Sabine Kasimir-Bauer

Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients.

IntroductionThe persistence of circulating tumor cells (CTC) in breast cancer patients might be associated with stem cell like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells also may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Here we evaluated 226 blood samples of 39 metastatic breast cancer patients during a follow-up of palliative chemo-, antibody – or hormonal therapy for the expression of the stem cell marker ALDH1 and markers for EMT and correlated these findings with the presence of CTC and response to therapy.Methods2 × 5 ml blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1 and HER2 transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist1, Akt2, PI3Kα] and separately for the tumor stem-cell markers ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample.Results97% of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts. CTC were detected in 69/226 (31%) cancer samples. In the CTC (+) group, 62% were positive for at least one of the EMT markers and 69% for ALDH1, respectively. In the CTC (-) group the percentages were 7% and 14%, respectively. In non-responders, EMT and ALDH1 expression was found in 62% and 44% of patients, in responders the rates were 10% and 5%, respectively.ConclusionsOur data indicate that a major proportion of CTC of metastatic breast cancer patients shows EMT and tumor stem cell characteristics. Further studies are needed to prove whether these markers might serve as an indicator for therapy resistant tumor cell populations and, therefore, an inferior prognosis.

Detection and characterization of circulating tumor cells in blood of primary breast cancer patients by RT-PCR and comparison to status of bone marrow disseminated cells

IntroductionThe role of circulating tumor cells (CTCs) in blood of primary breast cancer patients is still under investigation. We evaluated the incidence of CTCs in blood, we evaluated the correlation between CTCs and disseminated tumor cells (DTCs) in the bone marrow (BM), and we characterized CTCs for the expression of HER2, the estrogen receptor (ER) and the progesterone receptor (PR).MethodsBlood of 431 patients with primary breast cancer were analyzed for EpCAM, MUC1 and HER2 transcripts with the AdnaTest BreastCancer™ (AdnaGen AG, Germany). Expression of the ER and PR was assessed in an additional RT-PCR. BM aspirates from 414 patients were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.ResultsDTCs were found in 107/414 patients (24%), CTCs were detected in 58/431 (13%) patients. DTCs were associated with PR status of the primary tumor (P = 0.04) and CTCs significantly correlated with nodal status (P = 0.04), ER (P = 0.05), and PR (P = 0.01). DTCs in the BM weakly correlated with CTCs (P = 0.05) in blood. Interestingly, the spread of CTCs was mostly found in triple-negative tumors (P = 0.01) and CTCs in general were mostly found to be triple-negative regardless of the ER, PR and HER2 status of the primary tumor.Conclusions(1) Due to the weak concordance between CTCs and DTCs the clinical relevance may be different. (2) The biology of the primary tumor seems to direct the spread of CTCs. (3) Since the expression profile between CTCs and the primary tumor differs, the consequence for the selection of adjuvant treatment has to be evaluated.

Expression of stem cell and epithelial-mesenchymal transition markers in primary breast cancer patients with circulating tumor cells

IntroductionThe presence of circulating tumor cells (CTC) in breast cancer might be associated with stem cell-like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTC must be able to perform epithelial-mesenchymal transition (EMT). We studied the expression of three EMT markers and the stem cell marker ALDH1 in CTC from 502 primary breast cancer patients. Data were correlated with the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and with clinicopathological data of the patients.MethodsA total of 2 × 5 ml of blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1, HER2 and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [TWIST1, Akt2, phosphoinositide kinase-3 (PI3Kα)] and separately for the tumor stem cell marker ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Two BM aspirates from all patients were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.ResultsNinety-seven percent of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. CTC were detected in 97/502 (19%) patients. At least one of the EMT markers was expressed in 29% and ALDH1 was present in 14% of the samples, respectively. Interestingly, 5% of the ALDH1-positive and 18% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. DTC in the BM were detected in 107/502 (21%) patients and no correlation was found between BM status and CTC positivity (P = 0.41). The presence of CTC, EMT and ALDH1 expression was not correlated to any of the prognostic clinical markers.ConclusionsOur data indicate that (1) a subset of primary breast cancer patients shows EMT and stem cell characteristics and (2) the currently used detection methods for CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.

Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct.

Bispecific T-cell engager (BiTE) are a class of bispecific single-chain antibodies that can very effectively redirect cytotoxic T cells for killing of tumor target cells. Here, we have assessed the in vivo efficacy of one representative, called bscEp-CAMxCD3, with specificity for tumors overexpressing epithelial cell adhesion molecule (Ep-CAM) in human xenograft models. Cells of the human colon carcinoma line SW480 were mixed at a 1:1 ratio with unstimulated human peripheral mononuclear cells, s.c. injected in nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, and animals were treated with bscEp-CAMxCD3. Five daily i.v. injections of as little as 100 ng per mouse of bscEp-CAMxCD3 completely prevented tumor outgrowth when treatment was started at the day of tumor cell inoculation. BscEp-CAMxCD3 was also efficacious when administered up to 8 days after xenograft injection. Established tumors could be eradicated in all animals by five 10 microg doses given between days 8 and 12 after tumor cell inoculation. To test the efficacy of bscEp-CAMxCD3 in a more physiologic model, pieces of primary metastatic tumor tissue from ovarian cancer patients were implanted in NOD/SCID mice. Partial tumor engraftment and growth was observed with four of six patient samples. Treatment of established tumors with daily 5 microg doses led to a significant reduction and, in some cases, eradication of human tumor tissue. These effects obviously relied on the tumor-resident T cells reactivated by bscEp-CAMxCD3. Our data show that the class of single-chain bispecific antibodies has very high antitumor efficacy in vivo and can use previously unstimulated T cells at low effector-to-target ratios.

