A Novel Approach to the Comparative Genomic Analysis of Canine and Human Cancers

abstract: Study of canine cancer’s molecular underpinnings holds great potential for informing veterinary and human oncology. Sporadic canine cancers are highly abundant (~4 million diagnoses/year in the United States) and the dog’s unique genomic architecture due to selective inbreeding, alongside the high similarity between dog and human genomes both confer power for improving understanding of cancer genes. However, characterization of canine cancer genome landscapes has been limited. It is hindered by lack of canine-specific tools and resources. To enable robust and reproducible comparative genomic analysis of canine cancers, I have developed a workflow for somatic and germline variant calling in canine cancer genomic data. I have first adapted a human cancer genomics pipeline to create a semi-automated canine pipeline used to map genomic landscapes of canine melanoma, lung adenocarcinoma, osteosarcoma and lymphoma. This pipeline also forms the backbone of my novel comparative genomics workflow. Practical impediments to comparative genomic analysis of dog and human include challenges identifying similarities in mutation type and function across species. For example, canine genes could have evolved different functions and their human orthologs may perform different functions. Hence, I undertook a systematic statistical evaluation of dog and human cancer genes and assessed functional similarities and differences between orthologs to improve understanding of the roles of these genes in cancer across species. I tested this pipeline canine and human Diffuse Large B-Cell Lymphoma (DLBCL), given that canine DLBCL is the most comprehensively genomically characterized canine cancer. Logistic regression with genes bearing somatic coding mutations in each cancer was used to determine if conservation metrics (sequence identity, network placement, etc.) could explain co-mutation of genes in both species. Using this model, I identified 25 co-mutated and evolutionarily similar genes that may be compelling cross-species cancer genes. For example, PCLO was identified as a co-mutated conserved gene with PCLO having been previously identified as recurrently mutated in human DLBCL, but with an unclear role in oncogenesis. Further investigation of these genes might shed new light on the biology of lymphoma in dogs and human and this approach may more broadly serve to prioritize new genes for comparative cancer biology studies.Dissertation/ThesisDoctoral Dissertation Biomedical Informatics 201

Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.&nbsp

Computational Inferences of Mutations Driving Mesenchymal Differentiation in Glioblastoma

This dissertation reviews the development and implementation of integrative, systems biology methods designed to parse driver mutations from high- throughput array data derived from human patients. The analysis of vast amounts of genomic and genetic data in the context of complex human genetic diseases such as Glioblastoma is a daunting task. Mutations exist by the hundreds, if not thousands, and only an unknown handful will contribute to the disease in a significant way. The goal of this project was to develop novel computational methods to identify candidate mutations from these data that drive the molecular differentiation of glioblastoma into the mesenchymal subtype, the most aggressive, poorest-prognosis tumors associated with glioblastoma

Caracterización molecular de la Leucemia Aguda Linfoblástica B (LAL-B): Análisis genómico y funcional de la Leucemia Aguda Linfoblástica y un modelo in vitro de modificación genética dirigida.

Tesis por compendio de publicaciones[ES] Introducción: Los pacientes con leucemia aguda linfoblástica se caracterizan por la presencia de una serie de alteraciones genéticas que marcan el curso de la enfermedad. Estas alteraciones son la base de los actuales sistemas de clasificación y algunas de ellas han sido establecidas como importantes marcadores pronósticos. Además, el reciente desarrollo de las técnicas ómicas, como la secuenciación masiva y los ensayos de arrays, han posibilitado el estudio de un gran número de alteraciones genéticas recurrentes que podrían establecerse como nuevos marcadores de pronóstico, ayudando en la clasificación y estratificación del riesgo de los pacientes y en la identificación de predictores de la respuesta al tratamiento. A pesar de las altas tasas de supervivencia de estos enfermos, especialmente en la población pediátrica, un alto porcentaje de los pacientes siguen presentando recaídas, suponiendo así uno de los principales problemas en la clínica de la enfermedad, junto con la toxicidad al tratamiento. La recaída de la LAL-B se caracteriza por una gran heterogeneidad clonal, compuesta por un amplio número de alteraciones que podrían estar impulsando la evolución de la enfermedad. Aunque las recaídas se dan principalmente en los adultos, se ha reportado un alto porcentaje de niños, portadores del gen de fusión ETV6/RUNX1 que presentan recaídas tardías. Esta alteración es la traslación más frecuente de la infancia y aunque su papel en el desarrollo de la enfermedad parece estar claro, existe controversia acerca de la importancia de este en el mantenimiento del fenotipo leucémico. Mediante el desarrollo de esta tesis doctoral se emplea la combinación de las nuevas tecnologías ómicas y del sistema de edición genética CRISPR/cas9, con el objetivo de alcanzar una mayor comprensión de las anomalías genéticas de la LAL-B tanto en el diagnóstico como en la recaída. Además, el uso del sistema CRISPR/Cas9 proporcionará nuevos conocimientos sobre los mecanismos moleculares de las translocaciones involucradas en LAL-B, abriendo nuevas vías para el manejo de la enfermedad. Objetivos: I) Diseñar y validar un panel de NGS personalizado para la detección de las principales alteraciones moleculares asociadas con el riesgo genético de la LAL-B en el diagnóstico. II. Estudiar el perfil genético relacionado con la recaída de los pacientes de LAL-B a través del estudio integrativo de mutaciones y CNVs mediante el uso combinado de la NGS, MLPA y aCGH. III. Evaluar el papel del gen de fusión ETV6/RUNX1 en el mantenimiento del fenotipo leucémico mediante la generación de un modelo in vitro y un modelo de xenoinjerto. Los resultados de esta tesis se dividen en tres artículo publicados en revistas científicas, de acuerdo con los objetivos planteados: Artículo I: Montaño A. et al. J. Pers. Med. 2020, 10, 0137; doi:10.3390/jpm10030137. Artículo II: Forero-Castro M. & Montaño A. et al. Diagnostics 2020, 10(7), 455. doi:10.3390/diagnostics10070455. Artículo III: Montaño A. et al. Cells 2020, 9(1), 215. doi: 10.3390/cells9010215. Principales conclusiones: I) l uso de este panel personalizado de NGS permite detectar de forma rápida y eficaz las principales alteraciones genéticas presentes en LAL-B en un solo experimento (mutaciones, CNVs, aneuploidías y translocaciones). La aplicación del panel nos permitiría así acelerar el proceso de diagnóstico molecular de los pacientes, ayudando en la clasificación y estratificación de los enfermos, así como en la toma de decisiones terapéuticas. II) El presente estudio proporciona pruebas adicionales de que la clonalidad de la LAL-B es genéticamente dinámica desde el diagnóstico hasta la recaída. La combinación de análisis de NGS, aCGH y MLPA permitió además una mejor caracterización molecular del perfil genético en los pacientes con LAL-B en recaída. III) La eliminación del gen de fusión E/R reduce significativamente el potencial oncogénico de las células leucémicas (REH, E/R positivas) tanto in vivo como in vitro. Las células E/R KO también mostraron una mayor sensibilidad a los fármacos (copanlisib solo y en combinación con prednisolona), lo que sugiere que la expresión E/R podría estar implicada en la resistencia a la prednisolona observada en algunos pacientes. Estos resultados sugieren que el gen de fusión E/R juega un papel importante en el mantenimiento del fenotipo leucémico. Por consiguiente, el gen de fusión podría convertirse en una posible diana terapéutica específica para estos pacientes con la que se obtendrían mejores tasas de respuesta

Comprehensive Genomic Characterization of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a rare, aggressive and incurable type of cancer with a high incidence of systemic dissemination and a median survival time of 3-4 years. Understanding the genetics and biology of MCL are necessary for the development of better biomarkers and novel targets of therapies. A limited number of sequencing studies were performed on MCL recently, which improved the knowledge on causal mutations and molecular mechanisms of MCL. However, the small cohort sizes of these studies and use of outdated computational tools and databases limited the findings of these studies. The objective of our study was to identify the genetic alterations and molecular pathways that are associated with the development, progression, and dissemination of MCL by using leading-edge bioinformatics tools, up-to-date molecular biology databases, and a large sample size. Presented here is a meta-analysis of whole exome sequencing data from tumor biopsies of 67 MCL patients, which resulted in the identification of several novel gene targets of recurrent mutations in MCL such as SP140, S1PR1, PTPRD, HNRNPH1, LRP1B, FAT1, MAP3K14, and DST. Our analysis revealed four mutation hotspots in the first exons of MAP3K14 with predicted activating impact on the protein in MCL, and five other types of B-cell lymphoma and leukemia. We further proposed that the recurrent mutations of MAP3K14 are potentially associated with a higher likelihood of relapse, because of their emergence in relapse biopsies of four patients. This finding suggests that the recurrent mutations of MAP3K14 may have application for prediction of disease outcome, response to therapy, and transformation of the indolent variant of MCL to the more aggressive subtype.Finally, we detected a significant accumulation of mutations in signaling pathways with roles in mechanisms of tumor metastasis, such as Rho GTPase mediated signaling, focal adhesion, G-protein coupled receptor signaling, cAMP-PKA signaling, ERK-MAPK signaling, ROBO-Slit signaling, and JAK-STAT signaling. These findings offer new insights into the understanding of driver mutations and molecular mechanisms underlying the aggressive clinical course of MCL and may have implications for the development of therapies

Discovery and characterization of novel non-coding 3′ UTR mutations in NFKBIZ and their functional implications in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a very heterogenous disease that has historically been divided into two subtypes driven by distinct molecular mechanisms. The activated B-cell (ABC) subtype of DLBCL has the worst overall survival and is characterized by activation of the NF-κB signaling pathway. Although many genetic alterations have been identified in DLBCL, there remain cases with few or no known genetic drivers. This suggests that there are still novel drivers of DLBCL yet to be discovered. In this thesis I aimed to leverage whole genome sequencing data to identify novel regions of the genome that were recurrently mutated, with a specific focus on non-coding regions. Through this analysis we identified numerous novel putative driver mutations within the non-coding genome. One of the most highly recurrently mutated regions was in the 3′ untranslated region (UTR) of the NFKBIZ gene. Amplifications of this gene have been previously discovered in ABC DLBCL and this gene is known to activate NF-κB signaling. Therefore, we hypothesized that these 3′ UTR mutations were acting as drivers in DLBCL. The remaining portion of this thesis is focused on the functional characterization of NFKBIZ 3′ UTR mutations and how they drive DLBCL and contribute to treatment resistance. To this end, I induced NFKBIZ 3′ UTR mutations into DLBCL cell lines and determined that they cause both elevated mRNA and protein expression. These mutations conferred a selective growth advantage to DLBCL cell lines both in vitro and in vivo and overexpression of NFKBIZ in primary germinal center B-cells also provided cells a growth advantage. Lastly, I found that NFKBIZ-mutant cell lines were more resistant to a selection of targeted therapeutics (ibrutinib, idelalisib and masitinib). Taken together, this thesis highlights the importance of surveying the entire cancer genome, including non-coding regions, when searching for novel drivers. I demonstrated that mutations in the 3′ UTR of a gene can act as driver mutations conferring cell growth advantages and treatment resistance. This work also implicates NFKBIZ 3′ UTR mutations as potentially useful biomarkers for predicting treatment response and informing on the most effective treatment options for patients

Germline sequence variants contributing to cancer susceptibility in South African breast cancer patients

Thesis (PhD (Bioinformatics))--University of Pretoria, 2022.Breast cancer is increasingly a public health problem worldwide. It is the most commonly diagnosed cancer and the leading cause of cancer deaths in women. Breast cancer incidence and mortality rates are rising in transitioning countries in Africa, with some of the most rapid increases occurring in sub-Saharan Africa. Newly diagnosed breast cancer cases in South Africa accounts for 27.1% of female cancers in 2020, with age-standardized (World) incidence and mortality rates of 52.6 and 16 (per 100,000 women) respectively. Cancer results from a process of genetic changes, some inherited, some induced by environmental exposures and some occurring by chance. Early age of onset and a family history is a hallmark of hereditary breast cancer that is associated with germline variants in the high-penetrance genes, BRCA1 and BRCA2. An association with breast cancer susceptibility has also been reported for a further eleven high- to moderate-penetrance genes (TP53, PALB2, PTEN, STK11, CDH1, ATM, BRIP1, CHEK2, RAD51B, RAD51C, and RAD51D). In addition, pathogenic variants in genes from the mismatch repair pathway (MLH1, MSH2, MSH6 and PMS2) have been identified in breast cancer and ovarian cancer patients. This study screened 165 South African breast cancer patients of African ancestry (self-identified) for the presence of deleterious germline sequence variants in 94 genes associated with hereditary cancer. The patients were unselected for age at diagnosis or family history of cancer. We identified pathogenic/likely pathogenic variants in thirteen patients from genes (ALK, ATM, BRCA1, BRCA2, BUB1B, CHEK2, FANCG,PALB2, RB1 and XPC ). Furthermore, a set of 27 variants of unknown significance was identified and reported that may play an important role in the future of pathogenic variants in the African population. Lastly, fourteen significant non-coding pathogenic variants from upstream, downstream and intergenic introns around the exons were identified using a combination of variant effect, CADD-PHRED and FATHMM-MKL predictions. To our knowledge, only two studies in Africa, one on Nigerian women, and one on women from Uganda and Cameroon, have used multigene panel sequencing to test for germline variants in patients, unselected for family history or age at diagnosis. Although we investigated a relatively small cohort of patients, our study provides some insights towards the genetic breast cancer risk factors in South African women of African ancestry. Further studies of a larger patient cohort is warranted to assess the distribution of variants in clinically relevant cancer susceptibility genes.National research foundationCANSASouth African Medical research councilBiochemistryPhD (Bioinformatics)Unrestricte

Investigation of mutations, copy number variations and translocations by capture-based Next-generation sequencing in patients with chronic B-cell lymphoproliferative disorders

Patients with leukaemic indolent B-NHL who could not be assigned to a specific disease category were identified and 108 cases underwent extensive molecular characterisation using a capture-NGS sequencing approach designed by the EuroClonality NGS consortium. Without a clear diagnosis such patients have no evidence-based, standardised treatment pathways and have no or limited access to clinical trials and novel therapies as eligibility and approval are based on distinct histological categories or WHO-defined disease entities. Well-characterised translocations were found in 10 cases supporting a diagnosis of Mantle cell lymphoma in six patients, follicular lymphoma in two patients and atypical CLL in a further 2 cases. In a further case a rare, TRA-CDK6 fusion was detected which was recently confirmed as a recurrent, diagnostic finding in SMZL. Combination of variants in KLF2, deletion of 7q together with IGHV1-2*04 gene usage confirmed the suspected diagnosis of SMZL in one case. The most frequently mutated genes were TP53 in 16/101 (15%) and the hotspot MYD88 p.(Leu265Pro) variant in 13/101 (13%) followed by variants in genes associated with NF-B signalling. Integration of the molecular findings together with other (histo)pathological and clinical findings may enable the distinction between LPL and MZL-related disease categories in these patients. NGS was capable of identifying clonal IG-rearrangements in all cases. Further stereotyping analysis using the VLeader protocol and Sanger sequencing showed most cases carried IGHV3 (52%) and IGHV4 (27%) genes with IGHV4-34 the most common (17%). Evidence of Somatic hypermutations were found in 90% of cases with 77% showing <98% homology to the germline sequence. None of the IGHV rearrangements showed a CLL-stereotype. Preliminary analysis of copy-number variants showed a high frequency of trisomy 12 (17 cases), 17p deletion events in combination with TP53 variants as well as deletions of 7q, a recurrent feature of MZL. This prospective study clearly demonstrates the benefit of using NGS-panel approaches for the characterization of patient cohorts which based on routine 4 diagnostics evaluation cannot be assigned to a defined disease category. An additional 12 patients were given a formal diagnosis based on the molecular findings and further clinical review prompted by the results of this study will likely lead to an increase in this number. The detailed information obtained about the clonal structure, including the specific V(D)J-rearrangements and combinations of molecular aberrations in each case, will enable further research into the aetiology of these diseases. From a diagnostic laboratory perspective in particular, the NGS panel utilised here is capable of reducing the time and cost of the evaluations required by combining the analysis of clonality, single-nucleotide variants, copy-number variants and structural variants in a single assay. Given the breadth of results, this NGS panel is also a useful tool for the characterisation of cohorts in a research setting and the design can be extended in future as novel markers and areas of interest are described in the literature

Computational Biology-Driven Genomic and Epigenomic Delineation of Acute Myeloid Leukemia

Hematopoiesis is the deterministic process of blood cell formation taking place in the bone marrow. Mature blood cells are produced by a tightly controlled mechanism from hematopoietic stem cells (HSCs) residing in the bone marrow. Upon maturation blood cells are released into the peripheral blood and from this point onward can be transported to the different locations of the body. The mature blood cells exert different functions dependent on a strictly controlled path of maturation. The distinct leukocytes comprising granulocytes, monocytes, macrophages, natural killer cells and lymphocytes are essential for the defense against pathogens and foreign invaders, erythrocytes play a pivotal role in the transportation of oxygen to remote organs, and platelets confer the process of blood clotting. Mature blood cells are short-lived and require continuous replenishment. The control of the production and the total number of blood cells is conferred by multipotent progenitors and a small population of pluripotent HSCs (Figure 1). HSCs reside in the bone marrow of adult mammals at the apex of a hierarchy of progenitors which become progressively restricted to several and eventually single lineages of blood cells. Additionally these pluripotent stem cells have the unique ability to self-renew, generating a source for continuous replenishment of the complete blood cell system. The hematopoietic stem cell compartment contains stem cells with progressively decreased self-renewal capacity with the retention of multi-lineage reconstitution. The rare long term HSC (LT-HSC) is at the pinnacle of the hematopoietic hierarchy and is mainly quiescent. With the most conserved rate of self-renewal it prevents the depletion of the stem cell pool. The less rare short term HSC (ST-HSC) still retains a minimal ability for self-renewal and is the more active effector cell for hematopoietic replenishment in normal situations. The main constituent of the hematopoietic stem cell compartment is the multipotent progenitor (MPP) which lost its self-renewal capacity, however, kept the ability to give rise to daughter cells of different lineages. The daughter cells, common myeloid progenitor (CMP) and common lymphoid progenitor (CLP), are still oligopotent as they give rise to multiple blood cell types, e.g., lymphocytes, granulocytes, platelets and erythrocytes. The production of mature blood cells is a strictly controlled process that adapts to the needs of human physiology, e.g., erythrocyte production after blood loss. The control is asserted mainly by external stimuli, e.g., hematopoietic cytokines or growth factors, which are produced by constituents of the regulatory microenvironment within the bone marrow niche, other blood cells or cytokine secreting organs. The microenvironment plays a pivotal role in the formation of adequate numbers of blood cells of the correct type and the hematopoietic cytokines it produces allows the hematopoietic system to dynamically adapt to extramedullary events, e.g., blood loss, infection or cancer immunoediting