most effective TK inhibitors. This was also confirmed

in two phase III clinical trials evaluating these TK inhibitors with a significant effect on

progression free survival (PFS).39,40 Although these TK inhibitors show promising results,

only effects on PFS have been reported, while no benefits on overall survival (OS) have been

demonstrated yet. With vandetanib an estimated PFS of 30.5 months was reported versus 19.3


135

months in the placebo group. For cabozantinib a PFS of 11.2 months was observed versus 4

months in the control group, but this study included only progressive patients (i.e. showing

progression compared to imaging obtained within the prior 14 months). Targeted therapies are

generally better tolerated in comparison to cytotoxic chemotherapeutic regimens, although a

large proportion of patients develop serious side-effects which may have a great impact on the

quality of life. Furthermore, timing for initiation of these therapies has to be made with

consideration of the natural course of disease progression, as a large proportion of patients

have stable or slowly progressive disease. However, in patients presenting with progressive

disease there may be a role for (neo) adjuvant systemic therapy.

Besides the RET activated pathway, also other receptors and pathways can be potential

therapeutic targets in MTC. Indeed, over-expression of VEGF, VEGFR and MET have been

described in MTC.41-43 It also has been shown that inhibition of RET can lead to over

activation of signalling trough EGFR.44 Inhibiting multiple kinases can therefore be beneficial

to suppress such escape mechanisms. Most inhibitors currently in use are already multikinase

inhibitors, thus exerting their effects on multiple kinases. However, the optimal combination

of inhibition with regard to tumour regression and side effects may advocate the use of

multiple inhibitors. Combinations of TK inhibitors with classic chemotherapeutic agents may

also be beneficial as was reported in an in vivo study showing an additional effect of cisplatin

to sunitinib.45

In the meanwhile new TK inhibitors are being developed and tested. Ponatinib was shown

to be a potent inhibitor of RET kinases in a pre-clinical study.46 A phase II study with this

new drug is currently recruiting for patients with advanced MTC (clinicaltrails.gov). Other

TK inhibitors evaluated for MTC are lenvatinib and AZD1480.47,48 Not only tyrosine kinases

can be therapeutic targets for medullary thyroid carcinoma. A recent in vitro study showed

promising results with an agent targeting mitochondria in MTC tumour cells.49

In conclusion, the treatment options for patients with advanced metastatic MTC are being

extended. However, although results of clinical trials look promising, no definite

improvement of survival has been established. Further studies are needed to identify more

effective TK inhibitors, possible in combination with other TK inhibitors or with agents

blocking other potential targets. Careful consideration must be given to outweigh the benefits

of possible disease control versus side effects of these new therapies. Therefore treatment of

this rare neuroendocrine tumour should be performed in an experienced centre to further

enhance diagnostic and therapeutic strategies to ultimately improve the outcome of MTC

patients.

Chapter 8

136

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Nederlandse samenvatting


140


Medullair schildkliercarcinoom (medullair thyroïd carcinoom, MTC) gaat uit van de

calcitonine producerende C-cellen in de schildklier en is verantwoordelijk voor ongeveer 5%

van alle soorten schildklierkanker. MTC komt sporadisch voor (75%) of als onderdeel van

één van de erfelijke tumorsyndromen; familiair MTC (FMTC) en multipele-endocriene-

neoplasie-type 2 (MEN2). Bij het MEN2 syndroom worden een MEN2A en een MEN2B

variant onderscheiden, waarbij mutaties in het ‘REarranged during Transfection’ (RET) gen

ten grondslag liggen aan deze syndromen. Andere uitingen van het MEN2 syndroom zijn

bijniertumoren (feochromocytoom; MEN2A en 2B), hyperparathyreoïdie (MEN2A) of

neurofibromatose (MEN2B).

De meeste patiënten met MTC presenteren zich met een asymptomatische palpabele nodus

in de schildklier of in één of meerdere lymfeklieren. Als de diagnose wordt gesteld heeft 50%

van de patiënten uitzaaiingen in de lymfeklieren en 10% uitzaaiingen elders in het lichaam.

Om de diagnose MTC te stellen is een echogeleide dunne naald biopsie de eerste stap. Hierbij

kan immunohistochemische analyse de sensitiviteit voor het opsporen van MTC verhogen.

Omdat MTC uitgaat van de calcitonine producerende C-cellen, kan de bepaling van

calcitonine in het bloed ook worden gebruikt als een screenings instrument bij patiënten met

een schildkliernodus om MTC in een vroeg stadium op te sporen. Deze screening staat echter

nog ter discussie, omdat een aanzienlijk aantal patiënten een verhoogd calcitonine heeft op

basis van andere oorzaken dan een MTC.

Volgens de huidige richtlijn van de Amerikaanse Schildklier Associatie (American

Thyroid Association; ATA) bestaat de initiële behandeling van patiënten met een sporadisch

MTC zonder bekende metastasen bij diagnose uit een totale thyreoïdectomie met een

electieve lymfadenectomie van regionale lymfeklieren in het centrale compartiment (level VI)

(centrale compartiments dissectie (CCD)). Wanneer er sprake is van ziekte uitbreiding naar de

laterale lymfeklieren van de hals is er ook een indicatie voor een gemodificeerde radicale uni-

of bi- laterale dissectie (LND). Omdat chirurgische behandeling de enige kans biedt op curatie

en/of locoregionale controle, voeren sommige chirurgen naast een electieve CCD ook een

electieve LND uit. De keuze hiervoor wordt mede bepaald door het risico op micrometastasen

wanneer een ziekte zich locoregionaal presentereert. Bij meer dan 50% van de patiënten is de

ziekte zodanig uitgebreid dat geen curatie bereikt kan worden. In tegenstelling tot andere

vormen van schildkliercarcinoom hebben MTC patiënten geen baat bij adjuvante therapie met


141

radioactief jodium. Daarom is adequate chirurgie in MTC van cruciaal belang voor optimale

locoregionale controle en curatie.

Hoewel meer dan 50% van de patiënten ondanks uitgebreide chirurgie niet kan worden

genezen, is de levensverwachting van het merendeel van de patiënten met een beperkte

tumorrest goed. Zorgvuldige follow-up is van belang om tijdig ziekte progressie vast te

stellen, en bestaat uit regelmatige bepalingen van de tumormarkers calcitonine en

carcinoembryonic antigeen (CEA); een tweede tumormarker voor MTC. Wanneer deze

tumormarkers stijgen is verdere diagnostiek met anatomische en functionele beeldvorming

geïndiceerd. De verdubbelingstijden van de calcitonine en CEA waarden zijn vooralsnog de

meest betrouwbare markers voor ziekteprogressie. Om ziekteprogressie vast te stellen zijn

echter meerdere metingen nodig over een langere periode. Het vroegtijdig vaststellen van

ziekteprogressie kan van belang zijn, omdat eventuele therapeutische interventies verdere

ziekteprogressie kunnen vertragen. Afhankelijk van de uitgebreidheid van de ziekte en de

snelheid waarmee tumormarkers stijgen, kunnen verschillende therapeutische strategieën

worden overwogen; een ‘wait and see’ beleid met een zorgvuldig vervolg van de patiënt, een

chirurgische interventie (voor locoregionale controle) of behandeling met gerichte

systemische (targeted) therapie.

In de palliatieve behandeling van MTC is conventionele chemotherapie niet effectief

gebleken. Recent onderzoek met gerichte tyrosine kinase (TK) remmers bij MTC patiënten

heeft een langere progressie vrije overleving laten zien. Het is echter onbekend of het effect

van deze tyrosine kinase remmers afhankelijk is van de aanwezigheid van een (somatische of

kiembaan) RET mutatie in de tumor of patiënt. Verschillende RET mutaties zijn

verantwoordelijk voor specifieke configurationele veranderingen van het RET eiwit. De meest

voorkomende mutatie in sporadische MTC patiënten bijvoorbeeld, ‘de MEN2B mutatie’,

verandert het ATP pocket van het RET eiwit. Sommige TK remmers kunnen hier minder goed

aan binden. De effectiviteit van gerichte therapie kan daarom mogelijk verbeterd worden door

de TK remmer te selecteren aan de hand van de specifieke RET mutatie. Daarnaast hebben de

meeste TK remmers verschillende aangrijpingspunten die meerdere intracellulaire

signaalroutes kunnen activeren. Naast het effect op de tumor kunnen ook bijwerkingen

optreden, zoals cardiale toxiciteit of het hand-voet syndroom. Zorgvuldige selectie van

patiënten is belangrijk, en in de toekomst kan mogelijk mutatie specifieke therapie ontwikkeld

worden.


142

Dit proefschrift onderzoekt verschillende belangrijke klinische vraagstukken over de diagnose

en behandeling van patiënten met een primair en recidief MTC. Het doel was om de

diagnostische en therapeutische modaliteiten in de behandeling van MTC te evalueren en te

verbeteren door middel van: (1) het evalueren van de waarde van de calcitonine test voor het

opsporen van MTC in patiënten met een schildkliernodus; (2) het beoordelen van de huidige

ATA richtlijn ten aanzien van de chirurgische behandeling van MTC patiënten; (3) het

bepalen van de waarde van 18F-FDG PET en 18F-DOPA PET in de detectie van progressief en

recidiverend MTC en (4) het onderzoeken van de effectiviteit van verschillende tyrosine

kinase remmers bij de behandeling van MTC.

In Hoofdstuk 1 wordt een algemene introductie gegeven over MTC en worden het doel en de

achtergronden van dit proefschrift besproken.

In Hoofdstuk 2 presenteren we drie patiënten met verschillende ziektestadia om de variatie in

klinische presentatie en biologisch gedrag van het MTC te illustreren. Aan de hand van deze

patiënten wordt een korte beschrijving gegeven van de presentatie, diagnose, behandeling en

follow-up van MTC.

Omdat MTC patiënten bij wie de ziekte in een vroeg stadium wordt vastgesteld een betere

prognose hebben, zou vroege opsporing van waarde kunnen zijn. Deze tumoren zouden met

een calcitonine bepaling kunnen worden gedetecteerd omdat vrijwel alle MTC’s calcitonine

produceren. Er bestaat echter geen consensus over het routinematig bepalen van calcitonine in

bepaalde patiëntengroepen, bijvoorbeeld bij patiënten die zich presenteren met een

schildkliernodus. Om de diagnostische accuratesse van de calcitonine test te evalueren voor

het opsporen van MTC bij patiënten met een schildkliernodus is in Hoofdstuk 3 een meta-

analyse verricht. Zestien studies werden geïncludeerd, waarbij schattingen werden gemaakt

van de sensitiviteit en specificiteit voor verschillende cut-off waarden met en zonder

stimulatie. Bij een lage basale calcitonine cut-off waarde (10 pg/ml) werd een hoge

sensitiviteit gevonden. De gecombineerde basale en gestimuleerde calcitonine bepalingen

resulteerden ook in een hoge sensitiviteit. Wanneer gekozen werd voor een hogere cut-off

waarde of een gestimuleerde calcitonine waarde nam de specificiteit iets toe. Gezien de hoge

sensitiviteit en specificiteit is de totale diagnostische accuratesse van de calcitonine bepaling

hoog. Door de lage prevalentie van MTC is de calcitonine bepaling echter niet meteen

geschikt als screeningsinstrument. Omdat MTC een zeldzame ziekte is, is de positief


143

voorspellende waarde (PPV) laag. Daarmee is ook de klinische toepasbaarheid van de

routinematige calcitoninebepaling voor het opsporen van MTC beperkt in patiënten met een

schildkliernodus. De lage PPV maakt het risico op een het onnodig opereren van patiënten

groter. Ook blijft de kosteneffectiviteit van de calcitoninescreening, gezien deze lage

prevalentie, nog onduidelijk.

In Hoofdstuk 4 is geëvalueerd of de chirurgische behandeling van MTC zoals beschreven in

de richtlijn van de American Thyroid Association (ATA) patiënten een klinisch voordeel

oplevert. Voor de optimale chirurgische behandeling bestaat geen eenduidig bewijs, met name

niet ten aanzien van de uitgebreidheid van de laterale lymfeklier dissectie. Daarom worden

door verschillende experts en richtlijnen verschillende chirurgische strategieën voorgesteld.

De ATA richtlijn adviseert een totale thyreoïdectomie en lymfeklierdissectie van het centrale

compartiment (level VI) als initiële behandeling. Bij positieve regionale lymfeklieren is ook

een systematische lymfeklierdissectie van de laterale compartimenten (level II-V)

geïndiceerd. In een retrospectieve studie werd de klinische uitkomst van patiënten behandeld

volgens de ATA richtlijn vergeleken met patiënten niet behandeld volgens deze richtlijn.

Deze klinische uitkomst was gedefinieerd als aantal re-operaties, biochemische curatie,

overleving en complicaties. Daarnaast werd onderzocht in welke mate de klinische uitkomst

werd beïnvloed door (1) een operatie met curatieve intentie in één of twee sessies, (2) de

locatie van de initiële curatieve operatie (tertiair verwijzingscentrum versus niet-centrum

ziekenhuis) en (3) patiënt en tumorkarakteristieken. De resultaten van deze studie laten zien

dat patiënten die volgens de richtlijn adequaat zijn behandeld significant minder re-operaties

hadden vergeleken met de inadequaat geopereerde patiënten. Daarnaast hadden adequaat

geopereerde patiënten significant meer biochemische curatie. Er werd geen significant effect

aangetoond op de klinische uitkomst van patiënten geopereerd in een tertiair

verwijzingscentrum, maar deze patiënten ondergingen wel significant vaker een adequate

operatie. Tumorgrootte en lymfeklierbetrokkenheid waren de belangrijkste voorspellers van

ziektevrije en totale overleving.

In Hoofdstuk 5 wordt de rol besproken van beeldvorming met verschillende radioactieve

tracers met behulp van de positron emissie tomografie (PET) voor de stagering en follow-up

van patiënten met een papillair, folliculair en medullair schildkliercarcinoom. PET is

gebaseerd op het gebruik van isotopen die positronen uitzenden. Voor de detectie van

schildkliercarcinoom zijn meerdere radioactieve tracers beschikbaar. 18Fluorine-


144

Fluorodeoxyglucose (18F-FDG) is een glucose analoog, die van waarde is in de follow-up van

patiënten met een papillair en folliculair schildkliercarcinoom. Met name bij patiënten met

een negatieve 131Jodium scintigrafie maar een detecteerbare tumormarker (thyreoglobuline),

kan 18F-FDG ziekte activiteit lokaliseren. Andere beschikbare tracers voor gebruik in de

follow-up van het papillair en folliculair schildkliercarcinoom zijn 11C-Methionine PET (11C-

MET PET) en 124Iodine-PET (124I-PET). Hoewel de waarde van 11C-MET PET beperkt lijkt,

heeft 124I-PET mogelijk wel een toegevoegde waarde vergeleken met de standaard 131Jodium

scintigrafie, de huidige plaatsbepaling is echter nog onduidelijk.

Bij patiënten met MTC wordt 18F-FDG ook gebruikt, maar lijkt 18Flurorinedihydroxyphenylalanine (18F-DOPA) meer sensitief. Deze radiotracer maakt

gebruik van een sterk verhoogd transport van aminozuren via de ‘large amino acid

transporters’. Beeldvorming met behulp van PET is een belangrijk diagnostisch instrument bij

patiënten met een schildkliercarcinoom. De optimale radioactieve tracer is afhankelijk van het

type carcinoom en het doel van de beeldvorming (stageren/follow-up).

In Hoofdstuk 6 wordt een studie bij 47 patiënten met MTC beschreven, waarin de opname op

de 18F-FDG PET als 18F-DOPA PET wordt vergeleken met verdubbelingstijden van

calcitonine en carcinoembryonic antigeen (CEA). Vroege opsporing van progressieve ziekte

bij MTC patiënten kan belangrijk zijn, omdat behandeling verdere achteruitgang kan

vertragen. De calcitonine verdubbelingstijd is de belangrijkste biochemische marker voor

progressieve ziekte, maar hiervoor zijn meerdere metingen over een langere tijdsperiode

noodzakelijk. Positiviteit op de 18F-FDG PET en 18F-DOPA PET scan werd vergeleken met

de absolute calcitonine en CEA serum waarden, de verdubbelingstijden van deze

tumormarkers en overleving van de patiënt. Tevens werd in een subgroep aan de hand van een

gestandaardiseerde opname waarde van de tracers (standardized uptake value; SUV) en het

aantal laesies de zogenaamde ‘whole body metabolic burden (WBMTB)’ bepaald. Deze

WBMTB werd vergeleken met de bovengenoemde biochemische parameters. 18F-FDG PET positiviteit was significant gecorreleerd met zowel de absolute calcitonine

en CEA waarden als met de verdubbelingstijden van beide tumormarkers. 18F-DOPA PET

positiviteit was significant gecorreleerd met de absolute calcitonine en CEA waarden, maar er

was geen correlatie met verdubbelingstijden. Met de 18F-DOPA PET werden significant meer

laesies gedetecteerd dan met de 18F-FDG PET. Positiviteit op de 18F-FDG PET was een

belangrijke indicator voor een kortere overleving, in vergelijking met patiënten met een

negatieve 18F-FDG PET scan. Geconcludeerd kan worden dat beide scans van waarde zijn bij


145

de follow-up van patiënten met MTC; 18F-DOPA PET vooral om de uitgebreidheid van de

ziekte vast te stellen, 18F-FDG PET om de patiënten met progressieve ziekte beter te

identificeren.

In Hoofdstuk 7 wordt een studie beschreven waarin de effecten van vier verschillende

tyrosine kinase remmers in MTC en papillair schildklier carcinoom (PTC) cellijnen wordt

geëvalueerd. MTC en PTC kunnen worden veroorzaakt door een activatie of

herrangschikking in het ‘RE-arranged during Transfection’ (RET) gen. Dit gen codeert voor

de RET tyrosine kinase receptor, die betrokken is bij celgroei en proliferatie. Verschillende

tyrosine kinase remmers zijn getest in klinische trials, maar het is onbekend of er ook

specificiteit bestaat voor een bepaalde RET mutatie en welke tyrosine kinase remmer dan het

meest effectief is. Cellijnen met een MEN2A (MTC-TT) mutatie, een MEN2B (MZ-CRC-1)

mutatie en een RET/PTC (TPC-1) herrangschikking werden behandeld met vier verschillende

tyrosine kinase remmers (axitinib, sunitinib, vandetanib en cabozantinib (XL184)). Het effect

op celproliferatie, RET expressie en autofosforylatie werd bepaald. Ook werd het effect op

Mitogen-activated protein kinase (MAPK) signaalroute onderzocht (meer specifiek het effect

op ‘Extracellular Signal-regulated Kinase’ (ERK)); een onderliggende RET signaalroute

betrokken bij celdifferentiatie, proliferatie en overleving.

Alle vier onderzochte tyrosine kinase remmers lieten een dosisafhankelijke afname in

celproliferatie zien. Cabozantinib was de meest effectieve remmer in de MEN2A en

RET/PTC cellijn, terwijl vandetanib de meest effectieve remmer voor de MEN2B cellijn was.

Zowel cabozantinib als vandetanib waren in staat om de RET autofosforylatie en RET

expressie te verminderen in de MEN2A en MEN2B cellijn. Echter, alleen bij vandetanib

gebeurde dit door het remmen van de RET transcriptie. Bij behandeling met cabozantinib

werd een duidelijke afname in RET fosforylatie gezien in de RET/PTC cellijn, maar de

RET/PTC expressie was toegenomen. ERK fosforylatie en expressie waren zowel in MEN2A

als MEN2B cellijnen afgenomen na behandeling met cabozantinib of vandetanib. In

RET/PTC cellijnen werd geen verandering in ERK fosforylatie en expressie gezien. Deze

studie laat zien dat vandetanib en cabozantinib potente remmers zijn van tumorproliferatie in

MTC cellijnen. Daarnaast werd ook een specificiteit van de tyrosine kinase remmers

gevonden voor cellijnen met verschillende RET mutaties. Dit suggereert dat mutatie

specifieke therapie van waarde kan zijn in MTC en PTC patiënten.

146

147

Dankwoord

Het medullaire schildkliercarcinoom is een zeldzame tumor waarvoor de behandeling de inzet

vraagt van een ervaren en gemotiveerd behandelteam, met expertise vanuit verschillende

disciplines. Tijdens mijn onderzoek heb ik met veel mensen samengewerkt vanuit deze

verschillende vakgebieden. Dit proefschrift zou dan ook niet mogelijk zijn geweest zonder

hun hulp.

Prof. dr. T.P. Links, beste Thera, het was een voorrecht om onder jouw begeleiding dit

proefschrift te mogen schrijven. Zonder jouw enorme gedrevenheid zou dit project niet zijn

geslaagd. Je optimisme heeft me op de moeilijke momenten altijd weer kunnen motiveren.

Niet alleen op wetenschappelijk gebied heb ik enorm veel van je geleerd, maar ook op

persoonlijk vlak heb ik me onder jouw begeleiding verder ontwikkeld. Ik hoop dat we in de

toekomst nog veel kunnen samenwerken.

Dankwoord

148

Prof. dr. J.Th.M. Plukker, beste John, toen ik met mijn onderzoeksproject begon was ik erg

onder de indruk van jouw verschijning. Ik heb je gelukkig naast een enorm kundig chirurg,

ook leren kennen als een erg toegankelijke en vriendelijke begeleider. De chirurgische

precisie waarmee je mijn stukken las heeft me menigmaal doen zuchten, maar leidde

uiteindelijk altijd tot een beter eindresultaat. Tijdens onze discussies over mijn onderzoek heb

ik geleerd dat ondanks een totaal verschillende benadering, chirurgen en internisten heel goed

tot een gezamenlijke oplossing kunnen komen.

Prof. dr. R.M.W. Hofstra, beste Robert, bij jou begon dit wetenschappelijke avontuur. Mijn

proefproject bij jou op het lab heeft uiteindelijk geleid tot dit mooie resultaat. Jouw

enthousiasme en relativerende houding hebben me door het eerste deel van mijn promotie

geleid. De werkbesprekingen met jouw vrolijke noot en aanstekelijke lach waren altijd

motiverend, ook als er weer eens een experiment was mislukt. Daarnaast heb ik veel kunnen

leren van je kritische blik en inhoudelijk scherp commentaar.

Mijn dank gaat tevens uit naar de leden van de beoordelingscommissie, Prof. dr. E.G.E. de

Vries, Prof. dr. I.H.M. Borrel Rineks en Prof. dr. W.J.G. Oyen, voor de tijd en energie die zij

hebben gestoken in het lezen en beoordelen van mijn proefschrift.

Ook wil ik alle co-auteurs bedanken voor hun bijdrage; in het bijzonder bedank ik dr. J.W.B.

de Groot, dr. M.M. Alves, drs. J.A.A. Meijer en dr. W.J. Sluiter. Beste Jan Willem, als mijn

voorganger als onderzoeker naar het medullair schildkliercarcinoom, heb je vaak als mijn

voorbeeld gediend. Ik ben dankbaar voor de mogelijkheid om jouw werk op dit onderwerp uit

te breiden en verder te brengen. Dear Maria, without your help on the lab, a large part of my

preclinical work would not have been possible. It was great to work with you and thank you

for all your help. Beste Ton, menigmaal hebben we ‘s avonds uren aan de telefoon gezeten

om alle chirurgie- en pathologieverslagen te beoordelen. Ondanks de afstand en je drukke

agenda hebben we denk ik uiteindelijk een mooi eindresultaat behaald. Dank voor je kritische

inbreng en succes met het afronden van je eigen promotie. Beste Wim, regelmatig heb ik je

opgezocht voor statistische adviezen. Net als velen zat ik naast je op de stoel, waarbij je uitleg

altijd heel logisch leek, maar eenmaal weer terug op m’n eigen plek de toepassing hiervan

toch een stuk lastiger was.

Dankwoord

149

Tevens wil ik de volgende co-auteurs bedanken; Prof. dr. J. Kievit, Prof. dr. J.W. Smit, Prof.

E. van den Heuvel, dr. A.H. Brouwer, dr. A.C. Muller-Kobold. Beste Job, Jan, Adrienne,

Anneke en Edwin, dank voor jullie constructieve commentaar, adviezen en uitleg.

Al mijn collega’s en mede-onderzoekers van de verschillende afdelingen wil ik ook bedanken.

Speciaal wil ik Jan Osinga bedanken, die mij wegwijs heeft gemaakt in het laboratorium en

me de basistechnieken heeft bijgebracht. Daarnaast dank aan Wouter Zandee en Kelvin

Kramp voor het analyseren en beoordelen van de vele operatie- en pathologieverslagen.

Daarnaast ook dank aan mijn kamergenoten op de afdeling Endocrinologie voor de

gezelligheid en afwisseling naast de vele uren achter het computerscherm.

Daarnaast wil ik graag mijn vrienden bedanken voor de nodige ontspanning naast dit

promotietraject. De mooie ploma’s, borrels, feestjes, weekendjes weg en vakanties met de

SaBOOTeurs en Concept 3 waren een fantastische afleiding, die ik zeker niet had willen

missen!

Ook wil ik graag mijn broer en zussen bedanken. Lieve Carolien, Anton, Ingrid en Harriët;

het is geweldig om als jongste broer met jullie in een gezin op te groeien. Ondanks dat we

elkaar misschien minder vaak zien en spreken als we zouden willen, ben ik dankbaar voor de

fijne band die we met elkaar hebben!

Lieve Pap en Mam, dank voor jullie onvoorwaardelijke liefde, steun en interesse! Zonder alle

dingen die ik van jullie heb meegekregen en geleerd zou mijn leven er een stuk anders uit

zien. Ik ben jullie heel erg dankbaar!

Liefste Martine; met jou ben ik al vijf jaar heel gelukkig! Jij bent er voor mij altijd. Ik kijk uit

naar de toekomst om samen met jou te genieten van al het moois wat nog gaat komen!

150

151

Curriculum Vitae

Hans Verbeek werd op 1 september 1985 geboren te Emmen. In 2003 behaalde hij zijn VWO

diploma aan het Katholiek Drenths College te Emmen. Vervolgens begon hij zijn studie

Geneeskunde aan de Rijksuniversiteit Groningen. In 2005 haalde hij tevens zijn propedeuse

Natuurkunde. In aansluiting op een proefproject dat de effecten van tyrosine kinase remmers

op medullair schildkliercarcinoom cellijnen onderzocht begon Hans in 2008 aan een MD-PhD

traject. Dit MD-PhD traject, onder begeleiding van prof. dr. T.P. Links, prof. dr. J.T.M.

Plukker en prof. dr. R.M.W. Hofstra, onderzocht de diagnose en behandeling van primair en

recidiverend medullair schildkliercarcinoom. In januari 2013 rondde Hans zijn studie

Geneeskunde af. In juni 2013 startte hij als arts in opleiding tot internist in het Deventer

Ziekenhuis (opleider dr. C.G. Vermeij) waar hij op dit moment nog steeds werkzaam is.

152

Appendices

Appendices

154

Appendix 1

Search strategy

Search terms

Unless otherwise stated, search terms are free text terms.

Abbreviations:

'$': stands for any character; '?': substitutes one or no character; adj: adjacent (i.e. number of words

within range of search term); exp: exploded MeSH; MeSH: medical subject heading (MEDLINE

medical index term); pt: publication type; sh: MeSH; tw: text word.

The Cochrane Library

#1 MeSH descriptor Thyroid Neoplasms explode all trees

#2 MeSH descriptor Goiter, Nodular explode all trees

#3 ( (thyroid* in All Text near/6 neoplas* in All Text) or (thyroid* in All Text near/6 cancer in All Text)

or (thyroid* in All Text near/6 carcinoma* in All Text) or (thyroid* in All Text near/6

macrocarcinoma* in All Text) or (thyroid* in All Text near/6 microcarcinoma* in All Text) )

#4 ( (thyreoid* in All Text near/6 neoplas* in All Text) or (thyreoid* in All Text near/6 cancer in All

Text) or (thyreoid* in All Text near/6 carcinoma* in All Text) or (thyreoid* in All Text near/6

macrocarcinoma* in All Text) or (thyreoid* in All Text near/6 microcarcinoma* in All Text) )

#5 ( (thyroid* in All Text near/6 tumor* in All Text) or (thyroid* in All Text near/6 tumour* in All Text)

or (thyreoid* in All Text near/6 tumor* in All Text) or (thyreoid* in All Text near/6 tumour* in All

Text) )

#6 ( (thyroid* in All Text near/6 nodul* in All Text) or (thyroid* in All Text near/6 multinodul* in All

Text) or (thyreoid* in All Text near/6 nodul* in All Text) or (thyreoid* in All Text near/6 multinodul*

in All Text) )

#7 MTC in All Text

#8 MeSH descriptor Carcinoma, Medullary explode all trees

#9 (carcinoma* in All Text near/6 medull* in All Text)

#10 (#8 or #9)

#11 (thyroid* in All Text or thyreoid* in All Text)

#12 (#10 and #11)

#13 (#1 or #2 or #3 or #4 or #5 or #6 or #7 or #12)

#14 MeSH descriptor Calcitonin explode all trees

#15 (calcitrin* in All Text or calcitonin* in All Text or thyrocalcitonin* in All Text)

#16 (#14 or #15)

#17 (#13 and #16)

#18 MeSH descriptor Diagnostic Tests, Routine explode all trees

#19 MeSH descriptor Biopsy, Fine-Needle explode all trees

#20 MeSH descriptor Diagnostic techniques, endocrine explode all trees

#21 MeSH descriptor Magnetic resonance imaging explode all trees

#22 MeSH descriptor Ultrasonography explode all trees

#23 MeSH descriptor Biological markers explode all trees

#24 MeSH descriptor Carcinoembryonic antigen explode all trees

#25 MeSH descriptor Diagnostic imaging explode all trees

#26 MeSH descriptor Immunoassay explode all trees

#27 MeSH descriptor Chemiluminescent measurements explode all trees

Appendices

155

#28 diagnos* in All Text

#29 ( (tumor in All Text near/6 marker* in All Text) or (biological in All Text near/6 marker* in All

Text) or (tumour in All Text near/6 marker* in All Text) )

#30 ( (calcitonin* in All Text and test* in All Text) or (pentgastrin* in All Text and test* in All Text) )

#31 (PET-CT in All Text or RET in All Text or MRI in All Text)

#32 (fine in All Text and (needle in All Text near/6 biops* in All Text) )

#33 (MRI in All Text or FNAC in All Text or FNAB in All Text)

#34 CEA in All Text

#35 (cytology in All Text or immunohistochem* in All Text or ultrasonograph* in All Text or

echograph* in All Text)

#36 (imaging in All Text and technique* in All Text)

#37 (RIA in All Text or IRMA in All Text or ILMA in All Text)

#38 (#18 or #19 or #20 or #21 or #22 or #23 or #24 or #25 or #26 or #27 or #28 or #29 or #30 or #31

or #32 or #33 or #34 or #35 or #36 or #37)

#39 (#17 and #38)

MEDLINE

1 exp Thyroid Neoplasms/

2 exp Goiter nodular/

3 ((thyroid* or thyreoid*) adj6 (neoplas* or cancer or carcinoma* or macrocarcinoma* or

microcarcinoma* or tumo?r*)).tw,ot.

4 (nodul* adj3 (thyroid* or thyreoid* or goiter)).tw,ot.

5 ((thyroid* or thyreoid*) adj3 (multinodul* or multi nodul*)).tw,ot.

6 MTC*.ab.

7 or/1-6

8 exp Carcinoma, medullary/

9 (medullary adj6 (thyroid* or thyreoid*)).tw,ot.

10 8 or 9

11 (thyroid* or thyreoid*).tw,ot.

12 10 and 11

13 7 or 12

14 exp Calcitonin/

15 (Calcitrin* or calcitonin*).tw,ot.

16 thyrocalcitonin*.tw,ot.

17 9007-12-9.rn.

18 or/14-17

19 (screen* or detect*).tw,ot.

20 exp Diagnostic Tests, Routine/

21 exp Biopsy, Fine-Needle/

22 exp Diagnostic Techniques, Endocrine/

23 exp Magnetic Resonance Imaging/

24 exp Ultrasonography/

25 exp Biological markers/

26 exp Carcinoembryonic antigen/

27 exp diagnostic imaging/

28 exp Pentagastrin/du [Diagnostic Use]

29 exp Immunoassay/

30 Carcinoma, medullary/di [diagnosis]

31 chemiluminescent.mp.

32 ((tumo?r or biological) adj6 marker*).tw,ot.

33 diagnos*.tw,ot.

Appendices

156

34 (RIA or IRMA or ILMA).tw,ot.

35 calcitonin* test*.tw,ot.

36 PET-CT.mp.

37 RET.mp.

38 fine needle aspiration*.tw,ot.

39 pentagastrin-test*.tw,ot.

40 MRI.tw,ot.

41 (MRI or FNAC or FNAB).tw,ot.

42 (cytology or immunohistochem* or ultrasonograph* or echograph*).tw,ot.

43 CEA.tw,ot.

44 imaging technique*.tw,ot.

45 or/19-44

46 13 and 18 and 45

47 (animals not (animals and humans)).sh.

48 46 not 47

EMBASE

1 thyroid tumor/

2 exp thyroid cancer/

3 exp thyroid nodule/

4 exp thyroid medullary carcinoma/

5 exp nodular goiter/

6 ((thyroid* or thyreoid*) adj6 (neoplas* or cancer or carcinoma* or macrocarcinoma* or

microcarcinoma* or tumo?r)).tw,ot.

7 ((thyroid* or thyreoid*) adj6 medullary carcinoma*).tw,ot.

8 (nodul* adj3 (thyroid* or thyreoid*)).tw,ot.

9 ((thyroid* or thyreoid*) adj3 (multinodul* or multi nodul*)).tw,ot.

