Search

Background: Enumeration of CTCs in blood samples with the CELLSEARCH® system is a prognostic biomarker in metastatic breast, prostate and colorectal cancer (full intended use documents.cellsearchctc.com). Currently, the final CTC identification is performed by human reviewers through visual assessment, which is a time-consuming procedure that could be affected by subjective interpretations. Modern Artificial Intelligence (AI) methods can provide a solution by automating the process of CTC identification, thus eliminating subjectivity and producing faster and more reproducible results. We have recently presented an automated algorithm (for research use only) based on deep learning models for cell segmentation and classification which allows the automated identification of CTCs in CELLSEARCH® fluorescent images (Ansaloni et al., EACR 2022). The purpose of this study was to assess inter-rater variability among human reviewers and to compare the AI performance against them. Methods: Blood samples derived from metastatic cancer patients were processed with CELLTRACKS® AUTOPREP®. The AI algorithm was trained on fluorescent images of 7255 CTCs and 32876 non-CTC events (training set) acquired by CELLTRACKS ANALYZER II® (CTAII) from 90 breast, 122 prostate and 54 colorectal cancer samples. 10 human reviewers, qualified for CTC image analysis, performed blind labeling of a separate dataset independently (test set: 55 samples taken from 27 breast and 28 prostate cancer). We studied inter-rater agreement and variability using Fleiss Kappa and Coefficient of Variation (CV). The labels provided by 5 experienced reviewers (average experience ≅ 15 years) were used to generate the ground truth (GT) by majority voting. The CTC detection performance of the AI was ranked against the other 5 reviewers (average experience ≅ 5 years). Results: In the test dataset, 2990 out of 9554 events were identified as CTCs by the GT review on CTAII images. In the task of classifying each event as CTC or non-CTC, the inter-rater agreement among the 10 human reviewers measured by Fleiss Kappa was 0.85. These classification results provided the number of CTCs per patient counted by each reviewer. The inter-rater variability on the CTC enumeration among the 10 reviewers was evaluated for each sample (median CV = 17.9% and interquartile range = 17.6% , excluding 27 samples with mode = 0 CTC). Measured against the GT classification, the AI performance (accuracy = 94.7%; F1 = 91.6%) was at the top of the human reviewers’ ranking (accuracy = 92.4 - 93.6 - 93.8 - 94.0 - 95.8%; F1 = 86.8 - 89.2 - 89.2 - 89.5 - 93.1%). Conclusion: The AI algorithm reached top-ranking performance in CTC identification, surpassing 4 out of 5 expert reviewers. These results show the applicability of AI to cell classification to remove human subjectivity from the review process and to maximize standardization among different research centers. Citation Format: Luca Biasiolli, Pietro Ansaloni, Nicolò Gentili, Ramona Miserendino, Chiara Rossi, Giulio Signorini, Gianni Medoro. Deep learning for circulating tumor cell (CTC) identification with the CELLSEARCH system: achieving top human-level performance [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 3373.

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.

