BRCA2 monoclonal antibodies react with differentiating epithelium

The BRCA2 gene has previously been suggested to play a role in proliferation and DNA repair. Germline mutations in the BRCA2 gene predispose individuals to early onset, hereditary breast cancer. To better understand the expression pattern and function of the BRCA2 gene product, we have developed immunological reagents specific for BRCA2. These reagents recognize full-length (384 kDa) recombinant human BRCA2 proteins in transfected cell lysates as well as multiple smaller recombinant BRCA2 polypeptides. Detection of native BRCA2 protein in most tissue types, including breast epithelium, requires sensitive techniques such as immunoprecipitation-Western blot analysis. However, we have demonstrated strong reactivity of our immunological reagents with differentiating epithelium, including epidermis, thymic epithelium, and squamous cell carcinoma. These data suggest that BRCA2 may play a role in processes associated with cellular differentiation, in addition to its previously suggested roles in proliferation and DNA repair.     

Human stomach carcinoma-specific T cells derived from the tumour-draining lymph nodes.

In this paper we investigate the reactivity pattern of T cells from stomach carcinoma patients against autologous tumour cells. T cells obtained from the tumour environment, tumour-draining lymph nodes and peripheral blood were cloned in 78 patients with stomach cancer and anti-tumour cytotoxic T lymphocytes (CTLs) precursor frequencies were assessed in each sample by using limiting dilution analysis. When tumour-specific CTLs were tested for specific T-cell killing by using only low doses of Interleukin 2 (100 U ml-1), a moderate rate of proliferation frequency of T cells (0.047) and specific cytotoxicity (12%) were observed in lymph node populations. When both IL-2 and autologous tumour cells in mixed lymphocyte tumour cultures (MLTCs) were used for stimulation, a dramatic increase in number (0.1) and in specific lytic activity (46%) could be measured. No effect or specific activity to tumour cells was observed with peripheral blood lymphocytes and tumour-infiltrating lymphocytes.     

The use of lymphocytes from axillary lymph nodes of mastectomy patients to generate human monoclonal antibodies

Lymphocytes from axillary lymph nodes of breast cancer mastectomy patients were fused with murine non-immunoglobulin-secreting myeloma cells to generate hybridoma cell lines that synthesize human immunoglobulins. Lymph node lymphocytes from 21 patients were used to obtain 505 human-mouse hybrid cultures. From these, 62 cultures were established which synthesized immunoglobulins reactive in radioimmunoassays specific for either human IgG or human IgM. Some of these double-cloned hybrid cell lines produced human monoclonal antibodies for at least 6 months. Sodium dodecylsulfate polyacrylamide gel electrophoresis and immunodiffusion analysis demonstrated that the monoclonal antibodies possessed human heavy and light immunoglobulin chains. Levels of synthesis ranged from 0.1 to 20 μg of human immunoglobulin per ml of culture fluid. The immunoreactivity of some of these human monoclonal antibodies with mammary carcinoma cells is summarized and has been documented elsewhere (J. Schlom, D. Wunderlich and Y. A. Teramoto. Proc. Natl Acad Sci USA 1980;77: 6841); the reactivity of the majority of the immunoglobulins, however, is still unknown at this time. The studies reported here detail the procedures in which axillary lymph nodes from mastectomy patients are used in the generation of human-mouse hybridomas that synthesize human monoclonal antibodies.     

Detection of parasternal metastatic lymph nodes by sentinel lymph node methods in a patient with recurrence in the conserved breast

We herein report a case of second sentinel lymph node biopsy (SLNB). A 57-year-old woman underwent breast-conserving surgery including axillary clearance at Aichi Cancer Center on October 20, 2003. Recurrent tumor in the conserved breast was diagnosed in March 2006. She received SLNB using radioactive tracer. Preoperative lymphoscintigraphy detected 2 parasternal lymph nodes as hot spots. No abnormal lymph nodes were revealed on preoperative computed tomography. Salvage mastectomy was performed along with dissection of the Rotter and infraclavicular lymph nodes and biopsy of the detected parasternal lymph nodes. Micrometastases were discovered in both parasternal lymph nodes detected as sentinel lymph nodes. No more metastases were seen in the other lymph nodes. Reoperative SLNB offers the possibility of detecting metastasis in residual lymph nodes and determining whether chemotherapy should be used.     

Detection of micrometastases in sentinel lymph nodes of the breast applying monoclonal antibodies AE1/AE3 to pancytokeratins

Sentinel lymph node excision in breast cancer is a minimally invasive diagnostic procedure for accurate staging of the axilla and for avoiding unnecessary axillary dissection. In patients with palpable breast cancer we injected microcolloidal particles of human serum albumin labelled with technetium-99m the day before surgery. The sentinel node was detected intraoperatively with a handheld gammaprobe and then removed. Complete axillary dissection was performed and the nodes inspected by routine histological examination. The axillary lymph node status was correctly predicted by the sentinel node technique in 32 of 33 breast cancer patients. Two cases of micrometastases escaped routine histopathological detection but were identified by immunohistochemical analysis applying the antibody AE1/AE3 to pancytokeratins. Immunohistochemical examination of the sentinel node improves the diagnostic security of patients with breast carcinoma by detection of micrometastases.     

