Enhancing Estimates of Breakpoints in Genome Copy Number Alteration using Confidence Masks

Chromosomal structural changes in human body known as copy number alteration (CNA) are often associated with diseases, such as various forms of cancer. Therefore, accurate estimation of breakpoints of the CNAs is important to understand the genetic basis of many diseases. The high‐resolution comparative genomic hybridization (HR‐CGH) and single‐nucleotide polymorphism (SNP) technologies enable cost‐efficient and high‐throughput CNA detection. However, probing provided using these profiles gives data highly contaminated by intensive Gaussian noise having white properties. We observe the probabilistic properties of CNA in HR‐CGH and SNP measurements and show that jitter in the breakpoints can statistically be described with either the discrete skew Laplace distribution when the segmental signal‐to‐noise ratio (SNR) exceeds unity or modified Bessel function‐based approximation when SNR is <1. Based upon these approaches, the confidence masks can be developed and used to enhance the estimates of the CNAs for the given confidence probability by removing some unlikely existing breakpoints

Multi-omics Integration for Gene Fusion Discovery and Somatic Mutation Haplotyping in Cancer

Cancer is a disease caused by changes to the genome and dysregulation of gene expression. Among many types of mutations, including point mutations, small insertions and deletions, large scale structural variants, and copy number changes, gene fusions are another category of genomic and transcriptomic alteration that can lead to cancer and which can serve as therapeutic targets. We studied gene fusion events using data from The Cancer Genome Atlas, including over 9,000 patients from 33 cancer types, finding patterns of gene fusion events and dysregulation of gene expression within and across cancer types. With data from the CoMMpass study (Multiple Myeloma Research Foundation), we generated the largest gene fusion study in multiple myeloma (742 patients), which is the second most common type of blood cancer, and which is driven by recurrent translocations. We then developed a novel tool for analyzing the haplotype context of somatic mutations. Linked-read whole genome sequencing enables haplotype resolution for analyzing somatic mutation patterns, which is lost during typical short-read sequencing and alignment. We analyzed a cohort of 14 multiple myeloma patients across disease stages, phasing three-quarters of high confidence somatic mutations and enabling us to interpret clonal evolution models at higher resolution. Finally, we also studied the co-evolution of the multiple myeloma tumor and microenvironment using single-cell RNA-sequencing, finding distinct patterns of tumor subclone evolution between disease stages in 14 patients. Our methods and results demonstrate the power of integrating data types to study complex and dynamic evolutionary pressures in cancer and point to future directions of research that aim to bridge gaps in research and clinical applications

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

Data analysis methods for copy number discovery and interpretation

Copy number variation (CNV) is an important type of genetic variation that can give rise to a wide variety of phenotypic traits. Differences in copy number are thought to play major roles in processes that involve dosage sensitive genes, providing beneficial, deleterious or neutral modifications to individual phenotypes. Copy number analysis has long been a standard in clinical cytogenetic laboratories. Gene deletions and duplications can often be linked with genetic Syndromes such as: the 7q11.23 deletion of Williams-­‐Bueren Syndrome, the 22q11 deletion of DiGeorge syndrome and the 17q11.2 duplication of Potocki-­‐Lupski syndrome. Interestingly, copy number based genomic disorders often display reciprocal deletion / duplication syndromes, with the latter frequently exhibiting milder symptoms. Moreover, the study of chromosomal imbalances plays a key role in cancer research. The datasets used for the development of analysis methods during this project are generated as part of the cutting-­‐edge translational project, Deciphering Developmental Disorders (DDD). This project, the DDD, is the first of its kind and will directly apply state of the art technologies, in the form of ultra-­‐high resolution microarray and next generation sequencing (NGS), to real-­‐time genetic clinical practice. It is collaboration between the Wellcome Trust Sanger Institute (WTSI) and the National Health Service (NHS) involving the 24 regional genetic services across the UK and Ireland. Although the application of DNA microarrays for the detection of CNVs is well established, individual change point detection algorithms often display variable performances. The definition of an optimal set of parameters for achieving a certain level of performance is rarely straightforward, especially where data qualities vary ... [cont.]

