Claudio Forcato

Signaling Pathways Mediating the Induction of Apple Fruitlet Abscission

Apple (Malus × domestica) represents an interesting model tree crop for studying fruit abscission. The physiological fruitlet drop occurring in this species can be easily magnified by using thinning chemicals, such as benzyladenine (BA), to obtain fruits with improved quality and marketability. Despite the economic importance of this process, the molecular determinants of apple fruitlet abscission are still unknown. In this research, BA was used to obtain fruitlet populations with different abscission potentials to be analyzed by means of a newly released 30K oligonucleotide microarray. RNAs were extracted from cortex and seed of apple fruitlets sampled over a 4-d time course, during which BA triggers fruit drop, and used for microarray hybridization. Transcriptomic profiles of persisting and abscising fruitlets were tested for statistical association with abscission potential, allowing us to identify molecular signatures strictly related to fruit destiny. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via reactive oxygen species/sugar and hormones signaling cross talk, and followed by a block of embryogenesis and the consequent activation of the abscission zone.

Genomic profiles of primary and metastatic esophageal adenocarcinoma identified via digital sorting of pure cell populations: results from a case report

BackgroundWe report on a female patient who underwent primary radical resection for a stage 2B Her-2-positive Barrett’s-type esophageal adenocarcinoma (EAC). Despite Her-2 targeted therapy, her disease recurred and required repeated metastectomies.Case presentationDigital cell sorting and targeted sequencing of cancer sub-clones from EAC and metastases revealed a completely mutated TP53, whereas the sorted stromal cells were wild-type. Her-2 amplification was significantly lower in the metastases when the patient became therapy-resistant.ConclusionsThe mechanism of therapy resistance illustrated by this case could only be detected through accurate analysis of tumor sub-populations.Investigating tumor sub-populations of recurrent disease is important for adjusting therapy in recurrent EAC.

Abstract 1717: Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis

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, Nicolo 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

Abstract 5345: Complementary NGS approaches on digitally sorted pure tumor cells reveal hidden molecular characteristics in low tumor content FFPE specimens

Introduction: Precise characterization of tumor cell populations is an essential requirement for guiding the cancer care, allowing patients to receive personalized therapies. Poor biopsies with low-tumor content represent a significant barrier for sample enrollment in clinical trials. Here we describe a multi-level approach for precisely characterizing the genetic mutation landscape of pure tumor cell populations sorted by the DEPArray™ technology from low-cellularity FFPE samples. Methods: 50μm sections of FFPE from breast infiltrating ductal adenocarcinoma with 10% tumor content were processed by DEPArray sorting protocol. Illumina-compatible libraries were prepared from sorted stromal (n=497) and tumor (n=419) cell pools, and from the unsorted sample. An aliquot of these libraries was processed using SeqCap EZ MedExome enrichment kit (Roche) for whole-exome sequencing (WES) with a HiSeq 2500, reaching a mean coverage of 27x for tumor and 25x for stromal libraries. A second aliquot was used for low-pass (≈1M fragments per sample) whole-genome sequencing (WGS) on a MiSeq to analyze copy-number alterations (CNA). Other cell lysates from stromal (n=104, n=112) and pure tumor (n=75) populations were used in DEPArray OncoSeek amplicon-based assay for focused analysis of clinically relevant somatic variants and copy-number alterations. Results: In sorted pure tumor populations, WES analysis of B-allele frequencies of germline heterozygous SNPs clearly outlined an aberrant profile, precisely revealing several Loss-of-Heterozygosity (LOH) and copy-altered regions. Conversely, unsorted gDNA showed a flat profile non-distinguishable from sorted stromal cells, as expected for a low-cellularity tumor sample. Similar results were obtained by low-pass WGS, where the huge number of copy number aberrations (≈1.2 Gbp) in tumor is contrasted by lack of gains and losses in stromal cells and unsorted gDNA. Noteworthy, WES, low-pass and targeted sequencing by OncoSeek panel highlighted a focal amplification of ERBB2 gene (>13 copies), which was just barely detectable as a 1-copy gain in bulk gDNA. Genetic analyses showed a high concordance between WES and targeted panel data, with two non-synonymous homozygous somatic mutations found in TP53 (p.L111R) and ERBB2 (p.D769Y). In the unsorted sample, the TP53 mutation was missed because allelic frequency was below the limit of detection due to normal-cell dilution, while the ERBB2 mutation was still detectable only because of the high-level amplification. Conclusions: DEPArray sorting technology is an indispensable tool for accurately investigating cancer genomes, enabling multi-level applications for obtaining a fine-grain characterization of copy-numbers, LOH, and tumor-specific variants, independent of original tumor content. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Cassie Schumacher, Chiara Bolognesi, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Timothy Harkins, Nicolo Manaresi. Complementary NGS approaches on digitally sorted pure tumor cells reveal hidden molecular characteristics in low tumor content FFPE specimens [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 5345. doi:10.1158/1538-7445.AM2017-5345