Cancer is universally recognized as a heterogeneous disease. The different cell clones present in a tumor can display distinct molecular and phenotypic profiles, which may influence proliferation capabilities, metastatic potential and response to therapy. To better understand cancer progression and possibly design effective therapeutic strategies, it is fundamental to design cell-sorting technologies able to isolate rare, viable tumor cell clones from tissue or liquid biopsies. Fluorescence-activated cell sorting (FACS) is the most widely used technique to isolate cell populations from heterogeneous samples. However, FACS analysis presents technical limitations when applied to rare cell populations and/or samples with low cellularity and limitations associated with the low total number of target cells in the sample. DEParray™ PLUS (DA) is a highly automated image-based cell sorting technology capable of isolating pure, single and rare cells from heterogeneous samples. Here we evaluate the capabilities of the DA system to isolate a small number of viable tumor cells and assess the proliferation capacity of DA-sorted cells in comparison with FACS-sorted cells. Human Jurkat T lymphoblast cell line were cultured in RPMI medium and a double staining procedure was optimized to label cells with CellTraceFarRed (CTFR), division tracking dye and Live/Dead (LD) Fixable marker. CTFR/LD staining was performed for each experiment and, at days 0 and 7 of culture, CTFR and LD fluorescences were analyzed by means of BD CANTO II Flow cytometry. At day 0, FACS analysis discriminate dead from live cells, while at day 7 evaluate cell proliferation and viability after 7 days in culture. Comparison tests were carried out following two different recovery schemes: 1 recovery of 100 CTFR+/LD- cells pool and 10 recoveries of 10 CTFR+/LD- cells pool using DA Plus and BD FACSAria Fusion. For each experiment, the results were compared to cells seeded manually, which represent our unsorted control. When comparing the results obtained with a 100-cells pool, no significant difference in cell viability and proliferation were observed between DA-sorted and FACSAria-sorted cells after 7 days in culture. On the other hand, the 10 cells pool sorted with DA PLUS showed a significantly higher viability and proliferation rate compared to those sorted with FACSAria at day 7. Moreover, cells sorted by DA-technology showed less variation between experiments, highlighting a more robust performance when sorting a small number of cells. Overall, the results confirm the unique capability of the DA system to sort a small number of cells, which maintain a higher cell viability and proliferation capacity compared to those sorted with FACS. This opens up the possibility to isolate rare tumor cell clones for functional studies that can help to better understand tumor progression and response to therapy. Citation Format: Maximilian Sergio, Chiara Rossi, Valeria Stivani, Ilaria Maffei, Marco Rossi, Gianpiero Parisi, Gianni Medoro. Isolation of small number of tumor cells for proliferation studies using DEPArray technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5324.

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.

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.

Tumor heterogeneity is a hallmark of cancer and a major driving force for tumor progression, and emergence of resistance to cancer therapy. Single-cell studies have greatly contributed to unravelling tumour heterogeneity but wider adoption has been prevented by technical challenges. Isolation and analysis of single circulating tumor cells (CTCs) offer a unique minimally invasive approach to characterize and monitor dynamic changes in tumor heterogeneity at the genomic level. Standardization and reproducibility of the workflows are mandatory for translating CTC-based assays to the clinic and meet the regulatory requirements to enter routine practice. Recently, we and other groups have demonstrated the applicability of the CellSearch®/DEPArray™/ Ampli1™ workflow for the enumeration, isolation and molecular characterization of single CTCs. This workflow combines the high sensitivity and specificity of the CellSearch platform for CTC detection and enrichment with the DEPArray technology, which ensures a precise and accurate image-based isolation of single CTCs, 100% pure. Here, we validate the VRNxT™, an innovative technology that further harmonizes the CellSearch/DEPArray workflow by automating the volume reduction steps. The working principle of VRNxT instrument uses centrifugal forces to reduce the sample volume, eliminating the use of pipettes in washing steps and consequently, the risk of cell loss. Precision and accuracy of VRNxT were tested by comparing automatic and manual volume reduction (VR) procedures in a typical workflow. Briefly, after single cell isolation with the DEPArray, standard sample preparation for whole genome amplification analysis were carried out by four different skilled operators and compared to automatic VRNxT procedure. Each operator sorted 90 single CTCs using DEPArray and then performed manual VR in order to obtain a final volume of about 2 µl, compatible with the whole genome amplification procedure. Significant differences were observed in the volume reduction procedure performed by operators (2.65±0.79; 1.16±0.50; 1.26±0.96; 1.58±0.67, respectively). Conversely, VRNxT exhibited a higher reproducibility of data (1.82 µl±0.23 SD), by eliminating the user-dependent variability. Moreover, automatic VR exponentially reduced the time required to VR (about 30 minutes compared to 218 minutes of manual VR, for 96 single cell recoveries), and minimized single cell loss with a success rate never achieved so far (100% respect to 92.8% manual VR). VRNxT is thus an easy-to-use technology, that minimizes the variability and error rate in rare cell workflow and consequently reduces user-dependent variability and improve standardization. VRNxT technology coupled to CellSearch, DEPArray and Ampli1, offers an effective solution for a streamlined liquid biopsy workflow to characterize and monitor tumor heterogeneity. Citation Format: Mariano Di Trapani, Alex Calanca, Fabrizio Alberti, Gianpiero Parisi, Valeria Stivani, Chiara Maranta, Francesca Dattolo, Francesco Grasso, Michele Ricci, Alex Marchi, Gianni Medoro. Standardization and automation of volume reduction procedure for circulating tumor cells and rare cell workflows with VRNxT technology [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 5385.

