ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients

Occult hematogenous micrometastases are the major cause for metastatic relapse and cancer-related death in patients with operable primary breast cancer. Although sensitive immunocytochemical and molecular methods allow detection of individual breast cancer cells in bone marrow (BM), a major site of metastatic relapse, current detection techniques cannot discriminate between nonviable shed tumor cells and seminal metastatic cells. To address this problem, we analyzed the relevance of erbB2 overexpression on disseminated cytokeratin-18-positive breast cancer cells in the BM of 52 patients with locoregionally restricted primary breast cancer using immunocytochemical double labeling with monoclonal antibody 9G6 to the p185erbB2 oncoprotein. Expression of p185erbB2 on BM micrometastases was detected in 31 of 52 (60%) patients independent of established risk factors such as lymph node involvement, primary tumor size, differentiation grade, or expression of p185erbB2 on primary tumor cells. After a median follow-up of 64 months, patients with p185erbB2-positive BM micrometastases had developed fatal metastatic relapses more frequently than patients with p185erbB2-negative micrometastases (21 versus 7 events; P = 0.032). In multivariate analysis, the presence of p185erbB2-positive micrometastases was an independent prognostic factor with a hazard ratio of 2.78 (95% confidence interval, 1.11-6.96) for overall survival (P = 0.029). We therefore conclude that erbB2 overexpression characterizes a clinically relevant subset of breast cancer micrometastases.     

The fate of bone marrow micrometastases in patients with primary breast cancer

Using an immunocytochemical technique, micrometastases have been found in the bone marrow of approximately 26% of patients with primary breast cancer at the time of initial surgery. To determine the fate of these cells, both in patients receiving and not receiving adjuvant therapy, multiple bone marrow aspirates were repeated in 82 patients at a median time of 18 months after surgery but prior to overt relapse. In both treated and untreated patients micrometastases were only found in one of 45 (2%) and one of 37 (3%) patients, respectively. However, when multiple marrow aspirates were taken from patients with local recurrence the incidence of micrometastases was 19% (three of 16), and this increased to 30% (three of ten) in patients with disease at distant sites other than bone, and 100% (ten of ten) in patients with radiologically proven bony disease. Three of 11 (27%) patients in whom the primary tumor remained in situ while receiving adjuvant therapy before definitive surgery had micrometastases at the time of diagnosis and at follow-up 3 months later. These results suggest that many of the micrometastases from breast cancer patients are the result of "shedding" of cells from the primary carcinoma and that a proportion are not viable. The technique is currently insufficiently sensitive to accurately monitor adjuvant therapy in breast cancer patients.     

Detection of bone metastases in patients with breast cancer

Various methods have been evaluated for their ability to detect bone metastases in patients with breast cancer. Bone scanning and hydroxyproline measurements are insensitive and showed metastases in few patients with primary breast cancer despite the fact that most will develop bone metastases. We have therefore investigated the value of examining the bone marrow with immunocytochemical staining for breast carcinoma cells. Initial results in 68 patients with no evidence of bone metastases by conventional means indicated (a) that some patients have breast cancer cells in bone marrow despite having no evidence of dissemination using other tests, and (b) that patients with micrometastases relapse sooner than those patients with normal bone marrows.     

Detection of breast cancer micrometastases in bone marrow using reverse-transcriptase chain reaction and southern hybridization

Thirty-flveto40%ofallpatientswithbreastcarcinoma,includingupt024%ofpatientswithn0evidence0fmetastasesatthetimeofdiagnosis,willrelapseafterprimarytherapy.l"']Themostreliableprognosticindicesofaxillarylymphnodestatusandsizeofprimarytumorcannotpredictinwh0mthediseasewillrecur.Bonemarrowisafrequentandreadilyaccessiblesiteofmetastases.Inup8O%ofpatients,therelaPsedevelopsstariingfrombonemarrowmetastasesats0mepointinthepr0cessoftheirillness.l3]Currentmethodstodetectboneinvolvement,suchasX-rayandbon…     

In vitro and in vivo effects of a monoclonal antibody-toxin conjugate for use in autologous bone marrow transplantation for patients with breast cancer

We have devised a method utilizing a monoclonal antibody-toxin conjugate (LICR-LON-Fib75/abrin A-chain) for ridding bone marrow of infiltrating breast cancer cells to rescue patients with autologous bone marrow following high dose therapy. Initially we examined the activity of this conjugate in vitro. Five of seven human breast cancer cell lines were killed following exposure at 10(-8) M for 2 h; this concentration only reduced bone marrow colony formation to 83% (range, 50-100%) of control bone marrow. We then examined the pattern of bone marrow recovery after high dose melphalan (200 mg/m2) in patients with advanced breast cancer who were in remission following combination chemotherapy. To do this we compared the time of recovery of the blood count in three patients who received treated marrow and seven who received untreated marrow. Mean time to recovery of the peripheral white count (greater than 1.5 X 10(9)/liter) was 16.7 days (treated) and 18.3 days (untreated), respectively. Mean time to recovery of peripheral platelet count (greater than 50 X 10(9)/liter) was 23.7 days (treated) and 18.9 days (untreated), respectively. Patients continued in remission for 1-greater than 14 mo after high dose melphalan, and remission duration was similar in patients who received treated (6.2 mo) and untreated (7.3 mo) bone marrow. These findings indicate that treatment of bone marrow with LICR-LON-Fib75/abrin A-chain conjugate does not significantly impair bone marrow recovery, and it is, therefore, possible to rescue breast cancer patients with bone marrow that has been cleansed of infiltrating cancer cells. This may have an application in patients with poor-risk primary breast cancer who have micrometastases and who may benefit from intensive therapy, but it has minimal application in patients with more advanced disease.     

Molecular determinants of occult metastatic tumor cells in bone marrow

The success of mammographic screening for breast cancer is that it involves increasingly more patients with small primary tumors formerly thought to have an overall excellent prognosis. Yet, only approximately two thirds of these patients actually have this favorable prognosis, while the remaining third develops metastatic disease. Thus, there is emerging evidence that epithelial tumor cells can disseminate into secondary organs at an earlier stage of primary tumor development than appreciated by current risk classifications. Bone marrow is one of the most prominent secondary organs screened for the presence of disseminated tumor cells. The current data suggest that bone marrow micrometastases represent a selected population of dormant and heterogeneous cancer cells. The analysis of micrometastatic cells opens a new avenue by which to assess the molecular determinants of both early tumor cell dissemination and subsequent outgrowth into overt metastases. Moreover, identifying therapeutic target structures (e.g., HER2/neu), monitoring the elimination of bone marrow micrometastases, and assessing treatment-resistant tumor cell clones might help to understand the current limitations of adjuvant systemic therapy. This review summarizes the current knowledge of the biological characteristics of micrometastatic cancer cells in bone marrow of breast cancer patients.     

Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity.

PURPOSE: The incidence and biological characteristics of circulating tumor cells in the blood of patients with breast cancer were examined and subgroups were evaluated in the context of systemic treatment and the presence of disseminated tumor cells in bone marrow. EXPERIMENTAL DESIGN: Circulating tumor cells were isolated from the peripheral blood of patients with breast cancer using a gradient system designed for the enrichment of circulating tumor cells (OncoQuick). Circulating tumor cells were identified with the anti-cytokeratin antibody, A45-B/B3. In subsets of patients, expression of the proliferation-associated Ki-67 antigen in circulating tumor cells and the concomitant presence of micrometastases in bone marrow were examined. RESULTS: In patients with primary breast cancer (stage M(0)), circulating tumor cells were detected in 5 of 60 patients (8.3%) after surgery and before initiation of adjuvant chemotherapy; a positive correlation to the presence of disseminated tumor cells in bone marrow was observed (P = 0.030, n = 53). During the course of adjuvant chemotherapy, repeated analysis of 20 M(0) patients revealed the occurrence of circulating tumor cells in 7 of 16 patients that were initially negative. Patients with metastatic disease (stage M(1)) showed circulating tumor cells in 25 of 63 cases (39.7%, P < 0.0001 as compared with M(0) patients), and a positive finding was correlated with elevated concentrations of the serum tumor marker CA15.3 (P = 0.0093). Performing repeated analysis in a subgroup of 25 M(1) patients, circulating tumor cells were found more frequently in patients with progressive disease than in patients with stable disease or remission (87.5% versus 43.8% of patients with circulating tumor cells, respectively; P = 0.047). Independent of the disease-stage, none of the 47 patients examined for the proliferative status of their circulating tumor cells showed coexpression of Ki-67. CONCLUSIONS: Circulating tumor cells seem to be nonproliferating cells that persist during chemotherapy. Circulating tumor cell detection is linked to disease progression and elevated tumor marker concentrations in patients with metastatic breast cancer.     

骨髓微转移在胃癌中的意义及其与VEGF关系的初步探讨

目的:探讨骨髓微转移在胃癌中的意义,探讨骨髓微转移与VEGF之间的关系.方法:采用免疫组化SABC方法检测骨髓中的肿瘤细胞,ELISA方法检测血清VEGF表达.结果:骨髓微转移阳性胃癌患者较阴性患者肿瘤浸润深,腹腔播散及肝脏转移发生率高(P＜0.05).骨髓微转移阳性和阴性患者VEGF阳性率分别为77.8%(2l/27),43.4%(23/53)(P＜0.05).结论:骨髓微转移与胃癌临床生物学行为关系密切,与VEGF表达水平相关,对胃癌的治疗和预后有着指导性的意义.     

Monoclonal antibodies for detection of occult carcinoma cells in bone marrow of breast cancer patients

Metastases develop in 30% to 40% of patients with operable breast cancer. Investigators have reported on the detection of occult micrometastases in bone marrow using an antibody to epithelial membrane antigen (EMA) and have since reported prognostic significance for these antibody-detected cells. In this study, two anti-cytokeratin monoclonal antibodies (35 beta H11 and 34 beta E12) were used to examine bone marrow specimens from patients with breast cancer. The technique was first studied in a test system in which human or monkey bone marrow was seeded with MCF-7 cells, and was determined to be sensitive enough to detect fewer than one cancer cell in 10(4) hematopoietic cells. An immunoglucose oxidase method was used for patient specimen antibody localization and was found to be free of false-positive staining. Marrow specimens from 25 patients with breast cancer of various stages were examined. No correlation with disease stage was observed. We conclude that the technique is feasible, but prognostic import remains to be determined.     

Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases

We used monoclonal antibodies to identify occult micrometastases in the bone marrow of 49 patients with operable (stage I and II) breast carcinoma. Follow-up (mean, 29 months; median, 30 months) revealed that 12 patients recurred. The presence of bone marrow micrometastases (BMM) was significantly associated with early recurrence (P less than .04). The estimated 2-year recurrence rate for patients with no BMM detected (BMM-) was 3%; in patients with BMM, the 2-year recurrence rate was 33%. When BMM and axillary lymph node (LN) status were combined, groups of patients at low risk (LN-, BMM-; 2-year recurrence rate, 0%) and high risk (LN+, BMM+; 2-year recurrence rate, 42%) for early recurrence were identified. Bone marrow tumor burden was related to early recurrence. Among patients with BMM, those who did not recur had on average fewer extrinsic cells in their marrow than those who recurred (15 v 43 cells, respectively). Multivariate analysis comparing BMM, LN+ versus LN-, and tumor size (less than or equal to 2 cm v greater than 2 cm) revealed no factor independently associated with early recurrence. Peripheral tumor burden of BMM (0 or less than 10 extrinsic cells v greater than or equal to 10 extrinsic cells) was the only independent predictor of early recurrence (P less than .003). In conjunction with conventional prognostic factors, particularly axillary LN status, evaluation for BMM might be used to stratify patients for adjuvant treatment programs. Because this pilot study involved few patients with short-term follow-up, the results should be interpreted with caution. The examination of bone marrow for micrometastases remains an experimental procedure; the clinical usefulness of the test will be established through larger studies with long-term follow-up.     

Sequential immunochemotherapy and edrecolomab in the adjuvant therapy of breast cancer: reduction of 17-1A-positive disseminated tumour cells.

BACKGROUND: The aim of our study was to evaluate the efficacy of the monoclonal antibody edrecolomab after chemo- and radiotherapy in the elimination of disseminated tumour cells in bone marrow in the adjuvant therapy of breast cancer. PATIENTS AND METHODS: The bone marrow of 25 patients with breast cancer was tested for the presence of disseminated tumour cells using the pancytoceratine antibody and the alkaline phosphatase-anti-alkaline-phosphatase (APAAP) technique. To characterize tumour cells simultaneously, immunofluorescent double labelling of pancytoceratine and epithelial cell adhesion molecule (antibody 17-1A) was performed on tumour cells after magneto bead enrichment. Patients positive for the 17-1A antigen in bone marrow after chemotherapy were treated with edrecolomab (500 mg Panorex) initially, then 100 mg/month over 4 months) and investigated for the presence of micrometastases 6 weeks after the last treatment. RESULTS: Of the 17 patients showing bone marrow micrometastases (BM-MM), 14 tested 17-1A positive before adjuvant chemotherapy. After chemotherapy, nine patients remained positive for the 17-1A antigen and were treated with edrecolomab. The final investigation after immunotherapy showed a complete elimination of the 17-1A-positive BM-MM in seven patients and a significant reduction of these cells in two patients. CONCLUSIONS: Sequential treatment of breast cancer with edrecolomab after adjuvant chemotherapy can reduce disseminated tumour cells in the bone marrow and eliminate 17-1A-positive micrometastases.     

Micrometastases in breast cancer: Long-term follow-up of the first patient cohort

“Micrometastases” can be identified in the bone marrow of patients with apparently localised breast cancer using an immunocytochemical stain for epithelial membrane antigen (EMA). Of 39 women who had marrow samples examined at the time of initial presentation (37), or with locally recurrent disease (2), 13 (33%) had samples which contained small numbers of EMA positive cells. 10 out of 23 (44%) lymph-node positive patients were marrow positive, compared to 1 out of 14 (7%) lymph node negative cases (P = 0.03). Long-term follow-up (median 9.5 years) has shown that 11 out of 13 (85%) patients with micrometastases have developed metastatic disease compared to 8 out of 26 (31%) with negative bone marrow aspirates (P < 0.05). The small number of EMA positive cells detected in bone marrow samples probably reflects the high metastatic potential of primary or recurrent cancers rather than established microscopic deposits; it is not yet clear whether the finding of such micrometastases will act as an independent variable compared to established prognostic factors.     

Prognostic relevance of urokinase plasminogen activator detection in micrometastatic cells in the bone marrow of patients with primary breast cancer.

Patients with an elevated level of urokinase plasminogen activator (uPA) in breast cancer tissue have an adverse prognosis. This study evaluated the prognostic relevance of uPA detection in disseminated tumour cells in bone marrow. Bone marrow was sampled intraoperatively from both iliac crests in 280 patients with primary breast cancer. Interphase cells were enhanced and stained immunocytologically with two antibodies: 2E11, which detects TAG 12--a tumour-associated glycoprotein typically expressed by almost all breast cancer cells--and the anti-uPA antibody HD-UK9. Thirty-five of the 2E11-positive women (n = 132, 47%) developed metastatic disease (median follow-up time 44 months). Of these, most were uPA positive (n = 23, 65%) and only 12 were uPA negative. Patients with uPA-positive cells in bone marrow (n = 98, 35%) had a significantly shorter metastasis-free interval (36 months) than women who were uPA negative (44.5 months). The worst prognosis was seen in patients positive for both markers (29.5 months), followed by those who were uPA negative and 2E11 positive (37 months). The detection of uPA on disseminated tumour cells characterizes a subgroup of patients with an even worse prognosis, who should undergo more aggressive adjuvant systemic therapy. For the first time, it was possible to evaluate an important qualitative parameter involved in the process of breast cancer metastases.     

