Histopathological work-up and interpretation of sentinel lymph nodes removed for vulvar squamous cell carcinoma

Aims:  To evaluate the work-up of sentinel lymph nodes (SLNs) removed for vulvar pT1–pT2 squamous cell carcinoma (SCC). Inguinal lymphadenectomy yields metastases in only 30% of cases. Patients with missed inguinal disease, however, have a risk of dying from systemic disease. SLN dissections reduce morbidity, but work-up should reliably identify metastatic disease.
Methods and results:  All SLNs removed from 38 patients with pT1–pT2 SCC and clinically negative inguinal lymph nodes were submitted for frozen section analysis. When negative, SLN were formalin-fixed, sectioned entirely at 330-μm intervals to produce three slides per millimetre [two haematoxylin and eosin (H&E) stained slides; one slide for immunohistochemistry]. If screening of H&E-stained sections was negative, all remaining slides were subjected to immunohistochemistry with an antibody to cytokeratin. Twenty-five of 38 patients (66%) were pN0, 7/38 (18%) had metastases on frozen sections/H&E stains. Immunohistochemistry detected micrometastases in two patients and single tumour cells and anucleate cell structures in four patients. In 12/13 patients the SLN metastases, including all single-cell deposits, were from lichen sclerosus (LS)-associated SCC. Twelve of 13 patients with metastases had a pT2 SCC.
Conclusions:  Micrometastases and single tumour cell deposits in SLNs are typical of LS-associated vulvar SCC. Single tumour cell deposits in SNLs should be regarded as 'positive'. Identification requires serial sectioning and immunohistochemical analysis of all removed SLNs.     

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.     

Rapid intraoperative immunohistochemical evaluation of sentinel lymph nodes for metastatic breast carcinoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is an integral part of the surgical management of patients with breast cancer. Rapid immunohistochemistry (RIHC) has the potential to increase detection of metastatic carcinoma at the time of frozen section consultation. The authors assessed the accuracy and turnaround time of a newly developed RIHC method for pancytokeratin (RIHC-CK). METHODS: Sixty-six SLNs from 32 patients with breast carcinoma were examined for metastasis using the Zymed Sentinel Lymph Node Rapid IHC Kit. Intraoperative frozen sections (6 mum) of the SLNs were incubated with Zymed anti-pan-cytokeratin/HRP conjugate, diaminobenzidine (DAB), and stained with hematoxylin. Slides were ready within 8 minutes and were interpreted as positive or negative for metastatic carcinoma. Results were compared with previous intraoperative touch preparations, frozen sections, hematoxylin and eosin (Perm H&E), and AEl/3-immunostained permanent sections (Perm CK). RESULTS: Fourteen lymph nodes (19%) in 13 patients tested positive for metastatic carcinoma in Perm H&E, the gold standard. RIHC-CK had the highest sensitivity (92%) of the intraoperative tests, compared with touch preparations (64%) and frozen sections (80%). RIHC-CK showed 94% accuracy, compared with 96% (frozen section) and 93% (touch preparation). The RIHC technique took 8 minutes and was easy to perform and interpret. CONCLUSIONS: Zymed RIHC is a sensitive method for detecting breast cancer metastases in SLNs. The speed, accuracy, and ease of interpretation of the test allow for recognition of micrometastases (<2 mm) that might otherwise be undetectable by current methods of intraoperative evaluation. The prognostic significance and effect on surgical management of micrometastases in SLNs have yet to be determined.     

