RESUMO
Alternative splicing (AS) is an important mechanism in the development of many cancers, as novel or aberrant AS patterns play an important role as an independent onco-driver. In addition, cancer-specific AS is potentially an effective target of personalized cancer therapeutics. However, detecting AS events remains a challenging task, especially if these AS events are novel. This is exacerbated by the fact that existing transcriptome annotation databases are far from being comprehensive, especially with regard to cancer-specific AS. Additionally, traditional sequencing technologies are severely limited by the short length of the generated reads, which rarely spans more than a single splice junction site. Given these challenges, transcriptomic long-read (LR) sequencing presents a promising potential for the detection and discovery of AS. We present Freddie, a computational annotation-independent isoform discovery and detection tool. Freddie takes as input transcriptomic LR sequencing of a sample alongside its genomic split alignment and computes a set of isoforms for the given sample. It then partitions the input reads into sets that can be processed independently and in parallel. For each partition, Freddie segments the genomic alignment of the reads into canonical exon segments. The goal of this segmentation is to be able to represent any potential isoform as a subset of these canonical exons. This segmentation is formulated as an optimization problem and is solved with a dynamic programming algorithm. Then, Freddie reconstructs the isoforms by jointly clustering and error-correcting the reads using the canonical segmentation as a succinct representation. The clustering and error-correcting step is formulated as an optimization problem-the Minimum Error Clustering into Isoforms (MErCi) problem-and is solved using integer linear programming (ILP). We compare the performance of Freddie on simulated datasets with other isoform detection tools with varying dependence on annotation databases. We show that Freddie outperforms the other tools in its accuracy, including those given the complete ground truth annotation. We also run Freddie on a transcriptomic LR dataset generated in-house from a prostate cancer cell line with a matched short-read RNA-seq dataset. Freddie results in isoforms with a higher short-read cross-validation rate than the other tested tools. Freddie is open source and available at https://github.com/vpc-ccg/freddie/.
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Processamento Alternativo , Software , Transcriptoma , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Análise de Sequência de RNA/métodosRESUMO
BACKGROUND: The gut microbiome is a diverse network of bacteria which inhabit our digestive tract and is crucial for efficient cellular metabolism, nutrient absorption, and immune system development. Spinal cord injury (SCI) disrupts autonomic function below the level of injury and can alter the composition of the gut microbiome. Studies in rodent models have shown that SCI-induced bacterial imbalances in the gut can exacerbate the spinal cord damage and impair recovery. In this study we, for the first time, characterized the composition of the gut microbiome in a Yucatan minipig SCI model. We compared the relative abundance of the most dominant bacterial phyla in control samples to those collected from animals who underwent a contusion-compression SCI at the 2nd or 10th Thoracic level. RESULTS: We identify specific bacterial fluctuations that are unique to SCI animals, which were not found in uninjured animals given the same dietary regimen or antibiotic administration. Further, we identified a specific time-frame, "SCI-acute stage", during which many of these bacterial fluctuations occur before returning to "baseline" levels. CONCLUSION: This work presents a dynamic view of the microbiome changes that accompany SCI, establishes a resource for future studies and to understand the changes that occur to gut microbiota after spinal cord injury and may point to a potential therapeutic target for future treatment.
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Microbioma Gastrointestinal , Traumatismos da Medula Espinal , Animais , Bactérias , Medula Espinal , Suínos , Porco MiniaturaRESUMO
MOTIVATION: Cancer is a complex disease that involves rapidly evolving cells, often forming multiple distinct clones. In order to effectively understand progression of a patient-specific tumor, one needs to comprehensively sample tumor DNA at multiple time points, ideally obtained through inexpensive and minimally invasive techniques. Current sequencing technologies make the 'liquid biopsy' possible, which involves sampling a patient's blood or urine and sequencing the circulating cell free DNA (cfDNA). A certain percentage of this DNA originates from the tumor, known as circulating tumor DNA (ctDNA). The ratio of ctDNA may be extremely low in the sample, and the ctDNA may originate from multiple tumors or clones. These factors present unique challenges for applying existing tools and workflows to the analysis of ctDNA, especially in the detection of structural variations which rely on sufficient read coverage to be detectable. RESULTS: Here we introduce SViCT , a structural variation (SV) detection tool designed to handle the challenges associated with cfDNA analysis. SViCT can detect breakpoints and sequences of various structural variations including deletions, insertions, inversions, duplications and translocations. SViCT extracts discordant read pairs, one-end anchors and soft-clipped/split reads, assembles them into contigs, and re-maps contig intervals to a reference genome using an efficient k-mer indexing approach. The intervals are then joined using a combination of graph and greedy algorithms to identify specific structural variant signatures. We assessed the performance of SViCT and compared it to state-of-the-art tools using simulated cfDNA datasets with properties matching those of real cfDNA samples. The positive predictive value and sensitivity of our tool was superior to all the tested tools and reasonable performance was maintained down to the lowest dilution of 0.01% tumor DNA in simulated datasets. Additionally, SViCT was able to detect all known SVs in two real cfDNA reference datasets (at 0.6-5% ctDNA) and predict a novel structural variant in a prostate cancer cohort. AVAILABILITY: SViCT is available at https://github.com/vpc-ccg/svict. Contact:faraz.hach@ubc.ca.
