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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.

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.

The characterization of circulating tumor cells (CTCs) could substantially improve the management of cancer patients. However, their study is still a matter of debate, often due to lymphocyte contamination. In the present paper, an investigation of CTCs was carried out for the first time using DEPArray, a dielectrophoresis-based platform able to detect and sort pure CTCs. Analyses were conducted on peripheral blood (PB) samples from patients with metastatic colon cancer. After 100% pure cell recovery and whole genome amplification, KRAS gene mutation of CTCs was screened and compared to gene status in the primary tumor tissue. CTCs were found in 21 colon cancer patients (52.5%), with more than three tumor cells per 7.5 ml. KRAS gene mutation analysis, showed a mutational concordance between CTCs and primary tumor in 50% of matched cases. The present study demonstrates for the first time the feasibility of analyzing at the molecular level pure CTCs avoiding lymphocyte contamination using an innovative instrumentation, and a KRAS discordance between CTCs and primary tissue. Our results present dielectrophoresis-based procedures as a new standard in single cell analysis and recovery and invite careful reflection on the value of CTCs characterization.

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.