Fast and efficient microfluidic cell filter for isolation of circulating tumor cells from unprocessed whole blood of colorectal cancer patients

Liquid biopsy offers unique opportunities for low invasive diagnosis, real-time patient monitoring and treatment selection. The phenotypic and molecular profile of circulating tumor cells (CTCs) can provide key information about the biology of tumor cells, contributing to personalized therapy. CTC isolation is still challenging, mainly due to their heterogeneity and rarity. To overcome this limitation, a microfluidic chip for label-free isolation of CTCs from peripheral blood was developed. This device, the CROSS chip, captures CTCs based on their size and deformability with an efficiency of 70%. Using 2 chips, 7.5 ml of whole blood are processed in 47 minutes with high purity, as compared to similar technologies and assessed by in situ immunofluorescence. The CROSS chip performance was compared to the CellSearch system in a set of metastatic colorectal cancer patients, resulting in higher capture of DAPI+/CK+/CD45- CTCs in all individuals tested. Importantly, CTC enumeration by CROSS chip enabled stratification of patients with different prognosis. Lastly, cells isolated in the CROSS chip were lysed and further subjected to molecular characterization by droplet digital PCR, which revealed a mutation in the APC gene for most patient samples analyzed, confirming their colorectal origin and the versatility of the technology for downstream applications

Global Gene Expression Characterization of Circulating Tumor Cells in Metastasic Castration-Resistant Prostate Cancer Patients

BACKGROUND: Current therapeutic options in the course of metastatic castration-resistant prostate cancers (mCRPC) reinforce the need for reliable tools to characterize the tumor in a dynamic way. Circulating tumor cells (CTCs) have emerged as a viable solution to the problem, whereby patients with a variety of solid tumors, including PC, often do not have recent tumor tissue available for analysis. The biomarker characterization in CTCs could provide insights into the current state of the disease and an overall picture of the intra-tumor heterogeneity. METHODS: in the present study, we applied a global gene expression characterization of the CTC population from mCRPC (n = 9), with the goal to better understand the biology of these cells and identify the relevant molecules favoring this tumor progression. RESULTS: This analysis allowed the identification of 50 genes specifically expressed in CTCs from patients. Six of these markers (HOXB13, QKI, MAOA, MOSPD1, SDK1, and FGD4), were validated in a cohort of 28 mCRPC, showing clinical interest for the management of these patients. Of note, the activity of this CTC signature was related to the regulation of MYC, a gene strongly implicated in the biology of mCRPC. CONCLUSIONS: Overall, our results represent new evidence on the great value of CTCs as a non-invasive biopsy to characterize PC

Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer

<p>Summary</p> <p>Background</p> <p>A universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression.</p> <p>Methods</p> <p>In the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy.</p> <p>Results</p> <p>The majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells.</p> <p>Conclusion</p> <p>All these results validate the importance of <it>core-</it>fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets.</p

Detection and dynamics of circulating tumor cells in patients with high-risk prostate cancer treated with radiotherapy and hormones: a prospective phase II study

BACKGROUND: Circulating tumor cells (CTCs) are an established prognostic marker in castration-resistant prostate cancer but have received little attention in localized high-risk disease. We studied the detection rate of CTCs in patients with high-risk prostate cancer before and after androgen deprivation therapy and radiotherapy to assess its value as a prognostic and monitoring marker. PATIENTS AND METHODS: We performed a prospective analysis of CTCs in the peripheral blood of 65 treatment-naive patients with high-risk prostate cancer. EpCAM-positive CTCs were enumerated using the CELLSEARCH system at 4 timepoints. A cut off of 0 vs >/= 1 CTC/7.5 ml blood was defined as a threshold for negative versus positive CTCs status. RESULTS: CTCs were detected in 5/65 patients (7.5%) at diagnosis, 8/62 (12.9%) following neoadjuvant androgen deprivation and 11/59 (18.6%) at the end of radiotherapy, with a median CTC count/7.5 ml of 1 (range, 1-136). Only 1 patient presented a positive CTC result 9 months after radiotherapy. Positive CTC status (at any timepoint) was not significantly associated with any clinical or pathologic factors. However, when we analyzed variations in CTC patterns following treatment, we observed a significant association between conversion of CTCs and stages T3 (P = 0.044) and N1 (P = 0.002). Detection of CTCs was not significantly associated with overall survival (P > 0.40). CONCLUSIONS: Our study showed a low detection rate for CTCs in patients with locally advanced high-risk prostate cancer. The finding of a de novo positive CTC count after androgen deprivation therapy is probably due to a passive mechanism associated with the destruction of the tumor. Further studies with larger samples and based on more accurate detection of CTCs are needed to determine the potential prognostic and therapeutic value of this approach in non-metastatic prostate cancer. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT01800058

Extracellular vesicles-based biomarkers represent a promising liquid biopsy in endometrial cancer

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-e cient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly e cient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analysesThis research was funded by Instituto de Salud Carlos III, grant PI17/01919, co-financed by the European Regional Development Fund (FEDER), and by Fundación Científica de la Asociación Española Contra el Cáncer (AECC), Grupos Clínicos Coordinados 2018. Carolina Herrero is supported by a predoctoral i-PFIS fellowship from Instituto de Salud Carlos III (IFI17/00047); Laura Muinelo is supported by Asociación Española Contra el Cáncer (AECC)

Diagnostic and prognostic value of B4GALT1 hypermethylation and its clinical significance as a novel circulating cell-free DNA biomarker in colorectal cancer