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Algoritmos , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Análise Mutacional de DNA/métodos , Mutação , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Conjuntos de Dados como Assunto , Humanos , Masculino , Neoplasias da Próstata/genética , Sensibilidade e EspecificidadeRESUMO
MOTIVATION: Next-Generation Sequencing has led to the availability of massive genomic datasets whose processing raises many challenges, including the handling of sequencing errors. This is especially pertinent in cancer genomics, e.g. for detecting low allele frequency variations from circulating tumor DNA. Barcode tagging of DNA molecules with unique molecular identifiers (UMI) attempts to mitigate sequencing errors; UMI tagged molecules are polymerase chain reaction (PCR) amplified, and the PCR copies of UMI tagged molecules are sequenced independently. However, the PCR and sequencing steps can generate errors in the sequenced reads that can be located in the barcode and/or the DNA sequence. Analyzing UMI tagged sequencing data requires an initial clustering step, with the aim of grouping reads sequenced from PCR duplicates of the same UMI tagged molecule into a single cluster, and the size of the current datasets requires this clustering process to be resource-efficient. RESULTS: We introduce Calib, a computational tool that clusters paired-end reads from UMI tagged sequencing experiments generated by substitution-error-dominant sequencing platforms such as Illumina. Calib clusters are defined as connected components of a graph whose edges are defined in terms of both barcode similarity and read sequence similarity. The graph is constructed efficiently using locality sensitive hashing and MinHashing techniques. Calib's default clustering parameters are optimized empirically, for different UMI and read lengths, using a simulation module that is packaged with Calib. Compared to other tools, Calib has the best accuracy on simulated data, while maintaining reasonable runtime and memory footprint. On a real dataset, Calib runs with far less resources than alignment-based methods, and its clusters reduce the number of tentative false positive in downstream variation calling. AVAILABILITY AND IMPLEMENTATION: Calib is implemented in C++ and its simulation module is implemented in Python. Calib is available at https://github.com/vpc-ccg/calib. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Sequenciamento de Nucleotídeos em Larga Escala , Software , Algoritmos , Análise por Conglomerados , DNA , Análise de Sequência de DNARESUMO
MOTIVATION: Successful development and application of precision oncology approaches require robust elucidation of the genomic landscape of a patient's cancer and, ideally, the ability to monitor therapy-induced genomic changes in the tumour in an inexpensive and minimally invasive manner. Thanks to recent advances in sequencing technologies, 'liquid biopsy', the sampling of patient's bodily fluids such as blood and urine, is considered as one of the most promising approaches to achieve this goal. In many cancer patients, and especially those with advanced metastatic disease, deep sequencing of circulating cell free DNA (cfDNA) obtained from patient's blood yields a mixture of reads originating from the normal DNA and from multiple tumour subclones-called circulating tumour DNA or ctDNA. The ctDNA/cfDNA ratio as well as the proportion of ctDNA originating from specific tumour subclones depend on multiple factors, making comprehensive detection of mutations difficult, especially at early stages of cancer. Furthermore, sensitive and accurate detection of single nucleotide variants (SNVs) and indels from cfDNA is constrained by several factors such as the sequencing errors and PCR artifacts, and mapping errors related to repeat regions within the genome. In this article, we introduce SiNVICT, a computational method that increases the sensitivity and specificity of SNV and indel detection at very low variant allele frequencies. SiNVICT has the capability to handle multiple sequencing platforms with different error properties; it minimizes false positives resulting from mapping errors and other technology specific artifacts including strand bias and low base quality at read ends. SiNVICT also has the capability to perform time-series analysis, where samples from a patient sequenced at multiple time points are jointly examined to report locations of interest where there is a possibility that certain clones were wiped out by some treatment while some subclones gained selective advantage. RESULTS: We tested SiNVICT on simulated data as well as prostate cancer cell lines and cfDNA obtained from castration-resistant prostate cancer patients. On both simulated and biological data, SiNVICT was able to detect SNVs and indels with variant allele percentages as low as 0.5%. The lowest amounts of total DNA used for the biological data where SNVs and indels could be detected with very high sensitivity were 2.5 ng on the Ion Torrent platform and 10 ng on Illumina. With increased sequencing and mapping accuracy, SiNVICT might be utilized in clinical settings, making it possible to track the progress of point mutations and indels that are associated with resistance to cancer therapies and provide patients personalized treatment. We also compared SiNVICT with other popular SNV callers such as MuTect, VarScan2 and Freebayes. Our results show that SiNVICT performs better than these tools in most cases and allows further data exploration such as time-series analysis on cfDNA sequencing data. AVAILABILITY AND IMPLEMENTATION: SiNVICT is available at: https://sfu-compbio.github.io/sinvictSupplementary information: Supplementary data are available at Bioinformatics online. CONTACT: cenk@sfu.ca.