早期乳腺癌血清VEGF水平与骨髓微转移的相关性及临床意义

目的 检测早期乳腺癌患者血清中VEGF及骨髓标本中CK19的表达水平,探讨二者的相互关系及临床意义.方法 应用ELISA法分别检测6例乳腺纤维腺瘤及64例乳腺癌患者血清中VEGF的水平,同时抽取相应的骨髓血标本,应用实时定量逆转录-聚合酶链反应(qRT-PCR)方法检测标本中CK-19的表达水平,并进行相关性分析.结果 VEGF在乳腺癌患者血清中的表达水平明显高于乳腺纤维腺瘤患者,二者差异显著(P=0.012);在HER2阳性患者中,血清VEGF水平明显高于HER2阴性患者(P=0.016).CK19在乳腺纤维腺瘤患者骨髓标本中未见表达,在乳腺癌患者骨髓血中,阳性表达24例(37.5%),而骨髓中CK19阳性的乳腺癌患者血清VEGF水平显著高于CK19阴性的患者(110.46±90.65 vs 70.88±77.13,P=0.038),且二者的表达呈正相关.结论 早期乳腺癌患者血清VEGF水平与骨髓微转移密切相关,提示VEGF可能参与了肿瘤细胞的侵袭和播散,可作为早期乳腺癌微转移的血清学指标.     

Prognostic significance of an increased number of micrometastatic tumor cells in the bone marrow of patients with first recurrence of breast carcinoma

BACKGROUND: Using cytokeratin (CK) as a histogenetic marker of epithelial tumor cells in the bone marrow of patients with primary breast carcinoma, a subgroup of patients with decreased survival can be identified. This study was designed to evaluate the frequency and prognostic relevance of such cells in patients with recurrent breast carcinoma. METHODS: Bone marrow aspirates from 65 patients were analyzed immunocytochemically for the presence of CK positive cells. A quantitative immunoassay with monoclonal anti-CK antibody A45-B/B3 was used and 2 x 10(6) bone marrow cells per patient were evaluated. For prognostic evaluation the authors calculated a cutoff value of micrometastatic tumor cells by analogy to classification and regression tree (CART) analysis. Patients were monitored prospectively for a median of 37 months (range, 11-63 months). RESULTS: Bone marrow micrometastases were present in 5 of 32 patients (16%) with locoregional recurrence and in 24 of 33 patients (73%) with distant recurrence. The bone marrow status yielded no prognostic indication for patients with locoregional recurrence. In contrast, a cutoff value of 2.5 tumor cells per 1 million bone marrow cells analyzed (2.5 x 10(-6) tumor cells) correlated with a significantly different prognosis for women with distant disease. Patients with metastatic disease and a micrometastatic tumor load of > 2.5 x 10(-6) tumor cells survived for a mean of 6 months (95% confidence interval [95% CI], 2.0-9.1) compared with 17 months (95% CI, 11.6-22.0) for patients with < or = 2.5 x 10(-6) tumor cells (P < 0.0001). Multivariate analysis, allowing for hormone receptor status, disease free interval prior to recurrence, manifestation site of metastases, age, and micrometastases in bone marrow, revealed that bone marrow involvement was an independent risk factor, with a hazard ratio of 7.4 (95% CI, 1.6-13.3) for disease-related death. CONCLUSIONS: An increased number of micrometastases identified in the bone marrow of patients with metastatic breast carcinoma represents an independent prognostic factor that may influence future therapeutic strategies for patients with metastatic breast carcinoma.