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Molecular and Cellular Endocrinology 322 (2010) 8–28

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Molecular and Cellular Endocrinology

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olecular prognostic markers in papillary and follicular thyroid cancer: Currenttatus and future directions

aria Handkiewicz-Junaka, Agnieszka Czarnieckab, Barbara Jarzaba,∗

Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej4, 44-100 Gliwice, PolandDepartment of Oncologic Surgery, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 14, 44-100 Gliwice, Poland

r t i c l e i n f o

rticle history:eceived 7 July 2009eceived in revised form0 December 2009ccepted 9 January 2010

eywords:apillary thyroid cancerollicular thyroid cancerrognosis

a b s t r a c t

Gene expression profiling shows that, by gene signature, the difference between BRAF-positive and BRAF-negative PTC is so distinct that BRAF-positive cancer may be regarded as a molecular subtype of papillarythyroid cancer (PTC). Since much enthusiasm surrounds the BRAF-oncogene as a molecular prognosticfactor, a central focus of our consideration is to weigh the current arguments for and against applying BRAFmutation status of the tumor in clinical practice. The frequency of BRAF mutation in PTC is high—45% onaverage, with values over 70–80% in some populations. This will mean that implementing BRAF mutationas a factor of poor prognosis will shift many PTC patients, considered up to now as low risk ones, to themore extensive treatment. We estimate that 31% of all PTC patients and 39% of those diagnosed withstage I–II disease will face the risk of overtreatment if the decision will be based on the BRAF-positivity

RAFolecular markers

of their tumors. Also, the risk of undertreatment in the young patients with BRAF-negative tumors isevaluated with 26%. We think that, as of now, the evidence-based support for such consequences isstill weak. Thus, there is urgent need to look for genes or gene signatures which will be helpful in thestratification of BRAF-positive tumors to specify these with poor prognosis with higher accuracy, neededfor clinical decisions. Considering this, in the review we summarize the present status of knowledge onother prognosis-related gene expression changes in papillary and follicular cancer and relate them to he
tumor’s biology.
© 2010 Elsevier Ireland Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92. Pathological and clinical basis for evaluating prognosis in papillary and follicular thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.1. Histopathological and clinical characteristics of DTCs and their influence on disease prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.2. Clinicopathological staging of DTC and its limitations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.3. Molecular mechanisms of neoplastic transformation in papillary and follicular thyroid cancer: prognosis-related aspects . . . . . . . . . . . . . . 11

2.3.1. Activation of the RAS–RAF–MEK–ERK1/2 pathway and the PI3K/AKT pathway in thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113. Prognostic impact of the inititiating molecular events in papillary and follicular thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3.1. The role of BRAF proto-oncogene in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.1.1. BRAF mutation is the most frequent initiating molecular event in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.1.2. BRAF mutations are associated with poorer prognosis of PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.2. Is it the time to apply BRAF as clinically relevant prognostic/predictive factor in PTC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153.3. Other molecular initiating events specific for PTC—do they have prognostic potential? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.3.1. RET/PTC rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163.3.2. NTRK rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.3.3. Gene rearrangements are frequent in thyroid cancers . . .
3.4. Is there some multiclonality in papillary thyroid cancer? . . . . . . . . .3.5. Gene mutations important for development of both papillary and

3.5.1. RAS mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +48 322789339; fax: +48 322789325.E-mail addresses: [email protected] (D. Handkiewicz-Junak), aczarniecka@io

303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.mce.2010.01.007

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

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.gliwice.pl (A. Czarniecka), [email protected], [email protected] (B. Jarzab).

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D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28 9

3.6. Follicular thyroid cancer-specific gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.6.1. PAX8/PPAR� rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.6.2. RAS homolog 1 (ARH1) silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.7. Mutations of other genes and their importance for thyroid cancer and its outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.7.1. PIK3CA mutations and amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.7.2. PTEN mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.7.3. �-Catenin mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.7.4. TP53 mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4. Prognostic aspects of other gene expression changes in papillary and follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.1. Single genes expression changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.1.1. MET proto-oncogene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.1.2. Epithelial growth factor receptor (EGFR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.1.3. Mitogen-inducible gene-6 (MIG-6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.4. Vascular epithelial growth factor (VEGF) and VEGF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.5. E-cadherin and catenins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.6. Cyclin D1 and other cell cycle regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.7. Mucin 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.8. S100A4 calcium-binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.9. Osteopontin and CD44v6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.1.10. Immunity-related genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

4.2. Prognosis-related gene signatures in thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204.2.1. Gene expression profiling and prognosis of thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204.2.2. Metastasis-associated genes in thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204.2.3. Epithelial-to-mesenchymal transition in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4.3. Genetic germline background and the prognosis of papillary and follicular thyroid cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205. Final considerations on prognostic impact of genetic factors in papillary and follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.1. Relation between BRAF status and gene expression profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215.1.1. Is BRAF-positive DTC a distinct molecular subtype? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215.1.2. BRAF-mutated PTCs exhibit fewer thyroid-specific gene expression features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225.1.3. BRAF mutation is associated with the more aggressive tumor phenotype in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.2. Major questions in the therapeutic strategy in DTC which can be solved by molecular diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225.3. Conclusions from the transcriptome-wide analyses of DTCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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. Introduction

Papillary thyroid cancer (PTC) and follicular thyroid cancerFTC) are both described clinically as differentiated thyroid cancersDTCs) due to their tumor cells structurally and functionally resem-ling normal follicular cells and due to their sharing a relatively

ndolent natural history and good responsiveness to surgery andadioiodine in most – but far from all – cases. DTC constitutes theommonest endocrine malignancy, and among neoplastic diseases,ossesses the most favorable overall prognoses. However, over 10%f patients eventually die of DTC and an even greater proportionaces the morbidity of recurrences (Eustatia-Rutten et al., 2006;

azzaferri and Kloos, 2001; Sherman, 2003). In these respects, lit-le to nothing has improved in DTC management in the last twentyears (Clark and Duh, 1991).

As in other cancers, numerous studies in DTC have concentratedn relatively simple clinicopathological variables, e.g., primaryumor size, extent of disease, pathological histotype, patient’s aget diagnosis or sex, to formulate risk group stratification or stag-ng systems. The goal has been to select patients who will do well

ithout further therapy (i.e., to identify prognostic factors) or whoill do well with some treatments but not others (i.e., to identifyredictive factors).

In recent years, the impact of molecular diagnosis is becomingven more substantial in cancer care. This trend is already evident inany hematological malignancies, where the presence of particu-

ar chromosomal rearrangement influences the natural history andhe choice of treatment, i.e., is not only of prognostic significanceut also of predictive significance (Haferlach et al., 2003; Jabbourt al., 2008; Meijerink et al., 2009). A similar phenomenon is alsoccurring with solid tumors such as breast cancer, where the divi-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

sion into two molecular subtypes – luminal and basal – is slowlygaining influence in formulating disease management strategies(Sorlie et al., 2001; van’t Veer et al., 2005).

In the present review, we highlight that the molecular diag-nosis of thyroid cancer is already possible (Eszlinger et al., 2008;Giordano, 2008). We therefore examine whether, to improve prog-nostic/predictive ability, molecular diagnosis should now supplantor at least supplement the clinicohistopathological evaluation ofDTC. This question is increasingly pertinent, as in recent years,both research on single, cancer-specific mutations and genome-wide DNA microarray-based approaches have added greatly to ourunderstanding of DTC biology and clinical behavior. In our discus-sion of the prognostic relevance of molecular studies, we focus ondata regarding gene mutations, essential for neoplastic transforma-tion of follicular cells and, then, for gene expression changes, judgedboth by RNA and protein levels. We present details of microarray-based comparative genome hybridisation (CGH) studies on geneamplifications/deletions and of studies on epigenomic changesinsofar as this information is germane to the current role andfuture investigation of molecular prognostic factors in DTC. Sincethe BRAF-oncogene (v-raf murine sarcoma viral oncogene homologB1) is the major genetic factor in PTC (Puxeddu and Moretti, 2007;Xing, 2007), an important focus of the present review is to weighthe current arguments for and against applying BRAF mutation sta-tus in clinical practice. As we discuss in more detail below, the dataon the correlation of BRAF status with DTC clinicopathological fac-
tors are substantial and even show an impact on survival. However,from the clinical point of view, a factor present in 50% of cases is oflimited use in managing a disease which has a poor outcome in nomore than 10–15% patients, thus, a more detailed stratification isnecessary and we look for this possibility.

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Our review begins by summarizing the current understandingf histopathological and clinical prognostic factors in DTC. Next,e describe BRAF mutations and other molecular events occurring

arly in the thyroid neoplastic transformation in the context of theirelationship to thyroid cancer prognosis. Subsequently, we discusshe expression changes in other single genes other than BRAF thatre known to have prognostic utility in DTC. Lastly, we present thetatus of relevant genome-wide analyses and provide some closingemarks where we consider not only the prognostic value of geneticarkers but also comment briefly on their predictive contribution.

. Pathological and clinical basis for evaluating prognosisn papillary and follicular thyroid cancers

.1. Histopathological and clinical characteristics of DTCs andheir influence on disease prognosis

The follicular cells of the thyroid gland give rise to a varietyf tumors that differ markedly in their morphology and biologi-al and clinical behavior. Based on a number of diagnostic criteriand morphological hallmarks, these tumors may be divided intove major types: papillary, follicular, oncocytic, poorly differenti-ted, and undifferentiated (anaplastic), the first two of which inurn may be subdivided into a number of variants (LiVolsi and Asa,994; LiVolsi and Baloch, 2004). By far the most common is PTC,epresenting up to 80% of all thyroid malignancies. This prevalences distantly followed by that of FTC, ranging from less than 10% or5% to not more than 25%, depending in the series, while oncocyticistotype (either variant of PTC or FTC) constitutes not more than 2%f thyroid cancers (Kushchayeva et al., 2008). Several histopatho-ogical features of DTC have been claimed to influence its prognosis.n PTC, the tall cell variant has been shown to have a worse prog-osis than does the classical type (Ito et al., 2008). Concomitanthyroiditis, diffuse lymphocytic infiltration, or both may favorablynfluence the course of PTC as well as that of FTC (Kashima et al.,998; Loh et al., 1999; Modi et al., 2003).

