The regulatory effect of hyaluronan on human mesenchymal stem cells' fate modulates their interaction with cancer cells in vitro.

Metastatic spread of cancer cells into a pre-metastatic niche is highly dependent on a supporting microenvironment. Human bone marrow-derived mesenchymal stem cells (bmMSCs) contribute to the tumor microenvironment and promote cancer metastasis by inducing epithelial-to-mesenchymal transition and immune evasion. The underlying mechanisms, however, are incompletely understood. The glycosaminoglycan hyaluronan (HA) is a central component of the extracellular matrix and has been shown to harbor pro-metastatic properties. In this study we investigated the highly disseminating breast cancer and glioblastoma multiforme cell lines MDA-MB-321 and U87-MG which strongly differ in their metastatic potential to evaluate the impact of HA on tumor promoting features of bmMSC and their interaction with tumor cells. We show that adipogenic differentiation of bmMSC is regulated by the HA-matrix. This study reveals that MDA-MB-231 cells inhibit this process by the induction of HA-synthesis in bmMSCs and thus preserve the pro-tumorigenic properties of bmMSC. Furthermore, we show that adhesion of MDA-MB-231 cells to bmMSC is facilitated by the tumor cell-induced HA-rich matrix and is mediated by the HA-receptor LAYN. We postulate that invasive breast cancer cells modulate the HA-matrix of bmMSC to adapt the pre-metastatic niche. Thus, the HA-matrix provides a potential novel therapeutic target to prevent cancer metastasis.

A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

BACKGROUND: Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). METHODS: In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. RESULTS: All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. CONCLUSIONS: In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

Prognostic value of preoperative circulating tumor cells counts in patients with UICC stage I-IV colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. There is an urgent need to identify prognostic markers for patients undergoing curative resection of CRC. The detection of circulating tumor cells in peripheral blood is a promising approach to identify high-risk patients with disseminated disease in colorectal cancer. This study aims to evaluate the prognostic relevance of preoperative CTCs using the Cellsearch® system (CS) in patients, who underwent resection with curative intent of different stages (UICC I-IV) of colorectal cancer. Out of 91 Patients who underwent colorectal resection, 68 patients were included in this study. CTC analysis was performed in patients with CRC UICC stages I-IV immediately before surgery. Data were correlated with clinicopathological parameters and patient outcomes. One or more CTCs/7.5 mL were detected in 45.6% (31/68) of patients. CTCs were detected in all stages of the Union of International Cancer Control (UICC), in stage I (1/4, 25%), in stage II (4/12, 33.3%), in stage III (5/19, 26.3%) and in stage IV (21/33, 63.6%). The detection of ≥ 1 CTCs/ 7.5ml correlated to the presence of distant overt metastases (p = 0.014) as well as with shorter progression-free (p = 0.008) and overall survival (p = 0.008). Multivariate analyses showed that the detection of ≥ 1 CTCs/ 7.5ml is an independent prognostic indicator for overall survival (HR, 3.14; 95% CI, 1.18-8.32; p = 0.021). The detection of CTCs is an independent and strong prognostic factor in CRC, which might improve the identification of high-risk patients in future clinical trials.

Cysteine-Rich Angiogenic Inducer 61: Pro-Survival Function and Role as a Biomarker for Disseminating Breast Cancer Cells.

(1) Background: the early detection of cancer cells in the blood or bone marrow of breast cancer patients improves the understanding of metastasis. Disseminating tumor cells in the bone marrow with a pronounced manifestation of mesenchymal markers (mDTC) are difficult to detect by epithelial markers, but they are relevant in the initiation of metastasis. (2) Methods: the breast cancer mDTC cell line BC-M1 was analyzed by mass spectrometry, which revealed high levels of the protein-cysteine-rich angiogenic inducer 61 (Cyr61). The function of Cyr61 was investigated using shRNA and hypoxia. Peripheral blood samples from 35 breast cancer patients were investigated for CTCs defined as cytokeratin-positive/CD45-negative cells. (3) Results: the Cyr61 levels are elevated in mDTC lines from breast, lung, and prostate cancer patients. The loss of Cyr61 resulted in the diminished expression of hypoxia-inducible factor 1-alpha, and increased apoptosis. Cyr61 was present in 47 (43%) of the 109 detected circulating tumor cells (CTCs), while the blood and bone marrow cells from healthy controls were Cyr61-negative. (4) Conclusions: Cyr61 is expressed in mDTC lines, supports the viability of cancer cells, and classifies a new subset of cytokeratin-positive CTCs, which deserves further investigation.

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+ ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream.

