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ABSTRACT: Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. Assays based on RT-PCR are available; however so far they have not been used for routine detection of DTC. Therefore, the purpose of this study was to evaluate a newly established molecular method for the detection of DTC.
BM aspirates from 405 patients were examined. Half of the samples were immediately inserted into ICC and the other half was examined with our newly established molecular method based on RT-PCR. Immunocytochemistry was performed according to the Consensus Recommendations of the German, Austrian, and Swiss Societies of Senology and ISHAGE Working Group (A45B-B3 antibody). RT-PCR was conducted as a one-step real-time assay Cytokeratin 19-mRNA was amplified.
In 142 of 405 (35%) aspirates disseminated tumor cells were detected by RT-PCR. In 34% of patients DTC were detected by ICC. 48% of the BM samples were positive by at least one method. In 73% of the patients identical results were obtained (p<0,001).
Our newly established molecular assay for the detection of disseminated tumor cells, and thus minimal residual disease, is sensitive, fast and reproducible, and has a potential to be used as a confirmatory or alternative test for DTC detection.
Ginekologia polska 08/2012; 83(8):590-7. · 0.41 Impact Factor
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02/2012; , ISBN: 978-953-307-812-0
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ABSTRACT: The detection of disseminated tumor cells in bone marrow is a common phenomenon seen in 30-40% of primary breast cancer patients. The presence of disseminated tumor cells at diagnosis as well as the persistence of disseminated tumor cells is strongly associated with poor clinical outcome. Since bone marrow biopsies are not well tolerated by many patients, the evaluation of circulating tumor cells in the blood might become a desired alternative. Circulating tumor cells are routinely detected, depending on stage of the disease and methodology, in 10-80% of breast cancer patients. Recent studies have shown a prognostic potential of circulating tumor cells in both primary and metastatic settings. The evaluation of circulating tumor cells may become one of the crucial markers for prediction of survival and therapy monitoring, and its characterization might enable specific targeting of minimal residual, and metastatic disease.
Biomarkers in Medicine 02/2012; 6(1):109-18. · 0.86 Impact Factor
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ABSTRACT: The early spread of tumor cells in primary breast cancer patients may occur either through lymphatic or hematogenous dissemination. Lymph node (LN) status and presence of disseminated tumor cells (DTC) in bone marrow (BM) are independent predictors of poor outcome. It is unknown which factors determine one or the other route of tumor cell spread and whether lymphatic and hematogenous tumor cell dissemination are two independent processes. This study is aimed to compare the DTC status in clinically node-negative (N0) breast cancer patients with their sentinel LN status and to investigate predictors of BM and LN involvement. The DTC status of 1,345 clinically N0 breast cancer patients who underwent SLN biopsy during initial surgery was investigated. BM and LN status were compared and predictors of hematogenous and lymphatic tumor cell spread were investigated. DTCs were present in the BM of 181 (13%) patients. LN involvement was found in 348 (26%) patients. There was no correlation between LN and BM status: 137 of 997 nodal-negative patients (14%) had BM involvement and 44 of 348 nodal-positive patients (13%) were positive for DTCs (P = 0.649). The presence of DTCs was not influenced by tumorbiological factors. Conversely, a high correlation between nodal status and tumor size, histology, ER-status and lymph vessel invasion was found. Hematogenous and lymphatic tumor spread seem to be because of independent pathways of cancer progression.
Breast Cancer Research and Treatment 01/2012; 131(2):501-8. · 4.43 Impact Factor
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ABSTRACT: Recent studies have shown that the detection of circulating tumor cells (CTC) pre and postoperatively in the peripheral blood of primary breast cancer patients may be an indicator for poor survival. This study aimed to investigate the influence of removal of the primary tumor on incidence and phenotype of circulating tumor cells in primary breast cancer. 209 primary breast cancer patients could be included into this analysis. Blood sampling was performed both pre and postoperatively. The blood specimens were immunomagnetically enriched using AdnaTest BreastCancerSelect within 4 h after blood withdrawal, followed by RNA isolation and subsequent gene expression analysis by reverse transcription and multiplex PCR using AdnaTest BreastCancerDetect. Three breast cancer-associated tumor markers and two hormone receptor genes were amplified: GA733-2, Muc-1, Her-2, ER, PR. In addition, bone marrow (BM) status was intraoperatively determined. Forty-three of 209 patients (21%) had pre and/or postoperatively circulating tumor cells. The positivity rates after surgery were higher but did not differ significantly (12% pre and 16% postoperatively, P = 0.264). Disseminated tumor cells in BM were seen in 32 of 209 cases (15%). Patients with positive BM status had significantly higher CTC positivity rates both pre and postoperatively compared to those with negative BM status. The most common CTC phenotype was triple negative (24 patients) followed by HER2+/ER-/PR- subtype (10) and ER and/or PR positive (9). Interestingly, 41 of 43 primary tumors (95%) were ER and PR positive. Removal of the primary tumor did not alter the phenotype of CTC. Surgery does not significantly influence the tumor cell load in the blood stream. CTC phenotype before and after the surgery generally remains identical but may differ from that of the primary tumor.
