Intraoperative frozen section examination of axillary sentinel lymph nodes in breast cancer

The study presents the results from intraoperative frozen section assessment of axillary sentinel lymph nodes (SLNs) in breast cancer. Routine histological frozen sections from one level were used, two sections stained with haematoxylin and eosin. Immunohistochemistry for cytokeratins was applied to the permanent SLN paraffin sections only. Axillary dissection was performed on all SLN-positive cases regardless of the size of the metastatic deposits. With a detection rate of 83%, 272 patients entered the study over a period of 46 months. A total of 61 cases were SLN positive by frozen section analysis. The paraffin sections gave an additional 23 SLN-positive cases. The false-negative rate for frozen sections was then 27% (23/84). Micrometastases were found in 28 of 84 cases, and macrometastases in 56. The false-negative rate of frozen sections for micrometastases was 71% (20/28), and for macrometastases 5% (3/56). A total of 73% (61/84) of the patients underwent axillary surgery as a one-step procedure.     

Strategies for optimizing pathologic staging of sentinel lymph nodes in breast cancer patients

Due to the extensive pathologic evaluation of the sentinel lymph node (SLN), micrometastases are frequently observed. If micrometastases are clinically relevant, the histopathologic examination of SLNs should be sensitive enough to detect them. The probability of detecting micrometastases was calculated when examining the SLN according to the current Dutch pathology protocol and strategies evaluated to optimize the chance of detection. The dimensions of 20 consecutive axillary SLNs in patients with cT1-2N0 breast cancer were measured. In a mathematical model, the probability of detecting micrometastases in a SLN was calculated. Similarly, strategies to optimize the probability of detecting micrometastases were explored. When applying the pathology guidelines, the calculated probability to detect a micrometastasis was 18% for a 200-microm micrometastasis and 69% for a 2.0-mm metastasis in a median sized SLN. To detect the smallest micrometastasis in a median-sized SLN with a 95% probability, the interval between the sections must be decreased to 200 microm, and 20 levels from both halves must be examined. Given a prognostic significance of micrometastases, our current pathology guidelines are not sensitive enough. The number of sections should be increased, while the interval between cuts should be no more than 200 microm.     

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.     

Eight-year experience with the intraoperative frozen section examination of sentinel lymph node biopsy for breast cancer in a North-Italian university center

Sentinel lymph node biopsy (SLNB) completely changed the impact of breast surgery on patients psycho-physical wellness, reducing morbidity associated with complete axillary lymph node dissection (CALND) while granting an adequate breast cancer staging. We reviewed our experience with the SLNB in a University Clinic. We collected data about all breast cancer patients submitted to SLNB from 2002 to 2010, and analyzed them with R (version 2.15.2), considering significant p<0.05. We performed 615 SLNBs on 607 patients, with a mean age of 59.86 (±10.76). Sentinel node detection rate resulted 99,7%, with a mean number of biopsied nodes of 1.64 (±0.67), axillary localization in 98% of cases, and negative intraoperative histological finding in the 86.2% of cases. Prevalence of ITCs, micrometastasis, macrometastasis and pericapsular metastasis resulted respectively 0.6%, 4.9%, 7.5% and 8.8%. Among women who received CALND, mean number of examined nodes was 16.36 (±6.19) and mean number of metastatic non-sentinel nodes was 0.97 in case of micrometastasis, 2.65 in case of macrometastasis, and up to 9.88 when pericapsular invasion was described. To conclude, our data confirm the role of nodal metastasis size in the prediction of non-sentinel node involvement, but further studies are required in order to better assess the role of ITCs and micrometastasis in the diagnostic and therapeutic management of breast cancer, with the final aim to reduce the surgical complications of axilla demolition when unnecessary.     

影响乳腺癌前哨淋巴结数目的临床病理特征分析

目的分析影响乳腺癌前哨淋巴结数目的相关因素,探讨最佳的前哨淋巴结活检值。方法回顾性分析2007年1月-2011年12月中国医学科学院肿瘤医院乳腺癌前哨淋巴结活检病例578例。采用Logistic回归模型分析前哨淋巴结数目与临床病理特征的相关性。结果全组女性,平均年龄49.9(21～90)岁。总共获得2 222枚前哨淋巴结,平均每例3.8枚(1～15)。淋巴结转移率17.8%(103/578),转移组和无转移组淋巴结数目无差异。单因素分析显示,术式、显像方法和体质指数影响前哨淋巴结数目(P<0.05)。多因素分析中,单纯乳房切除、联合显像、BMI≤30者前哨淋巴结较多(P<0.05)。前哨淋巴结限于5枚时,转移病例检出率100%。18.7%(108/578)病例不必继续送检淋巴结,298枚淋巴结免于切除。结论乳腺癌前哨淋巴结活检数量受到显像方法、乳腺术式和体质指数的影响,5枚前哨淋巴结可能是一个比较合适的参考标准。     

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.     