Intraoperative evaluation of sentinel lymph nodes for breast carcinoma: current methodologies

Sentinel lymph node biopsy is an important new addition to the surgical management of patients with breast carcinoma. Sentinel nodes have a higher chance of containing metastases than do nonsentinel nodes. Sentinel lymph node biopsy provides an opportunity to stage breast carcinoma patients more accurately and to modify subsequent treatment. One of the most exciting current roles of sentinel lymph node biopsy is the ability to stage patients intraoperatively, allowing a one-step axillary lymph node dissection if the sentinel lymph node contains metastatic carcinoma. Currently, intraoperative evaluation of sentinel lymph nodes is performed using imprint cytology with or without rapid cytokeratin staining, frozen sectioning with or without rapid cytokeratin staining, scrape preparations, or some combination of these techniques. We review the relative strengths and weaknesses of these different methodologies. A great deal of controversy exists regarding the management of patients with metastatic breast carcinoma, particularly those patients with occult and micrometastatic disease. These issues are beyond the scope of this article.     

Modes of benign mechanical transport of breast epithelial cells to axillary lymph nodes

The status of axillary lymph nodes is a key prognostic indicator available for the management of patients with breast cancer. Sentinel lymph node (SLN) evaluation as a predictor of lymph node status has led to increased use of ancillary methods, principally immunohistochemistry, to increase the sensitivity of the SLN biopsy. So-called "occult" micrometastases detected by such methods have led to speculation that some may have reached the SLNs by benign mechanical transport (BMT) rather than a metastatic process. We review evidence suggesting two potential modes of BMT: lymphatic transport of epithelial cells displaced by biopsy of the primary breast tumor and by breast massage-assisted SLN localization. The biopsy techniques under most scrutiny include fine needle aspiration and large-gauge core biopsy. The evidence implicating breast massage prior to SLN biopsy as a mode of BMT has been supported by statistical analysis; however, no method of distinguishing massage-associated cells in SLNs from true occult micrometastases is available. The significance of small epithelial clusters in SLNs is currently unknown. Thus, deviation from current biopsy and SLN-localizing practices is unwarranted.     

Sentinel lymph node investigation in melanoma: detailed analysis of the yield from step sectioning and immunohistochemistry

AIMS: To evaluate in detail the extent to which step sectioning and immunohistochemical examination of sentinel lymph nodes (SLNs) in patients with melanoma reveal additional node positive patients, to arrive at a sensitive yet workable protocol for histopathological SLN examination. METHODS: The study comprised 29 patients with one or more positive SLN after a successful SLN procedure for clinical stage I/II melanoma. SLNs were lamellated into pieces of approximately 0.5 cm in size. One initial haematoxylin and eosin (H&E) stained central cross section was made for each block. When negative, four step ribbons were cut at intervals of 250 microm. One section from each ribbon was stained with H&E, and one was used for immunohistochemistry (IHC). RESULTS: When taking the cumulative total of detected metastases at level 5 as 100%, the percentage of SLN positive patients increased from 79%, 83%, 83%, 90% to 93% in the H&E sections through levels 1-5, and with IHC these values were 83%, 86%, 90%, 97%, and 100%, respectively. One of six patients in whom metastases were detected at levels 2-5 only had metastases in the subsequent additional lymph node dissection. CONCLUSIONS: Multiple level sectioning of SLNs (five levels at 250 microm intervals) and the use of IHC detects additional metastases up to the last level in melanoma SLNs. Although more levels of sectioning might increase the yield even further, this protocol ensures a reasonable workload for the pathologist with an acceptable sensitivity when compared with the published literature.     

Iatrogenically false positive sentinel lymph nodes in breast cancer: Methods of recognition and evaluation

With the introduction of sentinel lymph node (SLN) biopsy as a standard procedure for staging clinically node negative breast cancer patients, meticulous pathologic evaluation of SLNs by serial sections and/or immunohistochemistry for cytokeratins has become commonplace in order to detect small volume metastases (isolated tumor cells and micrometastases). This practice has also brought to the fore the concept of iatrogenically false positive sentinel nodes secondary to epithelial displacement produced largely by preoperative needling procedures. While this concept is well described in the clinical and pathologic literature, it is, in our experience, still under-recognized, with such lymph nodes frequently incorrectly diagnosed as harboring true metastases, possibly resulting in unwarranted further surgery and/or chemotherapy. This review discusses the concept of displaced epithelium in the histologic evaluation of breast surgical specimens and provides a stepwise approach to the correct identification of iatrogenically transported displaced epithelial cells in sentinel lymph nodes.     

Assessment of sentinel lymph nodes for breast cancer

Sentinel lymph node biopsy is standard of care for assessment of lymph node stage in early breast cancer in patients with clinically negative nodes. The limited clinical significance of low volume axillary metastatic disease has led to changes in surgical management of the axilla with a shift away from routine axillary lymph node dissection if the sentinel lymph node is found to contain metastatic tumour. This has led to a decrease in the use of intraoperative assessment of sentinel nodes. Specimen handling and histological assessment of sentinel lymph nodes is described, with the emphasis on identification of macrometastatic disease defined as metastases greater than 2 mm. Routine levels and/or cytokeratin immunohistochemistry is not recommended. The increasing use of neoadjuvant chemotherapy and growing evidence that sentinel lymph node biopsy is safe and accurate in this setting, including in patients with proven node positive disease, has resulted in new challenges in the interpretation of these specimens.     

Comprehensive examination of sentinel lymph node in breast cancer: a solution without a problem?

Although several methods have been devised to examine sentinel lymph node (SLN) specimens in breast cancer, the extent of examination and whether it should routinely include multilevel sectioning to detect micrometastases (MM) (<2.0 mm) is still debated. In this study all "positive' SLN biopsies from 67 consecutive patients with breast carcinoma and evaluated by means of an extended protocol were reviewed. Abnormal findings included micrometastases (MM) between 0.2 and 1.0 mm (14 cases), (MM) between 1.0 and 2.0 mm (8 cases), metastases>2.0 mm (22 cases), and isolated tumor cells (ITCs) (23 cases). The likelihood of finding metastatic deposits was comparable if sections were carried out at 100-, 150-, 200-, 250-, and 500-microm intervals. No metastatic foci>2.0 mm would have been missed. 1 MM (1.1 mm focus) was missed within the 250- and 500-microm levels on hematoxylin-eosin, but not complementary cytokeratin staining. Our data show that SLN step sectioning does not add significant yield if compared to standard examination carried on initial levels, if the minimal target of 1.0 mm micrometastatic deposit is sought.     

Strategies for management of axillary lymph nodes in breast cancer. Point of view of the Institut Bergonié.

Sentinel lymph node biopsy in breast cancer has changed pathology management of axillary lymph nodes by pathologists, but there is still no consensus either for serial sectioning nor for immunohistochemistry. We analyze: 1) data in the literature about the prognostic significance of micrometastases (pNlmi) and immunohistochemically detected infiltrating tumor cells (pN0(i+) in axillary lymph nodes. 2) management strategies at the Bergonié Institute and by other teams for axillary lymph nodes and sentinel lymph nodes. 3) questions and controversies on sentinel lymph node management and recommendations of the five main reference groups.     

Can Melan-A replace S-100 and HMB-45 in the evaluation of sentinel lymph nodes from patients with malignant melanoma?

The sentinel lymph node (SLN) biopsy has become an increasingly important procedure used in the primary staging of malignant melanoma. However, micrometastases in a lymph node can be easily missed on routine H&E-stained sections. Therefore, S-100 and HMB-45 IHC stains are standardly performed on grossly negative SLNs for detection of metastatic melanoma. Each of these IHC markers, however, is not ideal. The authors investigated whether the newer IHC marker Melan-A would improve the detection of metastatic melanoma in SLN biopsies. Forty lymph nodes previously diagnosed with metastatic melanoma were retrospectively evaluated for S-100, HMB-45, and Melan-A expression. In addition, 42 SLN biopsies for metastatic melanoma detection were prospectively collected and evaluated for S-100, HMB-45, and Melan-A expression. All lymph nodes with metastatic melanoma from the retrospective study demonstrated S-100 reactivity. Five of the lymph nodes with metastatic melanoma from the retrospective study failed to express either HMB-45 or Melan-A, all of which displayed a desmoplastic morphology. One of the metastases positive for S-100 and HMB-45 failed to show reactivity with Melan-A (3%). The prospective study found 10 lymph nodes from 42 cases to be positive for metastatic melanoma, which were positive for S-100 (100%). Nine of the involved lymph nodes were positive for HMB-45(90%), and nine were positive for Melan-A (90%). Melan-A, although very specific, cannot replace the use of S-100 and HMB-45 for the detection of metastatic melanoma in SLNs. It can, however, substitute for HMB-45 with equally good results.     

