Isolated Tumor Cells In Bone Marrow Research Articles

Hematogenous and lymphatic tumor cell dissemination may be detected in patients diagnosed with ductal carcinoma in situ of the breast

Tumor cell dissemination in bone marrow (BM) and lymph nodes is considered an important step in systemic disease progression and is associated with poor prognosis. Only invasive cancers are assumed to shed isolated tumor cells (ITC) into the bloodstream and infiltrate lymph nodes. However, latest studies indicate that tumor cell dissemination may occur before stroma invasion, i.e., in ductal carcinoma in situ (DCIS). Therefore, the purpose of this study was to examine the incidence of ITC in bone marrow and sentinel lymph nodes (SN) in patients diagnosed with DCIS and its correlation with clinicopathological factors. 266 patients who were treated at the Department of Gynecology and Obstetrics (University Hospital Tuebingen, Germany) between 2003 and 2009 with DCIS were included into this study. BM aspirates were analyzed by immunocytochemistry (pancytokeratin antibody A45-B/B3) using ACIS system (Chromavision) according to the ISHAGE evaluation criteria. SN were analyzed in 221 of these patients by extensive step sectioning and hematoxylin-eosin staining. In 34 of 266 patients (13%), ITC in BM could be detected. There was no correlation found between tumor size, grading, histology, or Van Nuys Prognostic Index and tumor cell dissemination. In two cases, metastatic spread into lymph nodes was observed (pN1mi), whereas in one case, ITC in lymph nodes were detected; however, additional sectioning and immunohistochemical staining of the primary lesion in the cases with positive SN did not reveal invasive cancer. Interestingly, all the three patients were BM negative. Tumor cell dissemination may be detected in patients diagnosed with DCIS. Either these cells have started already to disseminate from preinvasive mammary lesions or from occult invasive tumors or represent the earliest step of microinvasion in a preinvasive lesion. The clinical relevance of these cells has to be further evaluated.

Diagnostic accuracy of small breast epithelial mucin mRNA as a marker for bone marrow micrometastasis in breast cancer: a pilot study

Detection of isolated tumour cells (ITC) in the blood or minimal deposits in distant organs such as bone marrow (BM) could be important to identify breast cancer patients at high risk of relapse or disease progression. PCR amplification of tissue or tumour selective mRNA is the most powerful analytical tool for detection of this micrometastasis. We have evaluated for the first time, the diagnostic accuracy of small breast epithelial mucin (SBEM) as a potential marker for BM micrometastasis in breast cancer. A nested RT-PCR assay for detection of SBEM mRNA was compared with immunocytochemistry (ICC) with anticytokeratin AE1/AE3 antibody in paired samples obtained from the BM of breast cancer patients. Associations of SBEM mRNA detection in BM and clinical and pathological parameters were evaluated. SBEM mRNA status and time to breast cancer progression were analysed using Kaplan-Meyer curves. Fifty stages I-IV breast cancer female patients were prospectively included in our study. SBEM specific transcript was found in BM in 26% of the patients. Detection rate was similar to the percentage of patients with ITCs detected using ICC (24%). SBEM mRNA in BM aspirates were significantly associated with presence of clinically active disease, including locally advanced and metastatic patients (47%, P = 0.021) and tumours with positive hormonal receptors (36.7%, P = 0.035). In addition association with Her2/neu over-expression (44.4%, P = 0.051) and low proliferating tumours (36%, P = 0.067) were close to significant levels. When we analysed time to breast cancer progression adjusting for grade or hormone receptor status, presence of SBEM mRNA in BM defines distinct prognostic groups. SBEM might represent a suitable marker for molecular detection of ITCs in BM in breast cancer patients. Analysis of prognostic value for SBEM mRNA-based assay should take into account the heterogeneity and different molecular subtypes of breast cancer.

Hematogenous Metastasis in Gastric Cancer Requires Isolated Tumor Cells and Expression of Vascular Endothelial Growth Factor Receptor-1

Recent studies of cancer metastasis have focused on the role of premetastatic gene expression and circulating tumor cells. We did a blind prospective study in gastric cancer to assess the significance of isolated tumor cells (ITC) and to test the hypothesis that vascular endothelial growth factor receptor-1 (VEGFR-1) is expressed within the bone marrow at tumor-specific, premetastatic sites. Both bone marrow and peripheral blood samples from 810 gastric cancer patients were collected at the Central Hospital, National Cancer Center (Tokyo, Japan). The samples were transferred to Kyushu University Hospital (Beppu, Japan) where they were analyzed by quantitative real-time reverse transcription-PCR for three epithelial cell markers, carcinoembryonic antigen, cytokeratin-19, and cytokeratin-7, as well as VEGFR-1. ITCs were observed in peripheral blood and bone marrow even in early stages of gastric cancer. The frequency of ITC in bone marrow was significantly associated with the stage of disease by ANOVA (P < 0.01). Gastric cancer metastasized when ITCs were observed in the presence of VEGFR-1. In the 380 patients who were ITC negative and showed low VEGFR-1 expression, synchronous (at the time of surgery) and heterochronous (recurrent) metastases were not observed. ITCs circulate even in early stages of disease. Furthermore, elevated expression of VEGFR-1 facilitates the establishment of hematogenous metastases in gastric cancer. This study indicates that the simultaneous presence of ITC and VEGFR-1 expression at premetastatic sites is clinically significant for disease progression.

Zoledronsäure bei persistierenden isolierten Tumorzellen im Knochenmark bei Mammakarzinom

Recently the highest level of evidence indicated the prognostic value of isolated tumor cells (ITC) in bone marrow of patients with breast cancer, both at primary diagnosis and during recurrence-free follow-up. The aim of the present study was to investigate the therapeutic efficacy of zoledronate in reducing persistence of ITC in the bone marrow of patients with breast cancer after they had completed primary therapy. In a non-randomized phase II pilot study, 4 mg of zoledronate were administered once every four weeks for six months, after a initial loading dose of 8 mg, to 31 patients with persisting ITC in bone marrow. All patients had completed surgery and adjuvant chemotherapy, if indicated, at least 6 months previously. The bone marrow was re-examined after 7.9 months (std 0,89). ITC were detected by immunocytochemical staining, using the monoclonal pan-cytokeratin antibody A45-B/B3 and the APAAP technique. Patients were followed-up prospectively for a median of 39 months after the first aspiration. ITC were detected in all 31 patients at the time of first bone marrow aspiration, but 27 of them (87 %) were free of ITC 6 months after the end of zoledronate therapy. The reduction in cell numbers between first and second aspiration were statistically significance (P < 0,0001). Ten of 12 patients without detection of ITC in bone marrow after treatment and who had undergone additional aspirations, still had no ITC a median time of 19 months (range 4.7 - 38.7 months) after the end of treatment. Zoledronate treatment was well tolerated, bone pain having been the most common side effect in 45 % of patients (n = 14). These results indicate a potential antineoplastic effect of zoledronate, a cell-cycle independent drug, on persisting ITC in a dormant state. Our data provide the basis for investigating the efficacy of zoledronate on ITC in treating primary breast cancer in prospectively randomized trials. ITC in bone marrow represents a useful marker in selecting patients at risk for recurrence and for monitoring therapeutic efficacy.

The effects of preoperative chemotherapy on isolated tumour cells in the blood and bone marrow of gastric cancer patients

