Eva Obermayr

Detection of EpCAM positive and negative circulating tumor cells in metastatic breast cancer patients

Abstract Background. Immunomagnetic EpCAM based methods are used to enrich circulating tumor cells (CTCs) in metastatic breast cancer (mBC) patients. EpCAM negative CTCs may be missed. We addressed the question of the reliability of an EpCAM dependent assay to enrich CTCs. Methods. To elucidate this issue, our study has been designed to assess two different CTC enrichment technologies (i) in EpCAM positive (+) and EpCAM negative cell lines and (ii) in mBC patients in dependency on their respective EpCAM expression. These two technologies encompass one anti-EpCAM immunomagnetic enrichment technology, MACS HEA MicroBeads® (MACS), and one EpCAM independent density centrifugation method, OncoQuick® plus (OQ+). Furthermore, the coherence between EpCAM expression in the primary tumor tissue of mBC patients and the CTC detection rates in the corresponding patients is analyzed. Results. (i) MACS recovered significantly more EpCAM (+) than EpCAM (−) tumor cells (p < 0.001) in spiked blood samples. With OQ+ no significantly different recovery rates between EpCAM (+) and EpCAM (−) tumor cells (p = 0.796) were detected. (ii) In mBC patients MACS yielded a significantly higher (p = 0.024) detection rate of EpCAM (+) CTCs. No statistically significant difference (p = 0.070) was found concerning the EpCAM status-based detection rate of CTCs by OQ+. (iii) CTC detection rates are independent of the primary tumors’ EpCAM expression. Conclusions. EpCAM (−) CTCs can not be detected by immunomagnetic EpCAM dependent enrichment methods. EpCAM independent enrichment technologies seem to be superior to detect the entire CTC population. Evaluation of CTCs as prognostic marker should compromise EpCAM (+) and (−) subpopulations.

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

BackgroundThe presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.MethodsGene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.ResultsSix genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.ConclusionsThe panel of six genes was found superior to EpCAM and hMAM for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.

Molecular characterization of circulating tumor cells in patients with ovarian cancer improves their prognostic significance — A study of the OVCAD consortium

OBJECTIVE The study aims at identifying novel markers for circulating tumor cells (CTCs) in patients with epithelial ovarian cancer (EOC), and at evaluating their impact on outcome. METHODS Microarray analysis comparing matched EOC tissues and peripheral blood leucocytes (N=35) was performed to identify novel CTC markers. Gene expression of these novel markers and of EpCAM was analyzed using RT-qPCR in blood samples taken from healthy females (N=39) and from EOC patients (N=216) before primary treatment and six months after adjuvant chemotherapy. All samples were enriched by density gradient centrifugation. CTC positivity was defined by over-expression of at least one gene as compared to the healthy control group. RESULTS CTC were detected in 24.5% of the baseline and 20.4% of the follow-up samples, of which two thirds were identified by overexpression of the cyclophilin C gene (PPIC), and just a few by EpCAM overexpression. The presence of CTCs at baseline correlated with the presence of ascites, sub-optimal debulking, and elevated CA-125 and HE-4 levels, whereas CTC during follow-up occurred more often in older and platinum resistant patients. PPIC positive CTCs during follow-up were significantly more often detected in the platinum resistant than in the platinum sensitive patient group, and indicated poor outcome independent from classical prognostic parameters. CONCLUSIONS Molecular characterization of CTC is superior to a mere CTC enumeration or even be the rationale for CTC diagnostics at all. Ultimately CTC diagnostics may lead to more personalized treatment of EOC, especially in the recurrent situation.

Skeletal Muscle Depletion and Markers for Cancer Cachexia Are Strong Prognostic Factors in Epithelial Ovarian Cancer

Objective Tumor cachexia is an important prognostic parameter in epithelial ovarian cancer (EOC). Tumor cachexia is characterized by metabolic and inflammatory disturbances. These conditions might be reflected by body composition measurements (BCMs) ascertained by pre-operative computed tomography (CT). Thus, we aimed to identify the prognostically most relevant BCMs assessed by pre-operative CT in EOC patients. Methods We evaluated muscle BCMs and well established markers of nutritional and inflammatory status, as well as clinical-pathological parameters in 140 consecutive patients with EOC. Furthermore, a multiplexed inflammatory marker panel of 25 cytokines was used to determine the relationship of BCMs with inflammatory markers and patient’s outcome. All relevant parameters were evaluated in uni- and multivariate survival analysis. Results Muscle attenuation (MA)—a well established BCM parameter—is an independent prognostic factor for survival in multivariate analysis (HR 2.25; p = 0.028). Low MA—reflecting a state of cachexia—is also associated with residual tumor after cytoreductive surgery (p = 0.046) and with an unfavorable performance status (p = 0.015). Moreover, MA is associated with Eotaxin and IL-10 out of the 25 cytokine multiplex marker panel in multivariate linear regression analysis (p = 0.021 and p = 0.047, respectively). Conclusion MA—ascertained by routine pre-operative CT—is an independent prognostic parameter in EOC patients. Low MA is associated with the inflammatory, as well as the nutritional component of cachexia. Therefore, the clinical value of pre-operative CT could be enhanced by the assessment of MA.