BACKGROUND: Solid epithelial tumors like breast cancer are the most frequent malignancy in women. Circulating tumor cells (CTCs) are frequently released from hypoxic areas into the blood, where CTCs face elevated oxygen concentrations. This reoxygenation might challenge the use of CTCs for liquid biopsy. METHODS: We modeled this situation in vitro using the breast cancer cell lines-MCF-7, MDA-MB-468, MDA-MB-231-and the cell line BC-M1 established from DTCs in the bone marrow. Cells were cultured under hypoxia, followed by a reoxygenation pulse for 4 h, reflecting the circulation time of CTCs. Analyzed were gene products like EGFR, ErbB-2, EpCAM, PD-L1 on mRNA and protein level. RESULTS: mRNAs of erbb2 or pdl1 and protein levels of PD-L1 displayed significant changes, whereas ErbB-2 protein levels remained constant. The strongest discrepancy between protein and mRNA levels under hypoxia was observed for EGFR, supporting the idea of cap-independent translation of egfr mRNA. Analyses of the phosphorylation of AKT, Erk 1/2, and Stat3 revealed strong alterations after reoxygenation. CONCLUSIONS: CTCs reaching secondary sites faster than reoxygenation could alter the mRNA and protein levels in the cells. CTC and DTC with high PD-L1 levels might become quiescent under hypoxia but were easily reactivated by reoxygenation.

Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients.

Circulating tumor cells (CTCs) are promising tools for risk prediction and the monitoring of response to therapy in cancer patients. Within the EU/IMI CANCER-ID consortium, we validated CTC enrichment systems for future inclusion into clinical trials. Due to the known heterogeneity of markers expressed on CTCs, we tested the Parsortix® system (ANGLE plc) which enables label-independent CTC enrichment from whole blood based on increased size and deformability of these tumor cells compared to leukocytes. We performed extensive comparisons both with spiked-in blood models (i.e., MDA-MB-468 tumor cell line cells spiked at very low concentration into blood from healthy donors) and validated the protocol on actual clinical samples from breast, lung, and gastrointestinal cancer patients to define optimal conditions for CTC enrichment. Multiple parameters including cassette gap, separation pressure, and cell fixatives were compared in parallel. Also, the compatibility of blood collection tubes with whole genome amplification of isolated tumor cells was demonstrated and we furthermore established a workflow for semi-automated CTC detection using a quantitative cell imager. The established workflow will contribute to supporting the use of size-based CTC enrichment platforms in clinical trials testing the clinical validity and utility of CTCs for personalized medicine.

Tumor-Associated Release of Prostatic Cells into the Blood after Transrectal Ultrasound-Guided Biopsy in Patients with Histologically Confirmed Prostate Cancer.

BACKGROUND: Transrectal ultrasound-guided prostate biopsy (TRUS) is a standard procedure for prostate cancer diagnosis. Because prostate cancer is a multifocal disease in many patients, multiple sampling (n ≥ 10) is required, which may bear the risk of systemic spread of cancer cells. DESIGN: Using the standardized CellSearch® system that allows for the detection of single epithelial cell adhesion molecule-positive circulating tumor cells (CTCs) in blood, we investigated whether prostate biopsy is associated with release of prostatic tumor cells into the circulation. Peripheral blood was obtained before and within 30 min after performing prostate biopsy from 115 men with increased serum prostate-specific antigen. RESULTS: The number of CTCs significantly increased after biopsy in men with histologically confirmed prostate cancer (odds ratio, 7.8; 95% CI, 4.8-12.8), whereas no biopsy-related changes could be detected in men without confirmed prostate cancer. Multivariable analysis showed that biopsy-related increase of CTCs was significantly correlated with a worse progression-free survival (hazard ratio, 12.4; 95% CI, 3.2-48.6) within the median follow-up of 41 months. CONCLUSIONS: Prostate biopsies may lead to a tumor-associated release of CTCs into the blood circulation. Larger confirmatory trials with longer follow-up periods are required before any change in clinical practice can be recommended.

Intra-Patient Heterogeneity of Circulating Tumor Cells and Circulating Tumor DNA in Blood of Melanoma Patients.

Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.

Detection of Androgen Receptor Variant 7 (ARV7) mRNA Levels in EpCAM-Enriched CTC Fractions for Monitoring Response to Androgen Targeting Therapies in Prostate Cancer.

Expression of the androgen receptor splice variant 7 (ARV7) in circulating tumor cells (CTCs) has been associated with resistance towards novel androgen receptor (AR)-targeting therapies. While a multitude of ARV7 detection approaches have been developed, the simultaneous enumeration of CTCs and assessment of ARV7 status and the integration of validated technologies for CTC enrichment/detection into their workflow render interpretation of the results more difficult and/or require shipment to centralized labs. Here, we describe the establishment and technical validation of a novel ARV7 detection method integrating the CellSearch® technology, the only FDA-cleared CTC-enrichment method for metastatic prostate cancer available so far. A highly sensitive and specific qPCR-based assay was developed, allowing detection of ARV7 and keratin 19 transcripts from as low as a single ARV7+/K19+ cell, even after 24 h of sample storage. Clinical feasibility was demonstrated on blood samples from 26 prostate cancer patients and assay sensitivity and specificity was corroborated. Our novel approach can now be included into prospective clinical trials aimed to assess the predictive values of CTC/ARV7 measurements in prostate cancer.