Abstract 5349: Accurate molecular profiling of sequence and copy number alterations from sub-nanogram FFPE DNA amounts

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 g 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, Nicolo 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

Abstract 1675: Parallel genome and transcriptome-wide profiling of PD-L1 expressing tumor and infiltrating immune single cells in NSCLC

Introduction Targeting immune checkpoint has demonstrated contrasting clinical response in non-small cell lung cancer (NSCLC) due to the lack of robust biomarkers for patients stratification. This is partially due to a poor understanding of the role of the different cellular players (tumor, stromal, infiltrating immune cells) within the tumor microenvironment. Here we present an innovative workflow using the DEPArray™ sorting system, to isolate different classes of epithelial and hematopoietic viable single cells from fresh tissue biopsies for integrated genome and transcriptome sequencing analysis. Methods Freshly resected NSCLC tissue was dissociated mechanically and enzymatically down to single cell suspension, and labelled with immunofluorescent antibodies targeting PD-L1 (28-8), EpCAM, CD8, CD45 along with Hoechst 33342 for nuclei detection. Distinct pure single cells, along with pools of precise number of cells were digitally isolated using DEPArray™ platform based on marker expression. For each cell recovery, mRNA was physically separated from genomic DNA and then amplified using the Ampli1™ WTA Kit to generate a library of high quality, full-length double stranded cDNA. Amplified cDNA is used for NGS library preparation and sequencing on Illumina® MiSeq System. In parallel, genomic DNA is collected with magnetic beads and eluted directly into the reaction mixture for whole genome amplification by Ampli1™ WGA kit. The WGA products were used to profile genome-wide copy-number aberrations (CNA) using Ampli1™ LowPass kit. Results As a first step, we identified two main cell populations, the CD45+ infiltrating hematopoietic cells and the EpCAM+ epithelial cells. The hematopoietic fraction was further investigated to evaluate the presence of different subpopulations. Interestingly the PD-L1+ population, corresponding to immune suppressive hematopoietic cells, represented 47% of the total CD45+ fraction while CD8+ cells corresponding to cytotoxic T cells represented 9% of the total CD45. In the EpCAM+ population the PD-L1+ fraction represented 29% of the cells. Single viable cells along with matching pools of precise number of cells from each population were digitally isolated with DEPArray™ system for downstream analysis. Results of RNA-Seq and genomic DNA copy-number aberrations profiling will be presented at the conference. Conclusion We show here a new approach to isolate pure, single PD-L1+/- tumor and tumor infiltrating hematopoietic cells from NSCLC tissue for integrated genome and transcriptome sequencing. The direct comparison of gene expression data to its corresponding genomic data in the same tumor cell, along with the transcriptomic profiling of immune infiltrate and stroma, may add a powerful tool to disentangle the tumor microenvironment biology. This will potentially offer a novel tool to develop new biomarkers and identify therapeutic leads. Citation Format: Stefano Casonato, Giulio Bassi, Chiara Bolognesi, Chiara Mangano, Valentina del Monaco, Genny Buson, Claudio Forcato, Alberto Ferrarini, Farideh Bischoff, Gianni Medoro, Nicolo Menaresi, Francesca Fontana. Parallel genome and transcriptome-wide profiling of PD-L1 expressing tumor and infiltrating immune single cells in NSCLC [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 1675. doi:10.1158/1538-7445.AM2017-1675

Abstract 2914: Molecular characterization with single-cell resolution of CTCs and FFPE specimens from the same lung adenocarcinoma patients reveals the extent of intra-tumor heterogeneity

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, Nicolo 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

Abstract 3945: Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing

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, Nicolo 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