Comparison of pathologist-detected and automated computer-assisted image analysis detected sentinel lymph node micrometastases in breast cancer.

Sentinel lymph node biopsy has stimulated interest in identification of micrometastatic disease in lymph nodes, but identifying small clusters of tumor cells or single tumor cells in lymph nodes can be tedious and inaccurate. The optimal method of detecting micrometastases in sentinel nodes has not been established. Detection is dependent on node sectioning strategy and the ability to locate and confirm tumor cells on histologic sections. Immunohistochemical techniques have greatly enhanced detection in histologic sections; however, comparison of detection methodology has not been undertaken. Automated computer-assisted detection of candidate tumor cells may have the potential to significantly assist the pathologist. This study compares computer-assisted micrometastasis detection with routine detection by a pathologist. Cytokeratin-stained sentinel lymph node sections from 100 patients at the University of Vermont were evaluated by automated computer-assisted cell detection. Based on original routine light microscopy screening, 20 cases that were positive and 80 cases that were negative for micrometastases were selected. One-level (43 cases) or two-level (54 cases) cytokeratin-stained sections were examined per lymph node block. All 100 patients had previously been classified as node negative by using routine hematoxylin and eosin stained sections. Technical staining problems precluded computer-assisted cell detection scanning in three cases. Computer-assisted cell detection detected 19 of 20 (95.0%; 95% confidence interval, 75-100%) cases positive by routine light microscopy. Micrometastases missed by computer-assisted cell detection were caused by cells outside the instrument's scanning region. Computer-assisted cell detection detected additional micrometastases, undetected by light microscopy, in 8 of 77 (10.4%; 95% confidence interval, 5-20%) cases. The computer-assisted cell detection-positive, light microscopy-missed detection rate was similar for cases with one (3 of 30; 10.0%) or two (5 of 47; 10.6%) cytokeratin sections. Metastases detected by routine light microscopy tended to be larger (0.01-0.50 mm) than did metastases detected only by computer-assisted cell detection (0.01-0.03 mm). In a selected series of patients, automated computer-assisted cell detection identified more micrometastases than were identified by routine light microscopy screening of cytokeratin-stained sections. Computer-assisted detection of events that are limited in number or size may be more reliable than detection by a pathologist using routine light microscopy. Factors such as human fatigue, incomplete section screening, and variable staining contribute to missing metastases by routine light microscopy screening. Metastases identified exclusively by computer-assisted cell detection tend to be extremely small, and the clinical significance of their identification is currently unknown.     

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.     

Evaluating TIMP-1, Ki67, and HER2 as markers for non-sentinel node metastases in breast cancer patients with micrometastases to the sentinel node

The aim was to investigate whether the biochemical prognostic markers TIMP-1, Ki67, and HER2 could predict metastatic spread to non-sentinel nodes (NSN) in breast cancer patients with micrometastases to sentinel node (SN). We included all breast cancer patients with micrometastases to SN operated between 2001 and 2007 at the Department of Breast Surgery, Herlev Hospital. The study was designed as a matched case-control study with 25 cases with micrometastases to SN and, in addition, metastatic spread to NSN and 50 matched controls with micrometastases to SN, but without NSN metastases. Patient and tumor characteristics were retrieved from the Danish Breast Cancer Cooperative Group database. Immunohistochemical analyses of TIMP-1 and Ki67 and measurements of HER2 on formalin-fixed paraffin-embedded tumor tissue were performed. No significant differences in the immunoreactivity of TIMP-1 and Ki67 were found between patients with and without NSN metastases. Six of seven HER2 positive patients did not have NSN metastases, but the results did not reach statistical significance. Despite being prognostic markers in breast cancer, TIMP-1 and Ki67 could not predict NSN metastases in women with micrometastatic disease to SN. Larger studies are needed to further validate HER2 as a marker for NSN metastases in these patients.     

Cytokeratin immunohistochemical validation of the sentinel node hypothesis in patients with breast cancer

No standard method for handling and histopathologic examination of the sentinel node (SN) exists. We hypothesized that a focused examination of all nodes with serial sectioning and cytokeratin immunohistochemical staining would confirm the SN as the node most likely to harbor metastasis. Intraoperative lymphatic mapping and sentinel lymphadenectomy using blue dye and (99m)technetium-labeled sulfur colloid were performed. All nodes were stained with H&E. All tumor-free nodes underwent additional sectioning and staining with H&E and an immunohistochemical stain. Routine H&E examination detected SN metastases in 27.6% of cases. Occult SN metastases were identified in 12.7% of cases. None of the 724 non-SNs examined contained occult metastases. The SN false-negative rate was zero. This study confirms histopathologically that the SN has biologic significance as the axillary node most likely to harbor metastatic tumor Standardization of the handling, sectioning, and staining of the SN is necessary as lymphatic mapping and sentinel lymphadenectomy become integrated into the care of patients with breast cancer     

Lymphangiogenesis in breast cancer is associated with non-sentinel lymph node metastases in sentinel node positive patients