Determination of PD-L1 Expression in Circulating Tumor Cells of NSCLC Patients and Correlation with Response to PD-1/PD-L1 Inhibitors.

Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch® System. We tested an epitope-independent method (ParsortixTM system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45-, pankeratins (K)+ cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1+CTCs, 47% had PD-L1+ and PD-L1-CTCs, and only 7% displayed exclusively PD-L1-CTCs. The percentage of PD-L1+CTCs did not correlate with the percentage of PD-L1+ in biopsies determined by immunohistochemistry (p = 0.179). Upon disease progression, all patients showed an increase in PD-L1+CTCs, while no change or a decrease in PD-L1+CTCs was observed in responding patients (n = 11; p = 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1+CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

BACKGROUND: Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS: We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS: In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS: Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS: We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS: The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Improved detection of circulating tumor cells in non-metastatic high-risk prostate cancer patients.

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system, an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points, CTCs were detected in 37%, 54.9% and 58.7% of patients using CellSearch, CellCollector and EPISPOT, respectively. The cumulative positivity rate of the three CTC assays was 81.3% (87/107) with 21.5% (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66% before RP to 34% after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p < 0.0001) and clinical tumor stage (p = 0.04), while the other assays showed no significant correlations. In conclusion, CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer.

Accession of Tumor Heterogeneity by Multiplex Transcriptome Profiling of Single Circulating Tumor Cells.

BACKGROUND: Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. METHODS: CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. RESULTS: Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). CONCLUSIONS: Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Frequent detection of PIK3CA mutations in single circulating tumor cells of patients suffering from HER2-negative metastatic breast cancer.

Modern technologies enable detection and characterization of circulating tumor cells (CTC) in peripheral blood samples. Thus, CTC have attracted interest as markers for therapeutic response in breast cancer. First studies have incorporated CTC analyses to guide therapeutic interventions and stratification of breast cancer patients. Aim of this study was to analyze characteristic features of CTC as biomarker for predicting resistance to HER2-targeted therapies. Therefore, CTC from metastatic breast cancer patients with HER2-negative primary tumors screened for the prospective randomized phase III trial DETECT III were explored for their HER2 status and the presence of PIK3CA mutations. Detection and characterization of HER2 expression of CTC were conducted with the CellSearch(®) system. Fifteen of 179 CTC-positive patients (8.4%) contained ≥1 CTC with strong HER2 expression. Genomic DNA from individual CTC isolated by micromanipulation was propagated by whole genome amplification and analyzed for PIK3CA mutations in exons 9 and 20 by Sanger sequencing. One or more CTC/7.5 mL were detected in 179/290 patients (61.7%). In 109 patients (34.8%), ≥5 CTC/7.5 mL were found. We detected at least one CTC with the mutation p.E542K, p.E545K, p.H1047R, p.H1047L or p.M1043V in 12/33 patients (36.4%). Thirty six of 114 CTC (31.6%) harbored one of these mutations. CTC in individual patients exhibited heterogeneity concerning PIK3CA mutations and HER2 expression. In conclusion, clinically relevant genomic aberrations such as mutations in the hotspot regions of exon 9 and 20 of the PIK3CA gene can be detected in single CTC and might provide insights into mechanisms of resistance to HER2-targeted therapies.

Characterization of different CTC subpopulations in non-small cell lung cancer.

Circulating tumour cells (CTCs) serve as valuable biomarkers. However, EpCAM positive CTCs are less frequently detected in NSCLC patients compared to other epithelial tumours. First, EpCAM protein expression was analysed in primary and metastatic lung cancer tissue. In both groups 21% of the samples were EpCAM negative. Second, the CellSearch system identified 15% of patients (n = 48) as CTC positive whereas a multiplex RT-PCR for PIK3CA, AKT2, TWIST, and ALDH1 following EGFR, HER2 and EpCAM based enrichment detected CTCs in 29% of the patients. Interestingly, 86% of CTC positive patients were found to express ALDH1. Only 11% of the patients were CTC-positive by both techniques. CTC positivity was associated with patient disease state when assessed by the multiplex RT-PCR assay (p = 0.015). Patients harbouring tumours with an altered EGFR genotype were more frequently CTC-positive compared to patients with EGFR wildtype tumours. In subsets of patients, CTCs were found to express genes involved in resistance to therapy such as HER3 and MET. In conclusion, using multiple targets for CTC capture and identification increases the sensitivity of CTC detection in NSCLC patients, which can be explained by the presence of different CTC subtypes with distinct molecular features.