FTC, especially if widely invasive or oncocytic, was claimed toave a worse prognosis than does PTC (Gulcelik et al., 2007; Passlert al., 2004). However, a recent study of more than 1000 patientsound no difference in cancer-specific survival between PTC andTC when one accounted for clinical prognostic factors such as aget diagnosis, primary tumor size and the presence of extrathyroidalnvasion or of metastases (Verburg et al., 2009). In FTC, the prog-osis for the minimally invasive subtype is excellent (Thompsont al., 2001). Fig. 1 compares a variety of commonly used clin-cal prognostic factors with respect to the associated significantncreases in the risks of death or DTC recurrence that were foundn a recent large, long-term study of DTC patients conducted at ourenter (Czarniecka, 2004). As seen in that figure, the presence ofistant metastases is the strongest poor prognostic factor for over-ll survival in DTC (Elisei et al., 2008; Mazzaferri and Jhiang, 1994;azzaferri and Kloos, 2001). However, there are exceptions to this

ule, as distant metastases in young patients are not regarded as aoor prognostic factor. In fact, younger patients with distant metas-ases have a better prognosis than do older patients diagnosed withocally invasive tumors, lymph node metastases, or both (Jarzab etl., 2005a).

The prognostic impact of lymph-node involvement in DTC hasong been a matter of controversy, especially regarding survival.everal studies failed to demonstrate an influence on mortality
ates (DeGroot et al., 1990; Mazzaferri and Young, 1981) while oth-rs have identified an association between lymph node metastasesnd an increased risk of cancer-related death, especially when thenvolved lymph nodes are large, bilateral or located in the medi-stinum (Czarniecka, 2004; Mazzaferri and Kloos, 2001; Podnos
ellular Endocrinology 322 (2010) 8–28

et al., 2005; Tubiana et al., 1985; Zaydfudim et al., 2008). In fact,in the past, lymph-node involvement was even considered to bea favorable prognostic sign, until the effect of age was recog-nized: children and young adults with PTC have both far morefrequent lymph-node involvement and a far better outcome thando older patients. However, almost all studies unequivocally agreethat lymph-node involvement is associated with a higher risk oflocoregional recurrences (Beasley et al., 2002; Handkiewicz-Junaket al., 2007; Harwood et al., 1978; Sakorafas et al., 2009).

2.2. Clinicopathological staging of DTC and its limitations

At least 18 staging systems for PTC, FTC (Akslen, 1993; Cadyand Rossi, 1988; DeGroot et al., 1990; Greene et al., 2002; Hay etal., 1987, 1993; Mazzaferri and Jhiang, 1994; Pasieka et al., 1992;Shaha et al., 1994; Sherman et al., 1998) or both, have been formu-lated based on retrospective patient outcome evaluations (Lang etal., 2007a,b). In Europe, the UICC/TNM system is the most widelyadopted. It considers three main factors:

• T status—primary tumor size and extrathyroidal extension,• N status—presence or absence of lymph node metastases,• M status—presence or absence of distant metastases.

The UICC/TNM system also considers patient age at diagnosis,a factor not commonly used in other non-TNM-based DTC stagingsystems. All patients below 45 years of age who are diagnosed with-out distant metastases, are considered to have stage I disease, i.e.,belong to the “low-risk” group.

The clinicopathological factors upon which the majority ofstaging systems are based are summarized in Table 1. These vari-ables frequently overlap, e.g., older or very young patients tendto more often have lymph node or distant metastases or both;young patients with lymph node metastases more often sufferfrom distant metastases (Jarzab et al., 2005a). Such overlap is usu-ally addressed with multivariate analysis, which can help selectindependent variables while rejecting factors that are only surro-gates for those variables. Nonetheless, even with that statisticalapproach, results are sometimes contradictory, as in the evaluationof tumor histopathology (Gulcelik et al., 2007; Verburg et al., 2009)or of lymph node metastases (Bardet et al., 2008; McConahey et al.,1986). In the case of histopathology, the contradictory results maybe attributable to the somewhat subjective, not fully standardizedmethod of specimen evaluation. The rate of disagreement duringDTC evaluation can be high, especially in FTC where it can reach upto 50% of cases (Franc et al., 2003; Lange et al., 2006).

Table 2 provides the survival by disease stage or risk groupaccording to the most popular staging systems. As seen in Table 2,the systems show marked survival differences, especially for “high-risk” groups/advanced stages, in which, according to most systems,more than 50% of patients eventually succumb to disease (Duranteet al., 2006; Sampson et al., 2007; Schlumberger et al., 1996). Itis however, not established which patients in less advanced sub-groups do or do not require more intensive treatment to cure theircancer. This results in a very wide “gray zone” of DTC prognosis,which leads to a significant risk of over- or undertreatment in a sub-stantial number of patients (Gulcelik et al., 2007; Tuttle et al., 2008).The MACIS and TNM staging systems have been demonstrated to bethe most accurate prognostic systems in some (Lang et al., 2007a,b;Passler et al., 2003) but not all studies (Brierley et al., 1997). Never-theless, even if evaluated with the best score, they explained barely
about 20% of the observed variation in survival time (Lang et al.,2007a,b) and only a few studies yielded better results (D’Avanzo etal., 2004).
Exacerbating this problem, the extent of treatment (e.g., rad-icalness of surgery or use or activity of radioiodine) that is


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ig. 1. Clinical and pathological risk factors of overall and recurrence-free survivalyears) (Czarniecka, 2004).

ecommended in guidelines is not consistent between “high-risk”r “low-risk” groups according to different staging systems. It is, ofourse, well known that the extent of treatment can influence dis-ase outcome (Czarniecka, 2004; Gulcelik et al., 2007; Lang et al.,007a,b; Passler et al., 2003). In addition, since almost all currentlinicohistopathological prognostic factors are based on postsur-ical tumor and patient evaluation, none of these staging systemsan be applied pre-operatively, and hence help tailor the extent ofurgery.

In summary, then, the utility of the current clinicohistopatho-ogical staging systems for DTC is limited by the abundancef systems, their frequent overlap and sometimes contradictoryesults, their far from perfect match with short- or long-term prog-osis, and their inapplicability pre-operatively.

.3. Molecular mechanisms of neoplastic transformation inapillary and follicular thyroid cancer: prognosis-related aspects

From the molecular point of view, papillary and follicular thy-oid cancers are regarded as different diseases. This opinion isupported by the disparate molecular initiating events leading toeoplastic transformation. Also supporting this perspective are theifferences in DNA ploidy level (PTCs are generally diploid, FTCsneuploid) and the differences in localization and in the numberf gains and losses seen in comparative genomic hybridisationCGH)-based analysis. In PTC, aneuploidy, although observed in

inority of tumors (Rodrigues et al., 2007), is significantly associ-ted with cancer-specific death (Sturgis et al., 1999). The profoundifferences in gene expression will be described below. On thether hand, many subsequent down-stream molecular mecha-isms are very similar between PTC and FTC, which explains the

roid cancer (multivariate regression analysis of 1141 DTC patients, followed-up by

commonalities in the clinical course of both cancers and cre-ates a rationale for similar therapeutic approaches to both ofthem.

2.3.1. Activation of the RAS–RAF–MEK–ERK1/2 pathway and thePI3K/AKT pathway in thyroid cancers

DTC harbors several highly prevalent genetic alterations, someof which are seen only in this cancer or are characteristic of one of itshistotypes. In PTC, the most important oncogenic mechanism, seenin about 80% of tumors, is the activation of the mitogen-activatedprotein kinase (MAPK) pathway (Fagin, 2004; Fagin and Mitsiades,2008), which once constitutively activated, leads to tumorigene-sis (Hilger et al., 2002; Peyssonnaux and Eychene, 2001). Threemost important initiating events, RET/PTC (rearranged during trans-fection/papillary thyroid cancer), RAS (resistance to audiogenicseizures) and BRAF mutation, are regarded as mutually exclusive,alternative triggers for the activation of the pathway (Fagin, 2004).The mutual exclusivity between BRAF and the RAS mutation is char-acteristic not only of thyroid cancer but also of several other humancancers (Xing, 2005). The activating BRAF mutation has only beenrecognized relatively recently (Davies et al., 2002), but in fact is themost frequent trigger in PTC.

A single oncogenic alteration along the receptor tyrosinekinase–RAS–RAF–MEK–ERK1/2 pathway is likely sufficient to drivethyroid cell neoplastic transformation, although further supportivemolecular events are necessary. BRAF mutation and RET/PTC (and
NTRK1—neurotrophic tyrosine kinase, receptor, type 1) rearrange-ments differ to some extent in their effects on the shared oncogenicpathway, respectively, resulting more frequently in the classic vari-ant or the solid variant of PTC, while RAS mutations and RASSF1A(Ras association (RalGDS/AF-6) domain family member 1) methy-

12 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28

Table 1Clinical prognostic factors used in different staging systems for DTC.

Factor Positive prognostic indicator Negative prognostic indicator

Age Younger (usually below 40–45 years of age) OlderPrimary tumor—diameter Small (the best prognosis for tumors ≤1 cm) LargePrimary tumor—extension beyond thyroid capsule No YesLymph node metastasis Absent PresentDistant metastases Absent PresentSurgical resection Total thyroidectomy and appropriate extent of

lymphadenectomy ensuring complete removalof cancer tissues

Incomplete removal of cancer tissues and/orlack of total thyroidectomy and of appropriateextent of lymphadenectomy

Table 2Survival by disease stage or risk group in selected DTC staging systems.

Staging system DTC type (number of patients) Primary outcome end-point Disease stage or risk group

TNM stage (Greene et al., 2002) PTC (6590) 5-Year survival I97%

II 93% III84%

IV39%

FTC (1129) 5-Year survival 95% 90% 69% 41%

MACIS (Hay et al., 1993) PTC (1779) 20-Cause-specific survival rates I99%

II 89% III56%

IV24%

Ohio State University Stagingsystem (Mazzaferri andJhiang, 1994)

PTC and FTC (1355) Cancer-related deaths I0%

II 6% III15%

IV65%

AMES (Cady and Rossi, 1988) PTC and FTC (821) Cancer-related deaths Low1.8%

High46%

NTCTCS system (Sherman etal., 1998)

PTC and FTC (1607) 5-Year CSS I99.8%

II 100% III91.9%

IV48.9%

University of Chicago (DeGrootet al., 1990)

PTC (269) 10-Year CSS I100%

II 100% III87%

IV35%

GAMES Risk Groups (Shaha etal., 1994)

PTC and FTC (1038) 10-Year survival Low99%

Intermediate85% High57%

AGES (Hay et al., 1987) PTC (860) 25-Year CSS Low98%

High65%

DAMES (Pasieka et al., 1992) PTC (74) Cancer-related death or recurrence Low8%

Intermediate45% High100%

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ation are more prone to induce the follicular variant of PTC (Xing,005).