Recent studies in breast cancer suggest that monitoring the isolated tumour cells (ITC) may be used as a surrogate marker to evaluate the efficacy of systemic chemotherapy. In the present study, we have investigated the effects of preoperative chemotherapy on ITC in the blood and bone marrow of patients with potentially resectable gastric cancer. After sorting out the CD45-positive cells, the presence of ITC defined as cytokeratin-positive cells was examined before and after preoperative chemotherapy. The patients received two courses of preoperative chemotherapy with cisplatin (100 mg m−2, day 1) and 5-fluorouracil (1000 mg m−2, days 1–5), administered every 28 days. Fourteen of 32 (44%) patients initially diagnosed with ITC in blood and/or bone marrow were found to be negative (responders) after preoperative chemotherapy (P<0.01). The incidence of ITC in bone marrow was also significantly (P<0.01) reduced from 97 (31 of 32) to 53% (17 of 32). The difference between patients positive for ITC in the blood before (n=7, 22%) and after (n=5, 16%) chemotherapy was statistically insignificant. The overall 3-year survival rates were 32 and 49% in the responders and non-responders, respectively (P=0.683). These data indicate that preoperative chemotherapy can reduce the incidence of ITC in patients with gastric cancer.

Disseminated isolated tumor cells in bone marrow of esophageal cancer cases

In this study, we reviewed reports on the clinical significance of isolated tumor cells (ITC) in the bone marrow of patients with esophageal cancer and other solid tumor malignancies. We examined studies on bone marrow as a target organ for ITC identification and also looked at the appropriate sites and most efficient methodologies for bone marrow extraction to evaluate the presence of ITC. A diverse number of different cells exist in bone marrow, and it is important to distinguish disseminated cancer cells from other cell types, such as progenitor cells and endothelial cells. Among the various types of cells found in the bone marrow, we confirmed the presence of disseminated cells that can proliferate under specific cell culture conditions. Finally, we discuss recent studies related to the clinical significance of ITC in bone marrow that are associated with prognosis or recurrence in cases of solid cancers, including esophageal cancer. We concluded that the clinical significance of bone marrow as the target organ for identification of ITC varies among different types of cancer and that further study is required to determine a bona fide marker for predicting prognosis or recurrence in cases of esophageal cancer.

Prognostic Significance of Isolated Tumor Cells (ITC) in the Bone Marrow (BM) of Breast Cancer Patients During Recurrence-free Follow-up and Therapeutic Intervention with Zoledronate

Aims: If the persistence of ITC after adjuvant therapy is associated with risk for recurrence, it would be an indication to consider secondary adjuvant treatment. Cell-cycle independent agents might be effective in the elimination of dormant cells. Methods: We analyzed BM aspirates from 228 pts during recurrence-free follow-up at a median interval of 21.3mon after primary diagnosis. ITC were detected by monoclonal antibody A45-B/B3 against cytokeratin. Pts were followed for a median of 49.8mon. In a pilot study on 14 pts with evidence of persisting ITC, zoledronate was applied (4mg q4wx6mon, loading dose 8mg). Pts were to have completed surgery (R0) and adjuvant chemotherapy for at least 6mon. In a matched pair analysis, these pts were compared to 14 pts with persisting ITC in the BM, who received no further therapy. The BM was re-examined after a median of 8mon in the treatment group and 9mon in the control group. Results: Persistent ITC in BM were detected in 12.7% of pts. Positive BM status was more frequent (15.7%) within the first 21mon after primary diagnosis than after a follow-up>21mon (9.7%). Recurrence-free survival was 149mon in pts with negative and 86mon in pts with positive BM status (P=.0003). Pts who were without evidence of persistent ITCs had a significantly longer overall survival (162.1 vs. 98.7mon; P=.0008). In multivariate analysis ITC was an independent predictor for disease-free (RR 4.57; P<.0001) and overall survival (RR 5.57; P=.002). While ITC were detected in all 28 pts of the therapeutic intervention study at initial BM aspiration, no pt showed ITC in the BM after 6mon of zoledronate. ITC were detected in 4 pts (29%) of the control group (P=.03). Conclusions: Evidence of persistent ITCs in BM from pts with breast carcinoma indicated an increased risk for subsequent recurrence. Secondary adjuvant treatment intervention with zoledronate might have a potential antineoplastic effect on persisting ITC in dormant state.

The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence

The prognostic significance of isolated tumor cells (ITCs) in bone marrow (BM) from patients with breast carcinoma at the time of their primary diagnosis recently was been confirmed by a large pooled analysis. If the persistence of ITCs after adjuvant therapy confers a similar risk for recurrence, then it would be an indication to consider secondary adjuvant therapy. The authors analyzed BM aspirates from 228 patients during recurrence-free follow-up at a median interval +/- standard deviation (SD) of 21.3 +/- 29.1 months after a primary diagnosis of breast carcinoma (pathologic T1 [pT1]-pT2, pN0-pN3, pM0). Carcinoma cells were detected using a standardized immunoassay with monoclonal antibody A45-B/B3 directed against cytokeratin (CK). Patients were followed for a median +/- SD of 49.8 +/- 32.1 months after their primary diagnosis. Persistent ITCs in BM were detected in 12.7% of patients (n=29 patients). Positive BM status was more frequent (15.7%) within the first 21 months after primary diagnosis than after a follow-up > 21 months (9.7%). The Kaplan-Meier estimate for mean recurrence-free survival was 149.7 months (95% confidence interval [95% CI], 139.6-159.8 months) in patients with negative BM status and 86.5 months (95% CI, 65.7-107.4 months; P=0.0003) in patients with positive BM status at the time patients underwent follow-up BM aspiration. Patients who were without evidence of persistent ITCs had a significantly longer overall survival (162.1 months; 95% CI, 152.1-172.0 months) compared with patients who had positive BM status (overall survival, 98.7 months; 95% CI, 79.7-117.9 months; P=0.0008). In multivariate Cox regression analysis that included BM status, tumor size, lymph node status, and histopathologic grade, evidence of ITCs was an independent significant predictor for reduced disease-free survival (relative risk [RR], 4.57; P <0.0001) and overall survival (RR, 5.57; P=0.002). Persistent ITCs had the greatest prognostic relevance when they were detected between 25 months and 42 months after primary diagnosis (RR, 7.68). Evidence of persistent ITCs in BM from patients with breast carcinoma indicated an increased risk for subsequent recurrence. Prospective trials should investigate the benefit of secondary adjuvant treatment on the basis of BM marrow status.

Effect of zoledronate on persisting isolated tumor cells (ITC) in the bone marrow (BM) of patients without recurrence of early breast cancer

9515 Background: Patients with persisting ITC in the BM after primary therapy show an increased risk for distant relapse and shortened survival (Janni et al., Cancer 2001; Wiedswang et al., SABCS 2003). Adjuvant chemotherapy, however, seems to have only limited effect on ITC in dormant state (Braun et al., 2000). Aim of this study was to investigate the therapeutic efficacy of zoledronate on the persistence of ITC in BM after completion of primary therapy. Methods: Zoledronate was applied at 4 mg q4wx6mon (loading dose 8mg) to 14 breast cancer patients with persisting ITC in the BM. Patients were to have completed surgery, leading to R0 resection and adjuvant chemotherapy for at least 6 months and had no evidence for recurrence at this point of time. In a matched pair analysis, these patients were compared to 14 patients with persisting ITC in the BM, who received no further therapy. The BM was re-examined after a median of 8 months (range 6.5–9.83) in the treatment group and 9 months (range 2.33–29.17) in the control group. ITC were detected by immunocytochemical staining using the pan-cytokeratin antibody A45-B/B3 and the APAAP technique. Results: Primary tumor characteristics, i.e. tumor size (P=.66), axillary nodal status (P=1.0) and histopathological grading (P=.76), as well as surgical (P=.23), adjuvant systemic (P=.10) and irradiation treatment (P=.36) were well balanced between both patient groups. While ITC were detected in all 28 patients at the time of first BM aspiration, no patient showed ITC in the BM following 6 months of zoledronate therapy. In contrast, persisting ITC were detected in 4 patients (29%) of the control group without treatment (P=.03). Conclusions: These preliminary results indicate potential antineoplastic effect of the cell-cycle independent agent zoledronate on persisting ITC in dormant state. In our view, these data provide a hypothesis generating basis to investigate the therapeutic efficacy of zoledronate on ITC in a secondary adjuvant setting by prospectively randomised trials. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis

Effect of zoledronate on persisting isolated tumor cells (ITC) in the bone marrow (BM) of patients without recurrence of early breast cancer

9515 Background: Patients with persisting ITC in the BM after primary therapy show an increased risk for distant relapse and shortened survival (Janni et al., Cancer 2001; Wiedswang et al., SABCS 2003). Adjuvant chemotherapy, however, seems to have only limited effect on ITC in dormant state (Braun et al., 2000). Aim of this study was to investigate the therapeutic efficacy of zoledronate on the persistence of ITC in BM after completion of primary therapy. Methods: Zoledronate was applied at 4 mg q4wx6mon (loading dose 8mg) to 14 breast cancer patients with persisting ITC in the BM. Patients were to have completed surgery, leading to R0 resection and adjuvant chemotherapy for at least 6 months and had no evidence for recurrence at this point of time. In a matched pair analysis, these patients were compared to 14 patients with persisting ITC in the BM, who received no further therapy. The BM was re-examined after a median of 8 months (range 6.5–9.83) in the treatment group and 9 months (range 2.33–29.17) in the control group. ITC were detected by immunocytochemical staining using the pan-cytokeratin antibody A45-B/B3 and the APAAP technique. Results: Primary tumor characteristics, i.e. tumor size (P=.66), axillary nodal status (P=1.0) and histopathological grading (P=.76), as well as surgical (P=.23), adjuvant systemic (P=.10) and irradiation treatment (P=.36) were well balanced between both patient groups. While ITC were detected in all 28 patients at the time of first BM aspiration, no patient showed ITC in the BM following 6 months of zoledronate therapy. In contrast, persisting ITC were detected in 4 patients (29%) of the control group without treatment (P=.03). Conclusions: These preliminary results indicate potential antineoplastic effect of the cell-cycle independent agent zoledronate on persisting ITC in dormant state. In our view, these data provide a hypothesis generating basis to investigate the therapeutic efficacy of zoledronate on ITC in a secondary adjuvant setting by prospectively randomised trials. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis

Persistence of occult metastatic cells in bone marrow after adjuvant systemic therapy predicts relapse in localized breast cancer

657 Background: The presence of isolated tumor cells (ITC) in bone marrow (BM) in breast cancer patients has been evaluated in numerous studies. The majority of these studies have found correlation between the presence of ITC in BM and increased risk of relapse. In this study we assessed the association between the presence of BM and established prognostic factors in localizated breast cancer patients, and analyzed the influence of adjuvant therapies on the remove of these cells as a prognostic factor. Methods: BM aspirates from 296 patients with primary breast cancer with stages I-III (112 node negative, and 184 node positive), treated with adjuvant systemic chemotherapy (CT) with antracyclines, were evaluated. All patients underwent BM aspirate at diagnosis. In addition, 202 of these patients also underwent a second BM aspirate after CT. Carcinoma cells were detected using a standardized immunoassay with monoclonal antibody A45-B/B3 directed against cytokeratin. Detection of >= 2 tumor cells was scored as positive. Results: At diagnosis, 82 of 296 (28%) patients presented ITC in BM. The presence of ITC in BM did no correlate with standard prognostic variables such as lymph node status, tumor size, histological grade, hormonal receptor levels and expression of HER-2 neu. After a median follow up of 35 months (6–130), 53 patients (18%) relapsed (10 node negative, and 43 node positive). In the global group, the presence of ITC in BM at diagnosis showed a tendency to predict worse disease-free survival (DFS) and overall survival (OS). In the lymph node-positive group, 62% of patient showing ITC in the second BM aspirate after CT relapsed, whereas 21% of those with both negative BM aspirates presented relapse. The presence of ITC in the second BM aspirate was associated with inferior DFS (P=0,01) and OS (p=0,002). Conclusions: Our analytical approach allows the identification of micrometastatic BM disease in localized breast cancer patients that would be usually undetected using conventional methods. The persistence of occult metastatic cells in BM after adjuvant systemic therapy predicts relapse in localized breast cancer. No significant financial relationships to disclose.