Editorial

Sentinel Lymph Node Biopsy in Breast Cancer: a Technical and Clinical Appraisal

Gianpiero Manca1, Elisa Tadelli1, Domenico Rubello2, Marta Gennaro1,

Gary J Cook3, Duccio Volterrani1

1. Regional Center of Nuclear Medicine, University Hospital of Pisa, Pisa, Italy

2. Department of Nuclear Medicine, Radiology, NeuroRadiology, Medical Physics, Laboratory,

Microbiology, Pathology, Santa Maria della Misericordia Hospital, Rovigo, Italy

3. Department of Cancer Imaging, Division of Imaging Sciences and Biomedical Engineering,

King's College London, St Thomas' Hospital, London, UK

Running title: SLN biopsy in breast cancer. An update

Address correspondence to:

Domenico Rubello, MDHead Service of Nuclear Medicine, PET UnitDepartment of Nuclear Medicine, Radiology, NeuroRadiology, Medical Physics, Clinical Laboratory, Biomarkers Laboratory, Microbiology, PathologyVia Tre Martiri 140; 45100 Rovigo, Italy Santa Maria della Misericordia Hospital, Rovigo, ItalyEmail: domenico.rubello@libero.itEmail: rubello.domenico@azisanrovigo.it

Disclosure Statement: The authors have nothing to declare

1

Sentinel lymph node biopsy (SLNB) has replaced axillary lymph node dissection (ALND) as the

standard surgical procedure for staging clinically tumour-free regional lymph nodes in patients with

early-stage breast cancer1, 2. SLNB staging considerably reduces surgical morbidity, without

affecting diagnostic accuracy and prognostic information. Clinicians should not recommend ALND

for women with early-stage breast cancer who have tumour-free findings on SLNB because there is

no advantage in terms of overall survival (OS) and disease free survival (DFS). From the early

1990s, SLNB has been increasingly used in breast cancer management, with a gradual but steady

decrease in the list of clinical circumstances in which it was initially contraindicated. Today, more

than 20 years after it was first introduced, there is still strong debate over a number of aspects

concerning SLNB and ALND3. Recent multi-institutional trials have revealed an SLNB false

negative rate (FNR) between 5.5% and 16.7%, higher than the target set by the guideline

recommendations (<5%) established by the American Society of Clinical Oncology (ASCO) in

20054. The variability in the FNR stresses the need for a better standardization of the technique

itself and for its validation in various controversial clinical situations.

In this article, we report the aspects of SLNB that are well-established, those that are still debated,

and the novelties that have taken place over the last 20 years. We have tried to provide an update on

the methodology from both a technical and clinical point of view, considering the long-term clinical

outcome of patients who in the course of the years have undergone this procedure, which represents

the only tool for the development and wide acceptance of SLNB.

The sentinel lymph node (SLN) is the first node on the direct lymphatic pathway to drain from the

primary tumor. SLNB is based on the concept that a tumor-free SLN excludes metastatic

involvement of the other regional nodes. Although there is consensus about broad aspects of SLNB

in breast cancer, there is still a lack of standardization concerning the basic technical details of the

procedure. Controversies exist with regard to the optimal label for lymphatic mapping, modalities

of injection, preoperative scintigraphic imaging, intraoperative SLN detection, surgical removal and

SLN histopathological evaluation. The procedure for SLNB may employ a radiopharmaceutical for

2

pre-operative imaging and intraoperative γ-probe SLN identification, a blue dye for direct vision at

surgery, or both5. It has been shown that the identification rate is higher and the FNR is lower when

the two mapping techniques are used together, especially when there is a diffusely metastatic

involvement of the SLN6. The radiopharmaceuticals commonly used are 99mTc-sulfur colloid

(particle size 15-5000 nm), 99mTc-nanocolloid (5-100 nm), and 99mTc-antimony trisulfide (3-30 nm).

The choice of the radiotracer depends more on local availability than on the success rate of SLN

identification. However, there is a general agreement that a radiocolloid measuring 100 to 200 nm

should represent the best compromise between fast lymphatic drainage and optimal retention in

SLNs7. In order to achieve an improved SLN detection, literature supports the use of small volume

(0.3-0.4 mL) with high specific activity. In the last few years, studies have focussed on the

introduction of new imaging agents that can further help to detect the SLN. In this regard, an

alternative to radiocolloids is the radiopharmaceutical 99mTc-tilmanocept (7.1 nm), which was

approved by the U.S. Food and Drug Administration in 2013. Tilmanocept is mannosyl diethylene-

triamine-pentaacetate dextran that binds specifically to mannose receptors (CD206) expressed

within lymph nodes. Its potential advantages include fast drainage from the injection site with a

selective accumulation in the SLN and limited drainage to second-echelon nodes8. A further

technical innovation is that of hybrid tracers, which combine a radioactive with a fluorescent label

such as indocyanine green (ICG)-99mTc-nanocolloid. This bimodal tracer enables the direct

integration of conventional preoperative imaging with intraoperative radio and florescence-

guidance to the SLNs9. More recently, superparamagnetic iron oxides have been introduced as an

alternative to radiopharmaceuticals, with an identification rate that is not inferior to standard

techniques10. Following over 20 years’ experience, the optimal injection site has not yet been

defined, mostly because breast lymphatic drainage is not completely understood. Although axillary

drainage is the principal lymphatic path of the breast, there are alternative lymphatic drainage

pathways not necessarily passing through Sappey’s subareolar plexus11. These include the so-called

“perforating system”, which drains toward the internal mammary chain (IMC) with direct

3

anastomosis only with the deep lymphatic system12. The most widely used injection techniques are

deep (peritumoral, intratumoral) and superficial (intradermal, subdermal, periareolar, subareolar)

injections. The peritumoral injection is considered the most accurate technique because the tracer is

introduced near the lymphatic vessels that drain the tumor enabling identification of extra-axillary

drainage. On the other hand, superficial injections allow for almost exclusive identification of

axillary nodes. However, important advantages of superficial injections are simplicity, shorter time

between injection and SLN identification, and increased radiotracer nodal uptake, leading to an

improved SLN identification rate. The peri/subareolar approach should be favoured in non-palpable

and multicentric/multifocal (multiple) tumors and also in upper outer quadrant lesions for its ability

to prevent the “shine through phenomenon”5. A meta-analysis published in February 2015

confirmed that both superficial and deep injections of radioactive tracer and blue dye are effective

for axillary SLN identification and that deep injections are associated with significantly greater

extra-axillary SLN identification13. Therefore, it is reasonable to assume that the two modalities of

injection are often complementary and that their combination may improve SLN detection and

reduce the FNR5, 14. Preoperative imaging is recommended before surgery since it demonstrates the

lymphatic drainage into the axilla and extra-axillary regions (IMC visualization, intramammary and

infraclavicular lymph nodes, nodes in the contralateral axilla), increasing the accuracy of the

procedure and reducing the surgical morbidity5. Lymphoscintigraphic imaging should be performed

from 15 minutes after injection, and if necessary, repeated up to 18-24 hours, according to the

radiopharmaceuticals used, the injection site, and the patient characteristics. If the SLN is not

visualized, a second injection of radiocolloid may be useful. In case of unsuccessful axillary

mapping there is an increased risk of axillary involvement15 and it is recommended to use blue dye

during surgery in order to improve the sensitivity of SLN identification5. ALND is recommended if

no SLN is identified even during surgery16. Planar images display a 2-dimensional view and SLNs

can be detected and marked on the patient's skin by means of an external radioactive marker5. In

recent years, conventional SLN mapping based on lymphoschintigraphy has been improved by the

4

introduction of Single Photon Emission Computed Tomography combined with Computed

Tomography (SPECT/CT). The acquisition of SPECT scans allows to visualize “hot spots” (SLNs)

and the fusion of these images with CT images considerably improves the anatomical localization

of SLNs. SPECT/CT might be useful when planar imaging is negative or ambiguous, when it shows

unexpected drainage patterns, especially after previous breast surgery, and also in overweight

patients5, 17, 18. Another controversial issue concerns intraoperative SLN detection and removal.

Surgeons detect SLNs by combining the auditory signal (probe) with the visual one (blue dye).

Although it is recommended that all blue staining nodes should be harvested, there is no consensus

about the optimal surgical removal of the radioactive SLNs5. In patients with multiple radiolabelled

nodes it is difficult to distinguish between the “true” SLN and second tier nodes. Different

approaches have been developed according to the level of radioactivity detected within the nodes.

One of the most reported in the literature and used in clinical practice is based on an empiric

threshold according to which all nodes that are 10% or higher than the ex vivo radioactive count of

the hottest SLN should be harvested19-22. However, the removal of more than five axillary nodes

does not improve the sensitivity of SLNB5. For radioguided surgery, portable γ-cameras and the

Declipse system (SurgicEye, München, German), or so-called “free-hand SPECT” (fhSPECT) are

now available, which have led to a new “see and open” in contraposition to a former “open and see”

paradigm18, 23. Portable γ-cameras enable intraoperative images that provide an overview of all the

radioactive “hot spots” in the surgical field, helping to evaluate the completeness of the procedure24.

Portable γ-cameras have been recently combined with fluorescence cameras for simultaneous SLN

signal detection by using hybrid tracers25, but their role in breast cancer surgery is still to be

clarified. The fhSPECT permits a virtual reconstruction in a 3-dimensional environment in order to

provide a better road map for radio-guided surgery26, 27. A further development of fhSPECT

technology in breast cancer is the fusion of the 3-D images of fhSPECT with US28, 29. All these

image-guided techniques might further reduce the invasiveness of the surgical procedure,

contributing to the extension of the GOSTT (Guided intraOperative Scintigraphic Tumor Targeting)

5

concept30. Several histopathological methods for assessing the SLN status have been developed in a

20-year-long history. The rate of detection of metastases increases with the number of histological

sections examined and with the use of immunohistochemistry (IHC) in addition to conventional

Hematoxylin & Eosin staining31. Isolated tumor cells and clusters (ITCs, ≤0.2 mm) and

micrometastases (>0.2 mm to 2.0 mm) have been introduced as new definitions in addition to

macrometastases (>2.0 mm) for nodal staging in breast cancer1. However, standardization of

protocols for the SLN histopathological evaluation has not yet been achieved. Recently, one-step

nucleic acid amplification (OSNA, Sysmex, Kobe, Japan) has allowed to analyze the whole SLN by

using a fast molecular intraoperative procedure. This method is based on the quantification of

cytokeratin19 (CK19) mRNA as a surrogate of metastatic cells: - for CK19mRNA copy numbers

less than 250/µl, + for copy numbers between 250 and 5000/µl (micrometastases) and ++ for copy

numbers greater than 5000/µl (macrometastases). OSNA has the advantage of reproducibility

among institutions and seems to be more sensitive than conventional histology to detect small

volume metastases. This procedure is routinely used in many centres, while it is being validated in

others32-34. Regardless of the method of SLN assessment, pathologists should quantify nodal tumor

burden according to the American Joint Committee on Cancer (AJCC)/Union on International

Cancer Control (UICC) staging system (ITCs, micrometastases, macrometastases) to facilitate

uniform communication with clinicians and analysis of outcomes2.

Since the publication in 2005 of the ASCO original Guidelines on the use of SLNB for patients with

early-stage breast cancer16, there have been significant changes in clinical practice related to the

availability of additional randomized trial results and to the better knowledge of cancer biology.

The ASCO Committee of experts published the Updated Clinical Practice Guidelines on SLNB in

breast cancer in 20142, while the European Association of Nuclear Medicine (EAMN) Practice

Guidelines were updated and published in 20135. More research is needed on the potential role and

limitations of SLNB in some clinical circumstances, for which the SLNB procedure is still debated.

SLNB is recommended for T1 and T2 tumors, for multiple tumors, for ductal carcinoma in situ

6

(DCIS) when mastectomy is planned, for elderly or obese patients, for male patients with breast

cancer, and also for patients with prior breast or axillary surgery. The data are insufficient to

support the benefit of SLNB in women with large or locally advanced invasive breast cancers

(T3/T4) and inflammatory breast cancer. SLNB is not recommended for women with DCIS when

breast-conserving surgery (BCS) is planned, except when there is a high risk of invasive cancer at

final diagnosis (i.e. palpability of the lesion, presence of a mammographic mass, and calcifications

without a mass [spread, ≥ 5 cm). Until further data are available, SLNB is also not recommended in

the setting of clinically palpable axillary nodes. In any case, palpable nodes should be harvested for

histopathological evaluation, even when they are neither hot nor blue. SLNB may be offered before

or after neoadjuvant therapy (NACT), but in this case the FNR becomes higher after treatment.

Therefore, SLNB after NACT should not be recommended as routine procedure35 and it should be

routinely discouraged also in patients with prior cN+ disease who are downstaged to ycN0. Both the

SENTINA trial36 (Arm C) and the ACOSOG Z1071 trial37 demonstrated that, when 3 or more SLNs

are harvested and dual mapping techniques are used, the FNR and the detection rate for initially

cN+ patients after NACT are comparable to those of initially cN0 patients. These findings highlight

the importance of technical factors to ensure success in performing SLNB after chemotherapy38.

Another important controversy concerns the potential to perform SLNB in pregnant patients.

According to the EAMN Guidelines, pregnancy is not an absolute contraindication for SLNB. The

isotopic technique is considered safe because the calculated fetal dose is low. However, the data

currently available are insufficient to change the 2005 ASCO recommendations according to which

clinicians should not perform SLNB during pregnancy owing to an insufficient level of evidence. In

addition, the administration of blue dye during pregnancy is not accepted as a safe procedure.

SLNB of internal mammary nodes (IMN) is another debated issue. Controversy relates to the

technique of lymphatic mapping, safety of IMN SLNB, significance of positive IMN, and impact on

survival39. IMN drainage is more difficult than axillary drainage to be demonstrated and it is

significantly affected by the depth of radiopharmaceutical injection5. Spillane et al. found that

7

technical requisites such as peritumoral radiocolloid injection under ultrasound guidance and an

adequate learning curve of the team performing SLNB provided satisfactory IMN identification

rates40. SPECT/CT represents a further technical solution to increase the IMN identification rate,

and therefore to significantly reduce the FNR of the technique. According to the AJCC/UICC

Staging Criteria1, IMN metastases have the same prognostic significance as axillary nodal

involvement, but the clinical impact of the tumor positive IMN is not completely clear. Although

there is evidence that mapping of IMN could modify treatment planning, with respect to

radiotherapy and systemic therapy41-43, more evidence is necessary to support the idea that it will

improve the outcome of treatment and survival44. The decision to perform SLNB of IMN should be

determined on the basis of the clinical judgment of the treating physicians16.

Since the introduction of SLNB, ALND has long been regarded as the standard of care in breast

cancer patients with a positive SLN. Its role has been questioned in recent studies because SLNB

can supply the prognostic information to help physicians decide on adjuvant treatments. Breast

cancer is a systemic disease and the recognition of the complexity of tumor biology has led to the

treatment of cancer with more conservative surgery and an increased use of systemic therapy based

on prognostic factors and molecular subtypes. The utility of radiation as a management modality of

axillary metastases has also been studied. The AMAROS trial45 showed that axillary radiotherapy

could be an alternative to ALND in patients with cT1-2N0 breast cancer and positive SLN, with

comparable locoregional control and fewer side-effects. Therefore, SLNB has replaced ALND for

axillary staging in clinically node-negative breast cancer patients, but also for the management of

some patients with SLN metastases. The ACOSOG Z0011 study46, 47 compared ALND, by

following SLNB and SLNB alone in women with clinical T1-2N0M0 breast cancer and metastatic

SLNs. After a median follow-up of 6.3 years, no significant differences were reported between the

two groups in terms of DFS and OS rates. As with the ACOSOG Z0011 trial, the IBCSG 23-01 48

results showed that with minimal SLN involvement ALND is unjustified in patients receiving

whole breast irradiation and systemic adjuvant treatment. In the light of these recent results, the

8

ASCO Update Committee recommended that clinicians should avoid ALND in cases of women

affected by early-stage breast cancer with ≤2 positive SLNs, who will receive conservative surgery

and whole-breast radiotherapy2. ALND should still be recommended in clinically node-positive

patients, in patients with palpable nodes, in those receiving NACT, in case of ≥3 positive SLNs, in

patients with a positive SLN treated with mastectomy, in case of significant extra-nodal extension,

in patients that do not receive adjuvant systemic therapy or whole breast irradiation, and when the

number of positive nodes would be crucial for the choice of chemotherapy49, 50. Recently, the 14th St

Gallen International Breast Cancer Conference (2015) has reviewed new evidence on locoregional

and systemic therapies for early breast cancer. A clear majority of experts agreed that ALND can be

avoided for patients with 1 or 2 macrometastatic SLNs, provided BCS with standard tangent

radiotherapy is planned51. While ALND could be omitted in a subset of patients with

macrometastatic SLN involvement, it is no longer recommended in cases of SLN micrometastases,

regardless of the type of breast surgery carried out2, 52. The ACOSOG Z0010 trial53 and the NSABP

B-32 trial54 demonstrated that the prognostic relevance of ITCs and micrometastases is negligible

with respect to both OS and DFS. Therefore, the routine use of IHC or molecular testing to look for

low-volume SLN metastatic disease is not required, since micrometastases do not alter

management2, 55. IHC staining remains of value in lobular carcinoma, for which it is difficult to

detect SLN metastases with Hematoxylin & Eosin staining alone.

In conclusion, the introduction of SLNB in breast cancer management in the 1990s dramatically

changed surgical treatment, as it reduced morbidity without decreasing survival. SLNB permits

accurate staging, guides treatment options, and is an important prognostic indicator for OS. In the

course of the years, these advantages have enabled SLNB to gain widespread acceptance without

prospective randomized trials. Moreover, the lack of standardization of SLNB is likely responsible

for the variability in FNR of the procedure. High FNRs may have a direct adverse impact on patient

care, including accurate staging, treatment decision making and long-term outcomes. An important

requirement to ensure SLNB diagnostic accuracy is appropriate training by the performing team, so

9

as to monitor individual results, for example the proportion of successful mappings, FNRs and

complication rates, and the follow-up of all patients over time. In order to reduce the FNR, attention

to technical aspects is also necessary, in particular when applying the procedure to patients at

increased risk for node-positive disease. SLNB should be performed by a multidisciplinary team

with a continuous and coordinated effort for applying the procedure on the basis of individual

patients’ characteristics and prognostic factors such as tumor size, histopathological findings, tumor

grade, lymphovascular invasion, hormone receptor status and other biologic factors (HER2

overexpression, Ki-67 expression etc). Also fundamental is the development of clinical trials as the

only tools to ensure the diagnostic, prognostic and therapeutic value of SLNB in clinical practice

and to improve breast cancer care.

10

References

1. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Annals of surgical oncology. Jun 2010;17(6):1471-1474.

2. Lyman GH, Temin S, Edge SB, et al. Sentinel lymph node biopsy for patients with early-stage breast cancer: American Society of Clinical Oncology clinical practice guideline update. J Clin Oncol. May 1 2014;32(13):1365-1383.

3. Wiatrek R, Kruper L. Sentinel lymph node biopsy indications and controversies in breast cancer. Maturitas. May 2011;69(1):7-10.

4. Hindie E, Groheux D, Brenot-Rossi I, Rubello D, Moretti JL, Espie M. The sentinel node procedure in breast cancer: nuclear medicine as the starting point. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. Mar 2011;52(3):405-414.

5. Giammarile F, Alazraki N, Aarsvold JN, et al. The EANM and SNMMI practice guideline for lymphoscintigraphy and sentinel node localization in breast cancer. Eur J Nucl Med Mol Imaging. Dec 2013;40(12):1932-1947.