Intraoperative cytologic evaluation of sentinel lymph nodes in patients with breast carcinoma by scrape preparation

Intraoperative evaluation of sentinel lymph nodes (SLNs) in patients with breast carcinoma allows surgeons to complete axillary lymph node dissection in one procedure if any SLN shows metastasis. The accuracy of intraoperative pathological diagnosis is critical for decision-making. The purpose of this study was to evaluate our rapid intraoperative cytologic diagnosis of SLN through comparing with the final surgical pathologic diagnosis of the corresponding lymph nodes. A total of 454 SLNs from 159 consecutive female patients with a preoperative diagnosis of breast carcinoma over 3-year period were included in this study. After gross examination of each bisected lymph node, a scrape preparation was prepared for each submitted lymph node and was stained by the rapid Papanicolaou method. The intraoperative cytologic diagnosis was compared with the final surgical pathologic diagnoses. The overall sensitivity of intraoperative cytology was 52.5% with specificity of 100%. There were 17 false-negative cases. Of them, six nodes had isolated tumor cells, seven nodes had micrometastasis (0.2-2 mm), and four nodes had macrometastasis (>2 mm). There were no interpretive errors identified. The size of metastasis and tumor grade appeared to be significant factors in detecting metastasis by cytology. In addition, subsequent non-SLN involvement was 9% in patients with micrometastasis versus 50% in patients with macrometastasis (P < 0.05). Our study shows that the intraoperative cytologic evaluation of SLNs in breast carcinoma is a reasonably accurate method. The majority of false-negative cases were due to micrometastasis and isolated tumor cells.     

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.     

False-positive cells in sentinel lymph nodes

Melanoma sentinel lymph nodes (SLN) are carefully evaluated to maximize sensitivity. Examination includes hematoxylin and eosin (H+E) stained sections at multiple levels through the node, with subsequent immunohistochemical (IHC) stains for melanocytic markers if H+E sections are negative for melanoma. However, not all IHC-positive cells in SLN are metastatic melanoma, as evidenced by the presence of MART-1 positive cells in SLN from breast cancer patients with no history of melanoma (so-called 'false-positive' cells). These 'false-positive cells' could be nodal nevus, non-melanocytic cells with cross-reacting antigenic determinants, phagocytic cells containing melanocyte antigens, or possibly melanocytes or melanocyte stem cells liberated at the time of biopsy of the cutaneous melanoma. Examination of SLN requires careful correlation of H+E and IHC findings.     

Combined application of RT-PCR and immunohistochemistry on paraffin embedded sentinel lymph nodes of prostate cancer patients

The detection of tumor cells in the sentinel lymph node (SLN) is of great importance for the prognosis of cancer patients. At present, immunohistochemistry and RT-PCR for tumor marker expression are the most sensitive techniques available for this analysis. However, so far, most RT-PCR-based analyses of SLNs have been performed on fresh material, excluding a direct comparison with the (immuno)histologic results. In our view, this does not entirely aid routine diagnosis. We established an efficient method for RNA extraction and RT-PCR from paraffin sections of SLNs from prostate cancer patients and compared the results with the (immuno)histologic data of adjacent sections. Amplifiable RNA was obtained from 133 SLNs of 68 prostate cancer patients. Correlation of PSA-specific RT-PCR with (immuno)histologic findings showed a positive and negative predictive value of 83% and 100%, respectively, for the prostate cancer patients investigated. Four of 12 patients with biochemical relapse, but without (immuno)histologically detectable tumor cells were RT-PCR-positive for PSA. We found that single sections of paraffin-embedded SLNs are suitable for routinely performed RT-PCR. Combined with (immuno)histology, PSA-specific RT-PCR is a revealing supplementary technique for the detection of tumor cells in SLNs of prostate cancer patients.     

Reliability of intraoperative frozen section and imprint cytological investigation of sentinel lymph nodes in breast cancer

AIMS: The sentinel lymph node procedure enables selective targeting of the first draining lymph node, where the initial metastases will form. A negative sentinel node (SN) predicts the absence of tumour metastases in the other regional lymph nodes with high accuracy. This means that in the case of a negative SN, regional lymph node dissection is no longer necessary. Besides saving costs, this will prevent many side-effects of lymph node dissection. The aim of this study was to evaluate the reliability of intraoperative cytological and frozen section investigation of the SN to detect metastases. This would allow the axillary lymph node dissection to be performed in the same session as the SN procedure and the excision of the primary tumour in case of a positive SN. METHODS AND RESULTS: Seventy-four SNs were detected by gamma probe detection of nanocolloid and visual localization of Patent Blue accumulations in 54 women with stage T1-2N0M0 invasive breast cancer. The identified SN were immediately investigated by frozen section and imprint cytological investigation. Diagnoses were confirmed on the paraffin material, and in case of negative frozen section and paraffin haematoxylin and eosin sections, skip sections and immunohistochemistry were performed. Thirty-one SNs (42%) contained metastases, of which 27 were detected by the frozen section procedure (sensitivity 87%). There were no false positives (specificity 100%). The sensitivity of the imprints was 62% with a specificity of 100%. When evaluating the data per patient, for the frozen section procedure the sensitivity was 91% and the specificity 100%, and for the imprints, the sensitivity was 63% and the specificity 100%. There were no SNs in which the imprints showed metastases and the frozen section did not. CONCLUSIONS: Intraoperative frozen section analysis is a reliable procedure by which a high percentage of sentinel lymph node metastases can be detected in breast cancer patients without false positive results. This allows the surgeon to perform an immediate axillary lymph node dissection in case of positive SNs. In up to 10% of cases, the final paraffin sections will reveal micrometastases that were not detected by the frozen section, and in these patients axillary lymph node dissection will have to be performed in a second session. The imprint method is significantly less sensitive than the frozen section but may be used as an alternative when frozen section is not possible.     

A mathematical approach to the optimal examination of lymph nodes

There is no scientific evidence to support the idea that serial sectioning along the short axis of the lymph node is superior to a single bisection along the long axis. We mathematically evaluated methods of lymph node dissection and applied the result to six lymph nodes that had produced false negative results at the time of frozen examination. We simplified the geometry of a lymph node to that of a three-dimensional ellipse and compared two different cutting methods. Let A be the cross-sectional area obtained through a single bisection along the long axis, and let B be the sum of the cross-sectional areas of n fragments obtained via serial cutting along the short axis. The smallest n (n*) that makes a B larger than A can be calculated. n* = [3L + √9L2 + 16S2)/4S]. ([α], the smallest integer greater than or equal to α; L, long axis; S, short axis). The probabilities of tumor detection when the node is bisected along the long axis (P(D(A)|E)) and when serially cut along the short axis (P(D(B)|E)) are as follows. P(D(A)|E = {(3/2)S2 - 3ST + T2}T/(S - T)3. and P(D(B)|E) = (n - 1){(1 + 1/n)L2 - 3LT + T2}T/(L - T)3. (T, size of the tumor cell cluster). According to these formulas, three out of six lymph nodes were not examined in the most appropriate manner.     

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.     

Use of intraoperative stereomicroscopy for preventing loss of metastases during frozen sectioning of sentinel lymph nodes in breast cancer

Aims:  Optimal detection of metastases in sentinel lymph nodes (SLN) remains controversial. To determine the reliability of intraoperative frozen sections, SLN protocol with one frozen section was compared with macroscopic SLN evaluation with consecutive complete SLN embedding.
Methods and results:  SLN from 135 consecutive breast cancer patients were analysed under a sereomicroscope. Frozen sections were performed in suspicious or clearly involved SLN on cut surface. One control group ( n  = 143) underwent one intraoperative frozen section on each SLN. The second control group ( n  = 90) was subjected to stereomicroscopy and one intraoperative frozen section on each SLN. A conventional SLN protocol with cytokeratin immunohistochemistry was performed postoperatively in all cases. All groups were statistically comparable. In the study group metastases were suspected in 21 SLN (16%) under the stereomicroscope and all were confirmed histologically. The negative SLN rate was significantly lower in the study group than in the main control group (47% versus 64%, P  = 0.008), suggesting loss of metastases during frozen sections. More macrometastases were detected in the study group (30% versus 15%, P  = 0.006); there were no differences in isolated tumour cells or micrometastases. The false-negative rate was significantly lower in the control groups (29% versus 13% and 12%, P  = 0.001).
Conclusions:  Frozen sections potentially lead to loss or reduced size of metastatic deposits in SLN. Avoiding intraoperative frozen sections on grossly inconspicuous SLN may therefore be justified.     

Use of frozen sections in the examination of sentinel lymph nodes in patients with melanoma

This article examines the different views regarding the use of frozen sections and other rapid techniques in sentinel lymph nodes from patients with cutaneous melanoma. Special attention is given to the pros and cons of such techniques.     

乳腺癌前哨淋巴结活检术中不同染色淋巴结与肿瘤转移的关系

目的探讨乳腺癌前哨淋巴结活检术(SLNB)中不同染色情况的淋巴结与肿瘤转移的关系。方法选择我院2014年1月至2018年1月行前哨淋巴结活检的乳腺癌患者92例,以亚甲蓝为示踪剂,根据92例乳腺癌患者SLNB中淋巴结染色情况的不同分为无染色组、完全染色组和染色不均组,病理检测3组患者淋巴结的肿瘤转移情况并作比较。结果92例乳腺癌SLNB共取得淋巴结256枚,平均每例患者2.8枚,无染色组(80枚)肿瘤转移率为13.8%,完全染色组(112枚)肿瘤转移率为43.8%,染色不均组(64枚)肿瘤转移率为62.5%,3组间肿瘤转移率差异有统计学意义(P<0.05)。结论乳腺癌SLNB中染色不均的淋巴结最易出现肿瘤转移,其次为完全染色的淋巴结,染色淋巴结附近看到的未染色淋巴结也有肿瘤转移的可能,宜一并切除送检,有利于降低假阴性率。     

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.