10 MTC*.tw,ot.

11 or/1-10

12 exp calcitonin/

13 (calcitrin* or calcitonin* or thyrocalcitonin*).tw,ot.

14 9007-12-9.rn.

15 or/12-14

16 11 and 15

17 exp diagnostic test/

18 exp needle biopsy/

19 exp endocrine system examination/

20 exp nuclear magnetic resonance imaging/

21 exp echography/

22 exp biological marker/

23 exp carcinoembryonic antigen/

24 exp diagnostic imaging/

25 exp immunoassay/

26 exp chemiluminescent/

27 pentagastrin test.mp.

28 exp medullary carcinoma/di [Diagnosis]

29 ((tumo?r or biological) adj6 marker*).tw,ot.

30 diagnos*.tw,ot.

31 (RIA or IRMA or ILMA).tw,ot.

32 ((calcitonin* or pentagastrin*) adj6 test*).tw,ot.

33 (screen* or detect*).tw,ot.

Appendices

157

34 PET-CT.mp.

35 RET.mp.

36 needle biop*.tw,ot.

37 (MRI or FNAC or FNAB).tw,ot.

38 (cytology or immunhistochem* or ultrasonograph* or echograph*).tw,ot.

39 CEA.tw,ot.

40 imaging technique*.tw,ot.

41 or/17-40

42 16 and 41

43 limit 42 to human

Web of Science

# 1 Topic=(thyroid* tumor*) OR Topic=(thyroid* cancer) OR Topic=(thyroid* neoplas*) OR

Topic=(thyroid* carcinoma*) OR Topic=(thyroid* microcarcinoma*) OR Topic=(thyroid*

macrocarcinoma) OR Topic=(thyroid* medullary carcinoma*) OR Topic=(thyroid* nodul*) OR

Topic=(thyroid* multinodul*) OR Topic=(nodul* goiter*)

# 2 Topic=(thyreoid* tumor*) OR Topic=(thyreoid* cancer) OR Topic=(thyreoid* neoplas*) OR

Topic=(thyreoid* carcinoma*) OR Topic=(thyreoid* microcarcinoma*) OR Topic=(thyreoid*

macrocarcinoma) OR Topic=(thyreoid* medullary carcinoma*) OR Topic=(thyreoid* nodul*) OR

Topic=(thyreoid* multinodul*) OR Topic=(nodul* goiter*) OR Topic=(MTC)

# 3 Topic=(calcitonin*) OR Topic=(calcitrin*) OR Topic=(thyrocalcitonin*)

# 4 #1 OR #2

# 5 #3 AND #4

# 6 Topic=(diagnostic test*) OR Topic=(needle biopsy) OR Topic=(endocrine examination*) OR

Topic=(echograph*) OR Topic=(ultrasonograph*) OR Topic=(magnetic resonance imaging) OR

Topic=(MRI) OR Topic=(biological marker*) OR Topic=(diagnostic imaging) OR Topic=(immunoassay)

OR Topic=(chemiluminescent*) OR Topic=(tumor marker*)

# 7 Topic=(carcioembryonic antigen) OR Topic=(CEA) OR Topic=(pentagastrin test*) OR

Topic=(diagnos*) OR Topic=(calcitonin* test*) OR Topic=(screen*) OR Topic=(detect*) OR Topic=(RIA)

OR Topic=(IRMA) OR Topic=(ILMA) OR Topic=(PET-CT) OR Topic=(RET)

# 8 Topic=(FNAC) OR Author=(FNAB) OR Publication Name=(cytology) OR Topic=(immunhistochem*)

OR Topic=(imaging technique*)

# 9 #6 OR #7 OR #8

# 10 #5 AND #9

# 11 Topic=(animal*)

# 12 #10 NOT #11

'My NCBI' alert service

("thyroid nodule"[MeSH Terms] OR ("thyroid"[All Fields] AND "nodule"[All Fields]) OR "thyroid

nodule"[All Fields]) AND ("calcitonin"[MeSH Terms] OR "calcitonin"[All Fields])

Appendices

158

Appendix 2

Additional Table 2 Data extraction form

Design

Design:

Inclusion criteria:

Exclusion criteria:

Patient characteristics

and setting

Number of participants:

Number with NTD:

Number with NTD and calcitonin testing:

Sex (female%):

Age (mean/SD): range:

MTC:

Type of thyroid nodules:

Thyroid nodules detected by palpation or US:

Nodule size:

Number of nodules:

Sonographic morphology of thyroid nodules:

FNA procedures performed through ultrasound guidance or palpation:

Setting:

Country:

Index test

Index test:

Calcitonin as a triage or add-on test:

Used calcitonin assay:

Stimulated calcitonin performed:

Indication stimulated calcitonin:

Stimulative:

Dose:

Time:

Reported and extracted cut-off values

Basal:

Stimulated:

Reference standard

Target condition:

Reference standards:

Indication surgical treatment:

Type of surgical treatment:

Calcitonin negative (n)

Number FNA:

Number operated:

Calcitonin positive (n)

Number FNA:

Number operated:

Flow and timing

Follow-up calcitonin negative:

Type:

Duration:

Follow-up calcitonin positive:

Type:

Duration:

Appendices

159

Appendix 3

QU

AD

AS

-2 S

ign

all

ing

qu

est

ion

s fo

r b

ias

Do

ma

in

Sig

na

llin

g q

ue

stio

n

Cri

tera

Ye

s U

ncl

ea

r N

o

Pa

tie

nt

sele

ctio

n

1.

Co

nse

cuti

ve

or

ran

do

m s

am

ple

en

roll

ed

?

A c

on

secu

tive

or

ran

do

m s

am

ple

of

pa

tie

nts

we

re e

nro

lle

d in

th

e

stu

dy.

It is

un

cle

ar

wh

eth

er

a

con

secu

tive

or

ran

do

m s

am

ple

of

pa

tie

nts

wa

s e

nro

lled

in t

he

stu

dy.

Th

ere

wa

s n

o c

on

secu

tive

or

ran

do

m s

am

ple

incl

ud

ed

in

the

stu

dy

(e.g

. o

nly

pa

tie

nts

alr

ea

dy

in s

usp

icio

n o

f

(me

du

llary

) th

yro

id

ma

lign

an

cy a

nd

pa

tie

nts

wit

h

hig

h r

isk

for

(fa

mili

al)

MT

C.

2.

Ca

se c

on

tro

l d

esi

gn

av

oid

ed

?

Th

ere

wa

s n

o c

ase

co

ntr

ol d

esi

gn

. It

is u

ncl

ea

r if

th

ere

wa

s a

ca

se

con

tro

l de

sig

n

Th

ere

wa

s a

ca

se c

on

tro

l

de

sig

n

3.

Ina

pp

rop

ria

te

ex

clu

sio

ns

av

oid

ed

?

Th

ere

are

no

pa

tie

nts

ina

pp

rop

ria

te e

xclu

de

d (

e.g

.

pa

tie

nts

wit

h s

usp

icio

us

US,

wh

o

will

alr

ea

dy

be

op

era

ted

on

)

It is

un

cle

ar

if t

he

re w

as

avo

ida

nce

of

ina

pp

rop

ria

te

exc

lusi

on

s

Th

ere

is

ina

pp

rop

ria

te

exc

lusi

on

of

pa

tie

nts

(e

.g.

exc

lusi

on

of

pa

tie

nts

wit

h

hig

h r

isk

of

ma

lign

an

cy)

Ind

ex

te

st

1.

Ind

ex

te

st r

esu

lts

inte

rpre

ted

wit

ho

ut

kn

ow

led

ge

re

sult

s

refe

ren

ce s

tan

da

rd?

Th

is it

em

will

be

om

itte

d a

s o

nly

stu

die

s a

re in

clu

de

d in

wh

ich

th

e r

efe

ren

ce s

tan

da

rd

(his

top

ath

olo

gic

al e

xam

ina

tio

n)

is p

erf

orm

ed

aft

er

calc

ito

nin

te

stin

g.

Furt

he

rmo

re c

alc

ito

nin

te

stin

g i

s

a o

bje

ctiv

e t

est

(a

lth

ou

gh

inte

rpre

tati

on

de

pe

nd

s o

n t

he

th

resh

old

, b

ut

this

will

be

ass

ess

ed

in t

he

ne

xt it

em

)

2.

Pre

-sp

eci

fie

d

thre

sho

ld?

Th

ere

wa

s a

pre

-sp

eci

fie

d

calc

ito

nin

cu

t-o

ff l

eve

l.

It is

un

cle

ar

if t

he

re w

as

a p

re-

spe

cifi

ed

cu

t-o

ff l

eve

l

Th

ere

wa

s n

o p

re-s

pe

cifi

ed

calc

ito

nin

cu

t-ff

le

vel.

Appendices

160

Q

UA

DA

S-2

Sig

na

llin

g q

ue

stio

ns

for

bia

s

Do

ma

in

Sig

na

llin

g q

ue

stio

n

Cri

tera

Ye

s U

ncl

ea

r N

o

Re

fere

nce

sta

nd

ard

1.

Re

fere

nce

sta

nd

ard

lik

ely

to

co

rre

ctly

cla

ssif

y

targ

et

con

dit

ion

?

In p

ati

en

s re

ceiv

ing

su

rge

ry

the

re is

ad

eq

ua

te

his

top

ath

olo

gic

al e

xam

ina

tio

n

of

thyr

oid

tis

sue

.

In p

ati

en

ts r

ece

ivin

g f

oll

ow

-up

,

the

re is

at

lea

st t

hre

e y

ea

rs

follo

w-u

p y

ea

rs in

clu

din

g a

t

lea

st o

ne

US

exa

min

ati

on

an

d if

ind

ica

ted

FN

AC

.

In p

ati

en

ts r

ece

ivin

g s

urg

ery

it's

un

cle

ar

ho

w h

isto

pa

tho

log

ica

l

exa

min

ati

on

is p

erf

orm

ed

.

In p

ati

en

ts r

ece

ivin

g f

oll

ow

-up

the

tim

e a

nd

pro

toco

l fo

r

follo

w-u

p is

un

cle

ar

In p

ati

en

ts r

ece

ivin

g s

urg

ery

his

top

ath

olo

gic

al e

xam

ina

tio

n

is n

ot

ad

eq

ua

te.

In p

ati

en

ts w

ith

ou

t su

rge

ry,

follo

w-u

p is

to

sh

ort

or

do

es

no

t

incl

ud

e a

t le

ast

on

e U

S

exa

min

ati

on

an

d F

NA

C.

2.

Re

fere

nce

sta

nd

ard

resu

lts

inte

rpre

ted

wit

ho

ut

kn

ow

led

ge

resu

lts

ind

ex

te

st?

Th

e o

utc

om

e a

sse

sso

r o

f

his

top

ath

olo

gic

al a

nd

fo

llow

-up

resu

lts

wa

s n

ot

aw

are

of

calc

ito

nin

te

stin

g r

esu

lts

It is

no

t cl

ea

r if

th

e o

utc

om

e

ass

ess

or

of

his

top

ath

olo

gic

al

an

d f

ollo

w-u

p r

esu

lts

wa

s

aw

are

of

calc

ito

nin

te

stin

g

resu

lts

Th

e o

utc

om

e a

sse

sso

r o

f

his

top

ath

olo

gic

al a

nd

fo

llow

-up

resu

lts

wa

s a

wa

re o

f ca

lcit

on

in

test

ing

re

sult

s

Flo

w a

nd

tim

ing

1.