Comparison of estrogen and progesterone receptor status of circulating tumor cells and the primary tumor in metastatic breast cancer patients

OBJECTIVES The expression of predictive markers including the estrogen (ER) and progesterone receptor (PR) expression can change during the course of the disease. Therefore, reassessment of these markers at the time of disease progression might help to optimize treatment decisions. Metastatic tissue may be difficult to obtain for repeated analysis. In this context, characterization of circulating tumor cells (CTCs) could be of relevance. It was the purpose of the present study (1) to reevaluate the ER/PR expression by CTCs and (2) to compare the hormone receptor status expression profile of CTCs with the primary tumor. METHODS We evaluated 193 blood samples from metastatic breast cancer patients at the time of first diagnosis of metastatic disease or disease progression. All samples underwent immunomagnetic enrichment using the AdnaTest BreastCancerSelect (AdnaGen AG, Germany) within 4h after blood withdrawal followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR in separated tumor cells using the AdnaTest BreastCancerDetect. CTCs were analyzed for the three breast cancer-associated markers: EpCAM, Muc-1, Her-2 and actin as an internal PCR control. Expression of the ER and PR was assessed in an additional RT-PCR. The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. RESULTS The overall detection rate for CTCs was 45% (87/193 patients) with the expression rates of 71% for EpCAM (62/87 patients), 73% for MUC1 (64/87 patients), 48% for HER2 (42/87 patients), 19% for ER (17/87 patients) and 10% for PR (9/87 patients), respectively. Comparisons with the primary tumor were only performed in CTC+ patients (n=87). In 48/62 (77%) patients with ER+ tumors, CTCs were ER- and 46/53 (87%) patients with PR+ tumors did not express PR on CTCs. Primary tumors and CTCs displayed a concordant ER and PR status in only 41% (p=0.260) and 45% (p=0.274) of cases, respectively. CONCLUSION Most of the CTCs were ER/PR-negative despite the presence of an ER/PR- positive primary tumor. The predictive value of hormone receptor status expression profile of CTCs for palliative endocrine therapy has to be prospectively evaluated. STATEMENT: We recently demonstrated in more than 400 primary breast cancer patients that the expression profile between CTCs and the primary tumor with regard to ER/PR/HER2 positivity differs. The concordance rate between ER, PR and HER2 status of CTCs and the primary tumor was 29%, 25% and 53%, respectively (Fehm T et al., Breast Cancer Res Aug 10 2009, 11(4) pR59). Based on these results we studied blood samples of 193 metastatic breast cancer patients participating in the German DETECT study (1) to reevaluate the ER/PR expression by CTCs and (2) to compare the hormone receptor status expression profile of CTCs with the primary. As already shown for primary breast cancer, most of the CTCs were ER/PR-negative despite the presence of an ER/PR- positive primary tumor. In the metastatic setting the phenotype of CTC reflects the phenotype of metastatic disease. Therefore palliative treatment selected based on the expression profile may not be effective since the phenotype has changed during disease progression. To our knowledge, this study is one of the biggest to compare hormonal receptor expression on CTC and the primary tumor. We hope that our manuscript is suitable for publication in Gynecologic Oncology.

Screening for circulating nucleic acids and caspase activity in the peripheral blood as potential diagnostic tools in lung cancer

The focus of the current investigational study was to examine whether circulating nucleic acids (i.e., DNA and microRNAs) have the potential to become suitable blood‐based markers for diagnosis and progression of lung cancer. The concentrations of cell‐free DNA and four circulating microRNAs (miR10b, miR34a, miR141 and miR155) as well as the caspase activity were measured in serum of 35 lung cancer patients (19 non‐small‐cell lung cancer, 8 small cell lung cancer patients and 8 patients with indefinite cancer type), 7 patients with benign lung tumors and 28 healthy individuals by PicoGreen, TaqMan MicroRNA, and Caspase‐Glo®3/7 assay, respectively. The data were correlated with the established risk factors for lung cancer progression. The concentrations of cell‐free DNA (p = 0.0001), serum microRNAs (p = 0.0001) and caspase activities (p = 0.0001) significantly discriminated cancer patients from healthy individuals. Serum DNA, caspase activities and RNA levels could not distinguish between patients with benign lung disease and cancer patients. However, the levels of miR10b (p = 0.002), miR141 (p = 0.0001) and miR155 (p = 0.007) were significantly higher in lung cancer patients than those in patients with benign disease. As determined by the Spearman–Rho test, high levels of cell‐free DNA significantly correlated with elevated circulating caspase activities (p = 0.0001). In lung cancer patients high serum miR10b values associated with lymph node metastasis (p < 0.03) and elevated levels of TPA (tissue polypeptide antigen, p = 0.01), whereas high serum miR141 values associated with elevated levels of uPA (urokinase plasminogen activator, p = 0.02).

