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Background: EVs are secreted by cells in physiological and pathological conditions and carry selected molecules, such as proteins, DNA and RNA; specific EVs released by neoplastic cells are defined as “tumor-derived extracellular vesicles (tdEVs)”. tdEVs have been demonstrated to be involved in promoting cell growth and survival, shaping the tumor microenvironment and increasing invasiveness and metastatic activity. Thanks to their systemic availability in body fluids and their role in tumor pathogenesis, tdEVs have gained attention in the liquid biopsy area, as a possible biomarker for tumor diagnosis, prognosis and monitoring and also as possible therapeutic targets. Beyond being a target for the capture of CTCs, the Epithelial Cell Adhesion Molecule (EpCAM) was also recently employed for the detection of EVs released by tumors. Here we present a method (for research use only) based on the CellMag™ technology (Menarini Silicon Biosystems, MSB) to carry out parallel enrichment of EVs and CTCs from whole blood through an EpCAM-based magnetic enrichment. Method: Whole blood samples from stage III/IV prostate (n=5) or breast cancer patients (n=6) and healthy donors (n=4) were collected in CellRescue™ tubes (MSB). After centrifugation, EpCAM+ EVs and CTCs were enriched from the plasma and cell fraction respectively, using the CellMag™ Epithelial CTC kit (MSB); CTCs were counted at the CellTracks Analyzer II (MSB). EVs were enriched for qualitative evaluation by Transmission Electron Microscopy (TEM) and for miRNA analysis by Real-time PCR, using the TaqMan™ Advanced miRNA Human Serum/Plasma panel. Results: CTCs counts ranged from 0 to 68; TEM analysis showed a higher density of EpCAM+ extracellular microvesicles (0,1-1µm) in breast cancer patients’ samples (n=2) compared to controls (n=2). Endogenous hsa-miR-16-5p control was detected in all analyzed samples (n=9 patients’ samples, n=2 controls). One control and two patients’ samples were tested for a panel of 188 serum/plasma miRNAs. In both the breast and the prostate cancer patients a higher percentage of miRNAs was found with respect to healthy control (31%, 32% vs. 17%). Among these, several oncogenic miRNAs with diagnostic and prognostic value (such as hsa-miR-125b-5p or hsa-miR-26a-5p) were exclusively found in patients’ samples, and further confirmed to be related to ErbB2 pathway in breast cancer using the miRPathDB 2.0 database, despite the CTCs count was 0 for both these patients’ samples. Conclusions: The described multi-target liquid biopsy approach enabled the combined enrichment of EpCAM+ CTCs and plasma EpCAM+ EVs from a single blood sample, along with their molecular analysis. Citation Format: Giulia Bregola, Martina Masotti, Valentina Mattei, Martina Moras, Claudio Ratti, Francesco Picardo, Cecilia Simonelli, Nicolò Manaresi, Francesca Fontana. Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3364.

Circulating tumor cells (CTCs) are rare cells shed into the bloodstream by invasive tumors and their analysis offers a promising noninvasive tool to predict and monitor therapeutic responses. CTCs can be isolated from patient blood and their characterization at single‐cell level can inform on the genomic landscape of a tumor. All CTC enrichment methods bear a burden of contaminating normal cells, which mandate a further step of purification to enable reliable downstream genetic analysis. Here, we describe the DEPArray™ technology, a microchip‐based digital sorter, which combines precise microfluidic and microelectronic enabling precise, image‐based isolation of single CTCs, which can then be analyzed by Next Generation Sequencing (NGS) methods. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Despite the great scientific and technological advances in recent years, mixed profiles interpretation represents a persisting challenge in forensic genetics, routinely complicating expert witness reporting in court and fostering backlogs in evidence processing worldwide. Current solutions to the complexity of mixed profiles interpretation include either the physical separation of a mixture into its biological components before genotyping (e.g., differential lysis for sperm–epithelial mixtures, laser capture microdissection, and micromanipulation) or biostatistical data analysis exploiting dedicated semicontinuous and fully continuous algorithms. Moreover, complex mixtures deriving from the contribution of the same body fluid from different individuals determine a higher level of complication since no available method allows to phenotypically distinguish the admixed biological components. DEPArray™ digital sorting technology has been reported to enable the isolation of pure single cells from forensic mixed samples also in real-casework scenarios. The advent of single-cell analysis in forensics offers a promising approach to complex mixtures interpretation enabling the identification of each contributor’s profile through the collection of multiple single cells each purely representing one single contributor to the sample.

Gatekeeper mutations are identified in only 50% of the cases at resistance to Anaplastic Lymphoma Kinase (ALK)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in ALK-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six ALK-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1st and 3rd ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK+/cytokeratins-, 46 (56%) ALK-/cytokeratins+ and 4 (5%) ALK+/cytokeratins+. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial ALK-negative CTCs may drive resistance through activation of bypass signaling pathways, while ALK-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.

Introduction: Genomic profiling of circulating tumor cells (CTCs) is an approach to help to understand tumour heterogeneity and evolution in metastatic breast cancer (MBC) to inform prognosis and guide treatment options. We showed previously that single CTCs provide complementary information on hot spot mutations to those detected in circulating tumor DNA (ctDNA) and the primary tumour tissue, and now extend analyses to include genomic copy number changes. Herein, we report that identification of actionable targets through copy number profiling of single CTCs is achievable at any time point during metastatic progression and could be a valuable tool to guide treatment options. Methods: Fourteen patients (13 12 progressing and 1 2 responding) who were receiving treatment for MBC (12 13 HR pos, 1 HER2 pos, I1 TNBC) were recruited to the study with ethical consent and followed up with a single blood sample. A 7.5ml blood sample was collected into a CellSave Preservative tube and processed and counted within 96 hours of collection using the CELLSEARCH® system and the Circulating Tumor Cell Kit (Menarini Silicon Biosystems). The DEPArray™ System was used to identify and recover individual, intact CTCs of epithelial origin (Cytokeratin 8, 18, 19 +ve, CD45 -ve) and white blood cells (WBCs) as single cells or as a small pool of cells. Whole genome amplification was performed using the Ampli1 WGA kit. Libraries were generated using the Ampli1 LowPass Kit (Menarini Silicon Biosystems) and samples passing QC were analysed f by next generation sequencing on an S5 platform (Thermo fFisher). Results were analysed using the MSBiosuite software and data was compared with in- house informatics workfows. Results: From the 14 patients included in the study, we profiled a total of 97 samples using the Ampli1 LowPass sequencing workflow comprising 79 individual cells (across 14 patients) and 18 pools of approximately 10 cells. Successful genome- wide copy number profiles were generated in 12 of the 14 patients; samples from 2 patients had low QC scores and were inconclusive. In all, 12 patients individual CTCs from 12 patients showed similar copy number profiles, but with some heterogeneity, that revealed gene specific changes not detected by the cell pool. Moreover, 10 of the 12 patients had gene- specific changes (5 with CCND1 amplification, 2 with FGFR1 amplification, 1 with JAK2 amplification and 2 with STK11 deletion) that could have guided treatment decisions, for example offering an FGFR1 or JAK2 inhibitor. Conclusion: This study highlights the genomic heterogeneity in CTCs seen in MBC and the potential of monitoring gene specific changes to identify actionable targets in order to inform treatment decisions. The work requires further evaluation and validation but it may offer a new approach to managing treatment decisions in MBC for those patients with detectable CTCs. Citation Format: Daniel Fernandez Garcia, Georgios Nteliopoulos, Robert Hastings, Amelia Rushton, Karen Page, Luke J. Martinson, Molly Gray, David S. Guttery, Alberto Ferrarini, Nicolò Manaresi, Charles Coombes, Jacqui Shaw. Genomic copy number profiling of single CTCs reveals clonal evolution in metastatic breast cancer and identifies actionable targets for informing treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 587.

Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 598.

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles.

Background: Clonal evolution and heterogeneity are among the factors that make treatment of Multiple Myeloma (MM) challenging, and disease monitoring requires invasive bone marrow biopsies. Tumor heterogeneity has previously been demonstrated at copy-number level, showing patterns of recurring Copy-Number Alterations (CNA). Conversely, Loss-of-Heterozygosity (LoH) profiling has been investigated less extensively. Presence of genomic patterns of LoH has been associated with Homologous Recombination Deficiency (HRD) in MM, suggesting a role for therapy with PARP inhibitors. Here we report the genome-wide LoH profiling in single-CMMCs isolated from enriched peripheral blood of four different MM patients using a non-invasive approach that combines CellSearch® and DEPArray™ technology. Methods: CMMCs were obtained from peripheral blood of four MM patients using CellSearch for enrichment and DEPArray NxT for isolation. CMMCs enrichment was obtained using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. After single cell isolation, Ampli1™ Whole Genome Amplification (WGA) kit was used to amplify single-cell genomic DNA. Whole genome sequencing libraries were prepared from WGA products using Ampli1™ LowPass kit and low-pass sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBiosuite. Single-cell genome-wide LoH profiles were obtained by a custom algorithm based on the ratio between mono- and bi-allelic loci in non-overlapping segments of uniform copy-number. Results: Out of 315 cells analyzed from the four MM patients, 244 (77%) showed LoH events. Extensive LoH regions were detected in all patients, with 231 cells showing copy-neutral LoH events and 121 showing LoH events in copy-gain regions. Globally, 460 copy-neutral LoHs, and 292 LoHs in copy-gain regions were detected. Two highly conserved copy-neutral events (9p; 16q) were detected in one of the patients analyzed, suggesting they are early events. Interestingly, the first of the 2 events includes CDKN2A, a tumor suppressor which has been implicated in predisposition to MM. Notably, in a second patient, chromosome 11 is associated both to a copy-neutral LoH and to an LoH event in a copy-gain segment including the CCND1 gene, whose amplification in MM has been associated with multidrug resistance expression. Conclusions: Here we present a non-invasive workflow for the molecular characterization of single CMMCs isolated from peripheral blood. Specifically, we reported the presence of chromosomal or sub-chromosomal LoH events in all MM patients, suggesting a pervasive phenomenon in MM which deserves to be further explored as potential therapy target. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Paola Tononi, Marianna Garonzi, Valentina del Monaco, Andrea Raspadori, Steven Gross, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2702.

Background: Genome-wide analysis of Loss-of-heterozygosity (LoH) has been shown to be important in several contexts, including the assessment of BRCAness signature, a marker of homologous repair deficiency in several cancer types associated with efficacy of platinum therapy and PARP inhibitors. Methods to infer genome-wide LoH status require high-coverage whole-genome sequencing or low-pass sequencing of a large number of single cells. We present a method to infer genome-wide LoH status of as low as one single-cell from low-pass sequencing of libraries produced by Ampli1™ LowPass kit, at a fraction of the cost of current NGS-based methods. Methods: We used low-pass sequencing data generated from 294 single Circulating Tumor Cells (CTCs) from prostate, multiple myeloma and lung cancer patients, and from 24 single cells from 8 cell lines. DNA amplified with Ampli1™ WGA kit (based on ligation-mediated PCR) combined with Ampli1 LowPass kit for Illumina generates a reduced genome representation, whereby a significant fraction of fragments is expected to be covered by more than one read. We reasoned that, even at a mean coverage insufficient for variant calling (≤0.05x), a statistically significant decrease in loci showing 2 alleles should be expected in LoH regions. A pileup of mapped sequences at dbSNP loci was obtained (minor allele frequency ≥5%; read depth ≥2). Mono- and bi-allelic loci (pileup containing 1 or 2 different alleles, respectively), were counted in each uniform copy-number (CN) genome segment within a single chromosome arm. Regions with CN equal to cell ploidy were used as internal reference. A one-sided Fisher test on biallelic sites depletion (multiplicity corrected p-value threshold of 0.05) was used to call LoH on each region. Results: Compared with libraries obtained using random shearing, Ampli1 LowPass single-cell libraries showed an increase in mean coverage of covered genome from 1.1 to 1.9 starting from the same number of reads (1M) thus increasing the number of loci for LoH calling. Analysis of 10 patients' single cells with known LoH events, corresponding to regions at copy number 1, detected 1006 events out of 1142. Several (492) copy neutral LoH events or LoHs in copy gained regions were also detected. Representatives of such events were verified successfully by high pass sequencing of the same library. Performance was confirmed by analysis of 24 single cells from cell lines whose LoH status was inferred from CCLE database. Conclusions: This innovative method leverages reduced genome representation inherent in Ampli1 LowPass libraries, to compute genome-wide LoH along with copy number (CN) status, meeting the need for LoH/CN analysis at single-cell level on rare-cells such as CTCs. Citation Format: Alberto Ferrarini, Claudio Forcato, Marianna Garonzi, Genny Buson, Massimiliano Pellicano, Francesca Fontana, Nicolò Manaresi. Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1327.