Intraoperative evaluation of sentinel lymph nodes for metastatic breast carcinoma by imprint cytology.

BACKGROUND: The increasing utilization of lymphatic mapping techniques for breast carcinoma has made intraoperative evaluation of sentinel lymph nodes attractive. Axillary lymph node dissection can be performed during the initial surgery if the sentinel lymph node is positive, potentially avoiding a second operative procedure. At present the optimal technique for rapid sentinel lymph node assessment has not been determined. Both frozen sectioning and intraoperative imprint cytology are used for rapid intraoperative sentinel lymph node evaluation at many institutions. The purpose of this study is to evaluate experience with imprint cytology for intraoperative evaluation of sentinel lymph nodes in patients with breast cancer. METHODS: A retrospective review of the intraoperative imprint cytology results of 678 sentinel lymph node mappings for breast carcinoma was performed. Sentinel nodes were evaluated intraoperatively by either bisecting or slicing the sentinel node into 4 mm sections. Imprints were made of each cut surface and stained with H&E and/or Diff-Quik. Permanent sections were evaluated with up to four H&E stained levels and cytokeratin immunohistochemistry. Intraoperative imprint cytology results were compared with final histologic results. Results: The sensitivity of imprint cytology was 53%, specificity was 98%, positive predictive value was 94%, negative predictive value was 82% and accuracy was 84%. The sensitivity for detecting macrometastases (more than 2mm) was significantly better than for detecting micrometastases (相似文献   

Sentinel lymph nodes for breast carcinoma: an update on current practice

Sentinel lymph node (SLN) biopsy has been established as the standard of care for axillary staging in patients with invasive breast carcinoma and clinically negative lymph nodes (cN0). Historically, all patients with a positive SLN underwent axillary lymph node dissection (ALND). The ACOSOG Z0011 trial showed that women with T1–T2 disease and cN0 who undergo breast‐conserving surgery and whole‐breast radiotherapy can safely avoid ALND. The main goal of SLN examination should be to detect all macrometastases (>2 mm). Gross sectioning of SLNs at 2‐mm intervals and microscopic examination of one haematoxylin and eosin‐stained section from each SLN block is the preferred method for pathological evaluation of SLNs. The role and timing of SLN biopsy for patients who have received neoadjuvant chemotherapy is controversial, and continues to be explored in clinical trials. SLN biopsies from patients with invasive breast carcinoma who have received neoadjuvant chemotherapy pose particular challenges for pathologists.     

Pathologic examination of the axillary sentinel lymph nodes in patients with early-stage breast carcinoma: current and resolving controversies on the basis of the European Institute of Oncology experience

Several controversial aspects of sentinel lymph node biopsy (SLNB) for patients with early-stage, node-negative breast carcinoma have been dealt with and resolved in the past decade since its introduction. Unfortunately, however, there is still no consensus on how best to examine sentinel lymph nodes (SLN) histologically. As a consequence, the protocols for SLN examination are remarkably variable in different institutions, leading to a very poor reproducibility of the data stemming from investigations on series of patients whose SLNs have been evaluated according to diverse protocols. Patient outcomes, however, can be optimised only by standardization of the whole procedure of SLNB, with particular reference to the histopathologic scrutiny. Lack of a standardized histopathologic protocol likely derives also from the uncertainties about the clinical implications of minimal lymph node involvement (isolated tumour cells and micrometastases) with regard both to the risk of additional metastases to non-sentinel lymph nodes of the same basin and to the prognostic value for patients’ survival. This review aims at highlighting some of the controversial issues of the histopathologic examination of the SLNs, including the number of sections and cutting intervals, the use of immunohistochemistry and the role of molecular biology assays.     