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.

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.

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

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.

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: Immune system strongly affects cancer cell fate; indeed, immune-checkpoint blockade is emerging as an effective therapeutic strategy in a broad range of tumor types. The effectiveness of these therapies depends on different aspects of tumor biology such as the expression of immune checkpoint proteins, the presence of infiltrating active immune cells and many others. To date, a robust biomarker to predict therapeutic response to these drugs has not been found yet. Among all the strategies used to identify effective biomarkers, a single-cell level molecular profiling of tumor, stromal and infiltrating cells, has not been explored due to technical challenges. Here we present a method for the digital isolation of single PD-L1+ cells from FFPE tissues, along with their genetic characterization for the identification of novel biomarkers. Methods: FFPE tissue sections from two NSCLC patients, were independently tested for PD-L1 expression using IHC VENTANA PD-L1 (SP142) Assay and scored in terms of tumor content (30%, 85%), percentage of PD-L1 positive tumor cells (70%, 95%), PD-L1 intensity, and H-score (130, 175). From the same tissue blocks, additional FFPE sections were dissociated down to cell suspension, and immunofluorescently labelled using Keratin-AlexaFluor®488, Vimentin-AlexaFluor®647 and PD-L1 (clone SP263)-AlexaFluor®546. Four cell populations (Keratin+PD-L1-, Keratin+PD-L1+, Vimentin+PD-L1- and Vimentin+PD-L1+) were identified at the DEPArray™ platform and, for each, we isolated pools of precise number (range 43-121) of cells, along with pure single cells. Recovered cells were whole genome amplified (Ampli1™ WGA), and genome-wide copy-number aberrations (CNA) profiles were obtained by Ampli1™ LowPass kit and IonTorrent sequencing. Results: The expression of PD-L1 on cell membrane was attributed -by means of co-expression of either keratin or vimentin- to tumor or stromal cells, respectively. For both patients, in the tumor populations numerous gains and losses were clearly identified along the genome; Keratin+PD-L1+ and Keratin+PD-L1- pooled cells displayed comparable profiles. Notably, in both patients copy gains were identified on chromosome 9 region including the PD-L1 gene. A variable range of alleles (3 to 6) on the same PD-L1 locus was detected on single tumor cells, along with a high inter-cell heterogeneity in terms of gain extent and genomic regions involved, suggestive of the presence of different tumor clones, hidden in pooled cells data. All the stromal (Vimentin+PD-L1+/-) cells showed flat profiles. Conclusions: This study demonstrates an innovative approach for isolation and genetic profiling of single pure PD-L1+ cells from FFPE tissue. DEPArray™ sorting combined with low-pass whole genome sequencing enables high quality genome-wide profiling of pure single cells and pools isolated from phenotypically distinct populations in FFPE tumor tissue. Citation Format: Giulio Bassi, Chiara Bolognesi, Chiara Mangano, Claudio Forcato, Alberto Ferrarini, Valentina Del Monaco, Paola Tononi, Genny Buson, Gianni Medoro, Manaresi Nicolò, Francesca Fontana. Copy-number profiling of single, digitally-sorted PD-L1 positive cells in NSCLC FFPE tissues [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 5607. doi:10.1158/1538-7445.AM2017-5607