MEK–ERK1/2 pathway activation also plays a significant rolen FTC tumorigenesis (Liu et al., 2008). However, in FTC, neitherET/PTC nor the BRAF mutation can be found, while RAS mutationsre quite frequent, seen not only in follicular cancers, but also inollicular adenomas (FA). This pattern is repeated in the case ofAX8/PPAR� rearrangements, which however do not directly leado MEK–ERK activation and cause different down-stream effectsGiordano et al., 2006).

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway alsolays a fundamental role in the regulation both of normal cellrowth, mitosis and survival and, when disarranged, of tumorige-esis. PI3K/AKT alterations are found in every histotype of thyroidancer, frequently in FTC and, even more distinctly, in ATC. In FTC,hosphorylation of AKT, the key player in this pathway, was farore frequent than that of ERK (MAPK kinase effector) (Liu et al.,

008).

. Prognostic impact of the inititiating molecular events inapillary and follicular thyroid cancers

.1. The role of BRAF proto-oncogene in PTC

BRAF is a serine–threonine kinase, expressed in a tissue-specificanner and abundant in thyroid follicular cells, which among the

hree known RAF kinases, most potently activates the MAPK path-ay (Peyssonnaux and Eychene, 2001) (for reviews see Puxeddu

nd Moretti, 2007; Xing, 2005). Activating BRAF mutations are not

S I98.3%

II 88% III39%

ncer; PTC, papillary thyroid cancer.

thyroid cancer-specific. To the contrary, BRAF belongs to the com-monest oncogenes, mutated in 7–9% of all malignant solid tumors,with the highest prevalence (about 60%) in melanomas (DeLucaet al., 2008), and a frequent presence in ovarian, colorectal, andlung cancers and even some low-grade astrocytomas (Pfister et al.,2008). Interestingly, although the activation of the MAPK signalingpathway is regarded as frequent in human cancer, the evidence forthis is most compelling in thyroid cancer and melanoma (Johanssonet al., 2007; Knauf and Fagin, 2009).

3.1.1. BRAF mutation is the most frequent initiating molecularevent in PTC

After melanoma, PTC is the second human malignancy whereBRAF mutations are especially frequent, found in 29–87% (with amean frequency of about 45%) of cases (Kim et al., 2009; Kimuraet al., 2003; Puxeddu et al., 2004; Xing, 2007). BRAF mutations arenot found exclusively in PTC but also, albeit less often (not morethan 20–26% of cases, mainly those originating from PTC) in ATC(Nikiforova et al., 2003a; Smallridge et al., 2009; Takano et al.,2007).

Although more than 40 mutations have been identified in theBRAF gene, the most significant hot spot mutation, accounting forover 90% of all BRAF mutations, is a thymidine to adenine transver-sion at nucleotide 1799 (T1799A) in exon 15. This substitution
leads to a valine-to-glutamate transversion at residue 600 nearthe catalytic center of the protein (BRAFV600E) and is believed toproduce a constitutively active kinase by disrupting hydrophobicinteractions between residues in the activation loop and residuesin the adenosine triphosphate (ATP) binding site (Wan et al., 2004).

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RAFV600E potentiates the catalytic activity of the enzyme by morehan 500 times (Kim et al., 2005).

BRAF mutation is an early event in PTC tumorigenesis, whichan be found even in minute PTCs, less than 4 mm in diameterUgolini et al., 2007). The constitutively active kinase subsequentlyeads to tumorigenesis through aberrant activation of the MAPKathway (Davies et al., 2002; Garnett and Marais, 2004). Such acti-ation includes hyperphosphorylation of retinoblastoma proteinRB), which releases inhibition of E2F-dependent transcription fac-ors, allowing the cell to pass from G1 into S phase, increasingrowth and promoting survival (for a review of this process seenauf and Fagin, 2009). At the same time, apoptosis is inhibited,robably due to the action of NF�B transcription factor (Palonat al., 2006). Transgenic mouse studies clearly demonstrated theriving force of the BRAF mutation in promoting malignant trans-ormation of thyrocytes and extrathyroidal invasion of PTC (Knauft al., 2005). One important molecular mechanism involved in thisrocess is BRAF mutation-promoted methylation and hence silenc-

ng of the tissue inhibitor of metalloproteinase-3 (TIMP-3; Hu et al.,004) and resultant overexpression of metalloproteinases (Melillot al., 2005; Mesa et al., 2006; Palona et al., 2006).

The BRAF-oncogene induces chromosomal instabilityMitsutake et al., 2005), and is it not only an initiator of PTCut is necessary for the maintenance of PTC proliferation andumorigenicity (Liu et al., 2007b).

.1.2. BRAF mutations are associated with poorer prognosis ofTC

Based on molecular results, numerous studies have investigatedhe clinical significance of BRAF mutation in PTC. The general ten-ency of the studies to show the association of this mutation withactors related to poor prognosis is rather convincing (Table 3 ).owever, a careful overview of the literature indicates that the clin-

cal data on the link between BRAF mutation and DTC outcome areot very consistent. The first data were summed up in a large meta-nalysis of 1168 patients and showed that BRAF mutation correlatedith histologic subtype, presence of extrathyroidal extension, and

dvanced clinical stages, but not with age, sex, race, or tumor size ofTC patients (Lee et al., 2007). More recent studies addressed evenore carefully the correlation between BRAF status and PTC clini-

opathological factors: two such studies (Frasca et al., 2008; Wangt al., 2008a), which altogether included more than 400 patients,ound in multivariate analysis a positive correlation between BRAF

utation and extrathyroidal extension, while two other such stud-es totaling about twice as many patients did not (Kebebew et al.,007; Lupi et al., 2007). In the analysis of Lupi et al., encompassing00 consecutive PTC patients at the University of Pisa, the one-waynivariate correlations of BRAF mutation positivity with extrathy-oidal extension and tumor stage-related data were very distinct.owever, the multivariate analysis discarded all the factors except

ack of a tumor capsule (whether the tumor was single or multifo-al) (P = 0.0005 in multivariate analysis). In the Lupi study, lack ofncapsulation remained statistically significant in separate multi-ariate analyses for micro-PTC, larger tumors and follicular variantTC but not for classical variant PTC. Presence of tumor capsule,lthough relatively rare in PTC (present in 21.7% of the Lupi series),s a strong predictor of an especially good disease-free prognosis,een very distinctly in follicular variant PTC (Kakudo et al., 2004).

Summing up the analysis of the relationship of BRAF muta-ions with clinicopathological factors, there is only one factorhown by the majority of studies to correlate with BRAF
utations—extrathyroidal tumor extension, which is however
uestioned if tumor encapsulation is also included in the analy-is. For one perusing these studies, questions arise on the origins ofhe discrepancies between the study results. Many studies on theorrelation of BRAF with clinicopathological data (Costa et al., 2008;

ellular Endocrinology 322 (2010) 8–28 13

Elisei et al., 2008; Guan et al., 2009; Kebebew et al., 2007; Lee et al.,2009; Rosenbaum et al., 2005) were based on univariate analysesthat could not distinguish between overlapping significant factors.In that case what seems to be BRAF related in univariate analysis canresult from other unknown factors affecting studied outcome (e.g.,disease stage or recurrences) and be correlated with BRAF status.For example, BRAF mutations are relatively rare in PTC cases withconcommitant autoimmune thyroiditis (Nikiforova et al., 2002),and here the immune response may influence the clinical coursemore than do the molecular consequences of initiating mutationitself. Although the multivariate analysis may be somewhat help-ful in making the distinction between diverse confounding factors,it may only solve some but not all the problems, as only a limitednumber of factors can be included and other, theoretically deci-sive factors in the outcome of the disease can be unintentionallyomitted. Nevertheless at present, multivariate analysis seems to bethe best way to obtain the least biased results. A good example isthe already discussed study by Lupi et al., where BRAF mutationpositively correlated with extrathyroidal extension in univariateanalysis, but was abolished by the status of thyroid capsule in mul-tivariate approach.

A second issue in interpreting these studies is how preciselyclinicopathological factors are defined. For example, both mini-mal invasion beyond the thyroid capsule and gross invasion ofmuscles and other neck tissue can be regarded as extrathyroidaldisease extension, but their clinical significance differs substan-tially: tumors with gross invasion into surrounding tissues have aworse prognosis and a higher risk for recurrence. Differences in themethodology of BRAF detection can also cause conflicting resultsamong studies.

Surprisingly, distant metastases, the most significant factor ofpoor PTC outcome, were not associated with BRAFV600E mutationas a single factor. However, when that mutation was associatedwith other genetic events, the conjunction of events appeared asan independent predictor of distant metastasis (Costa et al., 2008).

A more definitive assessment of the impact of BRAF status onPTC prognosis can be derived solely from studies evaluating thelong-term outcome of the disease. Only recently have reports ondisease-free survival or overall survival in BRAF-positive versusBRAF-negative PTC patients been published (Table 4). Ito et al.(2009) evaluated disease-free survival and distant metastases-freesurvival with respect to BRAF mutation status in more than 600patients followed for a mean 83 ± 35 months. The BRAFV600E muta-tion was detected in 38% of their series, but these investigatorsdid not search for other far less frequent BRAF point mutations orrearrangements. The only significant difference among patient sub-groups was the frequency of BRAF mutations in micro-PTC (28%)versus tumors larger than 1 cm (41%) (P = 0.0175), however, BRAFmutation prevalence did not successively increase with size inpatients having tumors larger than 1 cm. In multivariate analysis,BRAF mutation was not linked to patient gender, disease stage, mas-sive extrathyroidal extension, or lymph node/distant metastases atdiagnosis. Importantly, the authors also did not find any differencein 5- or 10-year disease-free survival or metastases-free survival,which were about 90% in both BRAF-positive or BRAF-negativecases. This observation confirmed the findings of a previous mul-ticenter Italian report involving PTC of all types (Fugazzola et al.,2004) and a Korean study (Kim et al., 2006b) involving the clas-sic PTC variant. Contrary conclusions, supporting the correlation ofBRAF status with disease-free survival, were reported by Kebebewet al. (2007) (mean follow-up of 6 years) who in multivariate
analysis, noted a significant association of BRAF mutation withrecurrence risk.
The recent study by Elisei et al. (2008), who analyzed 102patients with a median follow-up of >10 years, is the only publishedstudy until now to address the relationship of BRAF mutation status

14D

.Handkiew

icz-Junaket

al./Molecular

andCellular

Endocrinology322 (2010) 8–28

Table 3Correlation of BRAF mutation and clinicopathological prognostic factors in papillary thyroid carcinoma: data from recent studies.