子宫颈癌患者血清、盆腔淋巴结HPV检测及其相关性探讨

目的:检测宫颈癌患者原发灶、血清及其盆腔淋巴结中HPV DNA及亚型,探讨其相关性及临床意义。方法:选取16例行广泛全子宫切除术和盆腔淋巴结清扫术宫颈癌患者的原发灶组织、术前静脉血与盆腔淋巴结石蜡组织,运用PCR方法对上述标本进行HPV DNA及亚型的检测。结果:宫颈癌原发灶组织、血清标本中HPV DNA阳性率为50%(8/16)。16例盆腔淋巴结石蜡组织中13例为HPV DNA阳性(13/16,81.25%),其中总共切除的133个淋巴结中60个为阳性(60/133,45.1%),8例淋巴结HPV DNA阳性的病例其对应的原发灶组织也为阳性且两者亚型相同。盆腔淋巴结中的HPV DNA阳性率为45.1%(60/133),显著高于病理证实的淋巴结转移率1.5%(2/133)。6例患者(6/16,37.5%)原发灶、血清、盆腔淋巴结同时均为HPV DNA阳性;2例患者(2/16,12.5%)原发灶、盆腔淋巴结中HPV DNA表达阳性,而血清为阴性;1例患者(1/16,6.25%)淋巴结、血清HPV DNA阳性,而原发灶为阴性;未发现原发灶、血清HPV DNA表达阳性而淋巴结为阴性的病例,而且以上HPV DNA阳性的病例同一个患者对应的HPV亚型也相同。结论:宫颈癌患者血清中HPV DNA检出率与临床分期无关。宫颈癌患者盆腔淋巴结的HPV DNA检测可提高病理诊断淋巴结转移的阳性率,并且淋巴结中HPV DNA的检出率与原发灶的分化程度相关。宫颈癌原发灶、血清、盆腔淋巴结中HPV感染可能存在相关性。     

Microinvasive breast carcinoma with extensive involvement of level III axillary lymph nodes: a case report

A 44-year-old woman presented with a right huge axillary mass. Both mammography and ultrasonography revealed a primary cancer of 2.8 cm maximum diameter in the right breast and metastases in the axillary lymph nodes, both being confirmed by aspiration cytology as ductal carcinoma. Right standard radical mastectomy with level III axillary lymph node dissection was carried out. Pathologically, the tumor was diagnosed as ductal carcinoma in situ with microinvasion (DCISM), histologic grade 3. The area of stromal invasion measured 1 mm at its widest point. Sixteen of the 17 resected axillary lymph nodes contained metastases, including six level III lymph nodes. Immunohistochemical studies of the tumor revealed overexpression of p53 protein, but not that of c-erbB-2 protein. The frequency of lymph node metastases from DCISM is reported to be very low. Therefore, the present case with extensive involvement of level III lymph nodes was unusual.      

A high number of tumor free axillary lymph nodes from patients with lymph node negative breast carcinoma is associated with poor outcome

BACKGROUND: Lymph node metastasis is the oldest and most reliable prognostic indicator in breast carcinoma. In the absence of tumor metastasis, draining lymph nodes can undergo hyperplasia, resulting in increases in the number and size of detectable lymph nodes. The prognostic value of this process has never been established. Lymph node negative breast carcinoma provides a unique opportunity to study the downstream effects of increased lymphatic drainage and lymph node hyperplasia. METHODS: The authors studied 290 cases of lymph node negative breast carcinoma and provided patients with a median of 103 months of follow-up. The number of tumor free lymph nodes in ipsilateral axillary resections, as well as 10 traditional histopathologic markers, were analyzed for their prognostic value. RESULTS: The cohort was divided into quartiles according to the number of tumor negative lymph nodes. The 5-year survival for patients with 20 or more tumor free lymph nodes (top quartile) was 84.7%, compared with 96.3% for patients with fewer than 20 tumor free lymph nodes. The 5-year relative risk of dying of metastatic disease in the top quartile was 3.61 (95% confidence interval, 1.37-9.52, P = 0.01), independent of necrosis, tumor size, patient age, nuclear and histologic grade, lymphocytic infiltrate, and lymphovascular invasion. The absolute lymph node number was highly associated with the presence of necrosis in invasive tumor. CONCLUSIONS: The number of tumor free lymph nodes is a novel, independent predictor of aggressive disease in cases of lymph node negative breast carcinoma. This finding may be a biologic function of host-derived, and possibly tumor-derived, lymphangiogenic cytokines.     