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: Presence of circulating tumor cells has prognostic value in multiple malignancies, and molecular analysis of CTCs is currently ongoing in numerous clinical trials. Most CTC enrichment methods rely on standard epithelial and leukocyte markers (CK+CD45-), so recovered cells are assumed to be of epithelial origin but never shown to be bona fide tumor cells. Conversely, atypical cells lacking the characteristic marker profile may not be analyzed, even though they may represent important tumor subpopulations. Here we evaluate a rapid, non-exhaustive, and cost-effective first-pass genomic analysis of individual candidate CTCs. This approach allows efficient upfront CNA-based confirmation that a given cell is of tumor origin, while leaving abundant DNA for deeper subsequent analysis in cells of interest. Methods: Whole peripheral blood of metastatic prostate cancer patients was enriched for CTCs using the CellSearch® system (Janssen Diagnostics) under an IRB-approved protocol, and 5 samples with >5 CTCs were selected for further study. Next, the DEPArray™ v2 system (Menarini Silicon Biosystems) was used to identify and isolate single CTCs (CK+CD45-DAPI+) and paired white blood cells (WBCs; CK-CD45+DAPI+) from the enriched samples. In addition, cells negative for both cytokeratin and CD45 but with characteristic malignant morphology (large with high nuclear-cytoplasmic ratio) were isolated. Recovered single cells were whole-genome amplified 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: Thirty-three single CTCs (CK+CD45-DAPI+) and 30 WBCs (CK-CD45+DAPI+), as well as 47 putative CTCs with non-conventional phenotype (CK-CD45-DAPI+) were isolated. Single-cell WGA products with high Genome-Integrity Index (QC score ≥3) were prioritized for CNA analysis. Ampli1 LowPass data demonstrated copy number gains/losses confirming tumor origin of the CK+ cells, while WBCs showed a normal profile. In addition, a portion of the cells having non-conventional phenotype also demonstrated copy number alterations consistent with tumor origin. Discussion: We demonstrate a WGA and low-pass WGS approach on single CTCs sorted from enriched peripheral blood, which offers a dual benefit: i) it allows rapid, non-exhaustive upfront identification of bona fide tumor cells for further study, and ii) it reveals genetic similarities and diversities (vis a vis copy number alteration) across CTCs of classical as well as non-conventional phenotypes, which may better represent clonal diversity. In a clinical setting, this molecular approach may be more effective for reliably identifying and characterizing heterogeneous CTCs, yielding profiles that more accurately reflect disease evolution and inform treatment strategies. Citation Format: Gareth Morrison, Valeria Sero, Yucheng Xu, Jacek Pinski, Sue Ingles, David Quinn, Claudio Forcato, Genny Buson, Chiu-Ho Webb, Kyle Horvath, Aditi Khurana, Gianni Medoro, Suman Verma, Matthew Moore, Philip Cotter, Nicolò Manaresi, Farideh Bischoff, Amir Goldkorn. Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis [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 1717. doi:10.1158/1538-7445.AM2017-1717

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

e12519 Background: The use of the DEPArray™ system to prepare pure tumor cell populations for more reliable and accurate downstream molecular sequence analysis has been previously demonstrated. To formally evaluate the utility of the DEPArray™ for sample preparation prior to molecular testing, we conducted a CLIA validation study to investigate the analytical performance of the instrument as well as accuracy in determining HER2 status in FFPE tumor specimens using a standard FISH assay. Methods: An initial cohort consisting of 93 FFPE samples (68 from Breast and 25 from Stomach) were selected based on defined inclusion criteria (tumor type and tumor content). For each sample, a single 50µm FFPE scroll was dissociated and then stained using fluorescently labeled Vimentin and Cytokeratin markers to distinguish between putative stromal and tumor populations, respectively. Following separation of these populations on the DEPArray™, a minimum of 100 single cells from each population was recovered and used for subsequent HER2 FISH testing. In addition, an H&E of each sample was evaluated by a Pathologist to confirm the presence of tumor content. Single-cell HER2-FISH analysis was then performed on the DEPArray™ processed samples to assess the number of signals present for each of the chromosome 17 and HER2 loci. Results were compared to the conventional tissue section FISH score. Results: Of the 93 specimens, 80 samples met pre-analytical acceptability criteria that were also confirmed by conventional methods to be either HER2-positive (n = 43) or HER2-negative (n = 37). Overall, a 95% concordance between HER2 results derived from the conventional as compared to the DEPArray™ method was observed. In addition, the instrument performance in terms of reproducibility and reliability was reported as 100%. Conclusions: DEPArray™ for preparation of FFPE-derived tumor cells was analytically validated and shown to yield high confidence in performing HER2-FISH analysis on recovered pure tumor cells. Current strategies to establish clinical utility and efficacy of this approach are underway for cases characterized as equivocal for HER2 or indeterminate by FISH.