Detection of structural variations during liver cancer progression

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world and accounts for the vast majority of all liver cancers. HCC develops in response to various factors including viral infections, aflatoxin, alcohol and metabolic diseases. Recent studies have highlighted substantial differences in the acquired genomic alterations depending on the causative agent. Despite such a mutagen-dependent genetic heterogeneity, HCC is almost invariably associated with an underlying inflammatory state, whose direct contribution to the acquisition of critical genomic changes is not yet clear. The aim of my PhD project has been to understand how chronic inflammation and fibrosis affect the cancer genome. We mapped the acquired genomic alterations in human and mouse HCCs induced by defects in hepatocyte biliary transporters. These HCCs arise as a result of chronic exposure to non-neutralized bile acids that cause the onset of chronic inflammation and develop into cancer in the absence of exogenous direct (viruses) or indirect (alcohol) mutagens. We first studied the mutational landscapes of human and mouse cancer genomes and found a surprisingly low number of somatic point mutations with no impairment of cancer genes. We next studied the acquisition of somatic copy number variations (CNVs) and used well-established approaches for detecting CNVs from SNP arrays and whole genome sequencing data. We also developed a novel method, GeneCNV, for the identification of CNVs from targeted re-sequencing screenings. Overall, we observed the acquisition of massive gene copy number gains and rearrangements in both human and mouse HCCs. Amplifications preferentially occurred at late stages of cancer development and frequently targeted the mitogen-activated protein kinase (MAPK) signalling pathway, in particular, direct regulators of c-Jun NH2-terminal kinases (JNKs). We showed that that pharmacological inhibition of JNK impairs the adenoma-to- carcinoma progression in mouse. This suggests that JNK inhibition may be a useful therapeutic approach to block HCC onset in bile salt export pump (BSEP) deficiency patients waiting for liver transplantation. Altogether, this study showed that human BSEP-HCCs and mouse Mdr2-KO HCCs acquire a similar genomic signature, thus highlighting the remarkable analogy between human and mouse tumours with similar etiopathogenesis. This genomic signature differs from that of other HCCs profiled so far, which were for the most part virus induced. This demonstrates that HCC in the absence of external agents develops through genomic alterations that can be clearly distinguished from those determined by other etiological factors

Statistical modelling of mitochondrial disease

PhD ThesisMitochondrial DNA mutations are a major cause of disease in the human population. Understanding the disease associated with these mutations is complicated by heteroplasmy, the mixture of wild-type and mutated mitochondrial DNA. Heteroplasmy can vary between cells, tissues, and organs, and the disease associated individual mutations is hugely varied on account of this. The mitochondrial genome encodes critical proteins of the oxidative phosphorylation system and mutation leads to energy deficits in cells and a wide range of secondary effects. The central and peripheral nervous system are commonly affected in mitochondrial disease and quality of life for patients is severely impaired. Although pathogenic mitochondrial genetic mutations were first identified over twenty five years ago, little progress has been made in understanding the expected progression of disease in patients. The aim of this study was to use statistical modelling to further understanding of disease progression in mitochondrial DNA mutations. The Medical Research Council Mitochondrial Disease Cohort provided the majority of patient data. Patients had been assessed using the Newcastle Mitochondrial Disease Adult Scale, which facilitates quantitative research on mitochondrial disease burden. This project comprises studies of two of the most common mitochondrial DNA mutations. The first study concerns patients with the m.3243A>G mutation, the most common pathogenic point mutation, and considers the effect of age and heteroplasmy on disease progression. Prediction models of both overall disease burden and specific phenotypic features were developed. Important features of the patient cohort were also examined, including heteroplasmy in different tissues and differences in disease expression between sexes. The second study looks at patients with single large-scale mitochondrial DNA mutations. The effect of deletion size, location of the deletion on the genome, and heteroplasmy were investigated, and all three predictors were found to be significant in understanding disease progression

Das MYCN-Onkogen als Marker für minimale Resterkrankung und therapeutisches Ziel beim Neuroblastom

Neuroblastoma, the most common extracranial solid childhood cancer, arises from precursors of the developing sympathetic nervous system. MYCN oncogene amplification is a determinant of high risk and occurs in ~25% of neuroblastomas. Despite intensive treatment, more than half these patients succumb to their disease, implying persistence of therapy-resistant MYCN-amplified minimal residual neuroblastoma cells. This thesis proposes a comprehensive concept for the specific diagnostic detection of the MYCN amplicon and evaluates new treatment options for MYCN-amplified neuroblastoma. Disease-relevant nucleotide changes, structural gene rearrangements and copy number alterations were detected in tumor material by next-generation sequencing of a customized hybrid capture-based targeted panel. Unique MYCN amplicon breakpoints in the rearranged gene constitute a target sequence for a personalized minimal residual disease (MRD) PCR diagnostic. MYCN amplicon breakpoints in neuroblastoma cell lines and tumors were identified and recovered by individual, semi-quantitative PCR assays and Sanger sequencing. The assay was further developed for highly sensitive, real-time quantitative and droplet digital PCR detection for selected MYCN breakpoints in cell lines. MRD level detected in bone marrow aspirates collected during therapy outlined different disease courses in patients, including MRD persistence until relapse and good response to the first treatment course. Combining multi-agent chemotherapy in current high-risk protocols with indirect MYCN inhibitors provides a potential route to improve poor cure rates for MYCN-amplified neuroblastomas. Different hyperactive biological networks in MYCN-amplified neuroblastoma were tackled using small molecule inhibitors of the bromodomain and extra-terminal (BET) domain-containing protein BRD4, phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (PLK1). BET (JQ1, OTX015 and TEN-010) and kinase (alpelisib, volasertib and rigosertib) inhibitors demonstrated anti-cancer activity by diminishing viability in cell line-based drug screens at nanomolar to low micromolar concentrations. Rigosertib treatment altered PLK1 and PI3K signaling and strongly impaired the cellular ability for wound healing and colony formation. In line with in vitro observations, rigosertib reduced tumor growth in patient-derived neuroblastoma xenografts in mice. Combining OTX015 and volasertib produced synergistic anti-tumor responses in two MYCN-amplified neuroblastoma cell lines. To prevent MYCN-driven proliferation of tumor cells, further indirect MYCN targets are also being considered. This is exemplified by a substrate of PLK1, ASPM, which is elevated in MYCN-amplified primary neuroblastomas. Knockdown of ASPM, a microtubule-associated protein involved in mitotic spindle assembly, in MYCN-amplified neuroblastoma cell lines reduced viability and proliferation, accompanying a neuronal differentiation phenotype with neurite-like outgrowth, cytoskeletal changes and increased expression of differentiation markers. This study presents clinical implementable molecular diagnostics to pinpoint unique MYCN-amplified neuroblastoma cells within non-invasively accessible biopsy material, and proposes indirect small molecule-based MYCN therapies and potentially new drug targets for a personalized treatment.Das Neuroblastom, der häufigste extrakranielle solide Krebs im Kindesalter, entsteht aus Vorläuferzellen des sich entwickelnden sympathischen Nervensystems. Eine Amplifikation des MYCN-Onkogens ist ein bestimmender Faktor für ein hohes Risiko und tritt bei ~25% der Neuroblastome auf. Trotz intensiver Behandlung erliegt mehr als die Hälfte dieser Patienten ihrer Krankheit, was die Persistenz therapieresistenter, MYCN-amplifizierter minimaler Restneuroblastomzellen impliziert. Diese Arbeit stellt ein umfassendes Konzept für den spezifischen, diagnostischen Nachweis des MYCN-Amplikons vor und evaluiert neue Behandlungsoptionen für MYCN-amplifizierte Neuroblastome. Krankheitsrelevante Nukleotidveränderungen, strukturelle Genrearrangements und Kopienzahl-veränderungen wurden im Tumormaterial mit Hilfe eines maßgeschneiderten, zielgerichteten hybrid-capture-basierten Next Generation Sequencing (NGS) Assays nachgewiesen. Einzigartige MYCN-Amplikon-Bruchpunkte im rearrangierten Gen stellen eine Zielsequenz für eine personalisierte PCR-Diagnostik der minimalen Resterkrankung (MRD) dar. MYCN-Amplikon-Bruchpunkte in Neuroblastom-Zelllinien und Tumoren wurden durch individuelle, semi-quantitative PCR-Assays und Sanger Sequenzierung identifiziert und wiedererkannt. Der Assay wurde für den hochsensitiven, quantitativen Echtzeit- und digitalen Tröpfchen-PCR-Nachweis für ausgewählte MYCN-Bruchpunkte in Zelllinien weiterentwickelt. Die MRD Level, die in den während der Therapie gesammelten Knochenmarkaspiraten nachgewiesen wurden, skizzierten die verschiedenen Krankheitsverläufe bei den Patienten, einschließlich der MRD-Persistenz bis zum Rezidiv und des guten Ansprechens auf den ersten Behandlungsabschnitt. Die Kombination der Multi-Wirkstoff-Chemotherapie in den aktuellen Hochrisikoprotokollen mit indirekten MYCN-Inhibitoren stellt einen möglichen Weg dar, die schlechten Heilungsraten für MYCN-amplifizierte Neuroblastome zu verbessern. Verschiedene, hyperaktive biologische Netzwerke in MYCN-amplifizierten Neuroblastomen wurden mit niedermolekularen Inhibitoren der Bromdomäne und des extra-terminalen (BET) domänenhaltigen Proteins BRD4, der Phosphoinositid-3-Kinase (PI3K) und der polo-ähnlichen Kinase 1 (PLK1) behandelt. BET (JQ1, OTX015 und TEN-010) und Kinase-Inhibitoren (Alpelisib, Volasertib und Rigosertib) zeigten eine krebshemmende Wirkung, indem sie die Viabilität in zelllinienbasierten Wirkstoff-Screens bei nanomolaren bis niedrigen mikromolaren Konzentrationen verminderten. Die Behandlung mit Rigosertib veränderte die PLK1- und PI3K-Signalübertragung und beeinträchtigte die zelluläre Fähigkeit zur Wundheilung und Koloniebildung stark. In Übereinstimmung mit In-vitro-Beobachtungen reduzierte Rigosertib das Tumorwachstum in von Patienten stammenden Neuroblastom-Xenografts bei Mäusen. Die Kombination von OTX015 und Rigosertib erzeugte synergistische antitumorale Aktivität in zwei MYCN-amplifizierten Neuroblastom-Zelllinien. Um die MYCN-gesteuerte Proliferation von Tumorzellen zu verhindern, werden weitere indirekte MYCN-Targets in Betracht gezogen. Ein Beispiel hierfür ist ein Substrat von PLK1, ASPM, das in MYCN-amplifizierten, primären Neuroblastomen erhöht ist. Das Herunterregulieren von ASPM, einem Mikrotubuli-assoziierten Protein, das an der mitotischen Spindelanordnung beteiligt ist, führte in MYCN-amplifizierten Neuroblastom-Zelllinien zu einer verminderten Viabilität und Proliferation, was mit einem neuronalen Differenzierungsphänotyp mit neuritenartigem Auswuchs, zytoskelettalen Veränderungen und erhöhter Expression von Differenzierungsmarkern einherging. Diese Studie stellt eine klinisch umsetzbare, molekulare Diagnostik vor, um einzigartige MYCN-amplifizierte Neuroblastomzellen in nicht-invasiv zugänglichem Biopsiematerial zu detektieren, und schlägt indirekte, niedermolekular-basierende MYCN-Therapien und potenziell neue Zielmoleküle für eine personalisierte Krebsbehandlung vor