Background Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers whose primary site cannot be determined after standard clinical and pathological evaluation. CUP patients are generally treated with empirical chemotherapy and have poor prognosis. As reported in recent studies, CUPs present an average of 4 genetic alterations per tumor and these genetic alterations can be detected in circulating tumor DNA (Kato 2017). Thus, a potential improvement in CUP outcomes could derive from targeted therapies directed toward actionable mutations, and Circulating tumor cells (CTCs) can provide valuable information. Here, we describe a case of a 51-yr-old Caucasian female (Pt#71) with metastatic CUP. CTCs were detectable in her blood and analyzed for genetic alterations. Methods Using CellSearch and DEPArray NxT, n=3 CTCs and n=1 leukocyte as a control were isolated from patient's peripheral blood. Single-cell whole genome amplification was obtained with Ampli1 WGA Kit and libraries were generated with Ampli1 OncoSeek Panel and Ampli1 LowPass kits. In parallel, formalin-fixed paraffin embedded (FFPE) tissue derived from a lymph node biopsy was analyzed with two different methods. One curl was sent for comprehensive genomic profile to FoundationOne assay testing (Roche). From another curl, DNA was extracted and directly processed with DEPArray LibPrep and DEPArray OncoSeek Panel kits. After sequencing on Illumina MiSeq platform, raw data were analyzed on MSBiosuite platform (Menarini Silicon Biosystems). Results CTCs genome-wide characterization allowed the detection of sub-chromosomal losses, including a LOH region comprising the APC gene, and patterns of extensive gains. Moreover, amplification signals were detected in correspondence of FGFR2 and CCNE1 genes. Targeted sequencing on both CTCs and bulk tumor DNA confirmed the FGFR2 amplification in all the samples and detected a somatic variant in APC gene (APC:p.T1556Nfs*3). FoundationOne assay on FFPE biopsy reported the same amplification on FGFR2 and CCNE1 genes, along with a somatic variant in ARID1A gene (ARID1A:p.R1276*), a gene not present in the OncoSeek Panel. On the other hand, APC somatic variant was not identified by FoundationOne, probably due to contamination by normal cells and/or tumor heterogeneity. Conclusions The comprehensive genomic profile of tumor FFPE tissue and CTCs was highly overlapping and allowed the characterization of genetic alterations in this CUP case, revealing potentially actionable mutations and copy number alterations. The advantage of isolating and analyzing single CTCs is clearly shown by the non-invasive procedure combined with a precise detection of druggable alterations due to cell purity. Citation Format: Elisa Porcellini, Francesco Gelsomino, Noemi Laprovitera, Mattia Riefolo, Marianna Garonzi, Paola Tononi, Francesca Fontana, Antonia D'Errico, Maria Pantaleo, Nicolò Manaresi, Andrea Ardizzoni, Manuela Ferracin. Isolation and genetic characterization of circulating tumor cells from cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5361.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

Background Characterization of individual CTCs helps investigating intra-tumour heterogeneity, and provides longitudinal information about temporal evolution of genomic lesions following therapeutic evolutionary pressure, missed by one-time, bulk, single-biopsy analysis. Here we present the validation of a complete workflow to detect hotspot mutations and CNAs in single cells. It combines ligation-mediated PCR (LM-PCR) Whole Genome Amplification (Ampli1™ WGA kit), best-in-class in terms of low allelic drop-out (ADO) and reproducibility of amplification bias, with a tailored, WGA-aware, next generation sequencing (NGS) targeted cancer panel (Ampli1™ OncoSeek) and a fully-automated cloud-based platform for bioinformatic analysis (MSBiosuite). Methods Single-cells (n=24) of breast, prostate, lung and neuroblastoma cancer cell lines spiked in healthy-donor blood, alongside 15 single White Blood Cells (WBCs) from 5 healthy donors and CTCs from prostate, lung and breast patients were enriched with CellSearch® System, sorted with DEPArray™ NxT technology and WGA’ed with Ampli1™ WGA kit (Menarini Silicon Biosystems). NGS reference materials (Seraseq™ Breast CNV and Seraseq™ Lung & Brain CNV) with known CNAs were also WGA’ed. WGA products were used as input for the Ampli1™ OncoSeek Panel assay (Menarini Silicon Biosystems), a single-tube, Illumina®-compatible kit that covers 60 clinically relevant genes including more than 1500 mutation hotspots and CNAs for a subset of 19 genes. WGA-tailored primer pairs design and their concentrations were optimized so that targeted amplicons for sequence variants are sufficiently represented. Redundant amplicons were included for robustness of focal CNA detection. Bioinformatic analysis was performed with an assay-specific, cloud-based pipeline (MSBiosuite, Menarini Silicon Biosystems). Results Results on Seraseq NGS reference materials with known copy number gains (3, 6, 12) on 6 genes showed accurate detection of expected CNAs and high linearity of response (R2 = 0.97 ± 0.04). We observed low ADO rate (12.7% ± 4.2%). The Ampli1™ OncoSeek Panel assay detected known mutations and CNAs from cell lines at high sensitivity and the analysis of polymorphic variants in WBCs showed high agreement between biological replicates (overall agreement = 0.94 ± 0.06). Sequencing of CTCs from patients is ongoing and will be presented at the conference. Conclusions Here we presented a complete solution to detect hotspot mutations and focal CNAs that meets the need for accurate tumour characterization at single-cell level, suitable for individual CTC analysis. Citation Format: Paola Tononi, Valentina del Monaco, Alberto Ferrarini, Genny Buson, Marianna Garonzi, Claudio Forcato, Andrea Raspadori, Nicolò Manaresi. Validation of a targeted sequencing workflow for sequence variants and focal copy number alterations (CNAs) in single circulating tumor cells (CTCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2517.

The Innovative Medicines Initiative (IMI) project CANCER-ID (www.cancer-id.eu) is a 5 year (2015-2019) international public-private partnership of currently 40 partners from 14 countries with the aim to evaluate technologies for Circulating Tumor Cell (CTC), circulating free tumor DNA (ctDNA), microRNA (miRNA) and exosome enrichment, isolation and analysis. At the core of CANCER-ID’s activities are establishment of harmonized best practice protocols from patient sample collection, pre-analytical sample handling, sample and bioinformatics analyses down to the actionable information guiding patient selection and personalized treatment. CANCER-ID is furthermore testing and supporting development of standards for liquid biopsy as well as clinical implementation of liquid biopsy based protocols in the clinical setting. This includes interaction with regulatory bodies in Europe (EMA Innovation Task Force) and the US (FDA Public-Private Partnership liaison) to support future approval of liquid biopsies in multi-centered worldwide clinical studies. During the clinical validation phase of the project, clinical-ready liquid biopsy protocols have been implemented in an observational study on the potential predictive value of monitoring treatment response towards Immune Checkpoint Inhibition (ICI) in 180 NSCLC patients at the UMC Groningen, The Netherlands, as well as in two ICI-chemotherapy combination studies in Triple-Negative Breast Cancer and Luminal B-type breast cancer, respectively, run by the University of Oslo, Norway (ALICE NCT03164993 and ICON NCT03409198). Within both studies, blood has been collected at baseline and at follow-up visits for ctDNA and CTC analysis, including technical evaluation of CTC PD-L1 protein expression. The aim is to assess whether the allelic frequency of mutations identified by plasma NGS as a potential measure for Tumor Mutational Burden or the number of PD-L1–positive/overall CTCs at different time points is indicative of treatment success. The studies aim at providing data to assess whether clinical predictive information could be inferred from baseline number of detected mutations and PD-L1 expressing CTCs. Preliminary data of these analyses will be presented. As a follow-up activity of the IMI CANCER-ID program, the European Liquid Biopsy Society (ELBS) is currently being established by Prof. Pantel at UKE Hamburg, Germany. The ELBS will be open to all interested liquid biopsy stakeholders worldwide as a platform for scientific exchange, further efforts to standardize technologies and protocols in the field as well as for the initiation of new basic and clinical research projects with the aim to make liquid biopsies an integral part of clinical studies and patient care. This work is supported by IMI JU & EFPIA (grant no. 115749, CANCER-ID). Samples from patients and healthy volunteers, respectively, were collected under signed informed consent. Citation Format: Klaus Pantel, Leon W. Terstappen, Nicolò Manaresi, Harry J. Groen, Ed M. Schuuring, Ellen Heitzer, Michael Speicher, Bjørn Naume, Jon Amund Kyte, Thomas Schlange, for the IMI CANCER-ID consortium. Standardization and clinical implementation of liquid biopsy assays - IMI's CANCER-ID [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3190.

Introduction: Multiple Myeloma (MM) evolution and heterogeneity is interesting for its potential translational relevance. Recent studies using bulk sequencing of Smoldering MM cells obtained from Bone Marrow (BM) report recurring CNA patterns. By analyzing single-CMMCs isolated from enriched peripheral blood, we show here, for the first time in MM, evidence of frequent convergent lesions, i.e. alterations developed independently across different evolutionary branches, including CNAs often found in MM and previously reported as common truncal alterations. Methods: Peripheral blood samples (4.0 ml) were obtained from n=3 patients with MM. CMMCs were enriched with CellSearch® AutoPrep® using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. Cell enumeration was based on the co-localization of nuclear DAPI staining and CD38-PE on CellSearch CTAII®. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated with DEPArray NxT system. Single-cell genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit, Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and the process was automated on a Hamilton STARLet Liquid handler. Multiplexed, low-pass whole-genome sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBio Suite (Menarini Silicon Biosystems). Results: 186/215 (86%) single CMMC in total passed QC criteria and were included in the analysis. Single WBCs were also included as normal controls. Cumulatively, CNA profiles of single CMMCs showed patterns typical of MM, including 1q gain, 13 monosomy, sub-chromosomal gain or trisomy 3, 5, 7, 9, 11, 15, 21. Of these, intra-patient single-cell profiling surprisingly revealed -in all three patients- convergent lesions, i.e. alterations developed independently across different evolutionary branches, along with conserved (common truncal), and divergent alterations (found only in specific sub-clusters). In addition, we found evidence that 1q gain, 13q deletion and 6p gain were actually subclonal, in contrast with recent publications reporting them as truncal early-onset lesions. Conclusion: Single CMMCs CNA profiling reveals patterns of frequent convergent alterations developed independently through branched evolution, undetectable through bulk sequencing. Citation Format: Claudio Forcato, Andrea Raspadori, Alberto Ferrarini, Mario Terracciano, Valentina del Monaco, Marianna Garonzi, Carrie Morano, Steven Gross, Genny Buson, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Genome-wide copy number profiling of single circulating multiple myeloma cells (CMMCs) reveals intra-patient convergent copy-number alterations (CNAs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2911.

Background: Classical Hodgkin Lymphoma (cHL) is generally highly curable with standard frontline therapies, although about 20% of the patients relapse or become refractory after initial treatment. cHL hallmark is the presence of morphologically characteristic malignant Hodgkin and Reed-Sternberg (HRS) cells that represent only a small fraction (about 1%) of the surrounding non-malignant environment. Genetic alterations of HRS cells are potentially a precious source of information to develop new treatments or prognostic biomarkers. In this perspective, low tumor cellularity, worsened by DNA degradation of FFPE samples, poses technical challenges to unravel malignant cells genetic alterations. Hereby we present new insights on purified HRS single cells obtained through highly automated platforms, providing precise observation of tumor genetic alterations. Methods: FFPE tissue sections from 5 cHL patients were dissociated down to single cell suspensions. Cells were immunofluorescently labeled using anti-CD30-FITC and anti-PD-L1-PE antibodies. HRS cells, along with normal leukocytes, were selected on the basis of morphological and immunofluorescence criteria, and isolated using DEPArray™ NxT (Menarini Silicon Biosystems, MSB). Customized, high-throughput automated protocols were developed and implemented on STARLet liquid handler (Hamilton Life Sciences) to amplify isolated purified single cells genomic DNA and to generate genome-wide copy-number alterations (CNAs) profiles using Ampli1™ WGA and Ampli1™LowPass kits (MSB), respectively. Results: More than 150 HRS cells were isolated from the 5 patient samples, from which CNA profiles were obtained. HRS cells presented extensive gains and losses across the whole genome, while leukocytes displayed flat profiles as expected. HRS cells clustered coherently with patients, revealing a high degree of heterogeneity of CNA profiles among different patients. However some commonalities across the patients genomes were identified. In particular, gains and amplification were detected in PD-L1, PD-L2 and JAK2 region (9p24), as well as gains and losses in regions where REL and other genes involved in NF-kB pathway map. Conclusions: Leveraging on high throughput automated platforms and single cells isolation, the described method enabled cHL genome-wide genetic analysis at a single cell level, overcoming the intrinsic limitations of low-frequency of HRS and DNA degradation due to FFPE samples. Furthermore, unprecedented data on single HRS cells were described, opening up to a new approach to understand tumor diversity and to potentially develop personalized therapeutic strategies for cHL patients. Citation Format: Andrea Raspadori, Paola Tononi, Chiara Mangano, Marianna Garonzi, Claudio Forcato, Chiara Bolognesi, Genny Buson, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3410.

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

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.

Introduction: Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. The high heterogeneity of MM cells is one of the major cause of disease relapse. Detection of circulating MM cells (CMMC) from peripheral blood is a useful procedure to investigate tumor heterogeneity and provides a painless alternative to the classic bone marrow biopsy to monitor disease progression. Here we demonstrate that the synergy between CellSearch® (CS) and DEPArray™ (DA) technologies can be used to identify, isolate and characterize at the genetic level single and pure CMMCs . Methods: 4.0 ml of peripheral blood samples were obtained from 3 patients with MM. Putative CMMCs were enriched with CS using anti-CD138 or anti-CD138/CD38 as positive selection marker and subsequently stained with CD38-PE, CD19/CD45-APC immunofluorescent probes. Cells detection and enumeration was performed based on the co-localization of nuclei DAPI staining and CD38-PE. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated using the DA NxT system. Single cells genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit and Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and a high-throughput, customized automated protocol using Hamilton STARLet Liquid handler. Highly-multiplexed, genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using HiSeq 2500 Illumina® platform. Results: CS and DA workflow* enabled the isolation of 215 single CMMC, selected for LPCNA analysis. 42 single WBCs were also included as normal controls. Copy-number profiles of single CMMCs showed relevant gains and losses of chromosomal segments, as result of a high-level genomic instability. Notably, intra-patient CMMCs revealed overall conserved CNA patterns with subclonal alterations, suggesting a certain level of branched tumor evolution. Conversely, a higher degree of heterogeneity in CMMCs CNA profiles was observed among different patients. Interestingly, CNAs detected in all patients are located in regions containing genes involved in cell cycle regulation (MAPK, NOTCH pathways) and cell signaling (IL6R), which might be involved in proliferative processes and immuno-surveillance escape. Conclusion: The combination of CS and DA workflow* with a streamlined automated protocol allowed to obtain hundreds of genomic libraries from pure single CMMCs. The presented workflow constitutes a non-invasive, rapid and high-throughput approach for characterizing MM tumor heterogeneity and progression, suggesting a possible future implementation in clinical applications. *For Research Use Only. Not for use in diagnostic procedures. Disclosures Raspadori: Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Del Monaco:Menarini Silicon Biosystems: Employment. Terracciano:Menarini Silicon Biosystems: Employment. Morano:Menarini Silicon Biosystems: Employment. Gross:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Connelly:Menarini Silicon Biosystems, Inc.: Employment, Other: Chief R&D Officer, USA. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.

Background: Multiple myeloma (MM) is a bone marrow derived cancer of plasma cells, which remains an incurable disease. Because of the invasive and painful nature of bone biopsy, an alternate tumor monitoring strategy is needed. We have previously shown that Circulating Multiple Myeloma Cells (CMMCs) isolated by CellSearch® (CS) are prognostic and indicative of disease burden through remission and relapse. Here we report, for the first time, the molecular characterization of pure single CMMCs isolated from a multiple myeloma patient, by integrating CS and DEPArray™ (DA) NxT systems, providing access to copy-number alteration (CNA) profiling. Methods: 4.0ml of peripheral blood from a patient with multiple myeloma was tested. On CS, target CMMCs were enriched using anti-CD138 for cell capture, and stained immunofluorescently with CD38-PE, CD19 and CD45-APC. Nuclei were stained with DAPI and detected target cells counted. The enriched sample was then analyzed using the DA NxT system: single CMMCs (CD38+/CD19- and CD45-/DAPI+), along with some single White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+), were isolated. The DNA of each single cell was amplified using the Ampli1™ WGA kit and used for highly-multiplexed, genome-wide single-cell CNA analysis using a Ampli1™ LowPass kit (LPCNA) on Illumina® MiSeq. Results: CS identified 128 CMMCs. From DA NxT we harvested 20 individual CMMCs for LPCNA analysis. Copy-number profiles of CMMCs confirmed their tumor origin, showing a high-level of genome instability with several gains and few losses of chromosomal segments. Moreover, an unsupervised hierarchical cluster analysis highlighted a conserved pattern of alterations, enabling the separation between CMMCs and WBCs groups. A pattern of copy-number gains shared by all CMMCs, coupled with gains and losses shared by a subset of CMMCs, suggests a branched evolution of different tumor subclones. WBC profiles were flat as expected. Conclusions: Cell enrichment by CS followed by individual cell sorting using DA NxT, enabled the isolation of single CMMCs with 100% purity. Ampli1™ single-cell analysis demonstrated CMMC molecular heterogeneity suggestive of tumor subclones presence. This platform combination provides a reliable and non-invasive method for MM characterization enabling translational research and future clinical application. Citation Format: Mario Terracciano, Claudio Forcato, Edoardo Petrini, Alberto Ferrarini, Valentina del Monaco, Andrea Raspadori, Carrie Morano, Steven Gross, Chiara Bolognesi, Genny Buson, Thai Bui, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. An integrated workflow for liquid biopsy of circulating multiple myeloma cells (CMMCs) with single cell resolution reveals tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2198.

Background: The rarity of in vivo and in vitro human prostate cancer (PCa) models has hampered progress in understanding disease pathogenesis, metastatic progression and drug resistance mechanisms. Using CTCs from a leukapheresis product of a patient with advanced PCa, we report the establishment of a CDX and an in vitro cell line derived from this CDX. The phenotypic and molecular characterization of patient tumor-biopsies, CTCs, CDX and CDX-derived cell-line are presented. Methods: Leukapheresis was performed in seven patients with advanced castration-resistant prostate cancer (CRPC). CTCs from the seven leukapheresis products were enriched by RosetteSep and implanted in Nod/Scid-IL2Rγ-/-mice. The CDX tumor was propagated in successive generations of mice. All samples, including eight tumor-biopsies performed at diagnosis two years prior leukapheresis and CTCs isolated at the single cell level during leukapheresis were characterized by immunofluorescence, immunohistochemistry, and whole-exome sequencing (WES). Results: Based on CellSearch® counts in leukapheresis products, the estimated median number of engrafted CTCs was 697 (range: 10-19988). A mouse engrafted with 19988 CTCs developed a tumor within 193 days. Immunohistochemistry performed on the CDX and two tumor-biopsies indicated that the CDX and biopsies were positive for EpCAM, CK5/6/8/18, negative for CK7 and vimentin, and weakly positive for synaptophysin. While biopsies expressed PSA and the androgen receptor, the CDX was negative for both indicating tumor evolution. In contrast to tumor biopsies, the CDX strongly expressed Ki67, NSE and chromogranin, evidencing emergence of a neuroendocrine phenotype. The in vitro cell line established by culturing dissociated CDX cells for five months, grew in microspheres and expressed epithelial and ALDH and CD133 cancer stem-cell markers. By WES, a high degree of intra-tumor heterogeneity was observed in the eight tumor biopsies and CTCs as already reported in this tumor type. Only 2.8% (58/2087) and 2.3% (49/2087) of the mutations present in the tumor biopsies were identified in CTCs and the CDX respectively, indicating that a very few number of mutations have the potential to support the dissemination and tumorigenic activity of CTC. Trunk mutations in TP53, NF1 and LRP1B genes were identified in all samples including the CDX while PTEN gene loss was acquired lately and detected only in CTCs and the CDX. Mutational similarity of the CDX and the in vitro cell line was 91%. The analysis of copy number variations is ongoing in all samples and will be presented. Conclusion: We report the first PCa CDX model, demonstrating the tumorigenicity of CTCs from CRPC. This CDX model represents a unique tool to identify clonal mutations associated with the tumor-initiating capacity of CTCs and explore the genetic and phenotypic basis of metastasis and drug resistance in advanced CRPC. Citation Format: Vincent Faugeroux, Emma Pailler, Olivier Deas, Virginie Marty, Kamélia Alexandrova, Kiki Andree, Jean-Yves Scoazec, Nikolas Stoecklein, Nicolo Manaresi, Dominique Tramalloni, Maud Ngo-Camus, Claudio Nicotra, Leon Terstappen, Valérie Lapierre, Karim Fizazi, Yohann Loriot, Jean-Gabriel Judde, Françoise Farace. Establishment and characterization of a unique circulating tumor cells-derived xenograft (CDX) in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5600.

12038Background: Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. ALK-rearranged NSCLC patients develop resistance ...

A hybrid valve for lab on chip applications is presented. The valve is assembled by bonding poly (methyl methacrylate), PMMA, and silicon-based elastomers. The process used to promote the hybrid bonding includes the deposition of an organosilane (TMSPM) on the thermoplastic polymer, PMMA to interface PMMA and elastomers. For this study, a membrane in ELASTOSIL® is bonded in correspondence of the end of two microfluidic channels of a fabricated PMMA microfluidic chip. Prior the bonding, a plasma etching process has been used to remove the TMSPM in a confined circular area. This process made possible to bond selectively the edge of a membrane leaving free to move its central part. Actuating the membrane with an external positive pressure or vacuum is possible, respectively, to obstruct or to connect the microfluidic channels. The microvalve may be simply integrated in microfluidic devices and permits the control of microvolumes of fluid in processes such as transport, separation, and mixing. The deposition of the TMSPM, the bonding of the valve and its actuation has been characterized and tested. The flow rate control of liquids through the valve has been characterized. The results have been discussed and commented. The valve can stand up to 14 psi without showing leakages.

Dielectrophoresis (DEP) is a very valuable approach for designing and developing laboratory-on-a-chip (lab-on-a-chip) devices that are able to manipulate microparticles and cells. Lab-on-a-chip technology will enable laboratory testing to move from laboratories using complex equipment to nonlaboratory settings. We used a lab-on-a-chip device, the SmartSlide, which carries 193 parallel electrodes and generates up to 50 cylinder-shaped DEP cages able to entrap microparticles and cells within DEP cages and move them along the chip. For lab-on-a-chip technology, the characterization of microparticles exhibiting a differential ability to be DEP-caged, levitated, and moved is important for the development of both diagnostic and therapeutic protocols. We determined whether the SmartSlide could be used to levitate and move tripalmitin-based lipospheres carrying increasing concentrations of dihexadecyl dimethyl ammonium bromide (DHDAB) as a cationic surfactant. The data obtained with this DEP-based platform showed that DEP caging, levitation, and movement of the cationic lipospheres depended on the percentage of DHDAB. Tripalmitin lipospheres containing 6% DHDAB could be DEP-caged and manipulated. On the contrary, lipospheres containing 12% DHDAB did not exhibit an efficient ability to be DEP-caged and moved throughout the chip. To our knowledge, this is the first report on the possible use of a DEP-based lab-on-a-chip device for guided manipulation of lipospheres. This information might be of interest in the fields of drug discovery, delivery, and diagnosis. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

This paper presents a novel approach to continuously measure the mechanical deformations of a tire due to contact with asphalt, by embedding capacitive–resistive sensors in it. A strain monitoring method is proposed, which adopts the tire itself as a sensing element. In this way, the sensing area is pushed towards the tread interface (the part of the tire in direct contact with the asphalt), where the information concerning tire state is actually generated. Tire deformation causes a change of the spacing between the steel wires inside the tire carcass and this change is translated into an impedance change of that region of the tire. By measuring such an impedance change, our approach makes it possible to determine the deformation of the tire. Static stress–strain measurements show a linearity of about 80% in the impedance–strain relation. A linear impulse of tensile strain has been used to validate the detection of instantaneous changes in the tread–asphalt interface. The mechanical solicitation resulted in a 1% deformation of the specimen’s main dimension. Experimental results report a change of about 40% in the real part of the impedance and a change of about 20% in the imaginary part of the impedance. The same measurements have been performed varying the temperature within a range of −20 to 90 ◦C, which is compatible with the operating conditions of a commercial car tire.

The selective recovery of particles is demonstrated after separation with dielectrophoretic (DEP) forces. Particles are separated based on their size using the so-called cage-speed separation protocol. A two-lane laminar flow enables the selective recovery of the particles. In order to prevent hydrostatic pressure flow, liquid reservoirs are absent. The sample liquid is thus replaced with air during recovery. Phaseguides are introduced in the system to control the liquid–air interface, so that the two-lane laminar flow profile is preserved.

The development of microfabricated devices manufactured in silicon, glass, or plastic materials is a well-known trend in the research of novel biological techniques and tools over the last two decades, resulting in a multitude of start-up companies serving the pharmaceutical, biotechnology, and diagnostics markets. However, the idea of implementing such devices on microelectronic substrates has been introduced only recently. This chapter aims to describe the state-of-the-art of microsystems for molecular and cell biology produced in general purpose CMOS (complementary metal oxide semiconductor) technology, emphasizing the advantages of this approach along with their challenges and limitations. This chapter discusses significant examples of fully tested devices in comparison with existing state-of-the-art techniques.

This paper presents a mixed-signal system-on-chip (SOC) for sensing capacitance variations, enabling the creation of pressure-sensitive fabric. The chip is designed to sit in the corner of a smart fabric such as elastic foam overlaid with a matrix of conductive threads. When pressure is applied to the matrix, an image is created from measuring the differences in capacitance among the rows and columns of fibers patterned on the two opposite sides of the elastic substrate. The SOC approach provides the flexibility to accommodate for different fabric sizes and to perform image enhancement and on-chip data processing. The chip has been designed in a 0.35-/spl mu/m five-metal one-poly CMOS process working up to 40 MHz at 3.3 V of power supply, in a fully reconfigurable arrangement of 128 I/O lines. The core area is 32 mm/sup 2/.

In this study we describe an original, efficient, and innovative printed circuit board (PCB) device able to generate dielectrophoresis-based, software-controlled cages that can be moved to any place inside a microchamber. Depending on their dielectrophoretic properties, eukaryotic cells can be “entrapped” in cages and moved under software control. The main conclusion gathered from the experimental data reported is that the PCB device based on dielectrophoresis permits levitation and movement of different tumor cells at different dielectrophoresis conditions. The results presented herein are therefore the basis for experiments aimed at forced interactions or separation of eukaryotic cells using “lab-on-a-chip.” In fact, because many cages can be controlled at the same time, and two or more cages can be forced to share the same or a different location, it is possible, in principle, either to bring in contact cells of a differing histotype or to separate them. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 474–479, 2003.

Introduction: Formalin-fixed paraffin-embedded (FFPE) specimens represent an invaluable source of material for precision oncology. However, FFPE samples pose significant challenges for molecular assays, such as targeted Next-Generation Sequencing (NGS), because of the highly variable DNA quality and often limited sample size. Here we present a complete workflow from sample quality control to targeted library preparation to reliably detect sequence and copy number alterations (CNA) through targeted NGS from extremely low input FFPE samples. Materials & methods: Three FFPE specimens from patients with breast (BrCa) or pancreatic cancer, with DNA quality varying over a broad range and with low tumor cellularity (down to 10%) were selected for targeted NGS profiling. The DNA quality was determined using the DEPArray™ FFPE QC Kit, a qPCR-based assay yielding a QC score defined as the ratio between the quantification of a 132 bp amplicon, corresponding approximately to the average length (116bp) of DEPArray OncoSeek amplicons, and a shorter amplicon of 54 bp. The QC scores of the 3 FFPE specimens ranged between 0.23 to 0.74. We then used QC score, multiplied by the ploidy (assessed on DEPArray during cell sorting) and by the number of cells recovered, to estimate the effectively amplifiable template (EAT). Thirty two pure cell populations (21 stromal and 11 tumor), with different EATs (80-300), corresponding to a wide range of number of cells per pool (31-214), were collected with the DEPArray digital sorter. Libraries were prepared from each cell pool, using the single-tube, Illumina-compatible DEPArray OncoSeek panel comprising 63 oncology relevant genes (average 740,000 sequenced amplicons per sample). Results: Variant calling showed sensitivity comprised between 93% and 99% and specificity > 99% for EATs ≥ 80 equivalent to as low as ≈250 pg of DNA. Analysis of CNAs in stromal cells was highly specific (zero false positive at 1.5 fold-change threshold). Moreover, analysis allowed to highly reproducibly identify CNAs in CCND1 (3x), ERBB2 (8x), MYC (3x) and PIK3KA (2x) in both replicates of one BrCa sample. Similarly, ERBB2 amplification (7x) was found in the other BrCa sample (10% tumor cellularity) across two replicates at different EATs (80, 120) corresponding to as low as 59 and 75 cells. As expected by the low tumor content, in the corresponding unsorted sample ERBB2 was below the 2-fold gain threshold with respect to the stromal control, which would not qualify a sample as Her2-amplified. Highlights: Starting from pure intact cells with well characterized DNA quality and ploidy, our workflow allows reliable molecular profiling of sub-nanogram DNA samples by determining with precision the extremely-low minimum amount of cells necessary to obtain highly reproducible sequence variant calling and CNAs detection by targeted NGS. Citation Format: Paola Tononi, Alberto Ferrarini, Genny Buson, Valentina del Monaco, Giulio Bassi, Chiara Mangano, Claudio Forcato, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Accurate molecular profiling of sequence and copy number alterations from sub-nanogram FFPE DNA amounts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5349. doi:10.1158/1538-7445.AM2017-5349

Introduction Intra-tumor heterogeneity can hide genomic and genetic features, which may be key driver of disease progression. Routinely, only one biological specimen per patient is generally analyzed, which may only partially represents the genetics of the tumor. Here we report a multi-approach analysis of Circulating Tumor Cells (CTCs) and formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived cells (TCs) obtained from the same patients, to investigate the underlying genetic heterogeneity. Methods Peripheral blood (PB) and FFPE tumor tissue were collected from two advanced lung adenocarcinoma patients, treated with cisplatin-pemetrexed and carboplatin-pemetrexed respectively as first line therapy. The first patient was previously diagnosed an ALK-traslocation and treated with an ALK-inhibitor. PB was enriched with either an EpCAM-based or EpCAM-independent method: the cell output of the latter was stained with Cytokeratin-PE, CD45-APC and DAPI. Matched FFPE sections were obtained from pleural effusion cell blocks for the first patient or from primary tumor tissue for the second one; after dissociation, cells were stained with Vimentin-APC, Keratin-FITC and DAPI. The DEPArray™ platform was used to detect and collect pure single CTCs or TCs, along with WBCs or stromal cells as controls. Whole genome amplified DNA of single CTCs and TCs was used to profile genome-wide copy-number aberrations (CNAs) using the Ampli1™ LowPass kit; single nucleotide variants were analyzed on CTCs WGA products and on pools of TCs using Ampli1™ CHP custom panel and DEPArray™ OncoSeek panel respectively. Results No clinically significant variants were detected in CTCs and FFPE samples; however the copy-number profiles of single TCs and CTCs revealed an overabundance of gains and losses, confirming the aberrant nature of tumor cells. In the first patient, all single cells showed a pattern of shared alterations, with a common amplification of the genome region comprising MYC gene (also confirmed by depth-of-coverage in targeted panel). A hierarchical unsupervised clustering clearly separated WBCs, from the group of TCs and CTCs, characterized by some emerging clones and low inter-cell heterogeneity. The analysis of the copy-number profiles of cells from the second patient showed an opposite situation; unsupervised clustering of low-pass profiles highlighted an independent group formed by single TCs clearly distinct from the highly heterogeneous cluster formed by CTCs. Conclusions The precision granted by analysis of pure cells derived from multiple specimens from the same patient, together with the combination of low-pass whole-genome sequencing and targeted sequencing, reveals unexpected genetic similarities and diversities, and provides fundamental information to understand intra-tumor heterogeneity. Citation Format: Mario Terracciano, Francesco Gelsomino, Francesco Bacchi, Francesca Fontana, Claudio Forcato, Alberto Ferrarini, Michelangelo Fiorentino, Valentina Del Monaco, Giulio Bassi, Chiara Mangano, Chiara Bolognesi, Paola Tononi, Genny Buson, Gianni Medoro, Nicolò Manaresi, Michele Tognetto, Andrea Ardizzoni. Molecular characterization with single-cell resolution of CTCs and FFPE specimens from the same lung adenocarcinoma patients reveals the extent of intra-tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2017-2914

Fluorescent in Situ Hybridization (FISH) is commonly used for assessment of chromosomal alterations. Guidelines for determining FISH-based classification of clinical biomarkers exist but are based on pre-analytical factors, including fixation/sectioning/thickness/age, that can greatly influence biomarker status determination. Here, we use single-cell image-based cell sorting by DEPArrayTM for the separation and recovery of pure distinct cell populations prior to FISH. Methods: A multi-center study to evaluate HER2-FISH based analysis on FFPE with and without DEPArrayTM pre-processing was conducted using breast tumors classified as infiltrating ductal carcinoma (n=12), metastatic (n=1) and ductal carcinoma (n=1). From each block, four 50-micron thick curls were sectioned. One curl from each sample was sent to each of four centers (3 US; 1 EU). Each site performed disassociation of curls to generate a single cell suspension. Cells were then stained and sorted using the DEPArrayTM platform for recovery of tumor (cytokeratin+/vimentin-/DAPI+) and stromal (cytokeratin-/vimentin+/DAPI+) cells. Dual-probe FISH for HER2 and centromere 17 was performed on the sorted cells and compared with conventional tissue section FISH. Results: Overall, ≥ 90% concordance between the sorted tumor cells and the conventional HER2 FISH result was observed. Among the 7 HER2+ cases, HER2 ratio scores for the sorted tumor cells ranged slightly higher, from 2.60 to 8.95, as compared to the conventional method (from 2.10 to 5.14). In all cases in which stromal cells were also recovered, an expected normal ratio was observed, thus verifying that the populations were efficiently separated. Discordance can be attributed to intra-tumoral heterogeneity and the fact that conventional FISH on FFPE requires only a 4-micron section for analysis. Conclusion: Today, a percentage of patients are likely misclassified for the biomarker of interest as result of pre-analytical factors. We demonstrate here the ability to overcome these pre-analytic factors and ultimately improve the accuracy in determining biomarker status using the DEPArrayTM Note: This abstract was not presented at the meeting. Citation Format: Amanda Gerber, Aditi Khurana, Lisa Koenig, Lindsay Strotoman, Lori Millner, Valeria Sero, Chiara Bolognesi, Sabine Kasimir-bauer, Gianni Medoro, Matthew Moore, Philip Cotter, Nicolo Manaresi, Farideh Bischoff. Image-based single cell-sorting to separate and recover distinct cell populations from complex heterogeneous mixed tissue: precise sample preparation upstream of FISH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2017-2730

Introduction Chromosome instability (CIN) is a hallmark of cancer, acting by boosting genetic alterations responsible of tumorigenesis, progression and heterogeneity. Whole-genome sequencing (WGS) protocols are established methods for studying copy-number alterations (CNA) in single-cells, following a necessary whole-genome amplification (WGA) step. We previously presented a method for single-cell CNA profiling of CTCs based on shallow WGS of LM-PCR based WGA products. Here for the first time, we show that the same method may be employed even on single FFPE cells, overcoming the challenges of DNA degradation and damage linked to this type of samples. Methods Two 50μm thick FFPE sections from pancreas ductal adenocarcinoma with Results The purity of DEPArray sorting was confirmed by the large number of chromosome alterations in sorted tumor and the parallel lack of gains and losses in sorted stromal pools and single-cells. A titration test was conducted measuring the consistency of CNA profiles of tumor WGA products starting from different number of cells, ranging from 117 down to single cells. For the purpose, we employed a Receiver Operating Characteristic (ROC) curve using the non-amplified tumor population as reference. Results showed an excellent performance level with a mean Area Under Curve (AUC) equals to 0.93. Interestingly, lower AUCs (0.87) were observed for single-cells, due to some level of inter-cell heterogeneity. Moreover, the proposed low-pass WGS method demonstrated a high resiliency to DNA degradation as quality of CNA profiles, measured by Derivative Log Ratio Spread (DLRS), only showed a weak correlation with GI level, with high-quality CNA profiles obtained also with the lowest GI value. Conclusions Presented approach for copy-number profiling of tumor single-cells isolated by DEPArray digital sorter and processed with Ampli1 workflow has proven to be a reliable and valuable application for the molecular characterization of tumor clones in degraded samples as FFPE tissues. Citation Format: Alberto Ferrarini, Genny Buson, Chiara Bolognesi, Claudio Forcato, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2017-3945

e23161 Background: DEPArray™ technology is based on Dielectrophoresis (DEP). High quality image-based cell selection enables users to identify, isolate and recover intact specific individual rare cells of interest from complex, heterogeneous tissues such as live or fixed cell suspensions. Here we demonstrate a validation workflow for application of molecular sequencing technologies and FISH downstream of rare cell recovery. Methods: FFPE scrolls (n = 93) were dissociated and stained with markers to distinguish putative stromal and tumor populations. DNA integrity was tested using a qPCR technique to predict the amount of DNA required for a successful downstream sequencing metrics. DEPArray™ recovered stromal and tumor cells underwent NGS analysis on an Illumina platform based DEPArray™ OncoSeek Panel. Separately, recovered stromal and tumor cells were also utilized for targeted HER2 FISH. CellSearch® cartridges (19 Breast and 5 Bladder Cancers) were processed through the DEPArray to isolate WBCs (CK-/CD45+/DAPI+; n = 30), CTCs (CK+/CD45-/DAPI+; n = 33) and atypical (CK-/CD45-/DAPI+; n = 47) cells. Recovered cells underwent whole-genome amplification with Ampli1™ WGA and quality controlled by Ampli1 QC. Ampli1 LowPass kit was then used to prepare NGS libraries for absolute CNA profiling by low-pass WGS. Results: Reproducibility and reliability was reported as 100% for instrument performance. For FFPE, the OncoSeek panel simultaneously detected SNPs, indels and CNAs of 63actionable and oncology relevant genes. For FISH, 95% concordance with conventional HER2 results was observed. For CTCs, Ampli1 LowPass detected copy number gains/losses confirming tumor origin of the CK+ cells. A portion of non-conventional cells also demonstrated copy number alterations consistent with tumor origin. Conclusions: The isolation of pure single or pooled tumor cells with the DEPArray™ technology can be used as a method to improve downstream MDx analysis using different techniques, thus to inform treatment decisions and provide valuable prognostic information.

7528 Background: Classical Hodgkin Lymphoma (cHL) is one of the disease in which the check-point inhibitors have been demonstrated to be more successful. Lately, it has been reported that in malignant Reed-Sternberg Cells (RSCs), PD-1 ligands (PD-Ls) are overexpressed and that chr.9 amplification correlates with advanced stages of the disease, when the standard therapy have already failed. Unfortunately, the detection of the genetic alterations in RSCs is challenging, as one of the hallmark of cHL is the presence of a small number of malignant cells sparse in an abundant and heterogeneous immune infiltrate. Here we present a method for the isolation and the genetic characterization of purified RSCs, which overcomes the limitations posed by the low-cellularity of cHL biopsies, and could be helpful for earlier detection of genetic alterations and adoption of immunotherapy. Methods: FFPE tissue sections from cHL patients were dissociated down to single-cell suspension and stained using anti-CD30 and anti-PD-L1 antibodies. Beyond the positivity to CD30 and PD-L1, RSCs were selected according to morphological criteria such as cell size and the presence of polylobate nuclei compared to surrounding lymphocytes. Target cells were isolated using the DEPArray™ cell sorter, as single cells or in small pools of cells. Recovered cells were whole genome amplified ( Ampli1™ WGA), and genome-wide copy-number aberrations (CNAs) profiles were obtained using Ampli1™ LowPass kit on IonTorrent platform. Results: After the dissociation, RSCs maintained cell morphology and therefore, we were able to discriminate them from the heterogeneous immune infiltrate. RSCs appeared as large multinucleated cells with a big central nucleolus surrounded by a clear halo; cell diameter and ploidy were computed from the images. Pools of lymphocytes and pools of CD30+/ PD-L1+ RSCs were isolated. Sequencing results confirmed the expected flat profile for lymphocytes, while RSCs showed an aberrant profile with multiple losses and gains. Conclusions: The analysis of purified RSCs, could offer a valuable tool to uncover genetic alterations hidden by cHL immune infiltrate, for earlier adoption of more effective treatment regimens.