Axillary lymph node dissection (ALND) is not suggested in breast cancer patients with negative sentinel lymph node (SLN) biopsies, and SLN is the only positive node in 40-70% of the remaining cases. To distinguish a subgroup in which ALND would be omitted, we investigated the role of lymphangiogenesis in primary breast cancer as a risk factor for distal lymph node involvements in patients with positive SLNs. 86 patients were included in this study. The frequency of proliferative lymphatic endothelial cells (LECP%) was evaluated in each specimen after immunohistochemical double staining for D2-40 and Ki-67. Larger primary tumor size, increased number of positive SLNs, lymphatic vessel invasion and LECP% were significantly associated with non-SLN metastases in the univariate analysis, but only LECP% retained significance in the multivariate model. A positive correlation between LECP% and lymphatic vessel invasion was also revealed. Our study confirmed the important role of lymphangiogenesis in tumor spread, and suggested that LECP% is a promising predictor for additional axillary lymph node involvements.     

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.     

Angiogenesis and mast cells in human breast cancer sentinel lymph nodes with and without micrometastases

AIMS: An increasing number of mast cells have been reported in angiogenesis associated with solid and haematopoietic tumours. Data concerning the number of mast cells in neoplastic lymph nodes and their relationship with microvessel density are controversial. The aim was to correlate the extent of angiogenesis with the number of mast cells reactive with tryptase in biopsy specimens of sentinel lymph nodes with and without micrometastases obtained from patients with breast cancer. METHODS AND RESULTS: Specimens from sentinel lymph nodes obtained from 80 patients (40 with and 40 without micrometastases) were investigated immunohistochemically by using anti-CD31 and anti-tryptase antibodies. Angiogenesis, measured as microvessel counts, increased in parallel with the number of tryptase-positive mast cells and their values were significantly higher in lymph nodes with micrometastases compared with those without. CONCLUSIONS: Tryptase-positive mast cells may contribute, at least in part, to angiogenesis occurring in sentinel lymph nodes with micrometastases from patients with breast cancer.     

Update on sentinel node pathology in breast cancer

Pathologic examination of the sentinel lymph nodes (SLNs) in patients with breast cancer has been impacted by the publication of practicing changing trials over the last decade. With evidence from the ACOSOG Z0011 trial to suggest that there is no significant benefit to axillary lymph node dissection (ALND) in early-stage breast cancer patients with up to 2 positive SLNs, the rate of ALND, and in turn, intraoperative evaluation of SLNs has significantly decreased. It is of limited clinical significance to pursue multiple levels and cytokeratin immunohistochemistry to detect occult small metastases, such as isolated tumor cells and micrometastases, in this setting. Patients treated with neoadjuvant therapy, who represent a population with more extensive disease and aggressive tumor biology, were not included in Z0011 and similar trials, and thus, the evidence cannot be extrapolated to them. Recent trials have supported the safety and accuracy of sentinel lymph node biopsy (SLNB) in these patients when clinically node negative at the time of surgery. ALND remains the standard of care for any amount of residual disease in the SLNs and intraoperative evaluation of SLNs is still of value for real time surgical decision making. Given the potential prognostic significance of residual small metastases in treated lymph nodes, as well as the decreased false negative rate with the use of cytokeratin immunohistochemistry (IHC), it may be reasonable to maintain a low threshold for the use of cytokeratin IHC in post-neoadjuvant cases. Further recommendations for patients treated with neoadjuvant therapy await outcomes data from ongoing clinical trials. This review will provide an evidence-based discussion of best practices in SLN evaluation.     

乳腺癌前哨淋巴结相关的临床病理问题

乳腺癌是全球女性发病率最高的恶性肿瘤,其手术方式从最初的乳腺癌根治术到改良根治术,并进一步发展至目前的保乳治疗,极大地提高了患者的生活质量.腋窝淋巴结转移是乳腺癌最重要的预后指标,通过确定淋巴结转移情况可对乳腺癌进行分期,从而确定患者的治疗方案.但传统的腋窝淋巴结清扫(axillary lymph node dissection,ALND)可造成患者上肢水肿、疼痛、手臂运动功能受损和肩部僵硬等,影响其生活质量.近年来,ALND正逐渐被乳腺癌前哨淋巴结(sentinel lymph node,SLN)活检所取代.SLN活检在国内的逐步推广也对病理工作者提出了新的要求,因此本文就乳腺癌SLN的临床病理相关问题进行介绍.     

乳腺癌前哨淋巴结相关的临床病理问题

乳腺癌是全球女性发病率最高的恶性肿瘤,其手术方式从最初的乳腺癌根治术到改良根治术,并进一步发展至目前的保乳治疗,极大地提高了患者的生活质量.腋窝淋巴结转移是乳腺癌最重要的预后指标,通过确定淋巴结转移情况可对乳腺癌进行分期,从而确定患者的治疗方案.但传统的腋窝淋巴结清扫(axillary lymph node dissection,ALND)可造成患者上肢水肿、疼痛、手臂运动功能受损和肩部僵硬等,影响其生活质量.近年来,ALND正逐渐被乳腺癌前哨淋巴结(sentinel lymph node,SLN)活检所取代.SLN活检在国内的逐步推广也对病理工作者提出了新的要求,因此本文就乳腺癌SLN的临床病理相关问题进行介绍.     