Prognostic significance of the number of lymph nodes examined in patients with lymph node-negative breast carcinoma.

BACKGROUND: A recent report suggested that the number of lymph nodes examined was a strong predictor of survival in patients with lymph node-negative breast carcinoma. Among women who had >or= 20 lymph nodes examined, the risk of dying from breast carcinoma within 5 years was increased nearly 4-fold compared with women who had fewer lymph nodes examined. Because these findings were based on a relatively small cohort of patients, corroborative studies with larger patient populations were needed. METHODS: The authors studied the relation between the number of lymph nodes examined and breast carcinoma survival among 911 women with lymph node-negative breast carcinoma with a median length of follow-up of 84 months. The association between other prognostic indicators and survival and the number of lymph nodes examined also was investigated. RESULTS: The number of lymph nodes examined was not found to be associated with either 5-year or long-term survival. The proportion of women dying from breast carcinoma was the same (8%) in both groups (those patients with >or= 20 lymph nodes examined vs. those in whom < 20 lymph nodes were examined) and the hazard ratio was 0.98 (95% confidence interval, 0.58-1.64). CONCLUSIONS: In this larger study population, the authors failed to confirm the findings of an earlier investigation in which having a larger number of lymph nodes examined was associated with poorer survival. This finding suggests that it is unlikely the number of lymph nodes examined is an important prognostic indicator in patients with lymph node-negative breast carcinoma.     

Intraoperative touch imprint of sentinel lymph nodes in breast carcinoma patients

BACKGROUND: Sentinel lymph node examination in patients with breast carcinoma has been gaining in popularity. Currently, there is no standard intraoperative assessment of sentinel lymph nodes. To assess the utility of an intraoperative touch imprint (TI) evaluation, the authors compared TI cytology with surface hematoxylin and eosin (H&E) histology in sentinel lymph nodes from patients with breast carcinoma. METHODS: Sixty five sentinel lymph node biopsy cases were identified. Diagnoses from TI and surface H&E histologic sections were compared. RESULTS: Touch imprint had a specificity of 100%, a negative predictive value of 88%, a sensitivity of 65%, and a false negative rate of 9% per sentinel lymph node biopsy case. Eighty three percent of the false negative TI cases were due to micrometastasis. Preoperative chemotherapy, primary tumor type, and primary tumor size did not significantly contribute to false negative events. Touch imprint identified 67% of the cases that required completion axillary dissection. CONCLUSIONS: Touch imprint is a reliable and accurate intraoperative technique, with the potential to save a significant number of patients morbidity and the cost of a second surgical procedure to remove axillary lymph nodes. The difficulty of identifying micrometastases appeared to be the major source of false negative events, a problem that is not unique to TI cytology.     

Histopathologic examination of axillary sentinel lymph nodes in breast carcinoma patients

The axillary sentinel lymph node biopsy (SLNB) has gained increasing popularity as a novel surgical approach for staging patients with breast carcinoma and for guiding the choice of adjuvant therapy with minimal morbidity. Patients with negative SLNB represent a subset of breast carcinoma patients with definitely better prognosis, because their pN0 status is based on a very thorough examination of the sentinel lymph nodes (SLNs), with a very low risk of missing even small micrometastatic deposits, as compared with routine examination of the 20 or 30 lymph nodes obtained by the traditional axillary clearing. The histopathologic examination of the SLNs may be performed after fixation and embedding in paraffin, or intraoperatively on frozen sections. Whatever is the preferred tracing technique and surgical procedure, the histopathologic examination of each SLN must be particularly accurate, to avoid a false-negative diagnosis. Unfortunately, because of the lack of standardised guidelines or protocols for SLN examination, different institutions still adopt their own working protocols, which differ substantially in the number of sections cut and examined, in the cutting intervals (ranging from 50 to more than 250 microm), and in the more or less extensive use of immunohistochemical assays for the detection of micrometastatic disease. Herein, a very stringent protocol for the examination of the axillary SLN is reported, which is applied either to frozen SLN for the intraoperative diagnosis, and to fixed and embedded SLN as well.     

