免疫组织化学和RT-PCR法检测乳腺癌骨髓和前哨淋巴结微小转移的临床研究

目的探讨免疫组织化学(IHC)和RT-PCR法检测乳腺癌骨髓和前哨淋巴结(SLN)微小转移的灵敏度及临床意义。方法留取乳腺癌改良根治术腋窝淋巴结HE染色证实阴性的病人的胸骨骨髓血和SLN,分别采用IHC和RT-PCR方法检测其微小转移情况。结果62例中,骨髓样本RT-PCR检测15例阳性表达,其中9例IHC检测也为阳性,二者结果有较好一致性(kappa=0.6945),检出率有统计学差异(P=0.0412);SLN样本RT-PCR法有13例KT19mRNA表达,其中7例IHC检出KT19阳性细胞,二者结果一致性较好(kappa=0.6483),检出率有统计学差异(P=0.0412);骨髓和SLN同时表达KT19mRNA仅3例,无显著相关(P=0.796);原发肿瘤大小和骨髓KT19mRNA表达率有关联(P=0.003)。结论常规检查未发现远处和腋窝淋巴结转移,骨髓和SLN可检出微小转移,RT-PCR较IHC更灵敏,肿瘤大小与骨髓微小转移有关联。由于骨髓和腋窝淋巴结微小转移不一定同步出现,选用灵敏方法对不同组织同时进行检测可能更具临床价值。     

乳腺癌组织与腋窝淋巴结中乳腺球蛋白的表达及其临床意义

目的:探讨乳腺球蛋白(MAM)在乳腺癌组织和腋窝淋巴结中的表达及其临床意义。方法:应用免疫组化法和RT-PCR法检测乳腺癌组织、非乳腺癌组织、乳腺癌腋窝淋巴结的MAM和MAM mRNA表达。结果:(1)MAM在乳腺癌组织表达的敏感度为88.8%,特异度为89.3%。MAM在乳腺癌组织和非乳腺癌组织中表达的差别有统计学意义(P<0.01)。(2)MAM的阳性表达与ER、PR状态(P=0.022和0.010)、组织分级(P=0.012)有关,与诊断时肿瘤大小、有无淋巴结转移、C-erbB-2状态、年龄无关。(3)MAMmRNA在乳腺癌腋窝淋巴结表达的灵敏度为90%,特异度为68.7%,阳性预测值为64.3%,阴性预测值为91.7%。结论:MAM是乳腺癌敏感且特异的标志物之一,有助于判断腋窝淋巴结转移存在。     

巢式RT-PCR检测乳腺癌前哨淋巴结微转移的研究

目的：探讨乳腺癌前哨淋巴站（SLN）定位和腋淋巴结微转移检测的临床意义,方法：对20例乳腺癌患者术中肿块周围注射美蓝定位前哨淋巴 结,用巢式RT－PCR法检测腋淋巴结中Mammaglobin mRNA的表达,结果：SLN定位成功率为85．0％（17／20）,SLN与非SLN组微转移的检出率具显著差异（P＜0．01）。在常规病检阴性的淋巴结中,巢式RT－PCR法微转移的检出率为15．6％（17／109）,结论：巢式RT－PCR法较常规病理检查更为敏感,通过SLN定位和巢式RT－PCR的联合使用,可明显提高乳腺癌腋淋巴结微转移的检出效率。     

Outcomes of a population-based series of early breast cancer patients with micrometastases and isolated tumour cells in axillary lymph nodes

BackgroundAxillary lymph node staging is traditionally important to provide prognostic information to guide further treatment. However, the relevance of isolated tumour cells (ITC) or micrometastases in axillary nodes and the need for adjuvant treatment remain uncertain.Patients and methodsData from 18 370 patients with pT1–2 breast cancer with pN0, pN0i+ or pN1mi were analysed. The primary end point was 5-year disease-free survival (locoregional recurrence, distant metastases or contralateral breast cancer).ResultsFive-year disease-free survival was 89.9% [95% confidence interval 89.5% to 90.4%]; and did not differ significantly between groups. After adjusting for prognostic factors (including treatment), patients with ITC had a comparable risk (hazard ratio = 1.12) as patients with node-negative disease, while patients with micrometastases had a 38% higher risk of recurrence.Conclusion(s)Patients with ITC and node-negative breast cancer appear to have similar prognosis, and those with micrometastases have a 38% higher risk of tumour recurrence. However, considering that disease-free survival is already high, we are reluctant to advise chemotherapy in all patients with ITC or micrometastases. In future, genomic tumour characteristics might predict the propensity of dissemination from the primary cancer better than the status of the axillary lymph nodes.     

Minimal axillary lymph node involvement in breast cancer has different prognostic implications according to the staging procedure

It is still controversial whether the identification of micrometastases and isolated tumor cells in the axillary lymph nodes of patients with breast cancer has any prognostic value. We evaluated the prognostic role of isolated tumor cells and micrometastases in the axillary lymph nodes in 3,158 consecutive patients pT1-2 pN0-N1mi (with a single involved lymph node) and M0, referred to the Division of Medical Oncology after surgery performed at the European Institute of Oncology from April 1997 to December 2002. Median follow-up was 6.3 years (range 0.1–11 years). Sentinel lymph node biopsy (SLNB) and axillary lymph node dissection (ALND) were performed in 2,087 and 1,071 patients, respectively. A worse metastasis-free survival was observed for patients with micrometastatic disease compared to node-negative patients, if staged with ALND (log-rank P < .0001; HR: 3.17; 95% CI 1.72–5.83 at multivariate analysis), but not for patients who underwent SLNB (log-rank P = 0.36). The presence of a single micrometastatic lymph node is associated with a higher risk of distant recurrence as compared to node-negative disease only for patients undergoing ALND for staging purposes. Treatment recommendations for systemic therapy should not take into account the presence of a single micrometastatic lymph node identified during complete serial sectioning of sentinel node(s).     

T1 breast cancer: identification of patients at low risk of axillary lymph node metastases

Objective. The status of the axillary lymph nodes is one of the most important prognostic factors in patients with breast cancer. A panel of molecular markers of tumor aggressiveness in addition to conventional clinical and histopathologic features were analyzed in an attempt to identify a subgroup of patients with a low risk of axillary lymph node metastases. Material and methods. Data from 358 patients with T1 breast cancer who underwent level I/II axillary lymph node dissection (ALND) were investigated. Hormone receptor status, Ki-67, S-phase fraction, DNA ploidy, HER-2/neu, p53, epidermal growth factor receptor, urokinase type plasminogen activator, plasminogen activator inhibitor-1, bone marrow micrometastases as well as patient age, menopausal status, tumor site, tumor size, histologic type, tumor grade, carcinoma in situ, multifocality, and lymph vascular invasion (LVI) were studied to predict axillary lymph node status. Results. In a multivariate logistic regression analysis LVI (present v.s. not present), Ki-67 (18% v.s. <18%), tumor size (1.1–2 cm v.s. 1 cm), and histologic grade (G3 v.s. G1/2) were identified as independent predictive factors of axillary lymph node metastases. Approximately 13% of patients (n = 47) with well or moderately differentiated tumors less than or equal to 1 cm, no lymph vascular invasion, and a low Ki-67 staining were identified as having a low risk of axillary lymph node metastases of 4.3%. However, 20 patients with all four unfavorable predictive factors had a 75% incidence of axillary lymph node involvement. Conclusion. Primary tumor characteristics can be used to identify a subgroup of patients with a low risk of axillary lymph node metastases in T1 breast cancer. Preoperative risk assessment might be used to omit routine ALND in those patients at low risk of axillary lymph node metastases.     

The prognostic significance of marrow micrometastases in women with early breast cancer

Twenty-five patients with early breast cancer (T0-T2, N0-N1, M0) have been studied prospectively to determine the relationship between marrow micrometastases, disease-free interval and survival. Marrow specimens were aspirated from three sites immediately prior to breast surgery. An immunocytochemical technique using monoclonal antibody LICR.LON.M8.4 was employed to detect micrometastases. The minimum follow-up was 38 months. Twelve of the 25 patients (48%) had micrometastatic lesions in their marrow at presentation. Four of these patients developed distal recurrence during follow-up, causing death in two of them. Five of the 13 patients with no evidence of micrometastases developed distant recurrence and four of them have died. There was no correlation between the state of the marrow and the development of metastatic disease, although axillary lymph node status, disease stage and tumour volume correlated significantly with outcome (all P less than 0.025). Micrometastatic lesions appear to be common in the marrow of patients with early breast cancer. We have been unable to demonstrate that they have prognostic significance.     

Axillary recurrence rate after tumour negative and micrometastatic positive sentinel node procedures in breast cancer patients,a population based multicenter study

Background

The sentinel lymph node procedure is a widely accepted method for staging of patients with early breast cancer. This study evaluates the incidence of axillary relapse after negative sentinel node biopsy in the seven hospitals in the central part of the Netherlands.

Methods

This study concerns all patients with a T1-2 breast carcinoma who were staged with a sentinel lymph node biopsy in one of the hospitals in the region. Patients with a tumour-free sentinel node without additional axillary lymph node dissection and patients with a sentinel node containing micrometastases were prospectively included and data concerning tumour and primary treatment were recorded. After a median follow-up period of 46 months supplementary data were collected of all patients.

Results

Between January 2002 and December 2003, 541 patients underwent a sentinel node biopsy of which the sentinel node was negative for metastatic disease. During the follow-up period three patients were diagnosed with an axillary recurrence. The incidence of axillary relapse after tumour negative sentinel node biopsy in this study is 0.6% (3/541). In 23 patients a distant metastasis developed. An event occurred in 11% of the patients with a micrometastasis in the sentinel node. This was not significantly different from the patients with a tumour-free sentinel node.

Conclusion

The results suggest that the sentinel lymph node procedure as performed in the region Middle Netherlands is a reliable and accurate instrument for staging of patients with early breast cancer. In our study we observed a non-significant different risk of distant disease in case of micrometastases compared to a tumour negative sentinel node.     

Prognostic significance of axillary lymph node micrometastases and microvessel count in breast cancer

Objective To investigate the influence of axillary lymph node micrometastases and the microvessel count on the prognosis of patients with breast cancer. Methods Forty-eight patients with breast cancer, who had no tumor cells in their regional lymph nodes based on conventional histopathologic examination, were re -examined with immunohistochemical LSAB techniques. H&E, anti-EMA, CK 19 and FVIII factor staining was used to identify tumor cells in both lymph nodes and tumor tissues and to count the mtcrovessels. A total of 882 lymph nodes were examined. Results Immunostaining-positive tumor cells were found in 9.0 %( 79/882) of the dissected lymph nodes. The positive rates were not significantly different between a surviving group and a deceased group (P>0.05). The microvessel count was significantly higher in group that had died (P<0.001). Conclusion The lymph node micrometastases did not show any correlation with patients’ survival, but the microvessel density had a negative correlation with the survival period in breast cancer patients who had negative axillary lymph nodes.     

Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients

The aim of this study was to search for specific and sensitive mRNA markers or a combination of markers for RT-PCR detection of micrometastases in axillary lymph nodes (LNs) from patients with breast cancer. LNs (n=177) from 17 patients were examined with Cytokeratin20 (CK20), melanoma-associated genes (MAGE1, MAGE3), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), mammaglobin (MGB1) and mammaglobin B (MGB2) as molecular markers. CK20, MAGE1 and MAGE3 were slightly positive in primary tumors and CEA, PSA, MGB1 and MGB2 were highly positive. MGB1 and MGB2 were 100% positive in HE-positive LNs while CEA and PSA were only 35.7% and 57.1% positive. MGB1 and MGB2 were also 30.1% and 17.8% positive in HE-negative nodes. Thus, MGB1 and MGB2 are specific and a combination of the two should be useful for detection of micrometastases in axillary LNs of breast cancer patients.     

乳腺癌微转移标记物研究进展

骨髓、淋巴结微转移的检出率与乳腺癌患者的预后密切相关,并已成为重要的独立预后因素,可早期诊断肿瘤复发和远处转移。目前乳腺癌微转移标记物的研究主要集中在乳腺小黏蛋白、人乳腺珠蛋白(hMAM)、黏蛋白1、丝氨酸蛋白酶抑制因子(maspin)、细胞角蛋白(CK)19等。     

CA15-3 RT-PCR检测乳腺癌腋窝淋巴结微小转移

目的：探讨检测乳腺癌淋巴结微小转移的新方法。方法：以RT－PCR检测CA15－3 mRNA中一段基因。总RNA是从乳腺癌细胞系MCF－7及原发性乳腺癌腋窝淋巴结提取。结果：CA15－3 mRNA可以MCF－7细胞和19例初发乳腺肿瘤患者中检测到，从良性乳腺疾病中取材的淋巴结不能检测到。通过倍比黧稀释发现CA15－3 RT－PCR是一种很敏感的方法，可以检测到1／10^6个转移的肿瘤细胞。检测的敏感性同免疫组化作了比较，19例患者取材的65个腋窝淋巴结，分别用RT－PCR和免疫组化作检测。在7个淋巴结中的肿瘤细胞微小转移只能通过CA15－3 RT－PCR方法检测到。CA15－3 RT－PCR方法发现2例HE染色和免疫组化染色不能发现的淋巴结有转移肿瘤患者。结论：CA15－3 RT－PCR是比免疫组化和HE染色更敏感的方法，可以避免已有淋巴结微小转移的患者被漏诊。     

Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors.

PURPOSE: We studied the prognostic and predictive value of immunohistochemically detected occult tumor cells (OTCs) in lymph nodes and bone marrow aspirates obtained from node-negative breast cancer patients. All were classified as distant metastases-free using conventional staging methods. PATIENTS AND METHODS: A total of 484 patients with pT1-2N0M0 breast cancer and 70 with pT1-2N1M0 breast cancer and a single affected lymph node participated in our trial. Ipsilateral axillary lymph nodes and intraoperatively aspirated bone marrow were examined. All samples were examined for OTCs using monoclonal antibodies to cytokeratins 8, 18, 19. Immunohistological findings were correlated with other prognostic factors. The mean follow-up was 54 +/- 24 months. RESULTS: OTCs were detected in 180 (37.2%) of 484 pT1-2N0M0 patients: in the bone marrow of 126 patients (26.0%), in the lymph nodes of 31 patients (6.4%), and in bone marrow and lymph nodes of 23 (4.8%) patients. Of the 70 patients with pT1-2N1MO breast cancer and a single involved lymph node, OTCs were identified in the bone marrow of 26 (37.1%). The ability to detect tumor cells increased with the following tumor features: larger size, poor differentiation, and higher proliferation. Tumors of patients with OTCs more frequently demonstrated lymph node invasion, blood vessel invasion, higher urokinase-type plasminogen activator levels, and increased PAI-1 concentrations. Patients with detected OTCs showed reduced disease-free survival (DFS) and overall survival (OAS) rates that were comparable to those observed in patients who had one positive lymph node. Multivariate analysis of prognostic factors revealed that OTCs, histological grading, and tumor size are significant predictors of DFS; OTCs and grading of OAS. CONCLUSION: OTCs detected by simultaneous immunohistochemical analysis of axillary lymph nodes and bone marrow demonstrate independent metastatic pathways. Although OTCs were significantly more frequent in patients with other unfavorable prognostic factors, they were confirmed as an independent prognostic factor for pT1-2N0M0, R0 breast cancer patients.     

Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy.

