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Simple Summary Circulating tumor cells (CTCs) are rare cells found in the bloodstream of oncologic patients with a central role in the metastatic spread. In this study, we aim at exploring their heterogeneity levels in metastatic breast cancer patients focusing on single cell single nucleotide variant (SNV) and copy number aberration (CNA) analyses. Our results show high levels of heterogeneity, especially concerning SNVs. Further analysis revealed the presence of CNAs associated with breast tumorigenesis, while longitudinal CNA profiling was demonstrated to track clonal selection of CTCs during treatment. Despite this heterogeneity, we found a group of CTCs from different patients sharing common genomic aberrations, such as losses on 15q. Collectively, our findings demonstrate that single-cell molecular analyses could be exploited in future to better address therapeutic strategies. Further investigations to better characterize this mixed population are needed to understand its role in MBC. Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q (CCND1) and 11q (WNT3A), loss of 22q (CHEK2). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q (CDK4). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated.

Simple Summary In the present work, we describe the evolution of circulating tumor cells (CTCs) released into the bloodstreams of 11 patients affected by locally advanced esophageal cancer (EC) during clinical treatments. We aimed at characterizing identified CTCs in depth both phenotypically as well as through single cell copy number aberrations profiles and to investigate the features of CTCs from relapsed patients, if present. We found that locally advanced EC spreads circulating tumor cells with both epithelial and mesenchymal phenotypes during the course of therapy. CTCs of relapsed patients display higher levels of genome disruption to those of disease-free patients. Specific enriched terms emerged from copy number aberration analysis of CTCs of relapsed patients. Abstract Background: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy. Methods: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis. Results: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients’ groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients. Conclusions: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients’ outcome.

High pCO2 habitats and their populations provide an unparalleled opportunity to assess how species may survive under future ocean acidification conditions, and help to reveal the traits that confer tolerance. Here we utilize a unique CO2 vent system to study the effects of exposure to elevated pCO2 on trait-shifts observed throughout natural populations of Astroides calycularis, an azooxanthellate scleractinian coral endemic to the Mediterranean. Unexpected shifts in skeletal and growth patterns were found. Colonies shifted to a skeletal phenotype characterized by encrusting morphology, smaller size, reduced coenosarc tissue, fewer polyps, and less porous and denser skeletons at low pH. Interestingly, while individual polyps calcified more and extended faster at low pH, whole colonies found at low pH site calcified and extended their skeleton at the same rate as did those at ambient pH sites. Transcriptomic data revealed strong genetic differentiation among local populations of this warm water species whose distribution range is currently expanding northward. We found excess differentiation in the CO2 vent population for genes central to calcification, including genes for calcium management (calmodulin, calcium-binding proteins), pH regulation (V-type proton ATPase), and inorganic carbon regulation (carbonic anhydrase). Combined, our results demonstrate how coral populations can persist in high pCO2 environments, making this system a powerful candidate for investigating acclimatization and local adaptation of organisms to global environmental change.

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients&rsquo, surveillance and follow-up.

Over the past 7 years, research funding has witnessed a changing trend. Besides available competitive research funding such as Horizon 2020 (Europe), National Institutes of Health (USA), National Health Service (UK) grants, and other public research funding programmes around the world, including those from charitable organisations, researchers have started to engage in crowdfunding activities to look for alternative sources of funding. Management

// Pietro Fici 1 , Giulia Gallerani 1 , Anne-Pierre Morel 2, 3, 4, 5, 6 , Laura Mercatali 7 , Toni Ibrahim 7 , Emanuela Scarpi 8 , Dino Amadori 9 , Alain Puisieux 2, 3, 4, 5, 6, 10 , Michel Rigaud 1 , Francesco Fabbri 1 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC), Italy 2 Inserm UMR-S1052, Centre de Recherche en Cancerologie de Lyon, Lyon, France 3 CNRS UMR5286, Centre de Recherche en Cancerologie de Lyon, Lyon, France 4 Centre Leon Berard, Lyon, France 5 UNIV UMR1052, Lyon, France 6 Universite de Lyon, Lyon, France 7 Osteoncology and Rare Tumors Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 8 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, FC, Italy 9 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, FC, Italy 10 Institut Universitaire de France, Paris, France Correspondence to: Pietro Fici, email: pietro.fici@irst.emr.it Keywords: EMT, early breast cancer, tumor aggressiveness, alternative splicing, EMT ratio Received: May 10, 2016 Accepted: November 21, 2016 Published: November 29, 2016 ABSTRACT Epithelial-to-mesenchymal transition (EMT) has been shown to be associated with tumor progression and metastasis. During this process in breast cancer, a crucial role is played by alternative splicing systems. To identify a new early prognostic marker of metastasis, we evaluated EMT-related gene expression in breast cell lines, and in primary tumor tissue from 31 patients with early breast cancer, focusing our attention on EMT-related splicing factors ESRP1, ESRP2 and RBFOX2. Results showed that the expression patterns of these genes were indicative of the onset of EMT in in-vitro models, but not in tissue samples. However, the ratio between ESRP1 or ESRP2 and RBFOX2 significantly decreased during EMT and positively correlated with the EMT-specific phenotype in cell models, representing a promising prognostic markers. Low ESRP1/RBFOX2 ratio value was associated with a higher risk of metastasis ( p < 0.005) in early breast cancer patients, regardless other clinical features. A cut-off of ratio of 1.067 was determined by ROC curve analysis (AUC 0.8375; 95% CI 0.6963–0.9787). Our study show evidence that a decrease in this ratio correlates with cancer progression. The results provide a rationale for using ESRP1/RBFOX2 ratio as a new prognostic biomarker for the early prediction of metastatic potential in breast cancer.

Liquid biopsy is a new diagnostic concept to investigate the molecular features of solid tumors by blood, saliva, urine, and any other body fluids which show a source of potential biomarkers. In cancer patients, it is a simple and less invasive mean, representing a sustainable alternative to interrogate all tumor cells longitudinally, quantifying and characterizing the biological materials (DNAs, RNAs, proteins) which originate from cancer tissues. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis from a simple blood draw received enormous attention for the related clinical research results. A rich scientific literature demonstrates that liquid biopsy is a valid instrument to assess the tumor biomarkers in real time and profile the cancer genotype in diagnostic and prognostic field, as well to quantify minimal residual disease, during patient follow-up. This could be a breakthrough for a companion diagnostic and personalized medicine. Liquid biopsy needs further implementation in the methodological aspects as well as cost-based assessment. The number of new molecular diagnostic assays increases day by day, but the standards for their adoption and clinical validation are still to be achieved.

Liquid biopsy is a new diagnostic concept to investigate the molecular features of solid tumors by blood, saliva, urine, and any other body fluids which show a source of potential biomarkers. In cancer patients, it is a simple and less invasive mean, representing a sustainable alternative to interrogate all tumor cells longitudinally, quantifying and characterizing the biological materials (DNAs, RNAs, proteins) which originate from cancer tissues. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis from a simple blood draw received enormous attention for the related clinical research results. A rich scientific literature demonstrates that liquid biopsy is a valid instrument to assess the tumor biomarkers in real time and profile the cancer genotype in diagnostic and prognostic field, as well to quantify minimal residual disease, during patient follow-up. This could be a breakthrough for a companion diagnostic and personalized medicine. Liquid biopsy needs further implementation in the methodological aspects as well as cost-based assessment. The number of new molecular diagnostic assays increases day by day, but the standards for their adoption and clinical validation are still to be achieved.

Although circulating tumor cells (CTCs) have been studied in early breast cancer (EBC), their value in this setting is still not fully understood. We isolated and studied CTCs in the peripheral blood (PB) of 48 EBC patients pre-surgery and one and 6 months post-surgery using an approach involving EpCAM-independent enrichment and a dielectrophoresis-based device. Method feasibility and the correlation between CTCs and primary tumor features were evaluated. CTCs were found in 27.1% of pre-surgery patients, 20.9% of patients one-month post-surgery, and about 33% of patients 6-months post-surgery. CTCs were recovered singly for further molecular characterization. Pre-surgery CTC-positive patients more frequently had negative prognostic features, i.e. high proliferation, large tumor dimension, lymph node positivity and negative receptor status than the other subgroup. In particular, vascular invasion showed a statistically significant correlation with CTC-positivity. Our procedure proved feasible and capable of recovering CTCs from EBC patients. Furthermore, our results suggest that CTCs may be linked to vascular invasion and to other known negative prognostic factors.

Introduction Immune-checkpoint blockade has emerged as an effective therapeutic strategy in solid tumor and in hematologic malignancies, including classical Hodgkin Lymphoma (cHL). cHL represents about 11% of all malignant lymphoma and it is generally highly curable with standard frontline therapies, although about 20% of the patients will relapse or become refractory after initial treatment. The hallmark of cHL is the presence of malignant Hodgkin and Reed-Sternberg Cells (HRS) that represent only a small fraction (about 1%) of the surrounding heterogeneous immune infiltrate. Despite this extensive inflammatory microenvironment, HRS are able to escape immune surveillance using several mechanisms, including the overexpression of PD-1 ligands (PD-Ls) that bind PD-1 on reactive T-cells, inhibiting their activity and proliferation and causing ultimately T-cell exhaustion. The PD-Ls expression is upregulated in a dose-dependent manner by copy number alterations of chromosome 9p24.1, a locus encoding for PD-L1/PD-L2 as well as JAK2, which further enhances PD-Ls expression through JAK2/STAT pathway. Here we present a method for the isolation and the genetic characterization of single purified HRS, which overcomes the limitations posed by the low tumor cellularity of cHL biopsies and gives an estimation of inter-tumor and intra-tumor heterogeneity which may be useful to guide immune treatment selection. Methods FFPE tissue sections from 4 cHL patients were dissociated down to single-cell suspension and stained using anti-CD30 and anti-PD-L1 antibodies. Since CD30 is not expressed exclusively by malignant cells, beyond the positivity to CD30 and PD-L1 HRS were selected according to morphological criteria, such as cell size and the presence of nuclei with ploidy higher than the surrounding lymphocytes. DEPArray™ NxT system (Menarini Silicon Biosystems) was used to isolate single target cells. After recovery, single cells were whole genome amplified (Ampli1™ WGA, Menarini Silicon Biosystems), and genome-wide copy-number alterations (CNAs) profiles were obtained using Ampli1™ LowPass kits (Menarini Silicon Biosystems) on Illumina® and Ion Torrent™ platforms. Results For each patient, at least 8 HRS cells and infiltrating lymphocytes were identified and isolated from lymphoid tissue using DEPArray™ NxT system. Copy-number analyses of recovered cells allowed us to precisely discriminate HRS, characterized by extensive gains and losses, from non-tumor cells, showing flat profiles as expected (Fig.1). Ploidy of HRS was automatically determined, based on best-fitting of profiles with underlying copy number levels. Hierarchical clustering showed that some alterations are highly conserved among patients, e.g. the region containing PD-L1/PD-L2/JAK2 has several copy gains in the majority of malignant cells. Interestingly, these alterations show high variable copy-number levels between different HRS even in the same patient, ranging from few copy-gains to amplifications, suggesting some level of heterogeneity. Different CNAs are also detected in regions containing genes belonging to pathways already known to be altered in cHL, like REL/NFKB and JAK/STAT pathways, which may be involved in the constitutive activation of proliferative and antiapoptotic phenotype of HRS. Conclusion Single HRS sorting combined with low-pass whole genome sequencing offer a valuable tool to uncover genetic alterations hidden by the massive cHL immune infiltrate and to estimate inter-tumor and intra-tumor heterogeneity in cHL patients. Considering that PD-Ls locus amplifications are associated with advanced stages of the disease and with a shorter progression free survival, the analysis of purified HRS could be helpful for patient stratification for the adoption of immune therapy. Disclosures Mangano: Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Garonzi:Menarini Silicon Biosystems: Employment. Lanzellotto:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Ceccolini:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fabbri:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fici:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Gallerani:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.

Circulating tumor cells (CTCs) are cellular elements of undeniable significance that spread from the tumor mass into the peripheral blood and constitute one of the main vehicles for disease diffusion. Their rarity, in addition to a number of molecular and cellular features, has severely impaired research and exploitation. CTCs have been evaluated in early breast cancer (EBC), although long from being fully accepted in this field also due to a lack of technical standardization. CTCs hold promise to be a powerful non-invasive real-time measurable biomarker in all disease stages. This hypothesis is particularly appealing in the adjuvant setting of breast cancer, as it still lacks a marker that could play a central role in monitoring disease-free intervals, predicting early relapse and guiding drug selection. This review aimed to discuss CTC characteristics and show the main results of CTC-research in EBC setting, stating the urgency to continue basic and translational research in this field to definitely translate this marker from bench to bedside.

e22028 Background: To date, studies conducted on circulating tumor cells (CTCs) and early breast cancer (EBC) have shown a CTC-positivity rate ranging from 9.4% to 48.6%. Although correlated with w...