Epigenetic modifications of glyco-genes have been documented in different types of cancer and are tightly linked to proliferation, invasiveness, metastasis, and drug resistance. This study aims to investigate the diagnostic, prognostic, and therapy-response predictive value of the glyco-gene B4GALT1 in colorectal cancer (CRC) patients. A Kaplan-Meier analysis was conducted in 1418 CRC patients (GEO and TCGA datasets) to assess the prognostic and therapy-response predictive values of the aberrant expression and methylation status of B4GALT1. Quantitative methylation-specific PCR (QMSP) and droplet digital quantitative methylation-specific PCR (dd-QMSP) were respectively used to detect hypermethylated B4GALT1 in metastasis and plasma in four cohorts of metastatic CRC cases (mCRC). Both the downregulated expression and promoter hypermethylation of B4GALT1 have a negative prognostic impact on CRC. Interestingly a low expression level of B4GALT1 was significantly associated with poor cetuximab response (progression-free survival (PFS) p = 0.01) particularly in wild-type (WT)-KRAS patients (p = 0.03). B4GALT1 promoter was aberrantly methylated in liver and lung metastases. The detection of hypermethylated B4GALT1 in plasma of mCRC patients showed a highly discriminative receiver operating characteristic (ROC) curve profile (area under curve (AUC) value 0.750; 95% CI: 0.592-0.908, p = 0.008), clearly distinguishing mCRC patients from healthy controls. Based on an optimal cut-off value defined by the ROC analysis, B4GALT1 yield a 100% specificity and a 50% sensitivity. These data support the potential value of B4GALT1 as an additional novel biomarker for the prediction of cetuximab response, and as a specific and sensitive diagnostic circulating biomarker that can be detected in CRC

EPAC-Lung:Pooled analysis of circulating tumor cells in advanced non-small cell lung cancer

Background: We assessed the clinical validity of circulating tumor cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a European pooled analysis of individual patient data. This is the largest study of its kind and the first to examine between-centre heterogeneity of CTC identification in NSCLC.Methods: Nine European NSCLC CTC centers were asked to provide reported/unreported anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 - March 2017. We used Cox regression models, stratified by centre, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinico-pathological models using likelihood ratio (LR) statistics and c-indices.Results: Seven out of nine eligible centers provided data for 550 eligible patients, including 209 patients whose prognostic information was previously unpublished. CTC counts of ≥ 2 and ≥5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: HR 1.72, p &lt; 0·001; ≥5 CTCs: HR 2.21, p &lt; 0·001) and overall survival (≥2 CTCs: HR 2·18, p &lt; 0·001; ≥5 CTCs: HR 2·75, p &lt; 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinico-pathological models (log-transformed CTCs p &lt; 0·0001; ≥2 CTCs p &lt; 0·0001; ≥5 CTCs p &lt; 0·0001), while more moderate improvements were observed with the use of c-index models. There was minor evidence of between-center heterogeneity in the effect on PFS, but not OS.No difference in CTC profile was observed between key NSCLC molecular subsets such as EGFR, ALK, and KRAS.Conclusions: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC. CTC count improves prognostication when added to full clinico-pathological predictive models. ≥2 CTCs is an appropriate cutoff to move towards establishing clinical utility

CTCs expression profiling for advanced breast cancer monitoring

The study of circulating tumor cells (CTCs) has a huge clinical interest in advance and metastatic breast cancer patients. However, many approaches are biased by the use of epithelial markers, which underestimate non-epithelial CTCs phenotypes. CTCs enumeration provides valuable prognostic information; however, molecular characterization could be the best option to monitor patients throughout the disease since it may provide more relevant clinical information to the physicians. In this work, we aimed at enumerating and performing a molecular characterization of CTCs from a cohort of 20 patients with metastatic breast cancer (MBC), monitoring the disease at different time points of the therapy, and at progression when it occurred. To this end, we used a CTC negative enrichment protocol that allowed us to recover a higher variety of CTCs phenotypes. With this strategy, we were able to obtain gene expression data from CTCs from all the patients. In addition, we found that high expression levels of PALB2 and MYC were associated with a worse outcome. Interestingly, we identified that CTCs with an EpCAM(high)VIM(low)ALDH1A1(high) signature showed both shorter overall survival (OS) and progression-free survival (PFS), suggesting that CTCs with epithelial-stem features had the most aggressive phenotype

EPAC-lung: pooled analysis of circulating tumour cells in advanced non-small cell lung cancer

Introduction: We assessed the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a pooled analysis of individual patient data. Methods: Nine European NSCLC CTC centres were asked to provide reported/unreported pseudo-anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 to March 2017. We used Cox regression models, stratified by centres, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinicopathological models using likelihood ratio (LR) statistics and c-indices. Results: Seven out of nine eligible centres provided data for 550 patients with prognostic information for overall survival. CTC counts of ≥2 and ≥ 5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: hazard ratio [HR] = 1.72, p &lt; 0·001; ≥5 CTCs: HR = 2.21, p &lt; 0·001) and overall survival (≥2 CTCs: HR = 2·18, p &lt; 0·001; ≥5 CTCs: HR = 2·75, p &lt; 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinicopathological models (log-transformed CTCs p &lt; 0·001; ≥2 CTCs p &lt; 0·001; ≥5 CTCs p ≤ 0·001 for both survival end-points), whereas moderate improvements were observed with the use of c-index models. There was some evidence of between-centre heterogeneity, especially when examining continuous counts of CTCs. Conclusions: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC and also reveal some evidence of between-centre heterogeneity. CTC count improves prognostication when added to full clinicopathological predictive models

Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer

Background: About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients. Methods: CTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn's post-test was used for comparisons between groups. Statistical significance was set at p < 0.05. Results: EpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis. Conclusions: Our results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing