Bioinformatics for precision medicine in oncology: principles and application to the SHIVA clinical trial

Precision medicine (PM) requires the delivery of individually adapted medical care based on the genetic characteristics of each patient and his/her tumor. The last decade witnessed the development of high-throughput technologies such as microarrays and next-generation sequencing which paved the way to PM in the field of oncology. While the cost of these technologies decreases, we are facing an exponential increase in the amount of data produced. Our ability to use this information in daily practice relies strongly on the availability of an efficient bioinformatics system that assists in the translation of knowledge from the bench towards molecular targeting and diagnosis. Clinical trials and routine diagnoses constitute different approaches, both requiring a strong bioinformatics environment capable of (i) warranting the integration and the traceability of data, (ii) ensuring the correct processing and analyses of genomic data, and (iii) applying well-defined and reproducible procedures for workflow management and decision-making. To address the issues, a seamless information system was developed at Institut Curie which facilitates the data integration and tracks in real-time the processing of individual samples. Moreover, computational pipelines were developed to identify reliably genomic alterations and mutations from the molecular profiles of each patient. After a rigorous quality control, a meaningful report is delivered to the clinicians and biologists for the therapeutic decision. The complete bioinformatics environment and the key points of its implementation are presented in the context of the SHIVA clinical trial, a multicentric randomized phase II trial comparing targeted therapy based on tumor molecular profiling versus conventional therapy in patients with refractory cancer. The numerous challenges faced in practice during the setting up and the conduct of this trial are discussed as an illustration of PM application

Toward a French cyber Galaxy ?

International audienceThe success of the open web based platform “Galaxy” is growing among scientific communities. The French Institute of Bioinformatics (IFB) wishes to initiate a collaborative work dedicated to scientific workflows and especially to the Galaxy platform. We report here the main items on which future collaborations could be build: (i) software and hardware architecture, (ii) tools integration and (iii) training. High throughput technologies advent significantly alters analysis behaviour and strategy with mobilization of new infrastructure, new tools and new skills. IFB decided to conduct a cross action on "workflows" data analysis solutions, and especially on the Galaxy platform. The first item called "software and hardware architecture" addresses the operational issues in production environments, the potential for automating deployment tasks and the monitoring solutions for Galaxy servers. With the second one, "Tools integration" we aim to provide processes facilitating tool interfacing in a Galaxy instance. Priority will be the development of a good practice guide, as well as a technology watch around the methods proposed by the international community. We also want to promote the sharing of training activities at national level (such as the Aviesan Bioinformatics school, January 2013 - http://galaxy-ecole.sb-roscoff.fr/) and ensure a smooth transition to new uses, such as E-learning. A first working group is already effective. Previous items will be improved in the coming months thanks to a specific dedicated wiki and the first French Galaxy Workshop this autumn

Towards a cyber Galaxy ?

National audienceThe success of the open web based platform “Galaxy” is growing among diverse scientific communities. The French Institute of Bioinformatics - IFB wish to initiate a collaborative work dedicated to scientific workflows and especially to the platform Galaxy. We report here the main items on which future collaborations could be build: (i) software and hardware architecture, (ii) tools integration and (iii) training

A Melanoma-Tailored Next-Generation Sequencing Panel Coupled with a Comprehensive Analysis to Improve Routine Melanoma Genotyping

International audienceBackground: Tumor molecular deciphering is crucial in clinical management. Pan-cancer next-generation sequencing panels have moved towards exhaustive molecular characterization. However, because of treatment resistance and the growing emergence of pharmacological targets, tumor-specific customized panels are needed to guide therapeutic strategies.Objective: The objective of this study was to present such a customized next-generation sequencing panel in melanoma.Methods: Melanoma patients with somatic molecular profiling performed as part of routine care were included. High-throughput sequencing was performed with a melanoma tailored next-generation sequencing panel of 64 genes involved in molecular classification, prognosis, theranostic, and therapeutic resistance. Single nucleotide variants and copy number variations were screened, and a comprehensive molecular analysis identified clinically relevant alterations.Results: Four hundred and twenty-one melanoma cases were analyzed (before any treatment initiation for 94.8% of patients). After bioinformatic prioritization, we uncovered 561 single nucleotide variants, 164 copy number variations, and four splice-site mutations. At least one alteration was detected in 368 (87.4%) lesions, with BRAF, NRAS, CDKN2A, CCND1, and MET as the most frequently altered genes. Among patients with BRAFV600 mutated melanoma, 44.5% (77 of 173) harbored at least one concurrent alteration driving potential resistance to mitogen-activated protein kinase inhibitors. In patients with RAS hotspot mutated lesions and in patients with neither BRAFV600 nor RAS hotspot mutations, alterations constituting potential pharmacological targets were found in 56.9% (66 of 116) and 47.7% (63 of 132) of cases, respectively.Conclusions: Our tailored next-generation sequencing assay coupled with a comprehensive analysis may improve therapeutic management in a significant number of patients with melanoma. Updating such a panel and implementing multi-omic approaches will further enhance patients' clinical management

Pros and cons of HaloPlex enrichment in cancer predisposition genetic diagnosis

Panel sequencing is a practical option in genetic diagnosis. Enrichment and library preparation steps are critical in the diagnostic setting. In order to test the value of HaloPlex technology in diagnosis, we designed a custom oncogenetic panel including 62 genes. The procedure was tested on a training set of 71 controls and then blindly validated on 48 consecutive hereditary breast/ovarian cancer (HBOC) patients tested negative for BRCA1/2 mutation. Libraries were sequenced on HiSeq2500 and data were analysed with our academic bioinformatics pipeline. Point mutations were detected using Varscan2, median size indels were detected using Pindel and large genomic rearrangements (LGR) were detected by DESeq. Proper coverage was obtained. However, highly variable read depth was observed within genes. Excluding pseudogene analysis, all point mutations were detected on the training set. All indels were also detected using Pindel. On the other hand, DESeq allowed LGR detection but with poor specificity, preventing its use in diagnostics. Mutations were detected in 8% of BRCA1/2-negative HBOC cases. HaloPlex technology appears to be an efficient and promising solution for gene panel diagnostics. Data analysis remains a major challenge and geneticists should enhance their bioinformatics knowledge in order to ensure good quality diagnostic results

DOT1L regulates chromatin reorganization and gene expression during sperm differentiation

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility

The histone methyltransferase DOT1L regulates chromatin reorganization and gene expression during the postmeiotic differentiation of male germ cells

Spermatozoa have a unique genome organization: their chromatin is almost completely devoid of histones and is formed instead of protamines which confer a higher level of compaction than nucleosomes and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in postmeiotic male germ cells called spermatids, and is indispensable for the production of functional spermatozoa and thus for male fertility. Here we show that the H3K79 (histone 3 lysine 79) methyltransferase DOT1L controls spermatid chromatin remodelling and subsequent reorganization and compaction of spermatozoon genome. Using a mouse model in which Dot1l is knocked-out in adult male germ cells, we found that the chromatin of Dot1l -knockout (KO) spermatozoa is less compact and characterized by a higher level of retained histones and of immature forms of protamine 2. Proteomic analyses performed on differentiating male germ cells reveal that Dot1l KO extensively modifies the spermatid chromatin prior to histone removal. By transcriptomics, we also show that Dot1l KO deregulates the expression of ~1500 genes, many of which are involved in other essential aspects of spermatid differentiation such as flagellum formation. As a consequence of all these chromatin and gene expression defects, Dot1l -KO spermatozoa have misshapen and less compact heads, and are less motile, which results in impaired fertility

Breakpoint Features of Genomic Rearrangements in Neuroblastoma with Unbalanced Translocations and Chromothripsis

<div><p>Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including <i>ALK</i>, <i>NBAS</i>, <i>FHIT</i>, <i>PTPRD</i> and <i>ODZ4</i>. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.</p></div

Characteristics of junctions defined at the base pair level.

<p>Characteristics of junctions defined at the base pair level.</p