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.

e23089 Background: Tumor infiltrating lymphocytes (TILs) are biomarkers that play a critical role in cancer diseases, including differential diagnosis, determination of prognosis, prediction of response to treatment, and evaluation of disease progression. Gene expression analysis in TILs derived from fresh tissue may not accurately depict the gene profile of the tissue microenvironment as it can change aggressively during lymphocyte isolation and RNA extraction. In addition, tissue sample size can limit the isolation of TILs with current technologies. In this study, we demonstrate the use of the DEPArray™platform to isolate pure populations of lymphocytes from a fixed mouse tissue for RNA analysi. Methods: Mouse splenocytes were activated in vitro with anti-CD3 and -CD28 for 72hs. Cells were harvested, fixed with 2% paraformaldehyde (PFA) for 20 min at RT, and stained for either CD4 or CD8 expression. Gene expression analysis of CD45, ADORA2A, GLS and GAPDH was performed in CD4+ and CD8+ DEPArray™sorted cells using the TaqMan PreAmp Cells-to-Ct kit. Results: The table below summarizes the Ct values for CD45, ADORA2A, GLS and GAPDH expression in 300 fixed unsorted control and DEPArray™sorted lymphocytes. Conclusions: We have demonstrated the feasibility of gene expression analysis on pure populations of CD4+ and CD8+ cells isolated from a fixed tissue using the DEPArray™ platform. The advantage of this approach is the DEPArray’s ability to identify and isolate subpopulations of cells from complex heterogeneous samples and/or specimens that are limited by size or content. This methodology will be applied for isolation of TILs in syngeneic and xenograft models of cancers for downstream RNA applications. [Table: see text]

Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version consisting of 320 × 320 arrayed electrodes generating >10,000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.

Body: Guidelines worldwide focus on the importance of precise, reproducible, and quality assurance of Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results due to pre-analytical sampling and tissue processing are observed. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise subsequent FISH analysis. Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n = 4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive tumor cells from each sample was achieved using the DEPArray™ platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Recovered cells were then cyto-spun onto poly-L coated glass slides prior to standard dual-color HER2/CEP17 FISH (Path Vysion Abbott/Vysis) analysis. Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed (HER2/CEP17 ratio >4.4) and consistent with levels reported in the literature. Among the patient samples, ≥75% of the DEPArray™ isolated tumor cells were recovered onto slides prior to FISH. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with observed HER2/CEP17 ratios only ranging from 1.1 to 1.4. Conclusion: We demonstrate feasibility in performing FISH for HER2/CEP17 on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray™ platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray™ sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway. Citation Format: Amanda Gerber, Trisky Clarin, Ambica Bhandari, Valeria Sero, Chiara Bolognesi, Gianni Medoro, Nicolò Manaresi, Mathew Moore, Philip D. Cotter, Farideh Bischoff. Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1374.

Introduction Several methods are currently available for Circulating Tumor Cells (CTCs) enrichment. Many studies compared CTC counts between FDA-approved CellSearch® system (based on EpCAM enrichment) and size-based selection methods. However, little is known about genomic differences across CTCs obtained from different platforms. For the first time, we report here a genomic characterization of copy-number and sequence variants in single CTCs enriched with different methods from the same patient. Methods Peripheral blood (PB) and FFPE tissue from a pelvis bone metastasis were collected from an advanced lung adenocarcinoma patient, treated with cisplatin-pemetrexed-necitumumab as first line therapy and with carboplatin-gentamicin as second-line. Two PB samples were collected at the same time: one was enriched with CellSearch® System, and one with Parsortix PR1 (size-based selection), followed by Cytokeratin-APC, CD45-PerCp and DAPI staining. Both samples were analyzed with DEPArray™ system and single CTCs along with single WBCs as controls, were isolated. Collected cells were whole genome amplified with Ampli1™ WGA kit and their Genome Integrity Index (GII) was assessed. On IonTorrent™ PGM Platform, we profiled Genome-wide copy-number alterations (CNA) by low-pass whole-genome sequencing with Ampli1™ LowPass Kit, and analyzed cancer-gene sequence variants with Ampli1™ CHP Custom Beta targeted panel. FFPE tissue was disaggregated down to single-cell suspension and labelled with Keratin and Vimentin to distinguish tumor from stromal cells. Aliquots of pure cells were recovered with DEPArray™ system; the analysis of CNVs and mutations on tissue-derived samples is in progress. Results Single CTCs (CK+, CD45-,DAPI), showing GII≥3, either isolated from CellSearch®-enriched or PR1-enriched blood, along with WBC, were selected for NGS. Ampli1™ LowPass Kit data showed several aberrations confirming tumor origin of all CTCs, while WBCs (n = 6) produced balanced profiles. Unsupervised hierarchical clustering segregated PR1-derived (n = 6) from CellSearch®-derived (n = 4) single CTCs in two separate branches. PR1-derived CTCs were very similar to each other, whereas CellSearch®-derived CTCs were more heterogeneous, although certain aberrations were common to all CTCs across platforms. At the sequence level, the targeted panel revealed somatic mutations shared by all CTCs (FLT3), a PTPN11 subclonal mutation (in 3/6 PR1 and 1/4 CellSearch), and other private mutations in single CTCs. Discussion The combination of low-pass whole-genome sequencing and targeted sequencing on single-CTCs sorted from PB enriched with different platforms reveals genetic similarities and diversities. Integration of results with genetic analysis of pure tumor cells isolated from the tumor tissue, will provide further insight of tumor diversity in different compartments. Citation Format: Francesca Fontana, Francesco Gelsomino, Claudio Forcato, Mario Terracciano, Chiara Bolognesi, Annalisa Altimari, Genny Buson, Chiara Mangano, Paola Tononi, Francesco Bacchi, Gianni Medoro, Michelangelo Fiorentino, Nicolò Manaresi, Michele Tognetto, Andrea Ardizzoni. Digital sorting and single-cell genomic profile comparison of lung adenocarcinoma CTCs between EpCAM and size-based enrichment methods. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2253.