Author Number ofpatients

Type of statisticalanalysis

Number ofBRAF-positive cases(%)

Tumor size Gender Age Extrathyroidalextension

Tumorcapsule

Lymph nodemetastases

Distantmetastases

Cancer stage

Rosenbaum et al. (2005) 85 Univariate 54 (64%) – – 0.0001 – – – – –

Kim et al. (2006a) 103 Univariate 34 (33%) 0.0001 ns 0.01 – – 0.0001 – 0.0001Multivariate ns – ns – – 0.0001 – ns

Kim et al. (2006b) 203 Univariate 149 (73%) 0.006 0.006 ns 0.06 – ns – nsMultivariate 0.03 0.04 0.005 (gross invasion) – 0.02 – –

Kebebew et al. (2007) 274 Univariate 133 (49%) ns ns 0.03 ns ns ns ns 0.04

Lupi et al. (2007) 500 Univariate 217 (44%) – ns ns 0.0001 <0.0001 0.0009 – 0.00001Multivariate – – – ns 0.0005 ns – ns

Lee et al. (2007) (meta-analysis) 1168 Univariate 570 (49%) ns ns ns 0.000 – ns – 0.000Costa et al. (2008) 49 Univariate 27 (55%) – – ns ns – ns ns nsElisei et al. (2008) 102 Univariate 38 (37%) ns ns 0.02 ns – ns ns 0.03

Wang et al. (2008a,b) 108 Univariate 54 (50%) ns – 0.02 0.02 – ns ns 0.04Multivariate – – – 0.005 – – – –

Frasca et al. (2008) 323 Univariate 125 (39%) 0.005 – – 0.0001 – 0.0001 – 0.05Multivariate – – – 0.0003 – 0.007 – ns

Lee et al. (2009) 64 Univariate 24 (38%) ns ns ns 0.001 – 0.003 – 0.001 (only T status)

Ito et al. (2009) 631 Univariate 242 (38%) – ns 0.05 ns – 0.005 ns nsMultivariate ns (for tumors >1 cm) 0.033 (for ≥ 55 years) – – 0.0001 (for N1b) – –

Guan et al. (2009) 1032 Univariate 639 (62%) – ns ns 0.003 – 0.005 – 0.001

ns, not significant.


Table 4Association of BRAF with disease-free and overall survival.

Author Number of patients Disease-free survival (decrease) Overall survival (decrease)

Kim et al. (2006a,b) 203 ns –Fugazzola et al. (2004) 260 ns –Kebebew et al. (2007) 274 0.03 by multivariate logistic regression analysis –Abubaker et al. (2008) 536 0.01 by Kaplan-Meier analysis –Elisei et al. (2008) 102 0.03 by multivariate logistic regression analysis 0.015 by log-rank analysis

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ith overall survival. A higher rate of persistent disease, mortality,r both in was reported in BRAF-positive PTC patients (P = 0.005).ultivariate logistic regression, that also included well-accepted

linicopathological factors, found significant correlation with nega-ive outcomes only for BRAF status. Log-rank analysis confirmed theegative prognostic impact of BRAF mutation on overall survival,ith deaths occurring practically exclusively in patients harbor-

ng BRAF-positive tumors. It must be stressed that patients in thelisei study were followed substantially longer than were patientsn studies negative for prognostic significance of BRAF mutationFugazzola et al., 2004; Ito et al., 2009; Kim et al., 2006b). However,enetic background differences also may explain the discrepantesults of the studies.

A recent study on the molecular biology of recurrent PTCHenderson et al., 2009) is far less convincing than the Elisei etl. study (Elisei et al., 2008). The authors report that the BRAFutation was found in 77.8% of recurrences, which far exceeds

he frequency seen historically in primary PTCs. However, in theajority of patients, these investigators did not compare the BRAF

tatus in the recurrent tumors versus in the primary tumors.dditionally, they analyzed only lymph node recurrences, and

he lower limit of their range of times-to-recurrence started atmonths, an interval short enough to suggest that the diseaseight be attributable to primary treatment failure rather than true

ecurrence.

.2. Is it the time to apply BRAF as clinically relevantrognostic/predictive factor in PTC?

The question, whether we are ready to implement the BRAF-ncogene as a molecular prognostic factor in PTC is answeredositively by the majority of recent publications. However, theuthors of this review would like to adopt a more cautious positionnd indicate what we believe is still necessary before BRAF mighte used as a clinically relevant factor of poor prognosis in DTC. Lets list our major doubts:

. As stated earlier, the frequency of BRAF mutation in PTC ishigh—45% on average, with values over 70–80% in some popula-tions. This high prevalence will mean that implementing BRAFmutation as a factor of poor prognosis will shift many DTCpatients, considered up to now as low risk patients, in the groupof patients who require more extensive therapy (Table 5). Ourevaluation indicates that as many as 31% of all PTC patientsand 39% of those diagnosed with stage I–II disease, will facethe risk of overtreatment if the decision will be based on theBRAF-positivity of their tumors, among them at least of (1/4) ofpatients with micro-PTC, who constitute up to 44% of patientswith tumor ≤2 cm (Bonnet et al., 2009) and who were consid-
ered until now as having an excellent prognosis. We think that,as of now, the evidence-based support for such consequencesis very weak and the risk of overtreatment is significant. As anexample, a 32-year-old pregnant woman (10th week of preg-nancy) with an incidentally detected hypoechoic nodule 8 mm
––

in diameter was referred to our department because of cyto-logic diagnosis of PTC. No enlarged lymph nodes were palpatedor seen by sonography. The recommendation based on currentguidelines (Cooper et al., 2009) would be to monitor carefullyand, if no signs of distinct progression are observed, to performthyroidectomy after delivery. However, for better evaluation,BRAF mutation was sought and confirmed to be present in thecytologic material obtained by fine needle aspiration biopsy. Dueto the presence of this molecular factor, the consulting surgeondecided to perform surgery in the second trimester. In our opin-ion, the risk of overtreatment based on molecular diagnosis inthis case was significant, since at least 20% of women of this agepresenting with PTC would bear BRAF-positive tumors, while theclinical data clearly show that the prognosis is excellent in thesecases.

On the other hand, the risk of undertreatment in the youngBRAF-negative patients should be considered.

2. The relevance of BRAF status to the indications for radioio-dine treatment seems still not well explained. On the one side,there are plenty of data showing that BRAF mutation corelateswith a poor response to radioiodine due to a lack of propermembrane sodium–iodine symporter expression (see furtherdiscussion below). On the other side, there are proposals toapply more radioiodine in BRAF-positive patients due to thepoorer prognosis. What we lack is evidence that more radioio-dine will be helpful in these patients. Approaches to increasetumor radiosensitivity or to apply complementary pharmaco-logical therapy in these patients would appear to be morereasonable.

For the evaluation of the prognostic significance of BRAF in thy-roid cancer, a comparison with one of the most aggressive cancers,melanoma, may be of value. As already mentioned, BRAF mutationsare frequent in both cancers and are found at early disease stage—inthyroid in micro-PTC and in skin, in the majority of benign nevi, theprecursors of melanomas. In benign nevi, the clonal activation ofMAPK by BRAF is followed by induction of senescence which pro-vides a barrier against tumor progression (Wajapeyee et al., 2008).Similarly, the expression of mutated BRAF in lung epithelium resultsin development of benign tumors that express senescence markersand only rarely progress to malignant tumors unless functional p53or p16 are absent (Dankort et al., 2007). BRAF-induced senescencehas not been analyzed in thyroid tissues and can difficult due to lackof benign counterpart to PTC. However, this hypothesis may explainwhy BRAF is surprisingly frequent in indolent micro-PTCs—only thenext molecular event(s) is/are necessary to accelerate its growth toclinically evident disease. In this case, we should focus on identi-fying events, secondary to BRAF mutation, which may be of better
prognostic/predictive value then BRAF itself. According to Knaufand Fagin (2009), in PTC, mechanisms for overcoming senescenceprobably do not require loss of functional p53, which occurs late inprogression to poorly differentiated thyroid carcinoma, nor rise inp16.

16 D. Handkiewicz-Junak et al. / Molecular and CTa

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Although molecular changes observed in other cancer cannotbe directly translated to PTC, we should also take into accountthat in other types of cancer (e.g., colorectal, ovarian, lung) BRAFmutation is not related to poorer prognosis, on the contrary, it isobserved in cases with better outcome. In fact, the generally morefavorable outcome of BRAF-induced cancers may be the cause ofthe generally good prognosis in PTC. This stresses once more theneed to search for molecular markers which shall stratify BRAF-positive PTC cases and help to specify patients with truly poorprognosis.

3.3. Other molecular initiating events specific for PTC—do theyhave prognostic potential?

3.3.1. RET/PTC rearrangementsRET is a transmembrane tyrosine kinase receptor, the consitu-

tive activation of which by point mutation in parafollicular C cellsleads to medullary thyroid cancer. RET/PTC rearrangements, wherethe 3′ or tyrosine kinase domain of the RET gene is fused with the5′ domain of one of several constitutively expressed genes, causethe constitutive activation of the RET gene, which otherwise is nor-mally silent in follicular cells (Santoro et al., 2004). The fusion leavesintact the tyrosine domain (TK) of the RET receptor and enablesthe RET/PTC oncoprotein to activate the MAPK cascade (Knauf etal., 2003b). Several studies suggest that the oncogenic effects ofRET/PTC rearrangements require signaling along the MAPK path-way in the presence of functional BRAF kinase (Knauf et al., 2003a;Mitsutake et al., 2006).

RET/PTC rearrangements may arise from fusion with a widerange of house-keeping genes (until now, 12 affected genes andcirca 15 rearrangements have been described), and constitute anevent specific both for organ (thyroid) and histotype (PTC) (Faginand Mitsiades, 2008; Santoro et al., 2004). However, some data indi-cate the possibility of RET/PTC rearrangements also in lymphocyticthyroiditis (Rhoden et al., 2006).