Ap

pro

pri

ate

in

terv

al

be

twe

en

in

de

x t

est

an

d

refe

ren

ce s

tan

da

rd?

Tim

e b

etw

ee

n c

alc

ito

nin

te

stin

g

an

d h

isto

pa

tho

log

ica

l

exa

min

ati

on

is <

3 m

on

ths

It is

un

cle

ar

wh

at

the

tim

e

pe

rio

d b

etw

ee

n r

efe

ren

ce

sta

nd

ard

an

d in

de

x te

st is

.

Tim

e b

etw

ee

n c

alc

ito

nin

te

stin

g

an

d h

isto

pa

tho

log

ica

l exc

ee

ds

3

mo

nth

s.

2.

All

pa

tie

nts

re

ceiv

ed

refe

ren

ce s

tan

da

rd?

All

pa

tie

nts

re

ceiv

ed

su

rge

ry,

an

d p

ati

en

ts w

ho

did

no

t

rece

ive

su

rge

ry h

ave

clin

ica

l

follo

w-u

p o

f a

t le

ast

th

ree

yea

rs.

It is

no

t cl

ea

r if

th

e w

ho

le

sam

ple

did

re

ceiv

e s

urg

ery

or

follo

w-u

p.

On

ly a

se

lect

ed

su

bse

t o

f th

e

pa

tie

nts

re

ceiv

ed

or

surg

ery

or

no

t a

ll p

ati

en

ts h

ave

clin

ica

l

follo

w-u

p.

3.

Pa

tie

nts

re

ceiv

ed

sam

e r

efe

ren

ce

sta

nd

ard

?

All

pa

tie

nts

we

re o

pe

rate

d a

nd

his

top

ath

olo

gic

al e

xam

ina

tio

n

of

the

th

yro

id w

as

pe

rfo

rme

d.

It is

no

t cl

ea

r if

all

pa

tie

nts

we

re

op

era

ted

an

d r

ece

ive

d

his

top

ath

olo

gic

al e

xam

ina

tio

n.

No

t a

ll p

ati

en

ts w

ere

op

era

ted

or

his

top

ath

olo

gic

al

exa

min

ati

on

wa

s n

ot

pe

rfo

rme

d in

all

pa

tie

nts

.

4.

All

pa

tie

nts

in

clu

de

d i

n

the

an

aly

sis?

All

pa

tie

nts

en

roll

ed

we

re

incl

ud

ed

in t

he

an

aly

sis

It is

no

t cl

ea

r if

all

pa

tie

nts

we

re

incl

ud

ed

in t

he

an

aly

sis.

No

t a

ll p

ati

en

ts e

nro

lled

we

re

incl

ud

ed

in t

he

an

aly

sis

(e.g

.

pa

tie

nts

lost

to

fo

llow

-up

)

Appendices

161

Appendix 4

Characteristics of included studies

Rieu 1995 Patient Selection

A. Risk of Bias

Patient Sampling Design: Prospective cohort study.

Inclusion criteria: Pts with thyroid nodules detected by clinical examination or with

abnormal TSH levels or both.

Exclusion criteria: Not reported.

Was a consecutive or random sample of patients enrolled? Yes

Was a case-control design avoided? Yes

Did the study avoid inappropriate exclusions? Unclear

Could the selection of patients have introduced bias? Low risk

B. Concerns regarding applicability

Patient

characteristics

and setting

Number of participants: 657 patients.

Number with NTD: 469 patients.

Number with NTD and calcitonin testing: 469 patients.

Sex (female%): 88,1% (579/657); only reported of whole study population.

Age (mean/SD): 45, SD: Not reported range: 15-87 years; only reported of whole study

population.

MTC: 4 patients.

Type of thyroid nodules: non toxic uninodular goitre (n = 136), autonomously functioning

thyroid nodule (n = 14), non toxic multinodular goitre (n = 224), toxic multinodular goitre (n =

15), nodular Hashimoto thyroiditis (n = 53), nodular Graves' disease (n = 25), nodular

formation related to subacute thyroiditis in a hyperthyroid phase (n = 2).

Thyroid nodules detected by palpation or US: both, number not specified.

Nodule size: not reported.

Number of nodules: Uninodular (n = 150), multinodular (n = 239), not reported (n = 80).

Sonographic morphology of thyroid nodules: A localized thyroid area was considered

nodular when it had a distinctive rim at two different incidences. When this rim was absent, a

thyroid area with round or oval patterns, and also echogenicity different from that of the

surrounding normal tissue, was classified as nodular.

FNA procedures performed through ultrasound guidance or palpation: Not

reported.

Setting: Not reported.

Country: France

Are there concerns that the included patients and setting do not match the

review question?

Low concern

Index Test

Index tests Index test: Basal and stimulated calcitonin.

Calcitonin as a triage or add-on test: Not reported.


01-1989 – 12-1989, RIA (Mallinckrodt Medical SA, Evry, France).

01-1990 – 12- 1993: IRMA (CIS-Oris International, Saint Quentin en Yvelins, France.

Stimulated calcitonin: Yes.

Indication: Patients with basal serum CT above normal range.

Stimulative: Pentagastrin.

Dose: 0.5 ug/kg.

Appendices

162

Time: 0, 3, 5, 15 and 30 min after injection.


Basal: reported: RIA: 35 ng/l, IRMA 10 ng/l; extracted 50 ng/l; 100 ng/l.

Stimulated: reported: 100 ng/l; extracted 100 ng/l.

A. Risk of Bias

If a threshold was used, was it pre-specified? Yes

Could the conduct or interpretation of the index test have introduced bias? Low risk


Are there concerns that the index test, its conduct, or interpretation differ from

the review question?

Low concern

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)

Target condition: MTC.

Reference standards: FNA in patients whose nodular formation was accessible to such a

procedure, in selected patients histopathological examination after surgery.

Indication surgical treatment: Patients with increased basal serum CT values, pentagastrin

stimulated CT levels > 100 pg/l, regardless of FNAC result, FNAC suggestive of thyroid

carcinoma (n = 7), compressive goitre (n = 4).

Type of surgical treatment: For suspected MTC total thyroidectomy and central neck

dissection. Bilateral modified neck dissection was performed only in the presence of evident

lymph node involvement in one or both sides. For all other types of thyroid carcinoma, total

thyroidectomy and lymph node dissection on the side of the thyroid nodule(s) or bilaterally

(as appropriate).

Calcitonin negative (n = 465)

Number FNAC: Not reported.

Number operated: 11 patients.

Calcitonin positive (n = 4)

Number FNAC: 4 patients.


Is the reference standards likely to correctly classify the target condition? Unclear

Were the reference standard results interpreted without knowledge of the results of

the index tests?

Unclear

Could the reference standard, its conduct, or its interpretation have introduced

bias?

Unclear risk


Are there concerns that the target condition as defined by the reference standard

does not match the question?

Low concern

Flow and Timing

A. Risk of Bias

Flow and timing Follow-up calcitonin negative: Not reported.

Type: -

Duration: -

Follow-up calcitonin positive: Not reported.

Type: -

Duration: -

Was there an appropriate interval between index test and reference standard? Unclear

Did all patients receive the same reference standard? No

Were all patients included in the analysis? Yes

Did all patients receive the reference standard? No

Appendices

163

Could the patient flow have introduced bias? High risk

User defined characteristics

Publication

details

LANGUAGE OF PUBLICATION: English.

FUNDING: Not reported.

PUBLICATION STATUS: Full article.

Stated aim of

study

Quote from publication: "to asses the prevalence of sporadic MTC in nodular and non -

nodular thyroid diseases by routine basal serum CT measurements"

Abbreviations FNAC: fine needle aspiration cytology, CT: calcitonin.

Notes

Appendices

164

Ozgen 1999

Patient Selection

A. Risk of Bias

Patient Sampling Design: Prospective study.

Inclusion criteria: Patients with nodular goiter.

Exclusion criteria: Patients with previously known medullary

carcinoma and their relatives.



Did the study avoid inappropriate exclusions? Yes



Patient

characteristics

and setting


Clinical features: Patients with nodular goiter.



Sex (female%)(n): 75,8% (586).

Age (mean/SD): range: 42.1 years, range 17-78.

Number with MTC: 4 patients.

Type of thyroid nodules: Multinodular goiter (nontoxic/toxic), solitary (nontoxic/toxic)

thyroid nodules.

Thyroid nodules detected by palpation or US: Both; number not specified.

Nodule size: Not reported.

Number of nodules: Not reported.

Sonographic morphology of thyroid nodules: Not reported.

FNA procedures performed through ultrasound guidance or palpation: Both (US guidance in

nonpalpable nodules), number not specified.

Setting: Outpatient clinic.

Country: Turkey.


review question?

Low concern

Index Test

Index tests Index test: Basal calcitonin.


Used calcitonin assay: Commercial kit; DSL-5200 Ultrasensitive calcitonin RIA kit, Diagnostic

System Laboratories Inc., Webster, Tx.

Sensitivity: Not reported.

Stimulated calcitonin: No

Indication: -

Stimulative: -

Dose: -

Time: -


Basal: Reported: 30 pg/ml; extracted:30 pg/ml, 50 pg/ml, 100 pg/ml.

Stimulated: -

A. Risk of Bias




Appendices

165



Low concern

Reference Standard

A. Risk of Bias

Target condition and

reference standard(s)


Reference standards: FNAB and in selected cases histopathological examination after

surgery,

Indication surgical treatment: Malignant or suspicious results in FNAB, or elevated

calcitonin levels regardless of the result of FNAB.

Type of surgical treatment: Thyroid surgery.


Number FNAB: 669 patients.






Were the reference standard results interpreted without knowledge of the results

of the index tests?

Unclear


bias?

Unclear risk


Are there concerns that the target condition as defined by the reference

standard does not match the question?

Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Described for 3 MTC patients.

Type: Basal and stimulated calcitonin levels.

Duration: 14-18 months







Publication

details




Stated aim of

study

Quote from publication: " To identify MTC by screening patients who have thyroid nodules

with basal calcitonin measurements and to determine whether basal serum calcitonin

measurement should be a part of the routine evaluation of a nodular goiter."

Abbreviations FNAB: Fine needle aspiration biopsy.

Notes

Appendices

166

Hahm 2001

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Cohort study.

Inclusion criteria: Patients with nodular thyroid disease.







Patient

characteristics

and setting




Sex (female%)(n): 80,3% (1163).

Age (mean/SD): 46 years Range: 14-86 years.


Type of thyroid nodules: Not reported.

Thyroid nodules detected by palpation or US: Not reported.




FNA procedures performed through ultrasound guidance or palpation: Not reported.

Setting: Thyroid clinic of Samsung Medical Center.

Country: Korea.


review question?

Low concern

Index Test


Used calcitonin assay: Two-site immunoradiometric assay commercial kit (MED-GENIX CT-

US.-IRMA kit) BioSource Europe S.A.,Belgium.

Sensitivity: 0.8 pg/ml.


Indication: Reported for 39 patients: basal calcitonin > 10 pg/ml (n=23), family members of

MEN2 or MTC patients (n=14) and FNAC findings suspicious for MTC (n=2).

Stimulative: Pentagastrin (Peptavlon, Ayerst Laboratories Ind. Philadelphia, PA).

Dose: 0.5 ug/kg body weight.

Time: Just before, 2, 5, and 10 min.


Basal: Reported: 10 pg/ml (n = 56); extracted: 10, 20, 30, 50, 100 pg/ml.

Stimulated: Reported: 100 pg/ml; extracted: 100, 200 pg/ml.