Introduction: We have previously shown reliability in isolating pure populations of rare cells from complex tissues using the DEPArray™ system. Several molecular techniques can be applied to a variety of histological samples, for example Macrodissection is the gross manual dissection of FFPE samples used to isolate areas of interest within a specimen for optimal downstream analysis, while Fine Needle Aspiration (FNA) is a safe procedure routinely used to examine a lesion helping to make a diagnosis. These techniques are also used to assess the effect of treatment. Here we demonstrate preliminary results showing 100% efficiency in recovering pure tumor cell populations from different samples using the DEPArray™ platform to overcome the issue of tumor heterogeneity. Method: FFPE macrodissected sections (n = 9;) and FNA (n = 5;) originating from prostate, breast, pancreatic and lung tumors were evaluated. Each was processed using a tissue disassociation and staining procedure followed by DEPArray™ sorting based on cytokeratin (Ker), vimentin (Vim) and nuclear staining. The recovered cell populations were lysed in the collection tube prior to PCR-based target enrichment for next generation sequencing using IonTorrent™ AmpliSeq CHPv2. Results: DEPArray™ analysis allowed identification of well separated cell populations, including Tumor (Vim-/Ker+) and Stromal (Vim+/Ker-) cells in all the samples analyzed. In the Macrodissection samples we were able to estimate the% of tumor cells (mean 23% range 4-54%), demonstrating an unexpected low frequency of tumor cells remaining following macrodissection. In FNA specimens analyzed only 21% (4.3% to 42.7% range) of the total (mean of 6335) cells analyzed were of tumor (KER+) origin. For subsequent NGS analysis, groups of pure cells (mean 174 cells, range from 37 to 280) for each population were recovered. Among the tumor cells isolated from the macrodissected and FNA specimens, we observed non-synonymous somatic variants and LOH events for different genes. This situation can not be highlighted in unsorted population. Conclusions: DEPArray™ technology can be used to isolate pure tumor cells from heterogeneous FFPE samples used in diagnostic application, such as Macrodissection or FNA specimens. Thus, the DEPArray™ platform brings digital precision to detection, quantification and recovery of pure target cells for subsequent downstream molecular analysis that can improve cancer diagnosis and treatment decisions. Citation Format: Valeria Sero, Claudio Forcato, Chiara Bolognesi, Genny Buson, Giulio Signorini, Paola Tononi, Gianni Medoro, Nicolò Manaresi, Farideh Z Bischoff. Efficiency in recovery of pure tumor cell populations from limited tumor tissue specimens intended for clinical application. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2417.

Introduction: Genome-wide profiling of copy-number alterations (CNA) in single cells enables the characterization and investigation of genomic heterogeneity and evolution in tumor cell populations such as individual circulating tumor cells (CTCs), or disaggregated solid tumors. Ampli1™ WGA is a commercially available kit for whole-genome amplification from single cells suitable for array comparative genomic hybridization (aCGH), which is currently the gold standard for genetic diagnosis of CNA. However, labor and reagent costs limit aCGH applicability. Here we introduce for the first time a rapid, high-throughput low-pass Whole Genome Sequencing (WGS)-based method for CNA analysis (LPCNA) on single cells, enabling simultaneous profiling of multiple cells at lower cost and effort. Methods: Six single cells from NCI-H23, NCI-H661 and NCI-H1650 cell lines and 19 white blood cells (WBCs) were amplified using Ampli1™ WGA Kit. Only 5μl (1/10th of total WGA product) were used as sample input for LPCNA. Purified WGA products were processed for each sample in a single-tube, one-step reaction resulting in IonTorrent-compatible indexed libraries. Samples were pooled and size-selected to recover fragments with a length ranging from 300 to 400 bp. Following purification and template preparation, pools were sequenced on Ion 318™v2 Chips. Array CGH was performed for comparison, using Agilent SurePrint 4×180k arrays as previously reported. CNA analysis was performed using Control-FREEC for low-pass WGS data, or Agilent Genome Workbench for aCGH. Results: By exploiting the deterministic nature of Ampli1™ whole genome amplification, we devised a method to generate IonTorrent compatible libraries in a single reaction, drastically reducing time of bench work and cost. Method accuracy has been assessed by comparing the copy number profiles generated by our approach with about 870,000 reads/sample (average genome coverage of 0.064x, range 0.051-0.073) with those obtained by a validated aCGH method. Very good agreement in gain and loss profiles was achieved for all single tumor cells analyzed. As expected WBC analysis resulted in normal copy-number profiles with autosomes copy number = 2, with minimal false-positive rate ( Highlights: This innovative low-pass sequencing approach for CNA detection on single cells enables high level multiplexing on different NGS platforms. The approach is highly scalable and flexible. From few (6-8) samples (IonTorrent PGM, 318™v2 Chip), up to 96 samples (Ion Proton, Ion PI™ Chip v3) can be processed in a single run. This approach meets the need for rapid results ( This work has been carried out within IMI CANCER-ID consortium (www.cancer-id.eu). Citation Format: Genny Buson, Paola Tononi, Claudio Forcato, Francesca Fontana, Gianni Medoro, Rui Neves, Birte Möhlendick, Nikolas Stoecklein, Nicolò Manaresi. Scalable, rapid and affordable low-pass whole genome sequencing method for single-cell copy-number profiling on LM-PCR based WGA products. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2394.

Introduction: The analysis of formalin-fixed paraffin embedded (FFPE) cancer specimens is particularly challenging due to minute amount of tissue, low-tumor cellularity and heterogeneity, associated with low quality DNA. This results in an imprecise characterization of somatic variants and copy-number alterations (CNA). Here we show how digital sorting combined with low-pass whole genome sequencing (WGS) can resolve genome-wide copy-number profiling even for the more challenging FFPE samples. Methods: An archival FFPE sample from one pancreatic cancer patient, rejected by the International Cancer Genome Consortium (ICGC) criteria due to low tumor content ( Results: A total of 11M paired-end reads and a mean coverage of 0.12x were obtained from low-pass WGS, enabling the detection of CNAs at good resolution. The resulting copy-number profiles clearly show the difference between the stromal and tumor populations, with the first characterized by a flat profile (0 gains, 0 losses) and the second presenting several somatic copy-number alterations (6 gains, 22 losses). As expected due to dilution by normal cells, CNA profiles of unsorted samples missed most of the gains and losses (only 5/28 = 18% of aberrant genomic regions were detected). At a single base level, the difference between unsorted and sorted samples was more significant as the unsorted fraction missed >99% of CNA bases found in the sorted tumor population. Conclusions: DEPArray™ sorting combined with Accel-NGS® 2S kits and Illumina® low-pass whole genome sequencing enables high quality genome-wide profiling of pure tumor and stromal populations. The capacity of this method to deal with low DNA input (0.5ng) from archival FFPE samples characterized by low cancer cell content ( Citation Format: Claudio Forcato, Julie Lalibertè, Chiara Bolognesi, Ivana Cataldo, Genny Buson, Chiara Mangano, Cinzia Cantù, Francesca Fontana, Paola Tononi, Gianni Medoro, Tim Harkins, Rita T. Lawlor, Aldo Scarpa, Nicolò Manaresi. Digital sorting rescues low-cellularity tumor FFPE samples for genome-wide copy-number profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3617.