Transfection of the RET/PTC1 gene in normal rat thyroid cellsresulted in loss of differentiation and of TSH growth dependency.However, the cells were totally transformed only after transfectionwith RET/PTC and mutated RAS genes, suggesting that simultaneousactivation of several genes was necessary for tumor progression.This observation, together with the frequent occurrence of RET/PTCrearrangements in papillary microcarcinoma, suggests that suchrearrangements are an early event in thyroid carcinogenesis (Jhianget al., 1996; Viglietto et al., 1995).

A wide range of RET/PTC prevalence in PTC, ranging from 3% to85% has been reported (Kondo et al., 2006; Nikiforov, 2002; Talliniand Asa, 2001). In general, RET/PTC prevalence is higher in youngpatients and in radiation-induced PTC, and decreases with patients’age (Collins et al., 2002; Fenton et al., 2000). However, apart fromstudy population variations, the wide range of RET/PTC prevalencealso reflects the different procedures used to identify RET/PTC rear-rangements (Zhu et al., 2006). RET rearrangement is frequentlyfound in micro-PTC, an observation that is usually interpreted asthe evidence in favor of an initiating role in PTC. However, doubtsabout this interpretation have been raised by the recent reports onRET/PTC multiclonality (Unger et al., 2004), which may suggest thatat least in some PTCs, the recombinations may have arisen later intumor progression.

The majority of RET/PTC studies did not find any correlationbetween the rearrangements and other clinicopathological factorsrelated to PTC course or outcome. In few of the studies, though,
an increased frequency of lymph node metastases was noticed inpatients with RET/PTC rearrangements (Adeniran et al., 2006). Ini-tially, this observation was interpreted as evidence for correlationwith poorer prognosis. Later on, when the more frequent occur-rence of RET/PTC in younger of PTC patients has been known, the

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igher occurrence of lymph node metastases was correlated withounger age rather than with RET/PTC rearrangements (Collins etl., 2002).

.3.2. NTRK rearrangementsNTRK1 is another membrane tyrosine kinase receptor which reg-

lates growth, differentiation and apoptosis in the peripheral andentral nervous system, with nerve growth factor acting as its lig-nd. Similarly to RET, NTRK-3 is activated by rearrangements withenes constitutively expressed in thyroid follicular cells. Activationf the oncoproteins TRK1-3 is ensured by autophosphorylation ofhe tyrosine kinase domain (Pierotti et al., 1996). NTRK rearrange-

ents are rare and are found in <10% of PTCs (Brzezianska et al.,007; Roque et al., 2001). Their rarity precludes the studies on theirrognostic roles.

.3.3. Gene rearrangements are frequent in thyroid cancersUntil recently, gene rearrangements were regarded as charac-

eristic of hematological malignancies. Among solid tumors, theyere found only in sarcomas and thyroid carcinomas. This picture

s now being changed by the detection of very small chromoso-al deletions/amplifications in other solid tumors, e.g., prostate

ancer (Carver et al., 2009; Tomlins et al., 2009). Nevertheless,redisposition of thyroid cells to undergo gene rearrangement isot completely understood (Teixeira, 2006; Wang et al., 2008b).he juxtaposing of the loci participating in RET/PTC and NTRK1earrangements in interphase nuclei of thyroid follicular cells haseen reported (Gandhi et al., 2006; Nikiforova et al., 2000; Roccatot al., 2005). Interestingly, these rearrangements are more fre-uently found in young patients (Collins et al., 2002; Fenton etl., 2000; Wang et al., 2008a). Of note, though, the decisive fac-or(s) explaining the good prognosis of PTC in young patients,emains unclear: is it the biology of the tumor (which is causedy a gene rearrangement, not BRAF mutation) or of the hostdifferent immunity in young patients) or of both (Jarzab et al.,005a).

.4. Is there some multiclonality in papillary thyroid cancer?

Many data indicate that, despite the general rule of non-overlapetween the main PTC-inducing mutations, which are regarded asutually exclusive (Fagin, 2004), there is a substantial degree ofulticlonality in this type of thyroid cancer. Both BRAF mutations

nd RET/PTC rearrangements can be found as non-clonal changesnd different types of RET/PTC rearrangement may be found in dif-erent tumor foci from the same patient (Aherne et al., 2008; Oler etl., 2005; Sugg et al., 1998; Unger et al., 2004; Vasko et al., 2005; Zhut al., 2006). Concomitant presence of BRAF mutation and RET/PTCearrangement in one tumor is possible and this is also true forther mutations (Nikiforova et al., 2004; Smyth et al., 2005; Wangt al., 2008b). This fact is of a great potential impact on the powerf these molecular changes as prognostic or predictive factors.

.5. Gene mutations important for development of both papillarynd follicular thyroid cancer

.5.1. RAS mutationsThe RAS–RAF–MEK–ERK1/2 pathway is hyperactivated in 30%

f human cancers (Balmanno and Cook, 2009). RAS is a membrane-ssociated, small protein with guanosine triphosphate (GTP)inding ability, involved in proliferation, differentiation and cell
urvival. Translocation of RAS to the cytoplasmatic membrane isn important step in the protein’s activation. Farnesylation of RASs the first obligatory step in a series of post-translational modifica-ions leading to membrane association, which, in turn, determineshe switch from an inactive to an active form. RAS is activated by

a variety of membrane receptors, mainly belonging to the tyrosinekinase receptor family, rarely G proteins. Non-membrane coupledtyrosine kinases can also to activate RAS, with the down-streamactivation of different effector pathways.

The RAS protein is encoded by three genes, H-, N- and K-RASand each of them may underlie activating point mutations, occur-ring most frequently at codon 12 or 61. Such mutations lead to thechange in conformation of the protein so that it remains boundto GTP, i.e., constitutively active (while in normal conditions GTP-ase activity of RAS leads to RAS deactivation). In some cancers, likecolon cancer, RAS mutation is regarded as the first oncogenic eventnecessary but not sufficient for the cell to cross over the benign ade-noma/cancer barrier (Takayama et al., 2006) as RAS mutations arefound with the same frequency in colonic adenomas as in coloniccancers. In some other types of cancer RAS mutations occur at laterstages of carcinogenesis.

In thyroid gland, RAS mutations are found already in follicu-lar thyroid adenomas (FAs) and the transition from the follicularadenoma to follicular cancer requires the next molecular step(although, unlike in colon cancer, adenomas are not regarded asprecancerous stage for FTC). RAS mutations were described in some19% of FAs and in up to about 50% of FTC (Esapa et al., 1999; Shi etal., 1991; Suarez et al., 1990), however, in more recent studies RASmutations are evaluated with an overall frequency of about 20%(Garcia-Rostan et al., 2003; Vasko et al., 2003).

Importantly, RAS mutations are not thyroid cancer histotype-specific and are also found in PTC, with the prevalence ranging from0% to 15% (Abrosimov et al., 2007b; Adeniran et al., 2006; Hou et al.,2007; Vasko et al., 2003). These mutations are rarest in the classicalvariant of PTC, and more frequent in the follicular variant, wheretheir prevalence may reach even 20–50% (Goutas et al., 2008; Zhuet al., 2003). Some studies indicate that N2-RAS mutations are par-ticularly frequent (Abubaker et al., 2008; Hou et al., 2007; Wanget al., 2007), other focus on K-RAS (Goutas et al., 2008) which areseen in FTC with lower frequency (Fagin and Mitsiades, 2008). Theirfrequency in anaplastic thyroid cancer is estimated as to be about20–25% (Smallridge et al., 2009).

RAS mutation can promote thyroid tumorigenesis throughthe RAF–MEK–ERK1/2 pathway or through its interaction withPI3K/AKT pathway (Xing, 2005). Garcia-Rostan et al. (2003)reported the association of RAS mutations with aggressive thyroidcancer phenotypes and poor prognosis: 55% (11/20) of patientswith RAS-positive well-differentiated thyroid cancers died in com-parison to 15% (9/58) RAS-negative ones (P = 0.016). There are noother reports on this issue, however, the observation that RAS muta-tions are rather frequent in poorly differentiated thyroid cancers(40–55%) supports this notion (Fagin and Mitsiades, 2008).

3.6. Follicular thyroid cancer-specific gene mutations

3.6.1. PAX8/PPAR� rearrangementsThis type of rearrangement results from the translocation

between the PAX8 (paired box gene 8) gene, which encodes a paireddomain transcriptional factor, and the peroxisome proliferator-activated receptor (PPAR�) gene. The resulting fusion protein (PPFP;PAX8/PPAR� fusion protein) encodes a nearly full-length PPAR�,the expression of which is under the transcriptional regulation ofthe PAX8 promoter (Kroll et al., 2000). The functional consequencesof this PAX8/PPAR� rearrangement are yet not fully understood.Some studies have shown negative effect of PAX8/PPAR� rearrange-ment on the function of wild-type PPAR� (Gregory et al., 2004),
however other have questioned this negative effect and showedthe up-regulation of many genes which are transcriptional tar-gets of PPAR� (Giordano et al., 2005). Another possible oncogenicmechanism of PAX8/PPAR� rearrangement is deregulation of PAX8function, which is critical for thyroid cell differentiation (Reddi

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t al., 2007). In FTC, PAX8/PPAR� rearrangement is expressed inome 35–47% of cases (Cheung et al., 2003; Fagin and Mitsiades,008; Sahin et al., 2005). PAX8/PPAR� rearrangement has also beenhown in follicular adenoma with a varying frequency (Cheung etl., 2003; Marques et al., 2004), summed up to 13% (Sahin et al.,005), thus may be regarded as an early event in follicular tumori-enesis, requiring additional mutations to induce FTC (Cheung etl., 2003; Gregory et al., 2004). Again, RAS and PAX8/PPAR� areegarded mutually exclusive (Nikiforova et al., 2003b). There areo very convincing data on PAX8/PPAR� rearrangement prognos-ic significance (Sahin et al., 2005). The rearrrangement does notppear to be expressed in classical PTC, while in follicular variantf PTC some authors describe it frequent occurrence (Castro et al.,006), while others (Sahin et al., 2005) negate this possibility.

.6.2. RAS homolog 1 (ARH1) silencingAs already mentioned, the mechanisms of transition from a

enign to a malignant tumor, from FA to FTC, are not well known.igh frequency of loss of heterozygosity in imprinted genomic

egions has been suggested to contribute to this transition (Sarquist al., 2006). One of the proposed mechanisms is the reduction of theumber of ARH1 copies. Weber et al. (2005a) showed that relatively

ew FAs but the majority of FTCs, including minimally invasive FTCs,how marked ARH1 mRNA underexpression, due to deletion of theon-imprinted second allele in conjunction with hypermethylationf the genomically imprinted allele.