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

Biological characterization of Philadelphia chromosome-positive acute lymphoblastic leukemia

The prognosis of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has significantly improved with the introduction of tyrosine kinase inhibitors (TKIs). As the incidence of Ph-positivity increases with age, a substantial number of elderly Ph+ ALL patients are ineligible for intensive treatment modalities. Currently, a proportion of patients experience prolonged survival with TKI-based therapies only, and many succumb eventually to non leukemia-related causes. The aim of this thesis was to identify potential predictive biomarkers for more personalized risk stratification in Ph+ ALL, including characterization of the immune microenvironment in ALL bone marrow (BM). We also wanted to assess the drug sensitivity of primary patient samples to identify potential novel or repurposed drugs, with especially non-fit patients in mind, and to study the prevalence of copy number alterations and other secondary mutations. In study I, we collected archived formalin-fixed and paraffin-embedded BM biopsies from Ph+ (n = 31) and Philadelphia chromosome-negative (Ph−; n = 21) ALL patients and non-leukemic controls (n = 14). The samples were constructed to tissue microarrays and analyzed with multiplex immunohistochemistry and automated image analysis. The immune contexture of Ph+ and Ph− ALL BM did not differ significantly. Instead, ALL BM was characterized by an increased amount of immune cells associated with immunosuppression when compared to healthy controls. Further, the higher proportion of CD4+PD1+TIM3+ T cells, older age, and lower platelet count at diagnosis segregated a group with poor survival. In study II, we analyzed the drug sensitivity of 18 primary B-ALL BM samples (Ph+ n=10, Ph− n=8) to a selection of 64 drugs by using a well-established drug sensitivity and resistance testing assay. The results were combined with whole transcriptome sequencing and publicly available gene expression data. Apoptosis-modulating BCL2 inhibitors and MDM2 inhibitors were widely effective. BCL2-selective venetoclax was more effective in Ph− samples, whereas BCL2, BCL-W, and BCL-XL targeting navitoclax showed uniform potency. BCL2 expression was significantly higher in Ph− ALL, whereas BCL-W and BCL-XL were overexpressed in Ph+ ALL, explaining the differential drug responses. In addition, the sequencing strategies recognized three previously undiagnosed Ph-like patients with a sensitivity to TKIs. In study III, we investigated the frequency and significance of copy number alterations (CNAs) and other secondary mutations in Ph+ ALL by applying targeted next-generation sequencing (NGS) gene panel and multiplex ligation-dependent probe amplification to diagnostic (n=40) and relapse-phase (n=11) BM samples. We also assessed the prevalence of subclonal T315I kinase domain mutations. The results were combined with clinical registry data. Deletions of IKZF1 together with deletions in CDKN2A/B and/or PAX5 were common, and they stratified a group with dismal outcome. Other secondary mutations at diagnosis were rare. In conclusion, this thesis shows Ph+ ALL BM immune contexture did not differ from Ph− ALL. Instead, ALL BM immune microenvironment differs from healthy controls, and immune profiling can serve as a tool in identifying novel prognostic biomarkers. Copy number alterations (CNA) defined a subset in Ph+ ALL with dismal outcome, and we recommend incorporating CNA analysis to routine diagnostic procedures. In addition, with ex vivo drug testing, we identified several potential compounds to be further tested in clinical trials.Tyrosiinikinaasiestäjät (TKE) ovat parantaneet merkittävästi Philadelphia-kromosomipositiivisen (Ph+) akuutin lymfaattisen leukemian (ALL) ennustetta. Koska Ph+ ALL :n yleisyys kasvaa iän myötä, merkittävää osaa näistä iäkkäämmistä tai heikkokuntoisemmista potilaista ei voida kuitenkaan hoitaa tavanomaisilla intensiivisillä hoito-ohjelmilla hoitoon liittyvien haittojen vuoksi. Toisaalta osa potilaista saa hyvän vasteen pelkälle TKE-pohjaiselle kevennetylle hoidolle, ja monet menehtyvät lopulta leukemiaan liittymättömiin syihin. Tämän väitöskirjatyön tavoitteena oli selvittää potentiaalisia biomarkkereita Ph+ ALL :n yksilöllisemmän riskinarvioinnin kehittämiseksi, sekä kuvata immuunijärjestelmän koostumusta ALL :n luuytimen mikroympäristössä. Analysoimme myös potilasnäytteiden herkkyyttä lupaaville lääkeaineille ajatellen erityisesti hauraampien potilaiden ilmeistä tarvetta tehokkaille ja samalla turvallisille lääkehoidoille. Arvioimme myös kopiolukumuutosten ja muiden sekundaaristen mutaatioiden esiintyvyyttä Ph+ ALL:ssa. Ensimmäisessä osatyössä keräsimme vanhoja luuydinbiopsioita Ph+ (n=31) ja Philadelphia-kromosominegatiivista (Ph−; n=21) ALL:ia sairastavilta potilailta sekä terveiltä kontrolleilta (n=14). Näytteistä koostetut kudosblokit värjättiin multipleksatulla immunohistokemialla ja analysoitiin käyttäen apuna automatisoitua kuva-analyysia. Ph+ ja Ph− ALL -potilaiden luuytimen immunologinen mikroympäristö ei eronnut merkittävästi toisistaan. Sen sijaan ALL-potilailla immuunivasteen heikentämiseen liittyvien solutyyppien osuus oli korostunut verrattuna terveisiin kontrolleihin. Lisäksi CD4+PD1+TIM3+ T-solujen suurempi osuus, korkeampi ikä sekä matalampi verihiutaleiden määrä diagnoosihetkellä erottelivat monimuuttujamallissa ALL-potilaista huonoennusteisen ryhmän. Toisessa osatyössä analysoimme 18 potilasnäytteen (Ph+ n=10, Ph− n=8) herkkyyttä 64 eri lääkeaineelle käyttämällä vakiintunutta lääkeherkkyystestausmenetelmää. Näytteistä tehtiin myös RNA-sekvensointi, sekä tulokset yhdistettiin julkisista tietokannoista saatavilla olevaan geenien ilmentymistä kuvaavaan dataan. Ohjelmoitua solukuolemaa edistävät BCL2:n ja MDM2:n estäjät olivat tehokkaita valtaosassa näytteitä. Valikoivasti BCL2:een kohdistuva venetoklaksi oli tehokkaampi Ph− näytteissä, kun taas laajemmin BCL2:een, BCL-W:een sekä BCL-XL:ään kohdistuva navitoklaksi oli tehokas lähes kaikissa näytteissä. BCL2-geenin ilmentyminen oli lisääntynyt Ph− ALL-potilailla, kun taas BCL-W- ja BCL-XL-geenien ilmentymistasot olivat korkeampia Ph+ ALL:ssa tarjoten samalla mekanistisen selityksen eroille lääkevasteissa. Sekvensointi tunnisti lisäksi kolmen Ph− potilaan näytteessä geneettisiä muutoksia, jotka aiheuttivat herkkyyttä TKE-lääkkeille. Kolmannessa osatyössä selvitimme kopiolukumuutosten ja muiden sekundaaristen geneettisen muutosten yleisyyttä ja merkitystä Ph+ ALL:ssa hyödyntämällä kohdennettua syväsekvensointia sekä MLPA-menetelmää (MLPA, multiplex ligation-dependent probe amplification) diagnoosi- (n=40) ja relapsivaiheen (n=11) luuydinnäytteissä. Arvioimme myös subklonaalisten T315I kinaasialueen mutaatioiden esiintyvyyttä. Tulokset analysoitiin yhdessä kliinisen rekisteridatan kanssa. IKZF1-geenin deleetiot yhdessä CDKN2A/B ja/tai PAX5-geenin deleetioiden kanssa olivat yleisiä ja erottelivat erityisen huonon ennusteen ryhmän. Muita sekundaarisia geneettisiä muutoksia esiintyi lähinnä relapsivaiheen näytteissä. Tässä väitöskirjatyössä osoitimme, että Ph+ ALL:ia ja Ph− ALL:ia sairastavien potilaiden luuytimen immunologinen mikroympäristö ei eronnut merkittävästi toisistaan. Sen sijaan ALL:n luuytimen immunologinen mikroympäristö erosi terveistä kontrolleista, ja immuunijärjestelmän profilointia voidaan hyödyntää etsittäessä uusia ennusteeseen vaikuttavia biomarkkereita. Yhdistelmä epäsuotuisia kopiolukumuutoksia erotteli huonon ennusteen alaryhmän Ph+ ALL:ssa, ja suosittelemme kopiolukumuutosten rutiininomaista määrittämistä diagnoosivaiheessa. Lisäksi tunnistimme ex vivo -lääkeherkkyystestauksella useita ALL:n kliinisiin lääketutkimuksiin soveltuvia, lupaavia lääkeaineita