Cyclin E1 (CCNE1) as independent positive prognostic factor in advanced stage serous ovarian cancer patients - A study of the OVCAD consortium

Cyclin E, coded by the genes CCNE1 and CCNE2, is the main regulator for transition from G1 to S phase determining cell division. CCNE1 and CCNE2 are known oncogenes in many cancer entities. Especially CCNE1 has frequently been associated with gene amplifications in various malignancies, emphasising its role as a putative oncogene. We determined gene expression and copy number of CCNE1 and CCNE2 by quantitative polymerase chain reaction (PCR) from 172 International Federation of Obstetrics and Gynecology (FIGO) II/III/IV stage serous epithelial ovarian cancer (EOC) tissues and analysed its impact on outcome. Furthermore, whole transcriptome gene expression changes correlating with CCNE1 expression were determined by microarray technology, interpreted by Signalling Pathway Impact Analysis (SPIA), Tool for Inferring Network of Genes (TINGe), and illustrated by hive plots. Protein-protein interaction (PPI) networks were also used for the interpretation. Interestingly, and contradictory to most reports and intuitive expectations, high CCNE1 expression correlated with better overall survival (p=0.005) if corrected for usual clinicopathologic parameters and a molecular subclassification. Using different grading systems or only high graded tumours had no impact on this correlation. Copy number of CCNE1 was increased in 25% of cases which correlated highly significantly with expression but showed no impact on outcome. CCNE2 had no impact on outcomes at all. Whole genome transcriptome analysis revealed 1872 differentially expressed genes correlated to CCNE1 expression, which were significantly enriched with genes from five pathways (e.g. cell cycle and viral carcinogenesis pathway were up-regulated and the Fanconi anaemia pathway was down-regulated). High CCNE1 gene expression is a significant and independent predictor for prolonged overall survival in FIGO III/IV EOC patients. This upside down impact of CCNE1 on survival probably reflects the special characteristic of EOC with tumour dissemination in the near anaerobic peritoneal cavity as the predominant cause of death, compared to other cancer entities where distant metastasis are predominantly lethal.

A combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer - a study of the OVCAD consortium

BackgroundThe immune system is a key player in fighting cancer. Thus, we sought to identify a molecular ‘immune response signature’ indicating the presence of epithelial ovarian cancer (EOC) and to combine this with a serum protein biomarker panel to increase the specificity and sensitivity for earlier detection of EOC.MethodsComparing the expression of 32,000 genes in a leukocytes fraction from 44 EOC patients and 19 controls, three uncorrelated shrunken centroid models were selected, comprised of 7, 14, and 6 genes. A second selection step using RT-qPCR data and significance analysis of microarrays yielded 13 genes (AP2A1, B4GALT1, C1orf63, CCR2, CFP, DIS3, NEAT1, NOXA1, OSM, PAPOLG, PRIC285, ZNF419, and BC037918) which were finally used in 343 samples (90 healthy, six cystadenoma, eight low malignant potential tumor, 19 FIGO I/II, and 220 FIGO III/IV EOC patients). Using new 65 controls and 224 EOC patients (thereof 14 FIGO I/II) the abundances of six plasma proteins (MIF, prolactin, CA125, leptin, osteopondin, and IGF2) was determined and used in combination with the expression values from the 13 genes for diagnosis of EOC.ResultsCombined diagnostic models using either each five gene expression and plasma protein abundance values or 13 gene expression and six plasma protein abundance values can discriminate controls from patients with EOC with Receiver Operator Characteristics Area Under the Curve values of 0.998 and bootstrap .632+ validated classification errors of 3.1% and 2.8%, respectively. The sensitivities were 97.8% and 95.6%, respectively, at a set specificity of 99.6%.ConclusionsThe combination of gene expression and plasma protein based blood derived biomarkers in one diagnostic model increases the sensitivity and the specificity significantly. Such a diagnostic test may allow earlier diagnosis of epithelial ovarian cancer.

Relaxin and gonadal steroid receptors in uterosacral ligaments of women with and without pelvic organ prolapse.