.7. Mutations of other genes and their importance for thyroidancer and its outcome

.7.1. PIK3CA mutations and amplificationPIK3CA mutation is not a common mechanism in the activation

f PI3K/AKT in thyroid carcinoma while amplification of this genes more frequent. In PTC, rare mutations and much more frequentmplifications, comprise alltogether 15–53% of tumors (Abubakert al., 2008; Hou et al., 2007). PIK3CA amplication is observed also inTC and even in FAs (Hou et al., 2007; Wu et al., 2005); however, its the most frequent in anaplastic thyroid cancer. Its down-streamarget, AKT, is phosphorylated in at least half of PTC, but indepen-ently of PIK3CA mutation or amplification (Abubaker et al., 2008).

.7.2. PTEN mutationsPTEN (phosphatase and tensin homolog) is a dual-specific

hosphatase, a negative regulator of the AKT/PI3K pathwayy dephosphorylation of phosphatytylinositol-3,4,5-triphosphatePIP3), which can also influence the MAPK pathway. PTEN is a tumoruppressor gene and its germline loss of function mutations pre-ispose to multiple tumors including FTC (Yeh et al., 1999). Singleases of loss of function somatic mutation of PTEN were describedn FTC (7%), somewhat more frequently in ATC (12–50%) and rarelyn PTC (2%) (Frisk et al., 2002; Garcia-Rostan et al., 2005; Gimm etl., 2000; Hou et al., 2007; Smallridge et al., 2009). A novel rear-angement involving PTEN and the H4 gene, has been described ashe non-clonal event in 14/18 PTCs investigated (Puxeddu et al.,005) and as a feature present in 4.8% of a larger series of PTCs inhinese patients (Wang et al., 2008b). All these rearrangements arearacentric inversions of chromosome 10q, the same chromosomehich is responsible for RET rearrangements.

.7.3. ˇ-Catenin mutations
The �-catenin protein, when bound to E-cadherin, mediates
ell skeleton/adhesion interactions, and when non-sequesterednd non-degraded, plays a role in gene transcription regulationf growth-promoting genes. It constitutes an element of Wnt sig-aling pathway and is in fact a Wnt nuclear effector (Fagin and


Mitsiades, 2008). The Wingless (Wnt) family of secreted glyco-proteins controls early developmental processes including cellularmigration, proliferation and differentiation. Non-canonical Wntsignaling is associated with tumorigenesis and Wnt5a has beenreported to be increased in the majority of PTCs but in only someFTCs (Kremenevskaja et al., 2005). Mutations of �-catenin gene(CTNNB1) are most frequent in poorly differentiated and anaplasticcancer (Garcia-Rostan et al., 1999; Garcia-Rostan et al., 2001).

3.7.4. TP53 mutationsTP53 encodes a multifunctional nuclear protein which is impor-

tant for cell cycle arrest at DNA damage, senescence or apoptosisand is one of the best known tumor suppressor genes. Contraryto what is seen in many other cancers, TP53 loss of functionmutations occur late in thyroid tumorigenesis and are practicallyabsent in DTC (0–9%), while the prevalence of these mutationsrises as the tumor grade worsens, reaching 17–38% in poorly dif-ferentiated and 55–88% (Fagin and Mitsiades, 2008; Smallridgeet al., 2009) in anaplastic cancers. At this moment, the featuresof poor differentiation are easily detected histologically, thus, theimmunohistochemical or molecular investigation of TP53, docu-mented widely in other tumors, has not gained clinical significancein thyroid cancer.

4. Prognostic aspects of other gene expression changes inpapillary and follicular thyroid cancer

Over the last two decades many gene expression changes havebeen described in papillary and follicular thyroid cancers andrelated to the stage of the disease or its outcome. The clear distinc-tion between the changes characteristic for papillary and follicularcancers is however not possible for many reasons: First, due tothe much higher frequency of PTC, most of the genes describedbelow were either not examined in FTC or investigated in smallgroups of FTC cases, not allowing for specific distinction. Second,many of those genes in which such comparison was performed,showed only minor differences between histotypes because theywere late consequences of the neoplastic transformation of the fol-licular thyroid cell. Thus, it is rationale to look for their prognosticsignificance independently from the histotype or variant of thyroidcancer. In the given below brief list of genes, expression changes ofwhich has putative or proved prognostic relevance, we inform onhistotype-specific changes only then, when they were investigatedwith sufficient power.

4.1. Single genes expression changes

4.1.1. MET proto-oncogeneThe MET proto-oncogene encodes a membrane tyrosine kinase

receptor for hepatocyte growth factor (HGF). HGF is a potent mito-gen for epithelial cells and promotes cell motility and invasion.About 50% of cases of PTC are characterized by MET overexpression(Di Renzo et al., 1992), which is believed to be a sign of more aggres-sive disease (Mineo et al., 2004; Ramirez et al., 2000). BRAF-positivetumors were associated wit MET overexpression in aneuploid PTCs(Rodrigues et al., 2007).

4.1.2. Epithelial growth factor receptor (EGFR)Among receptor tyrosine kinases EGFR belongs to the best

known. EGFR (HER1) protein levels are increased in PTCs in com-parison to normal thyroid tissue and the high expression of EGFR
has been suggested to be associated with worse outcome of PTCafter thyroidectomy (Akslen and Varhaug, 1995), an observationnot repeated in other studies (Ruan et al., 2008). Another earlystudy indicated on HER2 as the predictor of metastasis in PTC(Kremser et al., 2003). The immunohistochemical evaluation of

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GFRvIII showed its expression in the majority of PTCs (Omidfart al., 2009). In the study of Wiseman et al. (2008a), which investi-ated expression of HER1–4 genes, overexpression of at least onef them was observed in 76% of DTC and only HER3 was positivelyorrelated with DTC stage, while HER4 was inversely correlatedith T stage. In this context it is important that HER3 (ERBB3) is

haracteristic for BRAF-induced PTCs (Giordano et al., 2005).Until recently, it was assumed that activating mutations of EGFR

o not play any role in the molecular biology of DTC (Omidfar etl., 2009). However, the recent finding of Masago et al. (Masago etl., 2009) indicated that EGFR mutations commonly seen in pul-onary carcinoma may be observed in a subset of papillary or

oorly differentiated thyroid cancers which may make tumor cellsncogene-addicted.

.1.3. Mitogen-inducible gene-6 (MIG-6)MIG-6 is an immediate early response gene, expression of which

s induced by cellular stress, hormones or growth factors in manyells. It exerts a negative effect on EGFR signaling that is mediatedy the MAPK cascade (Ferby et al., 2006). Because MIG-6 expres-ion is induced by MEK–ERK activation, such expression may beegarded as a negative feedback loop signal in normal cells, lostn cancer. In PTC, MIG-6 directly correlated with EGFR expressionnd its higher mRNA level was associated with better overall sur-ival in a population of 106 PTC patients followed for six years.nterestingly, MIG-6 expression was independently predictive ofisease-free survival in BRAF-positive patients (Ferby et al., 2006).

.1.4. Vascular epithelial growth factor (VEGF) and VEGF receptorVEGF overexpression is a characteristic feature of malignant

umors, seen in thyroid cancer as in other cancers (Wiseman et al.,008b) and may be interpreted as marker of tumor hypoxia. How-ver, not in all cancers it is correlated with HIF-1alpha expression.hyroid cancer belongs to those tumors in which hypoxia-inducibleactor-1� is up-regulated at the same time that VEGF is overex-ressed (Jubb et al., 2004). The correlation of VEGF expression withhe expression of other angiogenic factors is strong (Tanaka et al.,002). Quantitative evaluation of VEGF expression in PTC showedhe association of this phenomenon with metastatic PTC (Klein etl., 2001). VEGF expression was closely correlated with tumor size,xtrathyroidal invasion and BRAF presence (Jo et al., 2006), whileian et al. (2008) observed the correlation with lymph node metas-ases both for VEGF and metalloproteinase MMP2. In the study of Jot al (Jo et al., 2006), VEGF was the only molecular marker associatedith tumor size, extrathyroidal invasion and cancer stage by uni-

ariate analysis and the association with extrathyroidal invasionas confirmed in multivariate analysis, while VEGFR expressionas only weakly associated with PTC recurrence in young patients

Fenton et al., 2000).

.1.5. E-cadherin and cateninsE-cadherin (CDH1) is a calcium-dependent transmembrane gly-

oprotein that functions as a cell-cell adhesion molecule. It has anntracellular domain that complexes with catenin proteins for regu-ation of further Wnt-mediated signal transduction to the nucleus.DH1 expression is reduced in thyroid carcinomas and has beenssociated with poor prognosis, not only in PTC (von Rhein et al.,997) but also in FTC (Brecelj et al., 2005) as well as with PTC-to-
TC transition (Wiseman et al., 2007). Dislocalization of catenins in
hyroid cancer may be secondary to loss of E-cadherin (Bohm et al.,000; Rocha et al., 2003). However, the study of Kapran et al. (2002)id not show any association of E-cadherin or catenins expressionith PTC recurrence.


4.1.6. Cyclin D1 and other cell cycle regulatorsAssociation of overexpression of cyclin D1, one of the target

genes of Wnt signaling pathway, with more advanced PTC, hasbeen adressed for a long time (Ferenc et al., 2005; Ito et al., 2005).Although cyclin D1 and other cell cycle regulators, such as cyclin E,p16, p21 were overexpressed in thyroid cancer, only p16 expres-sion correlated significantly with extrathyroidal tumor extensionand the presence of lymph node metastases in the study of Melcket al. (2007). Also, in the study of Pesutic-Pisac et al. (2008) lossof p27 was more essential than cyclin D1 overexpression, whileKhoo et al. (2002) suggested the contribution of both markers. Thestudy of Hoos et al. (2002) was negative for correlation of cyclinD1 expression with indices of prognostic relevance in oncocyticthyroid cancers.

4.1.7. Mucin 1Mucin 1 (MUC1) is a glycoprotein important for cell adhe-

sion, overexpressed in about 25% of PTC (Wreesmann et al., 2004).Preclinical studies support the role of MUC1 in promoting anaggressive phenotype of PTC (Patel et al., 2005). MUC1 overexpres-sion is especially frequent in the tall-cell variant of PTC, knownfor its poor prognosis (Ghossein and LiVolsi, 2008). Identified asan independent prognostic marker by genome-wide gene expres-sion profiling of PTC, MUC1 overexpression was described to besignificantly associated with the treatment outcome (Wreesmannet al., 2004), independent of extrathyroidal extension or of the PTCvariant. However, other reports question the correlation of MUC1overexpression with PTC prognosis (Abrosimov et al., 2007a; Minet al., 2008). The significance of MUC1 overexpression for canceroutcome may be more essential if we consider MUC1 contributionto the survival network, causing resistance to genotoxic agents inthyroid cancer (Siragusa et al., 2007).