Improved Detection of Circulating Tumor Cells in Metastatic Colorectal Cancer by the Combination of the CellSearch® System and the AdnaTest®.

Colorectal cancer (CRC) is one of the major causes of cancer-related death and reliable blood-based prognostic biomarkers are urgently needed. The enumeration and molecular characterization of circulating tumor cells (CTCs) has gained increasing interest in clinical practice. CTC detection by CellSearch® has already been correlated to an unfavorable outcome in metastatic CRC. However, the CTC detection rate in mCRC disease is low compared to other tumor entities. Thus, the use of alternative (or supplementary) assays might help to itemize the prognostic use of CTCs as blood-based biomarkers. In this study, blood samples from 47 mCRC patients were screened for CTCs using the FDA-cleared CellSearch® technology and / or the AdnaTest®. 38 samples could be processed in parallel. We demonstrate that a combined analysis of CellSearch® and the AdnaTest® leads to an improved detection of CTCs in our mCRC patient cohort (positivity rate CellSearch® 33%, AdnaTest® 30%, combined 50%). While CTCs detected with the CellSearch® system were significantly associated with progression-free survival (p = 0.046), a significant correlation regarding overall survival could be only seen when both assays were combined (p = 0.013). These findings could help to establish improved tools to detect CTCs as on-treatment biomarkers for clinical routine in future studies.

Heterogeneous PSMA expression on circulating tumor cells: a potential basis for stratification and monitoring of PSMA-directed therapies in prostate cancer.

The prostate specific membrane antigen (PSMA) is the only clinically validated marker for therapeutic decisions in prostate cancer (PC). Characterization of circulating tumor cells (CTCs) obtained from the peripheral blood of PC patients might provide an alternative to tissue biopsies called "liquid biopsy". The aim of this study was to develop a reliable assay for the determination of PSMA on CTCs. PSMA expression was analyzed on tissue samples (cohort one, n = 75) and CTCs from metastatic PC patients (cohort two, n = 29). Specific signals for the expression of PSMA could be seen for different prostate cancer cell line cells (PC3, LaPC4, 22Rv1, and LNCaP) by Western blot, immunohistochemistry (IHC), immunocytochemistry (ICC), and FACS. PSMA expression was found to be significantly increased in patients with higher Gleason grade (p = 0.0011) and metastases in lymph nodes (p = 0.0000085) or bone (p = 0.0020) (cohort one). In cohort two, CTCs were detectable in 20 out of 29 samples (69 %, range from 1 - 1000 cells). Twelve out of 20 CTC-positive patients showed PSMA-positive CTCs (67 %, score 1+ to 3+). We found intra-patient heterogeneity regarding the PSMA status between CTCs and the corresponding primary tumors. The results of our study could help to address the question whether treatment decisions based on CTC PSMA profiling will lead to a measurable benefit in clinical outcome for prostate cancer patients in the near future.

RHAMM splice variants confer radiosensitivity in human breast cancer cell lines.

Biomarkers for prognosis in radiotherapy-treated breast cancer patients are urgently needed and important to stratify patients for adjuvant therapies. Recently, a role of the receptor of hyaluronan-mediated motility (RHAMM) has been suggested for tumor progression. Our aim was (i) to investigate the prognostic value of RHAMM in breast cancer and (ii) to unravel its potential function in the radiosusceptibility of breast cancer cells. We demonstrate that RHAMM mRNA expression in breast cancer biopsies is inversely correlated with tumor grade and overall survival. Radiosusceptibility in vitro was evaluated by sub-G1 analysis (apoptosis) and determination of the proliferation rate. The potential role of RHAMM was addressed by short interfering RNAs against RHAMM and its splice variants. High expression of RHAMMv1/v2 in p53 wild type cells (MCF-7) induced cellular apoptosis in response to ionizing radiation. In comparison, in p53 mutated cells (MDA-MB-231) RHAMMv1/v2 was expressed sparsely resulting in resistance towards irradiation induced apoptosis. Proliferation capacity was not altered by ionizing radiation in both cell lines. Importantly, pharmacological inhibition of the major ligand of RHAMM, hyaluronan, sensitized both cell lines towards radiation induced cell death. Based on the present data, we conclude that the detection of RHAMM splice variants in correlation with the p53 mutation status could help to predict the susceptibility of breast cancer cells to radiotherapy. Additionally, our studies raise the possibility that the response to radiotherapy in selected cohorts may be improved by pharmaceutical strategies against RHAMM and its ligand hyaluronan.