Genny Buson

De novo transcriptome characterization of Vitis vinifera cv. Corvina unveils varietal diversity.

BackgroundPlants such as grapevine (Vitis spp.) display significant inter-cultivar genetic and phenotypic variation. The genetic components underlying phenotypic diversity in grapevine must be understood in order to disentangle genetic and environmental factors.ResultsWe have shown that cDNA sequencing by RNA-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar (Corvina) and the PN40024 reference genome. We detected 15,161 known genes including 9463 with novel splice isoforms, and identified 2321 potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. We also discovered 180 apparent private genes in the Corvina genome which were missing from the reference genome.ConclusionsThe de novo assembly approach allowed a substantial amount of the Corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations. The private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. Therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes.

Metabolite and transcript profiling of berry skin during fruit development elucidates differential regulation between Cabernet Sauvignon and Shiraz cultivars at branching points in the polyphenol pathway

BackgroundGrapevine berries undergo complex biochemical changes during fruit maturation, many of which are dependent upon the variety and its environment. In order to elucidate the varietal dependent developmental regulation of primary and specialized metabolism, berry skins of Cabernet Sauvignon and Shiraz were subjected to gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) based metabolite profiling from pre-veraison to harvest. The generated dataset was augmented with transcript profiling using RNAseq.ResultsThe analysis of the metabolite data revealed similar developmental patterns of change in primary metabolites between the two cultivars. Nevertheless, towards maturity the extent of change in the major organic acid and sugars (i.e. sucrose, trehalose, malate) and precursors of aromatic and phenolic compounds such as quinate and shikimate was greater in Shiraz compared to Cabernet Sauvignon. In contrast, distinct directional projections on the PCA plot of the two cultivars samples towards maturation when using the specialized metabolite profiles were apparent, suggesting a cultivar-dependent regulation of the specialized metabolism. Generally, Shiraz displayed greater upregulation of the entire polyphenol pathway and specifically higher accumulation of piceid and coumaroyl anthocyanin forms than Cabernet Sauvignon from veraison onwards. Transcript profiling revealed coordinated increased transcript abundance for genes encoding enzymes of committing steps in the phenylpropanoid pathway. The anthocyanin metabolite profile showed F3′5′H-mediated delphinidin-type anthocyanin enrichment in both varieties towards maturation, consistent with the transcript data, indicating that the F3′5′H-governed branching step dominates the anthocyanin profile at late berry development. Correlation analysis confirmed the tightly coordinated metabolic changes during development, and suggested a source-sink relation between the central and specialized metabolism, stronger in Shiraz than Cabernet Sauvignon. RNAseq analysis also revealed that the two cultivars exhibited distinct pattern of changes in genes related to abscisic acid (ABA) biosynthesis enzymes.ConclusionsCompared with CS, Shiraz showed higher number of significant correlations between metabolites, which together with the relatively higher expression of flavonoid genes supports the evidence of increased accumulation of coumaroyl anthocyanins in that cultivar. Enhanced stress related metabolism, e.g. trehalose, stilbene and ABA in Shiraz berry-skin are consistent with its relatively higher susceptibility to environmental cues.

The High Polyphenol Content of Grapevine Cultivar Tannat Berries Is Conferred Primarily by Genes That Are Not Shared with the Reference Genome

The Tannat grape berry is used to produce high-quality wines with an intense purple color and remarkable antioxidant properties. Through reference-guided assembly of the genome combined with de novo assembly of the transcriptome, we found that the variety-specific genes that might contribute substantially to the unique characteristics of the Tannat berry are not present in the reference genome. The grapevine (Vitis vinifera) cultivar Tannat is cultivated mainly in Uruguay for the production of high-quality red wines. Tannat berries have unusually high levels of polyphenolic compounds, producing wines with an intense purple color and remarkable antioxidant properties. We investigated the genetic basis of these important characteristics by sequencing the genome of the Uruguayan Tannat clone UY11 using Illumina technology, followed by a mixture of de novo assembly and iterative mapping onto the PN40024 reference genome. RNA sequencing data for genome reannotation were processed using a combination of reference-guided annotation and de novo transcript assembly, allowing 5901 previously unannotated or unassembled genes to be defined and resulting in the discovery of 1873 genes that were not shared with PN40024. Expression analysis showed that these cultivar-specific genes contributed substantially (up to 81.24%) to the overall expression of enzymes involved in the synthesis of phenolic and polyphenolic compounds that contribute to the unique characteristics of the Tannat berries. The characterization of the Tannat genome therefore indicated that the grapevine reference genome lacks many genes that appear to be relevant for the varietal phenotype.

Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

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.

Patchwork sequencing of tomato San Marzano and Vesuviano varieties highlights genome-wide variations

BackgroundInvestigation of tomato genetic resources is a crucial issue for better straight evolution and genetic studies as well as tomato breeding strategies. Traditional Vesuviano and San Marzano varieties grown in Campania region (Southern Italy) are famous for their remarkable fruit quality. Owing to their economic and social importance is crucial to understand the genetic basis of their unique traits.ResultsHere, we present the draft genome sequences of tomato Vesuviano and San Marzano genome. A 40x genome coverage was obtained from a hybrid Illumina paired-end reads assembling that combines de novo assembly with iterative mapping to the reference S. lycopersicum genome (SL2.40). Insertions, deletions and SNP variants were carefully measured. When assessed on the basis of the reference annotation, 30% of protein-coding genes are predicted to have variants in both varieties. Copy genes number and gene location were assessed by mRNA transcripts mapping, showing a closer relationship of San Marzano with reference genome. Distinctive variations in key genes and transcription/regulation factors related to fruit quality have been revealed for both cultivars.ConclusionsThe effort performed highlighted varieties relationships and important variants in fruit key processes useful to dissect the path from sequence variant to phenotype.

Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers.

AbstracBackgroundNon-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers.ResultsWe performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-rasLA1/p53R172HΔg and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-rasLA1/p53R172HΔg tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-rasLA1/p53R172HΔg mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our model p53 is inactivated, ADORA3 does not negatively affect tumor growth but remains expressed on tumor cells. Thus, it could represent an interesting target for the development of antibody-targeted therapy on a subset of NSCLC, which are p53 null and ADORA3 positive.ConclusionsOur study provided a complete transcription overview of the K-rasLA1/p53R172HΔg mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.

Slug/β-Catenin–Dependent Proinflammatory Phenotype in Hypoxic Breast Cancer Stem Cells

Cancer stem cell survival relies on the activation of inflammatory pathways, which is speculatively triggered by cell autonomous mechanisms or by microenvironmental stimuli. Here, we observed that hypoxic bone marrow stroma-derived transforming growth factor-β 1 promotes the growth of human breast cancer stem cells as mammospheres. The ensuing Slug-dependent serine 139 phosphorylation of the DNA damage sensor H2AX in breast cancer stem cells induces tumor necrosis factor-α and IL-8 mRNAs, whose stability is enhanced by cytoplasmic β-catenin. β-Catenin also up-regulates and binds miR-221, reducing the stability of the miR-221 targets Rad51 and ERα mRNAs. Our data show that the Slug/β-catenin-dependent activation of DNA damage signaling triggered by the hypoxic microenvironment sustains the proinflammatory phenotype of breast cancer stem cells.

MYC Amplification as a Potential Mechanism of Primary Resistance to Crizotinib in ALK-Rearranged Non-Small Cell Lung Cancer: A Brief Report

INTRODUCTION: Translocations of the anaplastic lymphoma kinase (ALK) can be effectively targeted in advanced non-small cell lung cancer by ALK-TKI inhibitors including Crizotinib. However, the development of acquired resistance often limits the duration of these therapies. While several mechanisms of secondary resistance have been already identified, little is known about molecular determinants of primary resistance. In our brief report we investigated the tumor molecular profile of a patient who failed to respond to Crizotinib. METHODS: Fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) were run on tumor specimen as well as search and characterization of circulating tumor cells (CTCs) in the blood. Confirmation of clinical findings was achieved using a translational cell-line in vitro model. RESULTS: We identified the amplification of MYC as a potential new mechanism of primary resistance to ALK inhibition. Human EML4-ALK rearranged cells infected with a lentiviral vector carrying full-length human MYC cDNA were treated in vitro with crizotinib and alectinib. Overexpression of MYC overexpression was associated with a reduced sensitivity to both ALK-inhibitors. MYC-overexpressing clones displayed also increased levels of both cyclin D and E and their growth was reduced by using Cdk4/6 inhibitors such as Palbociclib. CONCLUSIONS: We postulate that the MYC gene may be implicated in the mechanism of primary resistance to ALK inhibitors. We also suggest potential MYC-directed inhibition strategies to overcome primary resistance in advanced ALK-rearranged NSCLC.

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