超声造影对乳腺癌腋窝淋巴结转移的诊断价值

目的探讨超声造影对诊断乳腺癌腋窝淋巴结转移的应用价值。方法对141例浸润型乳腺癌患者行乳腺病灶及腋窝淋巴结常规超声检查后,再对腋窝淋巴结进行超声造影,先用目测法观察淋巴结超声造影增强模式,再用QontraXt软件分析超声造影时间-强度曲线参数。根据超声造影灌注特点,将腋窝淋巴结分为淋巴结转移组（有转移组）和无淋巴结转移组（无转移组）,并与病理检查结果相比较。增强模式之间的对比采用x2检验,造影参数用单因素方差分析。结果淋巴结有转移组灌注模式表现为不均匀增强型或无增强,淋巴结无转移组表现为均匀型增强,两组灌注模式之间的差异有统计学意义（P=0．000）。两组造影剂到达时间、达峰时间、峰值强度之间的差异无统计学意义（P值分别为0．129、0．094、0．140）。淋巴结实质内高灌注区与低灌注区的差值（SImax-SImin）有转移组大于无转移组（P=0．000）。以SImax-SImin）值大于28为最佳临界点,鉴别的灵敏度为93．3％,特异度为80．8％。结论超声造影对鉴别乳腺癌腋窝淋巴结转移有-定的临床价值。     

Prognostic value of micrometastases in sentinel lymph nodes of patients with breast carcinoma: a cohort study

Background: The prognostic meaning and thus indication for adjuvanttherapy of lymphogenic micrometastases in breast cancer patientsis still under debate. Patients and methods: From 1999 to 2007, 703 patients with _cT_1–2N₀breast cancer underwent surgery including sentinel lymph nodebiopsy. Examination of sentinel lymph nodes consisted of hematoxylinand eosin and immunohistochemistry staining following serialsectioning of the sentinel node. Patients were divided intofour groups: _pN₀ (n = 423), _pN_1micro (n = 81), _pN_1a (n = 130)and _pN_1b (n = 69). Median follow-up was 40 months. Results: At the end of follow-up, 53 patients had died and 64had recurrent disease. Compared with _pN₀ and following adjustmentfor possible confounders, including adjuvant systemic treatment,overall survival was not significantly different for _pN_1microwhile significantly worse for _pN_1a and _pN_1b {hazard ratio (HR)[95% confidence interval (CI)]: 0.59 [0.14–2.58], 4.31[1.85–10.01], 10.66 [4.04–28.14], respectively}.Likewise, disease-free survival was not significantly differentfor _pN_1micro and worse for _pN_1a and _pN_1b (HR [95% CI]: 1.43[0.67–3.02], 2.79 [1.37–5.66], 7.13 [3.27–15.54],respectively). Distant metastases were more commonly observedin the _pN_1micro than in the _pN₀ group, but still not as commonas in the _pN_1a or _pN_1b group (HR [95% CI]: 4.85 [1.79–13.18],10.34 [3.82–28.00], 23.25 [7.88–68.56], respectively). Conclusion: Although the risk of distant metastases was higherin patients in the _pN_1micro than in the _pN₀ group, no statisticallysignificant differences were observed in overall or disease-freesurvival between _pN₀ and _pN_1micro. Micrometastatic lymph nodeinvolvement in itself should not be an indication for adjuvantchemotherapy in breast cancer patients. Key words: breast cancer, micrometastases, prognosis, sentinel lymph node Received for publication March 11, 2008. Revision received June 25, 2008. Accepted for publication July 1, 2008.     

The detection of axillary lymph node metastases from breast cancer by radiolabelled monoclonal antibodies: a prospective study

In a prospective study to assess the accuracy of monoclonal immunoscintigraphy for the detection of axillary lymph node metastases in breast cancer, two murine monoclonal antibodies that react with human breast cancer (3E1.2 and RCC-1) were labelled with 131iodine, and the radiolabelled antibody was injected subcutaneously into the interdigital spaces of both hands of 40 patients, 36 of whom had breast cancer and the remaining four of whom had fibroadenoma (the normal, contralateral axilla was used as a control). Of the patients with breast cancer, the findings from the scintigraphy images were correlated with histopathology or cytology of the axillary lymph nodes; images were regarded as positive and hence indicative of lymph node metastases if the amount of background-subtracted radioactive count in axilla on the side of breast cancer exceeded the contralateral normal side by a ratio greater than or equal to 1.5:1.0 as assessed by computer analysis. Using this method, immunoscintigraphy had an overall sensitivity of 33% (23% with 131I-3E1.2 and 50% with 131I-RCC-1) for the detection of lymph node metastases and a specificity of 63% (67% with 131I-3E1.2 and 60% with 131I-RCC-1) with problems of non-specific uptake by presumably normal lymph nodes. The results of immunoscintigraphy obtained with 131I-RCC-1 (IgG) were superior to 131I-3E1.2 (IgM) although the accuracy of immunoscintigraphy using 131I-RCC-1 (56%) was not much better than preoperative clinical assessment (50%). However, there were cases when immunoscintigraphy using radiolabelled antibody (IgM or IgG) detected axillary lymph node metastases not suspected by clinical examination. Thus it appears that while immunoscintigraphy may be a useful adjunct to preoperative clinical assessment and is simple and safe, a major improvement in its accuracy is needed before it can replace axillary dissection and histological examination in the accurate staging of axilla in breast cancer.