Introduction and hypothesisThis study evaluates the expression of estrogen receptor isoforms alpha (ERα) and beta (ERβ), progesterone receptor (PR), and relaxin receptor isoforms 1 and 2 (LGR7, LGR8) in uterosacral ligament (USL) tissue of women with pelvic organ prolapse and controls.MethodsTissue samples of USL from women with and without pelvic organ prolapse (POP) were subjected to immunohistochemistry against ERα, ERβ, PR, and LGR7 proteins. The respective mRNA expression as well as of LGR8 was assessed by quantitative real-time polymerase chain reaction.ResultsThe cellular distribution of the receptor proteins was different due to cell types, independent of POP: ERα and PR were found in smooth muscle cells, but not in endothelial cells, whereas ERβ was found in endothelial cells, but not in connective tissue. ERα, ERβ, PR, and LGR7 mRNAs could be detected in all patients of both groups. ERα mRNA expression was significantly and ERβ mRNA borderline significantly higher in USL of patients with POP: ERα: p < 0.001, ERβ: p = 0.057.ConclusionsEnhanced effects of estrogen via altered mRNA expression patterns of ERα and ERβ—but not those of progesterone—may exist in USL of patients affected by POP. A local effect of relaxin needs to be further clarified because of this first report of prevalent ligamental expression of LGR7.

Circulating tumor cells: potential markers of minimal residual disease in ovarian cancer? a study of the OVCAD consortium

Purpose In 75% of ovarian cancer patients the tumor mass is completely eradicated by established surgical and cytotoxic treatment; however, the majority of the tumors recur within 24 months. Here we investigated the role of circulating tumor cells (CTCs) indicating occult tumor load, which remains inaccessible by established diagnostics. Experimental design Blood was taken at diagnosis (baseline samples, n = 102) and six months after completion of adjuvant first-line chemotherapy (follow-up samples; n = 78). CTCs were enriched by density gradient centrifugation. A multi-marker immunostaining was established and further complemented by FISH on CTCs and tumor/metastasis tissues using probes for stem-cell like fusion genes MECOM and HHLA1. Results CTCs were observed in 26.5% baseline and 7.7% follow-up blood samples at a mean number of 12.4 and 2.8 CTCs per ml blood, respectively. Baseline CTCs indicated a higher risk of death in R0 patients with complete gross resection (univariate: HR 2.158, 95% CI 1.111–4.191, p = 0.023; multivariate: HR 2.720, 95% CI 1.340–5.522, p = 0.006). At follow-up, the presence of CTCs was associated with response to primary treatment as assessed using RECIST criteria. Chromosomal gains at MECOM and HHLA1 loci suggest that the observed cells were cancer cells and reflect pathophysiological decisive chromosomal aberrations of the primary and metastatic tumors. Conclusions Our data suggest that CTCs detected by the multi-marker protein panel and/or MECOM/HHLA1 FISH represent minimal residual disease in optimally debulked ovarian cancer patients. The role of CTCs cells especially for clinical therapy stratification of the patients has to be validated in consecutive larger studies applying standardized treatment schemes.

Efficient leukocyte depletion by a novel microfluidic platform enables the molecular detection and characterization of circulating tumor cells

RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix™ technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach. In this study, we established a workflow including the micro-fluidic Parsortix™ technology for the molecular detection of CTC related transcripts. Background levels of EpCAM, PPIC, TUSC3, and MAL2 were efficiently removed due to an up to 106-fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix™-enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%). The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.

Immunobiochemical pathways of neopterin formation and tryptophan breakdown via indoleamine 2,3-dioxygenase correlate with circulating tumor cells in ovarian cancer patients– A study of the OVCAD consortium

OBJECTIVE Circulating tumor cells (CTCs) may represent a chronic stimulus for the immune system. In the present study we investigated the potential association of CTCs, the immune activation marker neopterin, and the ratio of kynurenine to tryptophan (Kyn/Trp) as a measure for tryptophan breakdown. METHODS Neopterin, tryptophan and kynurenine levels were measured in plasma samples from patients with benign gynecological diseases (n=65) and with primary advanced epithelial ovarian cancer (EOC) at diagnosis (n=216) and six months after adjuvant platinum-based chemotherapy (n=45) by an enzyme-linked immunosorbent assay and high performance liquid chromatography. The presence of CTCs had been assessed in a previous study by qPCR-based analysis of CTC-related transcripts in the blood. The respective plasma levels in EOC and benign samples were compared using a two-tailed Chi2 or Fishers exact test. The associations of the analytes and Kyn/Trp with clinicopathological parameters, platinum-sensitivity, and the presence of CTC-related transcripts were assessed using a two-sided t-test. Associations with patient outcome were evaluated using Cox regression analysis. RESULTS In EOC, elevated Kyn/Trp and neopterin levels were associated with advanced disease, peritoneal carcinomatosis, ascites, sub-optimal debulking, poor response to therapy and worse outcome. Likewise, neopterin and Kyn/Trp were elevated in CTC-positive patients, both at diagnosis and at follow-up in platinum-sensitive disease. CONCLUSIONS We observed concomitant alterations of CTCs and immune system related biomarkers suggesting that immune responses along with increase of neopterin and Kyn/Trp concentrations are not necessarily only located at the site of the tumor, but may also go on in the circulation.