Molecular profiling and prognostic relevance of circulating tumor cells in the blood of ovarian cancer patients at primary diagnosis and after platinum-based chemotherapy.

Objectives: Disseminated tumor cells (DTCs) in the bone marrow (BM) were shown to be of prognostic significance in gynecological cancers. Bone marrow aspiration is less accepted by patients compared with blood drawing. In this pilot study, we applied the AdnaTest BreastCancer based on immunomagnetic enrichment, targeting common antigens on epithelial gynecological cancers, followed by multiplex reverse transcriptase-polymerase chain reaction for selection and detection of circulating tumor cells (CTCs) in the blood of 122 ovarian cancer patients at primary diagnosis and/or after platinum-based chemotherapy. Results were compared with detection of DTC in BM. Methods: Ten-milliliter blood was obtained before surgery (n = 86) and/or after chemotherapy (n = 70) and analyzed for CTC with the AdnaTest BreastCancer for the detection of EpCAM-, MUC-1-, and HER-2-transcripts. CA 125 was assessed in an additional single-plex reverse transcriptase-polymerase chain reaction. Bone marrow aspirates were analyzed in duplicate by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results: Before surgery, CTCs were detected in 19% of patients, expressing EpCAM (31%), MUC-1 (50%), HER-2 (31%), and CA 125 (50%), respectively. After chemotherapy, the overall detection rate for CTC was 27%, thereof EpCAM (68%), MUC-1 (47%), HER2 (21%), and CA 125 (37%). The overall detection rate for DTC in the BM was 35% before surgery and 31% after therapy. A comparison between DTC and CTC resulted in a concordance rate of 59% before surgery and 56% after chemotherapy. CTC positivity significantly correlated with shorter overall survival before surgery (P = 0.0054) and after chemotherapy (P = 0.047). Conclusions: This methodological approach might help to identify molecular targets for specific biological therapies. Blood analysis could give additional information complimentary to that obtained by DTC.

Comparative evaluation of cell-free tumor DNA in blood and disseminated tumor cells in bone marrow of patients with primary breast cancer

IntroductionThe origin and clinical relevance of circulating cell-free tumor DNA in the blood of cancer patients is still unclear. Here we investigated whether the detection of this DNA is related to bone marrow (BM) micrometastasis and tumor recurrence in breast cancer patients.MethodsBM aspirates of 81 primary breast cancer patients were analyzed for the presence of disseminated tumor cells (DTC) by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. PCR-based fluorescence microsatellite analysis was performed for detection of loss of heterozygosity (LOH) at 6 polymorphic markers using cell-free serum DNA. The data were correlated with established risk factors, and patients were followed-up over 6-10 years.ResultsLOH was detected in 33.5% of blood samples. The occurrence of LOH at the entire microsatellite marker set correlated with histopathology (P = 0.05) and grading (P = 0.006) of the primary tumor. The genomic region characterized by marker D9S171 was only affected by LOH in patients with increased tumor stages (pT2-4, P < 0.05) and older age (≥ 55 years, P = 0.05). Kaplan-Meier analysis showed that LOH at D3S1255 (P = 0.009) and D9S171 (P = 0.001) were significantly associated with tumor relapse. In BM, DTC were detected in 39.5% of the patients, and this finding correlated with distant metastases (P < 0.05). Patients with DTC-positive BM had higher DNA yields in their blood than patients with DTC-negative BM (P < 0.05). However, no significant correlations were found between the presence of DTC in BM and the detection of marker-specific LOH on blood DNA.ConclusionsThe detection of LOH on cell-free tumor DNA in blood is unrelated to BM micrometastasis and provides independent information on breast cancer progression.

Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression

BackgroundAs cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases.MethodsThe concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer (M0, n = 31), metastatic breast cancer (M1, n = 32), and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo®3/7 Assay, respectively.ResultsPatients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001), whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008).ConclusionOur findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression.

In Acute Myeloid Leukemia only the Coexpression of at Least Two Proteins Including P-Glycoprotein, the Multidrug Resistance-Related Protein MRP, bcl-2, Mutant p53 and Heat-Shock Protein 27 is Predictive for the Response to Induction Chemotherapy

Purpose Determination of the potential resistance markers P-glycoprotein (Pgp), the multidrug resistance-related protein (MRP), bd-2, mutant p53 and the heat shock protein 27 (HSP27) in myeloid blasts for patients with de novo acute myeloid leukemia (AML) at the time of diagnosis. Evaluation whether these proteins alone or in combination are predictive for the response to induction chemotherapy with cytosine arabinoside (ara-C) idarubicin (AIDA).