4.1.8. S100A4 calcium-binding proteinThis small calcium-binding protein also called calvasculin has

been reported to promote metastasis and was indicated as a molec-ular factor of poor prognosis in PTC (Zou et al., 2004, 2005). Inpapillary microcarcinomas, the expression of S100A4 correlatedmore strongly with lymph node metastases than did any othermolecular factor investigated and was more significant for asso-ciation with stage than BRAF status (Min et al., 2008).

4.1.9. Osteopontin and CD44v6Osteopontin (SPP1) is a cytokine regulating cell trafficking

within the immune system, binding a splice variant of CD44,that is overexpressed in PTC and contributes to the mitogenesis(Figge et al., 1994), survival and motility of PTC thyrocytes and theirinvasion potential (Guarino et al., 2005). In PTC, the gene expres-sion level of SPP1 was associated with metastasis but not with theBRAF status of the tumor (Oler et al., 2008).

4.1.10. Immunity-related genesActivation of the MAPK pathway induces up-regulation of

chemokines and their receptors on tumor cells, relevant for theirsustained proliferation and cell motility. Tumors use molecules ofthe innate immune system for their growth, survival and metasta-sis (Melillo et al., 2005). Expression of toll-like receptor 3 (TLR3) ischaracteristic for PTC but not FTC (McCall et al., 2007). The basallyexpressed receptors are functional and able to activate the NF�Bpathway and to increase the expression of interferone IFN� andCXCL10 chemokine. Interestingly, TLR5 overexpression is coordi-
nated with WNT5a expression.
Chemokine receptors CXCR2–CXCR4 and CCR7 are expressed onPTC cells (Melillo et al., 2005; Sancho et al., 2006; Wagner et al.,2008) and CCR7 was reported to be associated with pathologic fac-tors of PTC aggressiveness such as extrathyroidal tumor extension,

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Interestingly, expression of HLA-DR and DQ molecules isnversely associated with PTC recurrence risk (Jo et al., 2008).

.2. Prognosis-related gene signatures in thyroid cancer

.2.1. Gene expression profiling and prognosis of thyroid cancerSeveral DNA microarray studies show distinct differences in the

olecular profiles of not only papillary versus follicular cancer, butlso between subtypes of papillary cancer (Aldred et al., 2004; Barist al., 2005; Chevillard et al., 2004; Finley et al., 2004a,b; Giordanot al., 2005; Oler et al., 2008). It is to be stressed that some genentology groups are characteristic of PTC, mainly cell adhesion-elated genes involved in cancer invasion and metastasis, or theene signature related to the immune response to this DTC his-otype (Huang et al., 2001; Jarzab et al., 2005b). While the genexpression profile of PTC differs from that of normal thyroid tissuey a huge number of overexpressed genes, in FTC, down-regulatedenes far outweigh up-regulated genes (Aldred et al., 2003). In theecent study by Oler et al. (2008) based on SAGE (serial analysis ofene expression) and followed by QPCR (quantitative polymerasehain reaction), there were 548 differentialy expressed genes whenTC was compared with FTC and FA. Of the 548, 131 genes had aore than 10-fold increase in expression in PTC, and 61 genes, an

ver 15-fold increase.Despite the very characteristic morphologic phenotype of

TC, surprisingly few single genes were described as unique forhis histotype. CITED1 (Cbp/p300-interacting transactivator, withlu/Asp-rich carboxy-terminal domain, 1), encoding a transcrip-

ional transactivator nuclear protein, is one of the few identifiedntil now (Prasad et al., 2004). Other examples are nicotinamide--methyl-transferase (NNMT), encoding a liver enzyme involved

n the biotransformation of many drugs and xenobiotics (Xu et al.,003), or chemokine (C–X–C motif) ligand 14 (CXCL14), cystatin/M (CST6) and DHRS3 (dehydrogenase/reductase (SDR family)ember 3), a protein involved in retinol storage, all three found

o be characteristic of PTC by Oler et al. (2008). It seems that ratherhan single genes, gene groups (gene clusters or gene signatures)re characteristic of PTC, among them, the already-mentioned genelasses related to adhesion/motility or invasion (Fig. 2). On the otherand, many genes have similarly altered expression in both PTCnd FTC, which helps to define these changes as common conse-uences of different neoplastic transformation pathways (Polanskit al., 2007).

In FTC, according to Aldred et al. (2003), three of the most down-egulated genes localized to previously reported regions of loss ofeterozygosity (LOH) were caveolin-1 and -2 and bone morpho-enetic protein 3b (GDF10). Takano et al. (2004) identified the trefoilactor 3 gene as down-regulated in FTC, but this gene should note regarded as FTC-specific, as it is down-regulated in PTC as wellHamada et al., 2005).

Despite the publication of numerous thyroid cancer genexpression profile analyses (see the meta-analysis of Griffith etl., 2006), only a few studies addressed prognosis-related geneignatures. Finley et al. (2004b) were the first to assess whetherene expression profiling was capable of differentiating aggressivehyroid carcinomas from non-aggressive ones. They characterizedheir group of 29 DTC tissue samples according to the patients’MES criteria and obtained a list of 339 differentially expressed
enes. By this criterion, all of an additional 6 samples for whichMES status was unknown were correctly clustered, while thereere 6/29 misclassified samples in the test group, yielding a sen-
itivity of 85.7% and a specificity of 80.9% for high-risk tumors.he same procedure done for extrathyroidal extension was even


more sensitive. Unfortunately, this gene signature has not yet beenverified by another group.

4.2.2. Metastasis-associated genes in thyroid cancerAlthough the survival rate in well-differentiated thyroid cancer

is excellent, that is not the case in the 5–10% of patients with dis-tant metastases. Thus, identification of a metastasis-related genesignature is desirable. One of the first approaches was carried outby Zou et al. (2004), who established a thyroid cancer cell line withhigh metastatic capacity and specified genes characteristic of thisline, among them MET, ezrin, integrin, motility-related protein-1, cadherin P, NEDD5 and S100A4. In their quantitative real-timePCR (QPCR) validation study, mentioned already above, they con-firmed S100A4 to be associated with poor thyroid cancer prognosis.Two other genes were listed by Oler et al. (2008) as metastasis-associated; CXCL14 and cystatin M. However, the definition ofmetastatic PTC was very poor in this study. Some comparisonswere also performed by Rodrigues et al. (2007) who reported lowexpression of many genes in M1 aneuploid PTCs.

4.2.3. Epithelial-to-mesenchymal transition in PTCIn the search for key molecular prognostic factors in thyroid

cancer, the mechanisms of tumor invasion must be considered.The process of epithelial-to-mesenchymal transition (EMT) hasbeen implicated in the conversion of early-stage tumors to inva-sive malignancy and has been extensively studied in invasive PTC(Vasko et al., 2007). After loss of cell-to-cell contacts and remodel-ing of the cytoskeleton, tumor cells are more prone to migration andthis process is hallmarked by loss of cadherin-E expression. Inte-grin pathway, Notch, MET, TGF-beta, NF�B, PI3K and p21-activatedkinase belong to EMT-regulating proteins, also fibronectin 1 isconsidered as EMT related. In the study of Vasko et al. (2007),microarray analysis of the central part and of the invasive frontof selected PTC tumors has been performed, showing in the lat-ter the overexpression of TGF-beta, NF�B and integrin pathway aswell as of small G protein regulators and CDC42. Overexpression ofvimentin, the hallmark of EMT, was associated with tumor invasionand nodal metastasis and was correlated with RUNX2 expres-sion, as judged by immunohistochemistry. Interestingly, there wasno correlation of expression of these invasion-related proteinswith tumor genotype, e.g., BRAF status. However, in the study ofWatanabe et al. (2009) silencing of BRAF reduced the increasedexpression of vimentin. For PTC cell invasion, enhanced AKT activ-ity is especially important, but there was no correlation of AKT withBRAF presence in PTC (Vasko et al., 2007). According to Wang et al.(2007), BRAF mutation and AKT pathway activation are mutuallyexclusive in PTC.

4.3. Genetic germline background and the prognosis of papillaryand follicular thyroid cancer

Some 5–10% of all cancers developing in follicular cells occur onthe background of familial predisposition, referred as familial non-medullary thyroid carcinoma (FNMTC), with autosomal dominantmode of inheritance and penetrance increasing with age (Cavacoet al., 2008; Handkiewcz-Junak et al., 2006; Malchoff and Malchoff,2006). This clinical entity develops most often (in over 85% ofcases) as familial PTC and differs from sporadic cancer by earlierage of onset, more frequent multifocal disease, higher degree ofaggressiveness and higher recurrence rate. Not all involved familymembers suffer from thyroid cancer, some develop multinodular
goiter or follicular adenomas. The diseases are genetically heteroge-nous, with some genes identified in single families but with onlya few general susceptibility genes described (Canzian et al., 1998;Gudmundsson et al., 2009; Jazdzewski et al., 2008; McKay et al.,2001; Ngan et al., 2009). Somatic BRAF or RAS mutations are found in


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ig. 2. A large cluster of cell adhesion-related genes are homogenously up-regulateenign tissues. Hierarchical cluster analysis based on 100 PTC and 99 FTC, FA and ny Giordano et al. (2005) and Weber et al. (2005b). The intensity of the color refershe downregulation. Color coding of tissues: blue, PTC; red, FTC; green, benign tum

amilial PTCs with similar frequency as in sporadic tumors (Cavacot al., 2008). In this aspect it is important to add that there are noata suggesting that any particular genetic background facilitateshe BRAF mutation what could be suspected considering the pres-nce of a unique BRAF mutation present in a large proportion of PTCases. However, environmental influences are also conceivable, asuggested by recent Chinese data showing that BRAF-positive PTCsre significantly more frequent in areas of extremely high iodinentake (Guan et al., 2009) and by comparison of different Sicilianreas (Frasca et al., 2008).

. Final considerations on prognostic impact of geneticactors in papillary and follicular thyroid cancer

.1. Relation between BRAF status and gene expression profile

.1.1. Is BRAF-positive DTC a distinct molecular subtype?The mutational status of PTC correlates well with its gene

xpression profile, allowing bioinformatical analysis of genome-ide data to clearly differentiate BRAF, RAS or RET/PTC mutants,

ven if an unsupervised approach, Principal Component Analysis,
s applied. The most important paper on this issue, published byiordano et al. (2005), showed distinctly that PTC mutational sta-
us was even more strongly correlated with gene expression profilehan with morphology as evaluated by subdivision into PTC vari-nts. When comparing RET/PTC mutants versus BRAF mutants, 3891

Cs. By their expression nearly all PTCs may be separated from follicular tumors andoplastic thyroid samples from own material (49 PTC tumors) and those publishede intensity of gene expression; red refers to the up-regulation, and green refers to

probesets with P < 0.01 were specified, with a similar distance ofboth of these mutant types from RAS mutants, the number of dif-ferent genes being on average approximately 20 times as many asobtained for permuted data sets. By further selection of genes forthose that yielded P < 0.01 as well as a fold-change <0.5 or >2 for anygroup compared to any other, the BRAF mutants were characterizedby 82 probesets (31 up-, 51 down-expressed). These probesets hada very strong significance, verified, as required in genomic studies,by the False Discovery Ratio (FDR), which was 0.5 (it is well acceptedin genomic studies that significant genes should show FDR under5–10%). Many genes were down-regulated in RAS mutants, whilemany increases were common between BRAF and RET/PTC mutanttumors. The most differentially expressed gene in the BRAF mutantswas TM7SF4. Its product is a dendritic cell protein, likely related tothe immune response in these tumors.

Although the other studies published were less powerful(Frattini et al., 2004; Musholt et al., 2006), they did not contra-dict the basic message of the Giordano study: the gene expressionprofile of BRAF-positive PTC is very distinct and easily recognizable.Oler et al. (2008) described that the expression of a newly discov-ered chemokine, CXCL14, and of cystatin M, a secreted inhibitor
of lysosomal cysteine proteases, was not only markedly elevatedin PTC, but also significantly higher in BRAF-positive versus BRAF-negative tumors. Both genes were simultaneously associated withmetastatic PTC. Also, expression of such PTC-characteristic genesas fibronectin 1, CITED1 and vimentin were positively correlated

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sorafenib, a tyrosine kinase inhibitor which is able to target BRAFas well, has not been shown dependent on BRAF mutation status


ith the expression of BRAFV600E (Watanabe et al., 2009). Theonditional expression of BRAFV600E and RET/PTC showed that theore invasive potential of BRAF-mutated tumors may be at least

artially explained by more distinct overexpression of metallopro-einases MMP3, MMP9 and MMP13 (Mesa et al., 2006).

All these data, especially regarding genome-wide differences,ubstantiate the hypothesis that BRAF-induced PTC is in fact a dis-inct molecular subtype of PTC, with a different, more aggressiveisease course and a poorer outcome, which demands a subtype-pecific treatment. However, prospective data still are necessary torove this theory.

.1.2. BRAF-mutated PTCs exhibit fewer thyroid-specific genexpression features

Many authors have confirmed the lower expression of thyroid-pecific genes in BRAF-positive PTCs: the gene expression profilingtudy by Giordano et al. (2005) noted a down-expression of TPOnd the study of Durante et al. (2007) confirmed the earlier obser-ations of the more intensive loss of gene expression of NIS (−82%),IT-B (−86%), TPO (−90%), and TG (−46%) in BRAF-mutated PTCs.hen BRAF-positive and BRAF-negative PTCs are compared, the

evel of NIS gene expression is at least 5 times lower in the formerDurante et al., 2007). Not only is there a BRAF-dependent down-egulation of NIS expression on the transcriptional level, but also,he trafficking of the NIS protein to the cell membrane is impaired inRAF-positive PTCs (Riesco-Eizaguirre et al., 2006). Moreover, sup-ression of the BRAF/MEK/MAPK pathway restores expression of

odide-metabolizing genes (Liu et al., 2007a). Unlike the case withIS or TPO genes, the expression of TSH-R and PAX8 seems not toe different in BRAF-positive tumors in comparison to in other PTCsDurante et al., 2007).

To interpret these data, some more general remarks seem neces-ary: the down-expression of thyroid-specific genes is not specifico BRAF-induced tumors, but is seen on the RNA level, the pro-ein level or both in every case of thyroid cancer (Ambroziak etl., 2005; Huang et al., 2001; Krause et al., 2007; Lazar et al., 1999;kubis-Zegadlo et al., 2005). Especially, the direct attribution of theoorer outcome of BRAF-positive PTC to the down-regulation ofhyroid differentiation-related genes seems precocious. These twoacts are only associated, the causative relation is not proven. Itppears to be more aggressive tumor biology rather than pooreresponse to radioiodine therapy which causes poorer outcomen BRAF-positive tumors. The impact of the lower expression ofIS protein in BRAF-mutated tumors is diminished by the fact

hat both BRAF-positive and BRAF-negative tumors exhibit mainlyytoplasmic localization of NIS, which precludes that protein’sunctionality. The down-expression of NIS in BRAF-positive PTCss seen also in microcarcinomas (Oler and Cerutti, 2009) andven in these low-risk tumors, BRAF mutation has been relatedo development of more advanced disease, while the prognosis isxcellent.

.1.3. BRAF mutation is associated with the more aggressiveumor phenotype in PTC

The exact molecular mechanisms of the invasive potential ofRAF-induced tumors are still not known. As mentioned above,he poorer outcome in patients bearing BRAF-positive tumors isith certainty not merely the result of poorer efficacy of radioio-ine treatment, due to impaired radioiodine uptake and retention.he prominent activation of the MEK–ERK1/2 pathway in BRAF-utated thyrocytes is well known, but the cascade of subsequent

vents is not well defined. Interestingly, MET overexpression, anvent related to poorer PTC prognosis, is seen mainly in BRAF-ositive tumors (Giordano et al., 2005). Also, the mean Ki-67 index,n indicator of tumor cell proliferation, has been reported to be sig-ificantly higher in BRAF-positive than in BRAF-negative patients


(1.01% versus 0.1%) (Nakayama et al., 2007); importantly, boththese Ki-67 values are rather low, which is a known feature of PTC(Saltman et al., 2006). The increased GLUT-1 expression in BRAF-positive tumors may be interpreted as the sign of such tumors’higher aggressivity. A multivariate analysis concluded that BRAF-positive tumors have higher VEGF expression (Jo et al., 2006).Recent reports indicate that prohibitin, a multifunctional protein,is overexpressed only in PTCs bearing a BRAF mutation. The mostplausible mechanism for prohibitin overexpression is the increasedgene promoter activity by the BRAF oncoprotein (Franzoni et al.,2009).

Nevertheless, recent publications indicate that some molecu-lar markers correlate more closely with clinicopathological factorsof poor prognosis than does BRAF-oncogene presence itself. Incontrast to BRAF-positivity, S100A4 and cyclin D1 overexpressionpredicted lymph node metastasis in a series of nearly 200 PTCmicrocarcinomas (Min et al., 2008). The S100A4 gene was alsoinvestigated in a more detailed study and shown to be significantlyassociated with metastases (Zou et al., 2005).

5.2. Major questions in the therapeutic strategy in DTC which canbe solved by molecular diagnosis

In fact, currently, the major clinically relevant problems we facein DTC are:

1. How to differentiate between indolent and progressing micro-PTC? BRAF estimation may help to find the progression-potenttumors, but obviously at the present status of knowledge is notpowerful enough to predict the need for more aggressive treat-ment than is presently recommended.

2. How to specify the more precise indications for lymphadenec-tomy in DTC?

Until now, the decision regarding lymphadenectomy is basedon the following rules: routine central lymphadenectomy andselective lateral lymphadenectomy meaning that the latter pro-cedure is performed only when evidence or at least strongsuspicion exists for the presence of lymph node metastases(Cooper et al., 2006; Pacini et al., 2006). Knowing the BRAF statusin the primary tumor may prompt omission of central lym-phadenectomy in BRAF-negative cases, however, this may leadto undertreatment of RET/PTC-positive young patients. On theother side, detection of a BRAF mutation would prompt a deci-sion to perform central lymphadenectomy in small tumors, astrategy that is not obligatory today, and hence could lead toovertreatment.

3. How to optimize the decision for adjuvant radioiodine aftersurgery, especially in the “gray zone” of T1-T2N0-N1a patients?

Basing the decision on BRAF status would mean that about in2/3 of the patients in this stage adjuvant treatment after surgerywould be necessary. However, the indications for a standardadjuvant treatment with radioiodine are doubtful in this groupin view of decreased expression of genes responsible for iodinemetabolism in BRAF-positive cancers.

4. How to treat patients with advanced DTC?We do not feel that the information on BRAF status changes

anyhow the present treatment rules—even the success of

(Kloos et al., 2009). On the other hand, data from other cancerssuggest that tumors bearing a BRAF mutation may be particularlyprone to respond to MEK inhibitors (Ball et al., 2007), an obser-vation that deserves confirmation in the context of advancedthyroid cancer.

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.3. Conclusions from the transcriptome-wide analyses of DTCs

. Among the genetic factors analyzed for their prognostic signif-icance, the presence of activating BRAF mutation in papillarythyroid cancer seems the only one to be considered in clinicalpractice. However, the putative significance of BRAF-positivityis limited by the fact of its frequent occurrence. Further studiesare necessary to stratifiy BRAF-positive papillary thyroid cancersand to indentifiy molecular factors related to the subset withpoor prognosis.

. By gene signature, the difference between BRAF-positive andBRAF-negative PTC is so distinct that BRAF-positive cancer shouldbe regarded as a molecular subtype of PTC. There is an urgentneed to investigate, which gene signature within the BRAF-related gene expression profile is specifically correlated withpoor prognosis.

. Differences between papillary and follicular cancers shown untilnow by gene expression profiling are profound, but they arestill not sufficiently well characterized, thus, their relation tothe putative differences in prognosis between these histotypesis precocious.

cknowledgments

The authors thank Robert J. Marlowe for very engaged and com-etent editorial assistance and Emilia Wilk and Monika Kowal forxcellent general assistance. Daria Handkiewicz-Junak was partlyupported by Polish Ministry of Science and Higher Educationrant no. N40110132/2138 and Barbara Jarzab was supported byuropean FP6 project no. 036495, acronym: Genrisk-T and Polishinistry of Science and Higher Education grant no. PBZ-MNiI-

/1/2005.

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