6. Kim T, Giuliano AE, Lyman GH. Lymphatic mapping and sentinel lymph node biopsy in early-stage breast carcinoma: a metaanalysis. Cancer. Jan 1 2006;106(1):4-16.

7. Mariani G, Moresco L, Viale G, et al. Radioguided sentinel lymph node biopsy in breast cancer surgery. J Nucl Med. Aug 2001;42(8):1198-1215.

8. Surasi DS, O'Malley J, Bhambhvani P. 99mTc-Tilmanocept: A Novel Molecular Agent for Lymphatic Mapping and Sentinel Lymph Node Localization. Journal of nuclear medicine technology. Jun 2015;43(2):87-91.

9. Van Den Berg NS, Buckle T, Kleinjan GI, et al. Hybrid tracers for sentinel node biopsy. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine. Jun 2014;58(2):193-206.

10. Ghilli M, Carretta E, Di Filippo F, et al. The superparamagnetic iron oxide tracer: a valid alternative in sentinel node biopsy for breast cancer treatment. European journal of cancer care. Sep 14 2015.

11. Tanis PJ, Nieweg OE, Valdes Olmos RA, Kroon BB. Anatomy and physiology of lymphatic drainage of the breast from the perspective of sentinel node biopsy. J Am Coll Surg. Mar 2001;192(3):399-409.

12. Suami H, Pan WR, Mann GB, Taylor GI. The lymphatic anatomy of the breast and its implications for sentinel lymph node biopsy: a human cadaver study. Annals of surgical oncology. Mar 2008;15(3):863-871.

13. Ahmed M, Purushotham AD, Horgan K, Klaase JM, Douek M. Meta-analysis of superficial versus deep injection of radioactive tracer and blue dye for lymphatic mapping and detection of sentinel lymph nodes in breast cancer. The British journal of surgery. Feb 2015;102(3):169-181.

14. Sadeghi R, Asadi M, Treglia G, Zakavi SR, Fattahi A, Krag DN. Axillary concordance between superficial and deep sentinel node mapping material injections in breast cancer patients: systematic review and meta-analysis of the literature. Breast cancer research and treatment. Apr 2014;144(2):213-222.

15. Brenot-Rossi I, Houvenaeghel G, Jacquemier J, et al. Nonvisualization of axillary sentinel node during lymphoscintigraphy: is there a pathologic significance in breast cancer? J Nucl Med. Aug 2003;44(8):1232-1237.

16. Lyman GH, Giuliano AE, Somerfield MR, et al. American Society of Clinical Oncology

11

guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol. Oct 20 2005;23(30):7703-7720.

17. Wagner T, Buscombe J, Gnanasegaran G, Navalkissoor S. SPECT/CT in sentinel node imaging. Nucl Med Commun. Mar 2013;34(3):191-202.

18. Valdes Olmos RA, Rietbergen DD, Vidal-Sicart S, Manca G, Giammarile F, Mariani G. Contribution of SPECT/CT imaging to radioguided sentinel lymph node biopsy in breast cancer, melanoma, and other solid cancers: from "open and see" to "see and open". Q J Nucl Med Mol Imaging. Jun 2014;58(2):127-139.

19. Martin RC, 2nd, Edwards MJ, Wong SL, et al. Practical guidelines for optimal gamma probe detection of sentinel lymph nodes in breast cancer: results of a multi-institutional study. For the University of Louisville Breast Cancer Study Group. Surgery. Aug 2000;128(2):139-144.

20. Liu LC, Lang JE, Jenkins T, et al. Is it necessary to harvest additional lymph nodes after resection of the most radioactive sentinel lymph node in breast cancer? J Am Coll Surg. Dec 2008;207(6):853-858.

21. McMasters KM, Reintgen DS, Ross MI, et al. Sentinel lymph node biopsy for melanoma: how many radioactive nodes should be removed? Annals of surgical oncology. Apr 2001;8(3):192-197.

22. Manca G, Romanini A, Pellegrino D, et al. Optimal detection of sentinel lymph node metastases by intraoperative radioactive threshold and molecular analysis in patients with melanoma. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. Nov 2008;49(11):1769-1775.

23. Vidal-Sicart S, Rioja ME, Paredes P, Keshtgar MR, Valdes Olmos RA. Contribution of perioperative imaging to radioguided surgery. Q J Nucl Med Mol Imaging. Jun 2014;58(2):140-160.

24. Vermeeren L, Valdes Olmos RA, Klop WM, Balm AJ, van den Brekel MW. A portable gamma-camera for intraoperative detection of sentinel nodes in the head and neck region. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. May 2010;51(5):700-703.

25. van den Berg NS, Brouwer OR, Schaafsma BE, et al. Multimodal Surgical Guidance during Sentinel Node Biopsy for Melanoma: Combined Gamma Tracing and Fluorescence Imaging of the Sentinel Node through Use of the Hybrid Tracer Indocyanine Green-(99m)Tc-Nanocolloid. Radiology. May 2015;275(2):521-529.

26. Wendler T, Herrmann K, Schnelzer A, et al. First demonstration of 3-D lymphatic mapping in breast cancer using freehand SPECT. Eur J Nucl Med Mol Imaging. Aug 2010;37(8):1452-1461.

27. Bluemel C, Schnelzer A, Okur A, et al. Freehand SPECT for image-guided sentinel lymph node biopsy in breast cancer. Eur J Nucl Med Mol Imaging. Oct 2013;40(11):1656-1661.

28. Freesmeyer M, Winkens T, Opfermann T, Elsner P, Runnebaum I, Darr A. Real-time ultrasound and freehand-SPECT. Experiences with sentinel lymph node mapping. Nuklearmedizin. Jul 24 2014;53(6).

29. Okur A, Hennersperger C, Runyan B, et al. FhSPECT-US guided needle biopsy of sentinel lymph nodes in the axilla: is it feasible? Med Image Comput Comput Assist Interv. 2014;17(Pt 1):577-584.

30. Valdes Olmos RA, Vidal-Sicart S, Giammarile F, Zaknun JJ, Van Leeuwen FW, Mariani G. The GOSTT concept and hybrid mixed/virtual/augmented reality environment radioguided surgery. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine. Jun 2014;58(2):207-215.

31. Cserni G, Amendoeira I, Apostolikas N, et al. Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines. Eur J Cancer. Aug 2003;39(12):1654-1667.

12

32. Hoon DS, Bernet L, Cano R, Viale G. Molecular analysis of sentinel lymph nodes and search for molecular signatures of the metastatic potential of breast cancer. Q J Nucl Med Mol Imaging. Jun 2014;58(2):180-192.

33. Tiernan JP, Verghese ET, Nair A, et al. Systematic review and meta-analysis of cytokeratin 19-based one-step nucleic acid amplification versus histopathology for sentinel lymph node assessment in breast cancer. Br J Surg. Mar 2014;101(4):298-306.

34. Cserni G. Intraoperative analysis of sentinel lymph nodes in breast cancer by one-step nucleic acid amplification. J Clin Pathol. Mar 2012;65(3):193-199.

35. Reimer T, Hartmann S, Stachs A, Gerber B. Local treatment of the axilla in early breast cancer: concepts from the national surgical adjuvant breast and bowel project B-04 to the planned intergroup sentinel mamma trial. Breast Care (Basel). May 2014;9(2):87-95.

36. Kuehn T, Bauerfeind I, Fehm T, et al. Sentinel-lymph-node biopsy in patients with breast cancer before and after neoadjuvant chemotherapy (SENTINA): a prospective, multicentre cohort study. Lancet Oncol. Jun 2013;14(7):609-618.

37. Boughey JC, Suman VJ, Mittendorf EA, et al. Sentinel lymph node surgery after neoadjuvant chemotherapy in patients with node-positive breast cancer: the ACOSOG Z1071 (Alliance) clinical trial. JAMA. Oct 9 2013;310(14):1455-1461.

38. Boughey JC, Suman VJ, Mittendorf EA, et al. Factors affecting sentinel lymph node identification rate after neoadjuvant chemotherapy for breast cancer patients enrolled in ACOSOG Z1071 (Alliance). Annals of surgery. Mar 2015;261(3):547-552.