Breast Cancer Research and Treatment 05/2011; 132(1):121-9. · 4.43 Impact Factor
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Malgorzata Banys,
Ines Gruber, Natalia Krawczyk,
Sven Becker,
Ralph Kurth,
Diethelm Wallwiener,
Jolanta Jakubowska,
Jürgen Hoffmann,
Ralf Rothmund,
Annette Staebler,
Tanja Fehm
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ABSTRACT: 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.
Breast Cancer Research and Treatment 04/2011; 131(3):801-8. · 4.43 Impact Factor
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ABSTRACT: Persistence of disseminated tumor cells (DTCs) is observed in 10 to 15% of breast cancer patients and is associated with poor prognosis. These patients might benefit from secondary adjuvant targeted therapy. The aim of this study was to assess HER2 status of persistent DTCs to determine whether the use of HER2-targeted agents might be a therapeutic option in patients with tumor cell persistence.
Bone marrow was obtained from 85 primary breast cancer patients intraoperatively and after completion of systemic treatment (median follow-up of 13 months; range: 6-30 months). Immunofluorescence double staining was used for identification of cytokeratin-positive, HER2-positive cells.
A total of 31 out of 85 (36%) patients had DTCs preoperatively. Out of 85 (16%) patients, 14 were DTC positive after completion of surgery and adjuvant cytotoxic therapy. Five of these patients had HER2-positive DTCs, however, the corresponding tumor was HER2 positive in only one case. The remaining nine patients with HER2-negative DTCs had HER2-negative primary tumors.
HER2-positive DTCs can be detected in patients with HER2-negative tumors, even after adjuvant therapy. Such patients may benefit from (secondary) HER2-targeted therapy in an adjuvant setting.
Anticancer research 10/2009; 29(10):4019-24. · 1.73 Impact Factor
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ABSTRACT: The presence of disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients is associated with poor prognosis. Several studies demonstrated that tumor cell dissemination may occur in gynecologic cancer and affect clinical outcome. The aim of our study was to evaluate the incidence of DTC and to assess their prognostic significance in patients with gynecologic malignancies.
Bone marrow aspirates from 377 patients with primary ovarian (112), endometrial (141), cervical (102), and vulvar cancer (22) undergoing surgery at the Department of Gynecology and Obstetrics, University Hospital, Tuebingen, Germany between November 2001 and November 2007, were included into the study. Disseminated tumor cells were identified by immunocytochemistry using the pancytokeratin antibody A45B/B3 and by cytomorphology.
Disseminated tumor cells were detected in 19% of BM aspirates from patients with gynecological malignancies. Incidences of DTC in ovarian, endometrial, cervical, and vulvar cancer were 25%, 16%, 19%, and 5%, respectively. For patients with ovarian and endometrial cancer, no correlation with established clinicopathological factors was observed. In case of cervical cancer, BM positivity was correlated with International Federation of Gynecology and Obstetrics stage, tumor size, and nodal involvement. Bone marrow positivity of ovarian cancer patients was correlated with significantly shorter disease-free survival (P = 0.035). For other tumor entities, no association between BM status and clinical outcome could be observed.
We conclude that up to 25% of patients with loco-regionally restricted gynecologic malignancies present with DTC at the time of diagnosis. For ovarian cancer patients, BM status affected clinical outcome.
International Journal of Gynecological Cancer 08/2009; 19(5):948-52. · 1.65 Impact Factor
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ABSTRACT: Isolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment.
Bone marrow (BM) aspirates from 254 patients with primary breast cancer were included in this study. A double immunofluorescence staining procedure was established for the identification of cytokeratin (CK) positive/Eralpha-positive cells. ERalpha status of the primary tumour was assessed immunohistochemically using the same antibody against ERalpha.
In 107 of 254 (42%) breast cancer patients, CK-positive cells could be detected in the BM. More than one DTC in the BM was observed in 38 of the 107 patients. The number of detected cells ranged between 1 and 55 cells per 2 x 10(6) mononuclear cells. DTCs demonstrated ERalpha positivity in 12% of the patients. The ERalpha expression was heterogeneous in 10 of the 38 (26%) patients with more than one DTC. The concordance rate of ERalpha status between primary tumour and DTC was 28%. Only 12 of 88 patients with ERalpha-positive tumours also had ERalpha-positive DTCs.
Primary tumours and DTCs displayed a concordant ERalpha status in only 28% of cases. Most of the DTCs were ERalpha negative despite the presence of an ERalpha-positive primary tumour. These findings further underline the distinct nature of DTCs and may explain the failure rates seen in conventional endocrine adjuvant therapy.
Breast cancer research: BCR 10/2008; 10(5):R76. · 5.24 Impact Factor
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ABSTRACT: Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC's) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. While assays based on RT-PCR are available, they have not been used for routine detection of DTC's.
To assess the quality of the assay, we performed a direct comparison of DTC detection rates in a large cohort of 385 patients using both standardized ICC and real-time RT-PCR protocols. Correlation rates were assessed, and results were compared with clinical data.
A significant correlation between ICC and RT-PCR was observed (P < 0.01). Positivity rates were similar (both 35%) and the results of both methods agreed in 73% of cases (280/385).
We describe a real-time RT-PCR based protocol for DTC-detection that has been specifically designed for routine clinical laboratory use. As such, RT-PCR has the potential to become an alternative testing method for BM evaluation in breast cancer patients.
Breast Cancer Research and Treatment 10/2008; 117(2):227-33. · 4.43 Impact Factor