PURPOSE: In node-negative patients, of whom up to 30% will recur within 5 years after diagnosis, markers are still needed that identify patients at high enough risk to warrant further adjuvant treatment. In the present study we analyzed whether a correlation exists between microscopic tumor cell spread to bone marrow and to lymph nodes and attempted to determine which route is clinically more important. PATIENTS AND METHODS: According to a prospective design, bone marrow aspirates and axillary lymph nodes of level I (n = 1,590) from 150 node-negative patients with stage I or II breast cancer were analyzed immunocytochemically with monoclonal anticytokeratin (CK) antibodies. We investigated associations with prognostic factors and the effect of micrometastasis on patients' prognosis. RESULTS: CK-positive cells in bone marrow aspirates were present in 44 (29%) of 150 breast cancer patients, whereas only 13 patients (9%) had such positive findings in lymph nodes; simultaneous microdissemination to bone marrow and lymph nodes was seen in merely two patients. No correlation of bone marrow micrometastases with other risk factors was assessed. Reduced 4-year distant disease-free and overall survival were each associated with a positive bone marrow finding (P =.032 and P =.014, respectively) but not with lymph node micrometastasis. Multivariate analysis revealed an independent prognostic effect of bone marrow micrometastasis on survival, with a hazards ratio of 6.1 (95% confidence interval, 1.2 to 31.3) for cancer-related death (P =.031) in our series. CONCLUSION: Immunocytochemical detection of micrometastatic cells in bone marrow but not in lymph nodes is an independent prognostic risk factor in node-negative breast cancer that may have implications for surgery and stratification into adjuvant therapy trials.     

Sentinel node micrometastases in breast cancer: clinical outcome

Aim: The sentinel lymph node biopsy has steadily replaced axillary lymph node dissection for staging clinically node‐negative breast cancer. This study assesses surgical and adjuvant practice in relation to micrometastases and isolated tumor cells found on biopsy in a single surgeon cohort. Methods: Clinicopathological characteristics were collated from 700 breast cancer patients undergoing sentinel lymph node biopsies between 1999 and 2007. The status and details of the node biopsies, continuing treatment and adverse outcomes were reported. Patient details at the time of diagnosis were entered into Adjuvant! online to look at likely prognosis. For both isolated tumor cells and micrometastases, data input was conducted twice, once as node‐negative and again as node‐positive, thus providing two predicted benefit data series. Results: A total of 665 women were eligible for inclusion, 67 with micrometastases and 20 with isolated tumor cells. Overall 33 patients developed recurrence with nine breast‐cancer related deaths. Women with isolated tumor cells or micrometastases were more likely to receive adjuvant radiotherapy to the axilla compared with women with node‐negative disease. Compared to those with isolated tumor cells, a higher number of women with micrometastases received systemic chemotherapy despite similar predicted benefits. Individual comparisons showed significantly higher rates of recurrence in women with isolated tumor cells than in node‐negative disease (P < 0.0001). Conclusion: The biological behavior of early breast cancer with isolated tumor cells on sentinel node biopsy is similar to both micrometastases and macrometastases, i.e. they behave in a node‐positive fashion. This is an early indication that these patients should be treated with more aggressive adjuvant therapy.     

Prognostic Significance of Axillary Lymph Node Micrometastases and Microvessel Count in Breast Cancer

Objective  To investigate the influence of axillary lymph node micrometastases and the microvessel count on the prognosis of patients with breast cancer. Methods  Forty-eight patients with breast cancer, who had no tumor cells in their regional lymph nodes based on conventional histopathologic examination, were re -examined with immunohistochemical LSAB techniques. H&E, anti-EMA, CK 19 and FVIII factor staining was used to identify tumor cells in both lymph nodes and tumor tissues and to count the mtcrovessels. A total of 882 lymph nodes were examined. Results  Immunostaining-positive tumor cells were found in 9.0 %( 79/882) of the dissected lymph nodes. The positive rates were not significantly different between a surviving group and a deceased group (P>0.05). The microvessel count was significantly higher in group that had died (P<0.001). Conclusion  The lymph node micrometastases did not show any correlation with patients’ survival, but the microvessel density had a negative correlation with the survival period in breast cancer patients who had negative axillary lymph nodes.