A. Risk of Bias






Low concern

Appendices

167

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAC in all patients with palpable or visible thyroid nodule by US, in

selected patients histopathological examination after surgery.

Indication surgical treatment: Abnormal findings suspicious of malignancy by FNAC, patients

who had basal or stimulated calcitonin concentrations of more than 100 pg/ml.

Type: Thyroidectomy.

Calcitonin negative (n = 1392 patients)



Calcitonin positive (n = 56 patients)





of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias

Flow and timing Follow-up calcitonin negative: Not reported

Type: -

Duration: -

Follow-up calcitonin positive: Not reported

Type:

Duration:







Publication

details

LANGUAGE OF PUBLICATION: English

FUNDING: OTHER FUNDING

PUBLICATION STATUS: PEER REVIEW JOURNAL / JOURNAL SUPPLEMENT / FULL ARTICLE /

CONFERENCE PAPER / OTHER)

Stated aim of

study

Quote from publication: "To evaluate the usefulness of routine measurement of serum

calcitonin concentration in patients with nodular thyroid diseases, and to identify the validity

of pentagastrin simulation test and FNAC "

Abbreviations US: Ultrasonography, FNAC: Fine needle aspiration cytology.

Notes

Appendices

168

Hatzl-Griesenhofer 2002

Patient Selection

A. Risk of Bias

Patient Sampling Design: Retrospective cohort study.

Inclusion criteria: Patients with nodular thyroid disease or with evidence of nodular growth

in follow-up examinations.


Was a consecutive or random sample of patients enrolled? Unclear



Could the selection of patients have introduced bias? High risk


Patient

characteristics

and setting




Sex (female%)(n): 78,8% (3073).

Age (mean/SD): 54.6 (+11.2) yrs range: 6 - 90 yrs.






Sonographic morphology of thyroid nodules: Only reported for MTC patients.


Setting: Outpatient ward of the department of Nuclear Medicine and Endocrinology of the

General Hospital Linz.

Country: Austria.


review question?

Low concern

Index Test



Used calcitonin assay: a two-site chemoluminesence immunoassay, Nichols institute diagnostics.

Sensitivity: 0.7 pg/ml


Indication: Patients with slightly or moderately elevated normal calcitonin levels (< 80 pg/ml).

Stimulative: Pentagastrin, Peptavlon, Zeneca, Vienna.

Dose: 0.5 ug/kg.

Time: before, 2, 5, and 8 min.


Basal: Reported: females: 4.6 pg/ml; males 11.5 pg/ml; extracted: females: 4.6 pg/ml; males 11.5

pg/ml.

Stimulated: Reported:100 pg/ml (males and females); extracted: 100 pg/ml.

A. Risk of Bias






Low concern

Appendices

169

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: Histological evaluation in patients referred to surgery.

Indication surgical treatment: Markedly elevated bCT and positive pentagastrin stimulation

tests.

Type: Hemithyroidectomy, (sub)total thyroidectomy, node dissection in selected patients.

Calcitonin negative ( n = 3639 patients)

Number FNA: -

Number operated: Not reported.

Calcitonin positive (n= 230 patients)

Number FNA: -

Number operated: 39 patients (?)



of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias

Flow and timing Follow-up calcitonin negative: Not reported

Type: -

Duration: -

Follow-up calcitonin positive: (1) Patients with pathological stimulation test in which the

consultant for internal medicine dissuaded against surgery because of elevated risks (n =2).

(2) patients with elevated basal calcitonin levels who declined pentagastrin stimulation (n

=41).

Type: (1) Ultrasound and determination of basal and stimulated calcitonin (2) clinical and

biochemical follow up.

Number with follow-up: 43 patients.

Duration: Not reported



Were all patients included in the analysis? No




Publication

details


FUNDING: Not reported

PUBLICATION STATUS: Full article

Stated aim of

study

Quote from publication: "To evaluate retrospectively the results of routine calcitonin

measurements in patients with nodular thyorid disease "

Abbreviations bCT: basal calcitonin,

Notes

Appendices

170

Elisei 2004

Patient Selection

A. Risk of Bias

Patient

Sampling









Patient

characteristics

and setting




Sex (female%)(n): 81.4% (8692).

Age (mean/SD): 49 yr SD not reported Range: 12 - 82 yr.

MTC: 44 patients.

Type of thyroid nodules: Single nodules, nontoxic multinodular goiter, autonomous

functioning thyroid nodules, and autoimmune thyroid disease associated with distinct cold

nodules.







Country: Italy


review question?

Low concern

Index Test



Used calcitonin assay: Solid phase 2 site immunoradiometric assay (ELSA-hCT, CIS, Gif Sur

Yvette, France).

Sensitivity: 14 pg/ml.


Indication: Patients with detectable levels of basal CTand twice confirmed.


Dose: 0.5 ug/kg.

Time: before, 2, 5, 15 and 30 min.


Basal: reported: 20 pg/ml; extracted: 20 pg/ml.

Stimulated: reported 60 pg/ml; extracted: 60, 100 pg/ml.

A. Risk of Bias






Low concern

Appendices

171

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAC in nodules > 1 cm or in nodules < 1 cm with suspicious aspects at

neck US, in selected patients histopathological examination after surgery.

Indication surgical treatment: Patients with elevated bCT levels (confirmed by abnormal Pg-

stimulated CT levels) regardless of the results of FNAC and in those with FNAC suspicious of

malignancy indepently from the results of serum CT.

Type:Total thyroidectomy and dissection of the central neck compartment









of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Only reported of MTC patients.

Type: Not reported.


Duration: Mean 6.2 + 2.5 yr (range, 3-10 yr).







Publication

details


FUNDING: Non-commercial.


Stated aim of

study

Quote from publication: "To asses whether we could confirm the results of our preliminiary

study of 1991 and to compare the outcome of patients diagnosed by serum CT measurement

with that of a historical group of MTC patients diagnosed and treated before the introduction

of serum CT screening".

Abbreviations CT: Calcitonin, FNAC: fine needle aspiration cytology, US: Ultrasonography.

Notes

Appendices

172

Karanikas 2004

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Cohort study

Inclusion criteria: Patients referred for the work-up of various suspected thyroid disorders.







Patient

characteristics

and setting




Sex (female%)(n): 79.5% (329) NB only reported for whole study population.

Age (mean/SD): 56 year; SD not reported range: 18 - 88 years, NB only reported for whole

study population.

MTC: 1 patients. (1 MTC patient in non-nodular study population)

Type of thyroid nodules: Uni and multinodular disease.


Nodule size: Only for patients with elevated CT; volume 21 + 10 ml.




Setting: Out-patient department.

Country: Austria.


review question?

Low concern

Index Test

Index tests Calcitonin as a triage or add-on test: Not reported.

Index test: Basal and stimulated calcitonin.

Used calcitonin assay: Commercial assay by a Nichols Advantage Chemiluminescence System

(Nichols Institute Diagnostics, San Juan Capistrano, Ca, USA)



Indication: Basal serum CT equal to or exceeding 10 pg/ml.

Stimulative: Pengagastrin Injection BP, Cambridge Laboratories, Tyne & Wear, UK

Dose: 0.5 ug/kg

Time: Before, 2 and 5 min after injection.


Basal: reported: 10 pg/ml; extracted: 10 pg/ml, 100 pg/ml.

Stimulated: reported: 100 pg/ml (abnormal), 500 pg/ml (pathological); extracted 100 pg/ml.

A. Risk of Bias






Low concern

Appendices

173

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: In selected patients histological examination after total thyroidectomy,

follow-up.

Indication surgical treatment: Patients with abnormal and pathological PG tests.

Type of surgical treatment: Total thyroidectomy and lymph node dissection along both

recurrent nerves in patients.


Number FNAC: -



Number FNAC: -

Number operated: 1 patient.



of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Only for MTC patient.

Type: Basal and stimulated CT.

Number with follow-up: 1.

Duration: Not reported







Publication

details




Stated aim of

study

Quote from publication: "To compare the distribution and relevance of elevated CT levels in

referrals with nonneoplastic and neoplastic thyroid disease (...)"

Abbreviations CT: calcitonin.

Notes

Appendices

174

Vierhapper 2005

Patient Selection

A. Risk of Bias

Patient

Sampling


Inclusion criteria: Patients with suspected thyroid disorders.

Exclusion criteria: Patients with a known elevation of hCT.






Patient

characteristics

and setting




Sex (female%)(n): 79.9% (8114).

Age (mean/SD): Not reported range: Not reported.

MTC: 36 patients.


Thyroid nodules detected by palpation or US: Both.

Nodule size: Only reported of MTC patients.




Setting: Thyroid outpatient clinic.

Country: Austria


review question?

Low concern

Index Test




1994-1999: A commercially available immunoradiometric assay (CIS-biointernational, Gif-Sur-

Yvette, France).

1999-2004: Acridinium-ester-labeled chemiluminescent immunoassay, running on the

‘Advantage’ auto-analyser (Nichols Institute Diagnostics, US).



Indication: All patients with basal hCT> 10.0 pg/ml.

Stimulative: Pentagastrin (CambridgeLaboratories, Wallsend, UK).

Dose: 0.5 ug/kg.

Time: Prior to, 2, 5 and 10 min.


Basal: Reported: 10 pg/ml extracted: 10 pg/ml.

Stimulated: Reported 100 pg/ml extracted: 100 pg/ml.

A. Risk of Bias




Are there concerns that the index test, its conduct, or interpretation differ from Low concern

Appendices

175


Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAB, in selected patients histopathological examination after surgery,

follow-up.

Indication surgical treatment: Elevated basal and/or pentagastrin stimulated serum

concentrations of hCT(>100 pg/ml) or cytological findings.

Type: In patients with elevated basal and/or stimulated calcitonin a total thyroidectomy was

performed. Routinely both recurrent nerves were dissected carfully and a systematic

microdissection of the central lymph node compartment. If MTC was documented

intraoperatively a systematic bilateral microdissection of the lateral lymph node

compartments was added.


Number FNA: Not reported.

Number operated: Not reported, at least 1 pt.


Number FNAB: 15 patients (only reported for MTC patients)




of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Only reported of MTC patients.

Type: stimulated hCTs.


Duration: 1-9 years.







Publication details LANGUAGE OF PUBLICATION: English.



Stated aim of study Quote from publication: Not reported.

Abbreviations hCT; serum calcitonin.

Notes

Appendices

176

Papi 2006

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Cohort study of consecutive patients.

Inclusion criteria: (1) male & female pts > 18 yrs (2) palpable nodules (3) non-palpable

nodules > 10 mm (4) non palpable nodule < 10 mm with malignant features on US.

Exclusion criteria: (1) patients < 18 years (2) non palpable nodules < 10 mm without

malignant US features (3) pts previously evaluated for nodular goiter by FNA and/or CT

measurement (4) patients reporting familial history of MEN and pts with known MTC (5) pts

with hyper- and hypothyroidism without thyroid nodules (6) patients in follow-up for thyroid

disease (7) patients not confirmed as having thyroid diseases.






Patient

characteristics

and setting




Sex (female%)(n): 80% (1144).

Age (mean/SD): 49.6(+ 6.8) yr range: 18 - 91 yr.

MTC: 9 patients.

Type of thyroid nodules: Euthyroid nodular thyroid disease (n = 1369), hypothyroid nodular

thyroid disease (n = 32), sub acute de Quervain's thyroiditis (n = 1), Graves' disease (n=2),

toxic nodular goiter (n = 21).

