ANALYSIS AND INTERPRETATION OF WHOLE EXOME SEQUENCING DATA OF LEUKEMIA PATIENTS

Leukemias are a cancer type which affects the leukocytes progenitor cells. These malignancies are highly heterogeneous in terms of molecular mechanisms involved in their onset and progression. Heterogeneity can be further observed within the same subgroup of disease at the inter-individual level, being reflected by different clinical outcomes and responses to treatment in different patients. Unfortunately, the exact leukemia aetiology is still poorly understood and consequently also related prevention, diagnostic, prognostic and follow up methods remain mainly unidentified. Therefore, early-diagnosis, together with specifically tailored approaches to leukemia treatment, still represents a key point in determining patients\u2019 health, life quality and estimated life. Several efforts have been started to improve diagnosis, treatment and disease monitoring of leukemia. In this regard, the work presented in my PhD thesis is part of an international project, named \u201cNGS-PTL: Next Generation Sequencing platform for targeted Personalized Therapy of Leukemia\u201d, whose objective is the development of technologies for the diagnosis and prognosis of haematological cancers. According to the project\u2019s objective, my thesis work aims to identify sequence variants from Whole Exome Sequencing data for the acute types of leukemia, to be used as potential biomarkers to improve therapeutic interventions and for personalize treatments. The work describes the setup and application of a bioinformatic pipeline able to identify the somatic mutations in the leukemia patients and the driver carrier genes, again with the result obtained by its application on all the samples of the project. The setup of the pipeline has required the identification of a set of tools to apply to Cancer sequencing data. In particular, selection of dedicated software to perform the initial pre-processing of the data guarantees the use of sequencing data of high quality and ensures that the subsequent analysis will be performed on well-generated data. Moreover, the selection of MuTect as variant caller has allowed us to overcome specific problems related to the heterogeneity of Cancer sample. The application of these software has led us to the identification of a large and reliable set of somatic variants to be evaluated for the identifications of new biomarkers and driver genes. Then, the interpretation of the somatic variants has required the use of specific database and resources to correctly interpret them and eventually to correlate the mutations with the driving or the development of the leukemia. Using the available biological knowledge, we were able to select likely highly damaging variants, some of which already connected with leukemia in cancer-related sources (COSMIC, ICGC and CIViC). At the end, the discover of genes that drives the development of the disease was performed using three statistical tools on the set of annotated mutations for each leukemia type, leading to the identification of a total of 32 biomarkers. In conclusion, the discovery of potential novel biomarkers, again with the additional biological information provided by the specific resources applied has demonstrated the importance of the application of NGS in the study of Leukemic patients

Optimized pipeline of MuTect and GATK tools to improve the detection of somatic single nucleotide polymorphisms in whole- exome sequencing data

Background: Detecting somatic mutations in whole exome sequencing data of cancer samples has become a popular approach for profiling cancer development, progression and chemotherapy resistance. Several studies have proposed software packages, filters and parametrizations. However, many research groups reported low concordance among different methods. We aimed to develop a pipeline which detects a wide range of single nucleotide mutations with high validation rates. We combined two standard tools – Genome Analysis Toolkit (GATK) and MuTect – to create the GATK-LODN method. As proof of principle, we applied our pipeline to exome sequencing data of hematological (Acute Myeloid and Acute Lymphoblastic Leukemias) and solid (Gastrointestinal Stromal Tumor and Lung Adenocarcinoma) tumors. We performed experiments on simulated data to test the sensitivity and specificity of our pipeline. Results: The software MuTect presented the highest validation rate (90 %) for mutation detection, but limited number of somatic mutations detected. The GATK detected a high number of mutations but with low specificity. The GATK-LODN increased the performance of the GATK variant detection (from 5 of 14 to 3 of 4 confirmed variants), while preserving mutations not detected by MuTect. However, GATK-LODN filtered more variants in the hematological samples than in the solid tumors. Experiments in simulated data demonstrated that GATK-LODN increased both specificity and sensitivity of GATK results. Conclusion: We presented a pipeline that detects a wide range of somatic single nucleotide variants, with good validation rates, from exome sequencing data of cancer samples. We also showed the advantage of combining standard algorithms to create the GATK-LODN method, that increased specificity and sensitivity of GATK results. This pipeline can be helpful in discovery studies aimed to profile the somatic mutational landscape of cancer genomes

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

Background and objective S ystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. Methods C hromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFNresponsive gene expression. Results 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFN\u3b1 induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. Conclusion SS c monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc

Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes

Genome-wide Membrane Protein Structure Prediction

Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome. - See more at: http://www.eurekaselect.com/114026/article#sthash.wXDa5z1g.dpu

Role of phage Φ1 in two strains of Salmonella Rissen, sensitive and resistant to phage Φ1

BACKGROUND: The study describes the Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1. RESULTS: Following this approach, we found that ϕ1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RRϕ1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes. CONCLUSIONS: Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle