Computational approaches for discovery of mutational signatures in cancer.

The accumulation of somatic mutations in a genome is the result of the activity of one or more mutagenic processes, each of which leaves its own imprint. The study of these DNA fingerprints, termed mutational signatures, holds important potential for furthering our understanding of the causes and evolution of cancer, and can provide insights of relevance for cancer prevention and treatment. In this review, we focus our attention on the mathematical models and computational techniques that have driven recent advances in the field

Helmsman: fast and efficient mutation signature analysis for massive sequencing datasets

Abstract Background The spectrum of somatic single-nucleotide variants in cancer genomes often reflects the signatures of multiple distinct mutational processes, which can provide clinically actionable insights into cancer etiology. Existing software tools for identifying and evaluating these mutational signatures do not scale to analyze large datasets containing thousands of individuals or millions of variants. Results We introduce Helmsman, a program designed to perform mutation signature analysis on arbitrarily large sequencing datasets. Helmsman is up to 300 times faster than existing software. Helmsman’s memory usage is independent of the number of variants, resulting in a small enough memory footprint to analyze datasets that would otherwise exceed the memory limitations of other programs. Conclusions Helmsman is a computationally efficient tool that enables users to evaluate mutational signatures in massive sequencing datasets that are otherwise intractable with existing software. Helmsman is freely available at https://github.com/carjed/helmsman .https://deepblue.lib.umich.edu/bitstream/2027.42/146537/1/12864_2018_Article_5264.pd

Identification of new candidate genes for germline predisposition to familial colorectal cancer using somatic mutational profiling

[eng] Colorectal cancer (CRC) is one of the malignant neoplasms with higher incidence and mortality in Spain, Europe and worldwide. As a complex disease, both environmental and genetic factors influence CRC predisposition. Up to 35% of CRC patients present familial aggregation for the disease, whereas only around 2-8% of cases are linked to a well-known hereditary syndrome associated to pathogenic germline alterations in specific genes, namely APC, MUTYH, POLE, POLD1 or the DNA mismatch repair genes. During last years, next generation sequencing (NGS) techniques such as whole exome sequencing (WES) have been used to address this gap of missing heritability. Characterization of somatic mutational profiles, performed by the application of NGS to both germline and tumor DNA, has also been recently established as a powerful tool to identify novel genes linked to CRC predisposition. However, although some bioinformatic packages have been developed to address this analysis, it remains inaccessible for a substantial proportion of the scientific community. Accordingly, the main purpose of this doctoral thesis was to identify new genes involved in germline predisposition to familial CRC, by using an integrated germline-tumor WES analysis and somatic mutational profiling, as well as facilitating the application of these genomic analyses to the scientific community. As a first step, a bioinformatic tool to deal with somatic mutational profiling was developed. Shiny framework was used to build MuSiCa, a user-friendly web application freely accessible and potentially useful for non-specialized researchers. Tumor mutational burden calculation and mutational signature refitting analysis according to the information present in COSMIC database is available, as well as different options for sample classification through clustering and principal component analysis. Subsequently, an integrated germline-tumor analysis was implemented in a cohort of 18 familial CRC unrelated patients. WES data of both germline and tumor DNA was available, allowing the identification of new potential tumor suppressor genes according to Knudson’s two-hit hypothesis. Benefitting from the development of MuSiCa application, somatic mutational profiling was also analyzed, uncovering five hypermutated samples. An enrichment of DNA repair-associated genes was found, as well as some genes previously linked to predisposition syndromes to other cancer types. BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were found as the most promising candidate genes for germline CRC predisposition. Interestingly, a germline mutation was found in the DNA repair gene RECQL in a patient with one of the hypermutated tumors, reinforcing the putative role of this gene in hereditary CRC. These findings could be helpful in clinical practice improving genetic counseling in the affected families.[spa] El cáncer colorrectal (CCR) es una de las neoplasias con mayor incidencia y mortalidad en España y el mundo. Aunque un 35% de los pacientes presentan agregación familiar, sólo un 2-8% se asocia con un síndrome hereditario conocido, causado por mutaciones germinales en genes como APC, MUTYH, POLE, POLD1 o los genes del sistema de reparación del ADN por mal apareamiento de bases. En los últimos años, las técnicas de secuenciación de nueva generación (SNG), como la secuenciación del exoma completo (SEC), han sido utilizadas para el descubrimiento de nuevos genes implicados en la predisposición al CCR. La caracterización de los perfiles mutacionales somáticos, aplicando SNG al ADN germinal y tumoral, también se ha utilizado recientemente en este proceso. Sin embargo, aunque se han desarrollado algunos paquetes bioinformáticos para su análisis, todavía permanece inaccesible para una gran parte de la comunidad científica. En consecuencia, el objetivo principal de esta tesis doctoral ha sido el de identificar nuevos genes implicados en la predisposición germinal al CCR familiar, utilizando un análisis de SEC germinal-tumoral y caracterización mutacional somática, así como facilitar la aplicación de estos análisis genómicos a la comunidad científica. En primer lugar, se llevó a cabo el desarrollo de una herramienta bioinformática denominada Mutational Signatures in Cancer (MuSiCa), una aplicación web de manejo sencillo y acceso libre desarrollada a través de la plataforma Shiny, que permite el cálculo de la carga mutacional tumoral y la caracterización de las firmas mutacionales según la información disponible en la base de datos COSMIC. Posteriormente, se implementó un análisis integrado de SEC germinal-tumoral en una cohorte de 18 pacientes de CCR familiar, complementado con una caracterización mutacional somática, gracias al desarrollo de MuSiCa. Se detectaron cinco tumores hipermutados, así como un enriquecimiento de mutaciones germinales en genes involucrados previamente en síndromes de predisposición a otros tipos de cáncer y a la reparación del ADN. Los genes BRCA2, BLM, ERCC2, RECQL, REV3L y RIF1 fueron priorizados como los más prometedores de cara a la predisposición al CCR. Estos descubrimientos podrían ser de utilidad en la práctica clínica, mejorando el consejo genético en las familias afectadas

The mutational landscape of normal urothelium

Genetic heterogeneity exists between cells within an individual. The accumulation of these somatic mutations can ultimately result in oncogenic transformation. Cancer development is often a multi-step process, the earliest stages of which remain incompletely understood. Recent technological advances have enabled detection of somatic mutations in healthy tissues, revealing that as we age many of our tissues are converted into a patchwork of mutant clones, despite retaining their histologically normal appearance. These clonal expansions are commonly driven by mutations in known oncogenes and tumour suppressor genes, with the driver landscape in a given tissue resembling its associated cancer. Bladder cancer exhibits complex landscapes of clonal selection and somatic mutagenesis. In order to characterise these features in normal urothelium, we developed an approach to sequence laser-dissected microbiopsies derived from transplant donors with no history of cancer and cystectomy specimens from bladder cancer patients. Using a combination of targeted and exome sequencing, we demonstrate that the driver landscape is dominated by chromatin remodellers, with mutations occurring less frequently in other classes of canonical bladder cancer genes, and is extensively heterogeneous between individuals. Whole-genome sequencing reveals that APOBEC mutagenesis, which accounts for the majority of mutations in bladder cancer, occurs in normal urothelium, with variable exposure levels between clones. Additionally, we identify three novel mutational signatures, one of which is associated with smoking, a major risk factor for bladder cancer the molecular basis for which was previously unknown. Overall, this dissertation provides a tantalising glimpse into the rich and diverse mutational landscape of normal urothelium. These findings may potentially contribute to the development of personalised risk models and tools for the early detection of cancer

Analysis of genomic alterations in morphologically normal tissue in prostate cancer patients reveals a potential role in tumour development.

Up to 80 % of cases of prostate cancer present with multifocal tumour lesions leading to the hypothesis of a field effect present in an apparently normal prostate that predisposes it to cancer development. In this thesis we explore the development of the field effect in the prostate by analysing normal tissues. We first applied Whole Genome DNA Sequencing (WGS) to morphologically normal tissue and benign prostatic hyperplasia (BPH) samples (n = 44) from men with and without prostate cancer. Substitutions (P =7.1x10-03, Wilcoxon rank sum test) and indels (P = 9.5x10-04) were significantly higher in morphologically normal samples, including BPH, from men with prostate cancer (median = 436) compared to those without (median = 141). Subclonal expansions under selective pressure were significantly associated with prostate cancer presence (P = 3.5x10-02, Fisher exact test). Phylogenies reveal lineages were sometimes shared between BPH and normal tissues but were completely distinct from tumour clones. Secondly, we gathered 95 samples from previously analysed normal tissue of a prostate cancer patient and performed deep targeted sequencing (> 500X) on a panel of 98 prostate cancer associated genes. We identified hundreds of mutations and validated the majority of the mutations previously found for this patient. Some genes showed repeated mutations in specific areas of the prostate whereas others were spread across the prostate. Apart from gene MUC3A, we did not find evidence of positive selection. Our results show that field characterisation of the human prostate is associated with selected clonal expansions in morphologically normal tissue/BPH that expand under selective pressure by mechanisms that are distinct from those occurring in adjacent cancer, but that are allied to the presence of the cancer. Expansions are characterised by lack of recurrent driver mutations, by almost complete absence of structure variants/copy number alterations and by distinct mutational processes

Normal and Aberrant TCRγδ+ T Cells and T Cell Large Granular Lymphocyte Leukemia

Throughout this thesis different aspects of T cells and T-LGL leukemias – with special emphasis on TCRγδ-variants – are studied