Detection of micrometastases in bone marrow and sentinel lymph nodes of breast cancer patients

Objective: To study the sensitivity and clinical significance of HE-staining,IHC and RT-PCR in detecting breast cancer micrometastases in bone marrow and sentinel lymph nodes (SLNs). Methods:After general anesthesia, all patients underwent bone marrow puncture and sentinel lymph node biopsy (SLNB) by 1% isosulfan blue, and then HE-staining,IHC and RT-PCR were used to detect micrometastases. Results:Of 62 patients with breast cancer whose axillary lymph nodes showed negative HE-staining results, 15 cases presented with positive RT-PCR and 9 cases showed positive IHC results positive in bone marrow micrometastases detection. PT-PCR and IHC showed good uniformity(kappa=0.6945)and there was significant difference in detective rate between these two methods (χ2=4.1667,P=0.0412). In SLN samples, 13 showed positive RT-PCR results, while 7 showed positive IHC results. PT-PCR and IHC showed good uniformity (kappa=0.6483)and significant difference was also found in detective rate between these two methods (χ2=4.1667,P=0.0412). Both bone marrow and SLN samples were RT-PCR positive in 3 cases,which indicated that bone marrow micrometastases did not always accompany SLN micrometastases(χ2=0.067,P=0.796). Conclusion: Even if no axillary lymph node involvement or distant metastases are present in routine preoperative examination, micrometastases can still be detected in bone marrow or SLNs. Because the bone marrow micrometastases and axillary node micrometastses are not present simultaneously, combination test of multiple indicators will detect micrometastases more accurately.     

Metastases in axillary sentinel lymph nodes in breast cancer as detected by intensive histopathological work up

AIM: To assess the value of the intensive histological work up of axillary sentinel lymph nodes (SLN) to demonstrate regional metastatic disease. METHODS: From a series of 58 successful lymphatic mapping procedures, 78 SLN were analysed by serial sections (mean of 49 levels/SLN) and by immunostaining to cytokeratin and epithelial membrane antigen, and the results compared with those obtained by assessing the central cross section. RESULTS: The central cross section would have failed to detect metastases in eight of 26 lymph nodes (31%) in patients with breast cancer metastasising to the SLN only. This would have led to a false negative node status in six of 21 patients (29%). Two micrometastases were detected with the aid of immunostains. CONCLUSIONS: The results suggest the need to examine SLN at multiple levels and to use immunohistochemistry in negative cases. Serial sections are also useful in the case of micrometastases, as some of these may convert to macrometastases at deeper levels. Multiple level investigation of SLN and immunohistochemistry in the event of the negativity of standard stains would result in improved staging and an increase in the proportion of node positive disease detected.     

Assessing second echelon lymph nodes during sentinel lymph node biopsy: can we have more accurate axillary treatment for breast cancer patients?

Sentinel lymph node biopsy (SLNB) is the standard treatment for breast cancer patients with clinically negative axilla. For patients with positive sentinel lymph nodes, axillary lymph node dissection (ALND) was required. However, approximately a half of the SLNs-positive patients were found to have clear axillary lymph nodes after ALND, indicating that they had received unnecessary ALND without therapeutic benefit. Therefore, we propose a hypothesis for solution of this clinical problem. We defined the second echelon lymph nodes (SELNs) as those nodes receiving lymphatic drainage directly from the SLNs. For patients with positive-SLNs, SELNs can be biopsy and assessed. If SELNs are negative, no more ALND was needed in these patients even if their SLNs are positive. If our hypothesis were confirmed to be true, we can tailored our axillary treatment to more breast cancer patients, avoiding unnecessary ALND and its complications.     

Non-sentinel lymph node involvement in patients with breast cancer and sentinel node micrometastasis; too early to abandon axillary clearance

AIMS: It has been suggested that patients with T1-2 breast tumours and sentinel node (SLN) micrometastases, defined as foci of tumour cells smaller than 2 mm, may be spared completion axillary lymph node dissection because of the low incidence of further metastatic disease. To gain insight into the extent of non-sentinel lymph node (n-SLN) involvement, SLNs and complementary axillary clearance specimens in patients with SLN micrometastases were examined. METHODS: A set of 32 patients with SLN micrometastases was selected on the basis of pathology reports and review of SLNs. Five hundred and thirteen n-SLNs from the axillary clearance specimens were serially sectioned and analysed by means of immunohistochemistry for metastatic disease. Lymph node metastases were grouped as macrometastases (> 2 mm), and micrometastases (< 2 mm), and further subdivided as isolated tumour cells (ITCs) or clusters. RESULTS: In 11 of 32 patients, one or more n-SLN was involved. Grade 3 tumours and tumours > 2 cm (T2-3 v T1) were significantly associated with n-SLN micrometastases as clusters (grade: odds ratio (OR), 8.3; 95% confidence interval (CI), 1.4 to 50.0; size: T2-3 tumours v T1: OR, 15; 95% CI, 2.18 to 103.0). However, no subgroup of tumours with regard to size and grade was identified that did not have n-SLN metastases. CONCLUSIONS: In patients with breast cancer and SLN micrometastases, n-SLN involvement is relatively common. The incidence of metastatic clusters in n-SLN is greatly increased in patients with T2-3 tumours and grade 3 tumours. Therefore, axillary lymph node dissection is especially warranted in these patients. However, because n-SLN metastases also occur in T1 and low grade tumours, even these should be subjected to routine axillary dissection to achieve local control.     

Sentinel lymph node biopsy in breast cancer: creating controversy and defining new standards

Sentinel lymph node (SLN) biopsy is an exciting and promising new addition to surgical management and pathologic evaluation of breast cancer. Sentinel nodes are more likely to contain metastases than nonsentinel nodes providing an opportunity to more accurately stage breast cancer patients. In the future, surgical management of the axilla may be approached as a staged procedure, perhaps eliminating axillary dissection in SLN-negative patients, should clinical trials demonstrate the safety of this approach. The advent of SLN biopsy has propelled an old question back to the forefront of clinical investigation: do occult metastases in lymph nodes have prognostic, predictive, and clinical significance? This article provides one opinion and a review on the pathology and future of SLN biopsy in breast cancer. Practicing pathologists will need to stay abreast of the issues and facts in order to participate in the debate and shape the future of pathologic management of sentinel nodes.     

Implementation of step sectioning in the examination of sentinel lymph nodes to improve the detection of micrometastases in breast cancer patients

The object of this study was to examine whether a new protocol for examination of sentinel lymph nodes (SLNs) would lead to the detection of more metastases. Sections of 1 mm would identify most SLN macrometastases, and step sections at intervals of 200-250 μm would identify most micrometastases. A total of 111 breast cancer patients who underwent the SLN procedure at St. Olavs University Hospital in Trondheim, Norway in 2008 were included in the study group. Their SLNs were processed according to a new standardized protocol with sections of 2-3 mm being step sectioned at intervals of 200-250 μm. A total of 109 breast cancer patients undergoing the SLN procedure in 2007 were used as a reference group. Metastases were found in 29% of the cases, compared with 26% in the reference group. Step sectioning of SLNs revealed metastases in five cases initially found to be negative. The metastases of the study group were smaller, with a median value of 1.25 mm compared with 4.25 mm in the reference group. Step sectioning led to the detection of metastases in SLNs initially found to be negative. The median size of the metastases was considerably smaller in the study group than in the reference group.