Thyroid nodules detected by palpation or US: both.

Nodule size: 21.8 + 4 mm



FNA procedures performed through ultrasound guidance or palpation: Ultrasound.


Country: Italy.


review question?

Low concern

Index Test



Used calcitonin assay: Two-site chemiluminesence assay, Nichols instute diagnostics, San

Juan Capristano, CA92675, USA).



Indication: When basal serum CT concentrations exceeded 5 pg/ml, but not exceeded 100

pg/ml.

Stimulative: Pentagastrin (Cambridge laboratories, Walsend, Tyne and Wear, NE289NX).

Dose: 0.5 ug/kg.

Time: Before, 2, 5 and 10 min after injection.


Basal: Reported: 5 pg/ml extracted: 5, 10, 15, 20, 30, 50,100 pg/ml.

Stimulated: Reported: 100 pg/ml extracted: 100, 200 pg/ml.

A. Risk of Bias

Appendices

177






Low concern

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAC and in selected patients, histopathological examination after

surgical treatment.

Indication surgical treatment: Basal serum CT concentrations > 5 pg/ml < 100 pg/ml and Pg-

stimulated CT levels > 100 pg/ml or basal serum CT concentrations > 100 pg/ml; patients with

a suspicious or repeatedly non-diagnostic FNAC and patients with a benign FNAC and

compressive symptoms.

Type: In patients with benign FNAC and compressive symptoms a lobectomy or near-total

thyroidectomy. In patients with suspicious or non-diagnostic FNAC, surgical extent depended

on histological examination of frozen sections. In patients with malignancy other than MTC a

total thyroidectomy and in case of lymph node involvement a lymphadenectomy. In MTC

patients a total thyroidectomy and a systematic microdissection of both central neck and

bilateral neck compartments. Contralateral lymph node dissection was omitted in MTC

patients with a unilateral thyroid tumor and no ipsilateral and central lymph node

involvement.




Calcitonin positive: (n = 23)





of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -


Type: -

Duration: -






Appendices

178


Publication

details




Stated aim of

study

Quote from publication: " To assess the prevalence of hypercalcitoninemia and MTC in NTD

patients, to compare the ability of CT measurement and fine needle aspiartion cytology (FNAC)

to predict MTC, to identify age groups of NTD patients who should be better candidates than

others to undergo routine measurement of CT."

Abbreviations CT: calcitonin, FNAC: fine needle aspiration cytology.

Notes

Appendices

179

Schuetz 2006

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Cohort study

Inclusion criteria: Patients with Hashimoto's thyroiditis with documented positivity for TPOAb,

Negativity for anti-TSH receptor antibodies and thyroid ultrasound imaging suggestive of a

chronic thyroiditis.







Patient

characteristics

and setting




Sex (female%)(n): 88.0% (500) NB only reported for whole study population.

Age (mean/SD): 55 + Not reported range: 18 - 88 years, NB only reported for whole study

population.

MTC: 0 patients. (1 MTC patient in non-nodular study population)


Thyroid nodules detected by palpation or US: US.

Nodule size: Only reported for patients with elevated calcitonin (13 + 5 mm; range 4.5-17 mm).


Sonographic morphology of thyroid nodules: Only reported for patients with elevated

calcitonin; all nodules were hypoechic/circumscribable.


Setting: Out-patient department.

Country: Austria.


review question?

Low concern

Index Test



Used calcitonin assay: Commercial assay by a Nichols Advantage Chemiluminescence System

(Nichols Institute Diagnostics, San Juan Capistrano, Ca, USA)



Indication: Basal serum CT equal to/or exceeded 10 pg/ml.

Stimulative: Pengagastrin Injection BP, Cambridge Laboratories, Tyne & Wear, UK

Dose: 0.5 ug/kg

Time: Before, 2,3 and 5 min after injection.



Stimulated: reported: 100 pg/ml; extracted 100 pg/ml.

A. Risk of Bias




Appendices

180



Low concern

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: In selected patients histological examination after total thyroidectomy,

follow-up.

Indication surgical treatment: Patients with abnormal and pathological PG tests.

Type of surgical treatment: Total thyroidectomy and lymph node dissection along both

recurrent nerves in patients with a abnormal PG tests and an additional lateral lymph node

dissection in patients with a pathological PG test or intraoperatively verified MTC.


Number FNAC: -







of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -


Type: -

Number with follow-up: -

Duration: -







Publication

details




Stated aim of

study

Quote from publication: " To evaluate the relevance of routine CT measurements for

detection of MTC or its premalignant associated conditions (micro MTC and neoplastic C cell

hyperplasia) in HT patients."


Notes

Appendices

181

Costante 2007

Patient Selection

A. Risk of Bias

Patient

Sampling


Inclusion criteria: Patients diagnosed with thyroid nodules.

Exclusion criteria: Renal failure, persistent or recurrent MTC, or a family history of MTC.






Patient

characteristics

and setting




Sex (female%)(n): 80.9% (4706).

Age (mean/SD): 49.7 + 16.6 yr range: 11 - 72 yr.

MTC: 15 patients.

Type of thyroid nodules: Euthryoid nodular/multinodular goiter (n = 4894), hashimoto's

thyroiditis with nodules (n = 436), autonomously functioning thyroid nodules (n = 276), toxic

nodular goiter ( n = 211).






Setting: A national healthcare system hospital (outpatient and inpatient) sectors.

Country: Italy.


review question?

Low concern

Index Test



Used calcitonin assay: Chemiluminesence assay (Nichols advantage Calcitonin

Chemiluminesence assay, San Juan Capistrano, CA).



Indication: Basal calcitonin levels > 20 pg/ml and < 100 pg/ml.


Dose: 0.5 ug/kg.

Time: 2 and 5 min after iv injection.


Basal: 10 pg/ml, 20 pg/ml, 50 pg/ml and 100 pg/ml; extracted 10, 20, 50 and 100 pg/ml

Stimulated: 100 pg/ml; extracted: 100 pg/ml.

A. Risk of Bias






Low concern

Appendices

182

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAB in patients with nonautonomous nodules exceeding 10 mm in

diameter; in selected patients histological examination after surgery, follow-up.

Indication surgical treatment: 1) FNAB indicative or suggestive of thyroid malignancy, 2)

multinodular autonomous or toxic goiters, 3) large euthyroid goiters causing compression, 4)

serum CT levels (basal or PG stimulated) more than 100 pg/ml.

Type: Not reported.


Number FNAB: Not reported.







of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: In patients with basal CT between 10-100 pg/ml and negative

Pg testing.

Type: Basal CT in patients with basal CT between 10-20 pg/ml; yearly stimulated CT in

patients with basal CT between 20-100 pg /ml.


Duration: 2-4 yr.







Publication

details




Stated aim of

study

Quote from publication: " To evaluate the diagnostic accuracy of systematic CT

measurement in non-multiple endocrine neoplasia type 2 patients with nodular thyroid

disease."

Abbreviations CT: calcitonin, FNAB: fine needle aspiration biopsy, PG: pentagastrin.

Notes

Appendices

183

Rink 2009

Patient Selection

A. Risk of Bias

Patient

Sampling


Inclusion criteria: Nodular thyroid disease diagnosed by high-resolution US.







Patient

characteristics

and setting




Sex (female%)(n): 76.9% (16857).

Age (mean/SD): not reported range: 8 - 97 yrs.

MTC: 28 patients.



Nodule size: Only reported for MTC patients.




Setting: Four sites in central Germany.

Country: Germany.


review question?

Low concern

Index Test



Used calcitonin assay: Radioimmunoassays Calcitonin-IRMA (IBL GmbH, Hamburg, Germany)

and Calcitonin-IRMA magnum (Medipan GmbH, Dahlewith/Berlin, Germany).

Sensitivity: 0.7 ng/l (IBL), 1.5 ng/L (Medipan).


Indication: If basal CT > 10 ng/l and if renal insufficiency as well as proton pump inhibiotr

medication could be ruled out.

Stimulative: Pentagastrin (Pentagastrin Injection BP, Cambridge Laboratiries Ltd., Tyne &

Wear, England).

Dose: 0.5 ug/kg.

Time: Not reported.


Basal: reported: 10 ng/l; extracted: 10, 15 ng/l.

Stimulated: reported males: 80 ng/l, females 50 ng/l.; extracted 50, 80 ng/l.

A. Risk of Bias






Low concern

Appendices

184

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: In selected patients histopathological examination after thyroid

surgery, follow-up.

Indication surgical treatment: An abnormal PGT, a basal CT exceeding 30 ng/l, increasing

basal or progressive morphologic alterations during follow-up.

Type: Not reported.


Number FNA: -



Number FNA: -




of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: In patients not having surgery and without renal insufficiency.

Type: Ultrasound and determination of basal CT.


Duration: mean observation 21 months, median 17 months, range 3-87 months.







Publication

details




Stated aim of

study

Quote from publication: " the calculation and validation of upper limits for basal and

stimulated pCT-Cs in a large patient population with nodular thyroid disease, to distinguish

bewteen the subgroups with and without MTC."

Abbreviations pCT-C: plasma calcitonin concentration, PGT: pentagastrin test.

Notes

Appendices

185

Hasselgren 2010

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Retrospective study.

Inclusion criteria: Patients with non-toxic nodular goiter.

Exclusion criteria: Patients with missing data or with wrongly registered diagnosis.






Patient

characteristics

and setting




Sex (female%)(n): 84.6% (811) NB of whole study population (n=959).

Age (mean/SD): mean not reported; median 49 yrs range: 13-93 yrs NB of whole study

population (n=959).

MTC: 6 patients.

Type of thyroid nodules: Non toxic nodular goiter.


Nodule size: Only reported of MTC patients.



FNA procedures performed through ultrasound guidance or palpation: Ultrasound.

Setting: Secondary/tertiarry referral centre.

Country: Denmark.


review question?

Low concern

Index Test

Index tests Index test: Basal calcitonin


Used calcitonin assay: (1) double-antibody radioimmunoassay technique (MediLab A/S,

Copenhagen, Denmark) (n =668) (2) solid-phase, enzyme-labelded, 2-site chemiluminescent

immunometric principle (Immulite 2000, Calcitonin, Siemens Medical Solutions Diagnostics,

Erlangen, Germany) (n=14).

Sensitivity: Not reported (1 and 2).

Stimulated calcitonin: No.

Indication: -

Stimulative: -

Dose: -

Time: -


Basal: reported 0.10 ug/l, 0.20 ug/l, 0.50 ug/l (= 100 ng/l(pg/ml)).

Stimulated: -

A. Risk of Bias




Are there concerns that the index test, its conduct, or interpretation differ from Low concern

Appendices

186


Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAB, in selected patients histopathological examination after surgery,

cross-linkage with the Danish Thyroid Cancer Database.

Indication surgical treatment: Based on the composition of the following variables: (1) clincal

evaluation including age, comorbidity, thyroid size, suspicion of malignancy; (2) sonographic

appearance; (3) result of FNAB; (4) result of serum calcitonin measurement; (5) patient

preference.

Type: Not reported.

Calcitonin negative: (n = 663)






Is the reference standards likely to correctly classify the target condition? Yes


the index tests?

Unclear

Could the reference standard, its conduct, or its interpretation have introduced bias? Unclear risk




Low concern

Flow and Timing

A. Risk of Bias

Flow and timing Follow-up calcitonin negative: All patients.

Type: Cross linkage with Danish Thyroid Cancer Database.