e13003Background: Often, the amount of tumor tissue needed for clinical determination of patient’ mutation status exceeds what is actually available. Macrodissected FFPE tumor specimens can be small, difficult to recover and/or remain compromised by low tumor content. Though the less invasive fine needle aspiration (FNA) procedure yields cells with good quality, tumor cell frequency is often also very low. We have previously reported the utility of the DEPArray in overcoming intracellular tumor heterogeneity, which limits downstream analysis. Here, we demonstrate 100% efficiency in recovering pure tumor cell populations from tumor-derived macrodissected FFPE and FNA using the DEPArray platform. Methods: FFPE macrodissected sections and FNA (n = 14) originating from prostate, breast, pancreatic and lung tumors were evaluated. Each was processed using a disassociation and staining procedure followed by DEPArray sorting based on cytokeratin (Ker), vimentin (Vim) and nuclear staining. The recovered cells were...

Introduction: We have previously shown reliability in isolating pure populations of cells from complex tissues using the DEPArray™. Fine Needle Aspiration (FNA) is a quick and simple procedure often performed to make a diagnosis or rule out conditions such as cancer. Although FNA is also used to assess response to treatment, the procedure is often deemed insufficient in yield and purity of tumor cells. Here we provide preliminary results showing 100% efficiency in recovering pure tumor cell populations from FNA samples of patients affected by Metastatic Breast Cancer and known to have low tumor burden ( Method: FNA paraffin embedded sections (50 microns thickness) from metastases originating from breast (n=3) primary tumors were evaluated. Each FFPE curl was processed to yield single cells followed by DEPArray™ sorting based on cytokeratin (Ker), vimentin (Vim) and nuclear staining. The recovered cell populations were directly lysed in the collection tube prior to PCR-based target enrichment for next generation sequencing using Ion AmpliSeq™ CHPv2. Results: DEPArray™ analysis allowed identification of 3 well separated cell populations, including tumor (Ker+/Vim-), stromal (Vim+/Ker) and putative EMT (Ker+/Vim+) cells. Overall, only 21% (4.3% to 42.7% range) of the total (mean of 6335) cells analyzed were of tumor (KER+/Vim-) origin. Groups of pure cells (mean 105 cells, range 15-200) for each population were recovered for sequence analysis. In one breast cancer FNA sample, we observed TP53 LoH but only in the recovered tumor (KER+) cells and not in the unsorted, stromal (VIM+), or EMT (KER+/VIM+) populations. In addition, a PIK3CA missense somatic heterozygous variant was identified in both the tumor and putative EMT populations but not in stromal cells, confirming this as a somatic mutation. Conclusion: DEPArray™ allows resolution of two main limitations associated with FNA samples obtained for genomic analysis: too few target cells and unwanted admixture of normal cells. DEPArray™ allows for phenotypic distinction between the sorted cells prior to recovery; thus, enabling sequence analysis that is suitable for detecting genomic aberrations such as CNVs and LoH, which cannot be evaluated as precisely in an unsorted sample. Clearly, the DEPArray™ platform brings precision to detection, quantification and recovery of pure target cells that are suitable for subsequent downstream molecular analysis that can improve cancer diagnosis and personalized treatment strategies for breast cancer patients. Citation Format: Sero V, Forcato C, Bolognesi C, Buson G, Medoro G, Yazdani M, Blevins A, Manaresi N, Bischoff FZ. DEPArray™ enables recovery of pure tumor cells from heterogeneous fine needle aspirates for routine downstream NGS analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-11.

A microelectronic chip for the ultra-rapid detection of cells in few micro-liters drop samples is presented. The architecture is based on a two-dimentional 640times480 array of microsites. Dielectrophoresis (DEP) is the physical phenomenon used to manipulate the cells. A corresponding array of photodiodes is used for integrated optical detection. The chip implements a column-parallel, reconfigurable read-out and A/D conversion. The single readout channel is composed by a pre-amplifier with programmable gain and an algorithmic analog-to-digital converter with a 1.5-bit/stage architecture. The chip is implemented in 0.35 mum CMOS technology with a 3.3 V supply.