39. Manca G, Volterrani D, Mazzarri S, et al. Sentinel lymph node mapping in breast cancer: a critical reappraisal of the internal mammary chain issue. Q J Nucl Med Mol Imaging. Jun 2014;58(2):114-126.

40. Spillane AJ, Noushi F, Cooper RA, Gebski V, Uren RF. High-resolution lymphoscintigraphy is essential for recognition of the significance of internal mammary nodes in breast cancer. Ann Oncol. Jun 2009;20(6):977-984.

41. Bourre JC, Payan R, Collomb D, et al. Can the sentinel lymph node technique affect decisions to offer internal mammary chain irradiation? Eur J Nucl Med Mol Imaging. May 2009;36(5):758-764.

42. Madsen E, Gobardhan P, Bongers V, et al. The impact on post-surgical treatment of sentinel lymph node biopsy of internal mammary lymph nodes in patients with breast cancer. Ann Surg Oncol. Apr 2007;14(4):1486-1492.

43. Coombs NJ, Boyages J, French JR, Ung OA. Internal mammary sentinel nodes: ignore, irradiate or operate? Eur J Cancer. Mar 2009;45(5):789-794.

44. Madsen EV, Aalders KC, van der Heiden-van der Loo M, et al. Prognostic Significance of Tumor-Positive Internal Mammary Sentinel Lymph Nodes in Breast Cancer: A Multicenter Cohort Study. Annals of surgical oncology. Dec 2015;22(13):4254-4262.

45. Donker M, van Tienhoven G, Straver ME, et al. Radiotherapy or surgery of the axilla after a positive sentinel node in breast cancer (EORTC 10981-22023 AMAROS): a randomised, multicentre, open-label, phase 3 non-inferiority trial. The Lancet. Oncology. Nov 2014;15(12):1303-1310.

46. Giuliano AE, McCall L, Beitsch P, et al. Locoregional recurrence after sentinel lymph node dissection with or without axillary dissection in patients with sentinel lymph node metastases: the American College of Surgeons Oncology Group Z0011 randomized trial. Ann Surg. Sep 2010;252(3):426-432; discussion 432-423.

47. Giuliano AE, Hunt KK, Ballman KV, et al. Axillary dissection vs no axillary dissection in women with invasive breast cancer and sentinel node metastasis: a randomized clinical trial. JAMA. Feb 9 2011;305(6):569-575.

48. Galimberti V, Cole BF, Zurrida S, et al. Axillary dissection versus no axillary dissection in patients with sentinel-node micrometastases (IBCSG 23-01): a phase 3 randomised controlled trial. Lancet Oncol. Apr 2013;14(4):297-305.

13

49. Caudle AS, Hunt KK, Tucker SL, et al. American College of Surgeons Oncology Group (ACOSOG) Z0011: impact on surgeon practice patterns. Ann Surg Oncol. Oct 2012;19(10):3144-3151.

50. Gangi A, Essner R, Giuliano AE. Long-term clinical impact of sentinel lymph node biopsy in breast cancer and cutaneous melanoma. Q J Nucl Med Mol Imaging. Jun 2014;58(2):95-104.

51. Coates AS, Winer EP, Goldhirsch A, et al. Tailoring therapies-improving the management of early breast cancer: St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2015. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. Aug 2015;26(8):1533-1546.

52. Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn HJ. Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. Aug 2011;22(8):1736-1747.

53. Hunt KK, Ballman KV, McCall LM, et al. Factors associated with local-regional recurrence after a negative sentinel node dissection: results of the ACOSOG Z0010 trial. Ann Surg. Sep 2012;256(3):428-436.

54. Weaver DL, Ashikaga T, Krag DN, et al. Effect of occult metastases on survival in node-negative breast cancer. N Engl J Med. Feb 3 2011;364(5):412-421.

55. Nowikiewicz T, Srutek E, Zegarski W. Application of immunohistochemistry for detection of metastases in sentinel lymph nodes of non-advanced breast cancer patients. Polish journal of pathology : official journal of the Polish Society of Pathologists. Mar 2015;66(1):22-29.

14