Number with follow-up: 663 patients

Duration: Median follow-up 7 years (range 3-10 years) (whole study population).

Follow-up calcitonin positive: All patients.

Type: Cross linkage with Danish Thyroid Cancer Database.


Duration: Median follow-up 7 years (range 3-10 years) (whole study population).


Did all patients receive the same reference standard? Yes


Did all patients receive the reference standard? Yes

Could the patient flow have introduced bias? Unclear risk


Publication

details




Stated aim of

study

Quote from publication: "To estimate the validity of serum calcitoin for detection of MTC in a

consecutive population of patients with nontoxic nodular goiter, living in a mild to moderate

iodine-deficient area."

Abbreviations

Notes

Appendices

187

Herrmann 2010

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Retrospective cohort study.

Inclusion criteria: Patients with nodular thyroid disease found by sonography, living in central

Germany (an area with endemic goitre due to previous iodine deficiency).

Exclusion criteria: Known elevation of hCt, Graves' Disease, autoimmune thyroid disease.






Patient

characteristics

and setting




Sex (female%)(n): 56.3% (567)

Age (mean/SD): 55 + 14, range: not reported.

MTC: 2 patients.



Nodule size: Only reported for patients with elevated bCT.




Setting: Division of Endocrinology, Technology Center Bochum, Germany.

Country: Germany.


review question?

Low concern

Index Test

Index tests Index test: basal and stimulated hCT.


Used calcitonin assay: Solid-phase, enzyme labeled, two site chemiluminescent assay, with

the Immulite 2000, (Siemens Immulite 2000, Munich, Germany).



Indication: Basal CT > 10 and < 100 pg/ml.

Stimulative: Pentagastrin (Peptavlon; Laboratoires SERB, Paris, France).

Dose: 0.5 ug/kg bodyweight.

Time: 2 and 5 min after injection.


Basal: 10 pg/ml; extracted: 10, 15, 20, 30, 50 and 100 pg/ml.

Stimulated: 100 pg/ml; extracted:100 and 200 pg/ml.

A. Risk of Bias






Low concern

Appendices

188

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)

Target condition: MTC

Reference standards: FNAB, in selected patients histolopathological examination after

surgery, follow-up.

Indication surgical treatment: Elevated stimulated hCT > 100 pg/ml.

Type: Total thyroidectomy, with careful dissection of both recurrent nerves and a systematic

microdissection of the central lymph node compartments along both nerves from the upper

thoracic outlet up to the larynx.




Calcitonin positive: (n = 17)





of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias

Flow and timing Calcitonin negative: Not reported.

Type: -

Duration: -

Calcitonin positive: Patients with stimulated hCT < 100 pg/ml.

Type: Re-testing.


Duration: Not reported.







Publication

details


FUNDING: Other funding.


Stated aim of

study

Quote from publication: "Shedding further light on the hCT measurement and its testing

after pentagastrin stimulation in patients with thyroid nodule disease."

Abbreviations hCT: serum calcitonin.

Notes

Appendices

189

Schneider 2012

Patient Selection

A. Risk of Bias

Patient

Sampling


Inclusion criteria: Patients diagnosed with thyroid nodules > 2 mm based on the spatial

resolution of ultrasound equipment.

Exclusion criteria: Patients referred with elevated or previously determined CT values, MTC

or a family history of MTC, renal insufficiency, bacterial infection, alcohol abuse, proton-

pump inhibitor therapy, Graves'disease or autoimmune thyroid disease.






Patient

characteristics

and setting




Sex (female%)(n): Not reported.

Age (mean/SD): Not reported range: Not reported.

MTC: 12 patients.


Thyroid nodules detected by palpation or US: Ultrasound.

Nodule size: Only reported of MTC and PTC patients.


Sonographic morphology of thyroid nodules: Only reported of patients with elevated basal

CT levels.



Country: Germany


review question?

Low concern

Index Test



Used calcitonin assay: Solid-phase, enzyme labeld, two-site chemiluminescent assay with

Immulite 2000 (Siemens Immulite 2000, Munich, Germany).



Indication: bCT > 13 and < 100 pg/ml (if PG was available and the patients physical condition

allowed testing).

Stimulative: Pentagastrin (Pentagastrin injection BP, Ireland, UK)

Dose: 0.5 ug/kg bodyweight.



Basal: Reported 13 pg/ml; extracted: 13, 15, 20, 30, 50, 100 pg/ml.

Stimulated: Reported 100 pg/ml; extracted: 100, 200 pg/ml.

A. Risk of Bias



Appendices

190


Are there concerns that the index test, its conduct, or interpretation differ from the

review question?

Low concern

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)

Target condition: MTC

Reference standards: Histopathological examination after thyroid surgery, follow-up.

Indication surgical treatment: 1) basal CT > 100 pg/ml, PG-stimulated CT > 100 p/ml, 3)

suspicious thyroid nodules based on the patients history, sonography (hypoechogencity,

irregular margins, microcalcifications) or scintigraphy (cold nodules have been consistently

associated with malignancy).

Type: Not reported.


Number FNAC: -



Number FNAC: -




the index tests?

Unclear

Could the reference standard, its conduct, or its interpretation have introduced bias? Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Patients with no SCT > 100 pg/ml, patients with no SCT

performed.

Type: Repeated bCT testing.


Duration: Mean 7 months.







Publication

details


FUNDING: Not reported (no conflicts of interest).


Stated aim of

study

Quote from publication: "The positive predictive value (PPV) of a slightly elevated basal

calcitonin for the diagnosis of medullary thyroid cancer is still under debate."

Abbreviations

Notes

Appendices

191

Giovanella 2012

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Prospective cohort study.


Exclusion criteria: Pulmonary or pancreatic tumors, kidney failure, Graves' disease,

autonomously functioning thyroid nodules, autoimmune thyroid diseases, sepsis, alcohol

abuse, smoking or the use of proton-pump inhibitor therapy in the last month.






Patient

characteristics

and setting




Sex (female%)(n): 54.5% (674).

Age (mean/SD): 53 + 17 yrs range: Not reported.

MTC: 2 patients.








Country: Switzerland


review question?

Low concern

Index Test



Used calcitonin assay: Immulite 2000XPi platform (Siemens Healthcare Diagnostics, Erlangen,

Germany).



Indication: Patients with true high CT value.

Stimulative: Pentagastrin (Pengagastrin Injection BP, Cambridge Laboratories, Wallsend, UK)

Dose: 0.5 ug/kg.



Basal: reported: 10 pg/ml; extracted: 10, 15, 20, 30, 50, 100 pg/ml.

Stimulated: reported 100 pg/ml; extracted: 100, 200 pg/ml.

A. Risk of Bias






Low concern

Appendices

192

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAC, in selected patients histopathological examination after surgery,

follow-up.

Indication surgical treatment: Basal and/or stimulated CT levels > 100 pg/ml, patients with

indeterminate, suspicious or malignant cytological outcome.

Typeof surgical treatment: Thyroidectomy with bilateral dissection of the central

compartment (in patients with basal and/or stimulated CT levels > 100 pg/m)l.




Calcitonin positive (n =14)





of the index tests?

Unclear


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Patients with benign cytology.

Type: US, basal CT and PCT.


Duration: 24-35 months.







Publication

details


FUNDING: Other funding

PUBLICATION STATUS: Full article

Stated aim of

study

Quote from publication: " To prospectivevly evaluate the role of routine PCT measuremnet in

detecting MTC among patients with thyroid nodules, and to assess the potential

improvement provided by adding PCT to increased basal CT."

Abbreviations PCT: procalcitonin, CT: calcitonin.

Notes

Appendices

193

Grani 2012

Patient Selection

A. Risk of Bias

Patient

Sampling

Design: Not reported

Inclusion criteria: At least one one discrete nodular lesion of the thyroid or a multinodular

goitre and were referred to our institution to undergo FNAC because of clinical or

ultrasonographic suspicion (irregular margins, micro calcifications, and chaotic pattern

vascularization).







Patient

characteristics

and setting




Sex (female%)(n): 83.2% (893)

Age (mean/SD): 55.7 + 13.4 yrs range: Not reported.

MTC: 2 patients.

Type of thyroid nodules: One discrete nodular lesion of the thyroid or a multinodular goitre

with clinical or ultrasonographic suspicion (irregular margins, microcalcifications, and chaotic

pattern vascularization).

Thyroid nodules detected by palpation or US: Both.



Sonographic morphology of thyroid nodules: Irregular margins, microcalcifications, and

chaotic pattern vascularization.

FNA procedures performed through ultrasound guidance or palpation: Ultrasonography.

Setting: The Thyroid Center of Sapienza University of Rome.

Country: Italy.


review question?

Low concern

Index Test


Index test: Basal calcitonin.

Used calcitonin assay: Automated two-site immunochemiluminometric assay.


Stimulated calcitonin: No.

Indication: -

Stimulative: -

Dose: -

Time: -



Stimulated: -

A. Risk of Bias



Appendices

194




Low concern

Reference Standard

A. Risk of Bias

Target condition

and reference

standard(s)


Reference standards: FNAC, in selected patients histological examination after total

thyroidectomy, follow-up.

Indication surgical treatment: Not reported.

Type of surgical treatment: Total thyroidectomy.









of the index tests?

No


bias?

Unclear risk




Low concern

Flow and Timing

A. Risk of Bias


Type: -

Duration: -

Follow-up calcitonin positive: Patients with unexplained hypercalcitonemia.

Type: basal CT.


Duration: 12-36 months.




Did all patients receive the reference standard? Yes



Publication

details


FUNDING: Other funding.


Stated aim of

study

Quote from publication: " To evaluate the basal CT values in patients with and without

thyroid autoimmunity".


Notes

Appendices

195

Appendix 5

Table Characteristics of excluded studies

Study Reason for exclusion

Lepage 1992 Only abstract available (meeting abstract).

Pacini 1994 (Partially) same study population as Elisei 2004.

Henry 1995 Article in French, (partially) same study population as Henry 1996.

Henry 1996 Article in French, (partially) same study population as Nicolli 1997, Iacobone 2001,

Mirallie 2004.

Shong 1996 Article in Korean.

Vierhapper 1997 (Partially) same study population as Vierhapper 2005.

Niccoli 1997 Evaluation of preoperative calcitonin determination (not part of this review).

Kaserer 1998 Included patients with thyroid disease; number of patients with nodular thyroid

disease not specified. (Partially) same study population as Vierhapper 2005.

Mariss 2001 Only abstract available (meeting abstract).

Mariss 2001 (2) Only abstract available (meeting abstract).

López-Guzmán 2002 Article in Spanish.

Iacobone 2002 Included patients with thyroid disease; number of patients with nodular thyroid

disease not specified.

Mirallie 2004 Included patients with thyroid disease; number of patients with nodular thyroid

disease not specified.

Gibelin 2005 Evaluation of preoperative calcitonin determination (not part of this review).

Papi 2010 Calcitonin testing in isthmic thyroid nodules.

Chambon 2011 Evaluation of preoperative calcitonin determination (not part of this review).

Lipp 2011 Only abstract available (meeting abstract).

Zaplatnikov 2012 Only meeting abstract available

Marui 2012 Only meeting abstract available

Appendices

196

Appendix 6

Differences between protocol and review

No separate meta-analyses for calcitonin testing as a triage or as an add-on test was

performed because all included studies were considered as performing calcitonin testing

as a triage test.

No email requests to contact persons of included studies were send

No studies written in non-English language were included.

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