Development and evaluation of a robust algorithm for computer-assisted detection of sentinel lymph node micrometastases

Clarke G M, Peressotti C, Holloway C M B, Zubovits J T, Liu K & Yaffe M J
(2011) Histopathology 59 , 116–128 Development and evaluation of a robust algorithm for computer‐assisted detection of sentinel lymph node micrometastases Aims: Increasing the sectioning rate for breast sentinel lymph nodes can increase the likelihood of detecting micrometastases. To make serial sectioning feasible, we have developed an algorithm for computer‐assisted detection (CAD) with digitized lymph node sections. Methods and results: K‐means clustering assigned image pixels to one of four areas in a colourspace (representing tumour, unstained background, counterstained background and microtomy artefacts). Four filters then removed ‘false‐positive’ pixels from the tumour cluster. A set of 43 sections containing tumour (a total of 259 foci) and 59 sections negative for malignancy was defined by two pathologists, using light microscopy, and CAD was applied. For the clinically relevant task of identifying the largest focus in each section (micrometastasis in 22/43 sections), the sensitivity and specificity were 100%. Isolated tumour cells (ITCs) were identified in one slide initially considered to be negative. Identification of all 259 foci yielded sensitivities of 57.5% for ITCs (<0.200 mm), 89.5% for micrometastases, and 100% for larger metastases, with one false‐positive. Reduced sensitivity was ascribed to variable staining. Nine additional metastases (<0.01–0.3 mm) that were not initially identified were detected by CAD. Conclusions: This algorithm is well suited to the task of sentinel lymph node evaluation and may enhance the detection of occult micrometastases.     

Intra-operative examination of the sentinel node in breast cancer

Intra-operative sentinel node analysis allows immediate progression to axillary clearance in patients with node positive breast cancer and reduces the need for re-operation. Despite this, intra-operative sentinel node analysis is infrequently performed in Ireland. We report our experience using this technique. Sentinel node biopsy was performed in 47 consecutive patients with symptomatic T1-T2 clinically node negative breast cancer. Sentinel nodes were examined intra-operatively by frozen section and imprint cytology and definitive histological assessment was performed on paraffin-embedded tissue. The sentinel node was identified in 46 (98%) patients. Twelve patients had axillary metastases. The sensitivity of intra-operative analysis in identifying nodal metastases was 92%. False negative rate was 8%, negative predictive value 97%, and specificity 100%. Intra-operative analysis of the sentinel node allowed re-operation to be avoided in 92% of patients with axillary node metastases. In our experience this technique can be readily introduced with reliable outcomes.     

Axillary staging of breast cancer and the sentinel node

Pathological aspects of axillary nodal staging of breast cancer and in particular sentinel lymph node (SLN) biopsy are reviewed. SLN biopsy seems an almost ideal staging procedure because it has both high accuracy and a low false negative rate. It may also allow a cost effective use of more sensitive methods of metastasis detection. However, the biological relevance of metastases detected only by modern tools remains to be elucidated. This review focuses on standard axillary staging and the histopathological investigation of SLNs, with emphasis on the intraoperative setting. Future trends including ancillary studies, quality control issues, prediction of non-SLN involvement, and suggestions concerning the minimum requirements for the histology of axillary SLNs are also discussed.     

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.     

乳腺癌前哨淋巴结术中分子诊断的临床实用性初探

目的 探讨GeneSearchTM乳腺淋巴结检测试剂盒(以下简称GeneSearch)在乳腺癌前哨淋巴结(SLN)术中诊断的临床实用性.方法 对复旦大学附属肿瘤医院2009年2月至6月诊治的88例乳腺癌患者行SLN活检.首先垂直长轴将所得淋巴结切成数块厚约2 mm的组织块,对各切面进行术中细胞印片后,奇数号组织块用于术后连续切片检查,偶数号组织块采用GeneSearch进行检测,应用即时荧光定量逆转录聚合酶链反应检测SLN中CK19和乳腺球蛋白表达的Ct值.将GeneSearch以术后连续切片的诊断为准,与术中细胞印片、术后连续切片的病理结果分别进行比较.结果 88例共获得225枚SLN,其中宏转移淋巴结27枚,微转移淋巴结9枚,阴性淋巴结189枚(其中5枚为孤立肿瘤细胞).从切割淋巴结开始到最终形成报告,GeneSearch耗时范围为35～45 min(平均40 min).基于淋巴结数目,GeneSearch与术后连续切片的总体符合率为95.6%(215/225),其检测敏感度为86.1%(31/36),均高于术中细胞印片[分别为94.7%(213/225)和72.2%(26/36)].SLN转移灶大小与CKl9和乳腺球蛋白的Ct值存在统计学相关性(P＜0.01).结论 GeneSearch用于SLN术中诊断时,其检测敏感度高于术中细胞印片,达到比较满意的效果,但在应用中仍存在一些问题.