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Análise Mutacional de DNA/métodos , DNA de Neoplasias/sangue , Mutação INDEL , Neoplasias/genética , Mutação Puntual , Software , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Neoplasias/sangue , Sensibilidade e EspecificidadeRESUMO
FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.
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Fator 3-alfa Nuclear de Hepatócito , Neoplasias de Próstata Resistentes à Castração , Semaforinas , Humanos , Masculino , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Mutação , Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Fatores de Transcrição/metabolismo , Semaforinas/genética , Semaforinas/metabolismoRESUMO
The current paradigm of cancer care relies on predictive nomograms which integrate detailed histopathology with clinical data. However, when predictions fail, the consequences for patients are often catastrophic, especially in prostate cancer where nomograms influence the decision to therapeutically intervene. We hypothesized that the high dimensional data afforded by massively parallel sequencing (MPS) is not only capable of providing biological insights, but may aid molecular pathology of prostate tumours. We assembled a cohort of six patients with high-risk disease, and performed deep RNA and shallow DNA sequencing in primary tumours and matched metastases where available. Our analysis identified copy number abnormalities, accurately profiled gene expression levels, and detected both differential splicing and expressed fusion genes. We revealed occult and potentially dormant metastases, unambiguously supporting the patients' clinical history, and implicated the REST transcriptional complex in the development of neuroendocrine prostate cancer, validating this finding in a large independent cohort. We massively expand on the number of novel fusion genes described in prostate cancer; provide fresh evidence for the growing link between fusion gene aetiology and gene expression profiles; and show the utility of fusion genes for molecular pathology. Finally, we identified chromothripsis in a patient with chronic prostatitis. Our results provide a strong foundation for further development of MPS-based molecular pathology.
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Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Hormônio-Dependentes/genética , Células Neuroendócrinas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Idoso , Processamento Alternativo , Biomarcadores Tumorais/sangue , Colúmbia Britânica , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Análise por Conglomerados , Técnicas de Apoio para a Decisão , Dosagem de Genes , Fusão Gênica , Predisposição Genética para Doença , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias Hormônio-Dependentes/terapia , Células Neuroendócrinas/patologia , Nomogramas , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Prognóstico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Interferência de RNA , TransfecçãoRESUMO
Next-generation sequencing is making sequence-based molecular pathology and personalized oncology viable. We selected an individual initially diagnosed with conventional but aggressive prostate adenocarcinoma and sequenced the genome and transcriptome from primary and metastatic tissues collected prior to hormone therapy. The histology-pathology and copy number profiles were remarkably homogeneous, yet it was possible to propose the quadrant of the prostate tumour that likely seeded the metastatic diaspora. Despite a homogeneous cell type, our transcriptome analysis revealed signatures of both luminal and neuroendocrine cell types. Remarkably, the repertoire of expressed but apparently private gene fusions, including C15orf21:MYC, recapitulated this biology. We hypothesize that the amplification and over-expression of the stem cell gene MSI2 may have contributed to the stable hybrid cellular identity. This hybrid luminal-neuroendocrine tumour appears to represent a novel and highly aggressive case of prostate cancer with unique biological features and, conceivably, a propensity for rapid progression to castrate-resistance. Overall, this work highlights the importance of integrated analyses of genome, exome and transcriptome sequences for basic tumour biology, sequence-based molecular pathology and personalized oncology.
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Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Genômica , Neoplasias da Próstata/genética , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Terapia Combinada , DNA de Neoplasias/análise , Amplificação de Genes , Dosagem de Genes , Perfilação da Expressão Gênica , Fusão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/patologia , Prognóstico , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Complex genome rearrangements are frequently observed in cancer but their impact on tumor molecular biology is largely unknown. Recent studies have identified a new phenomenon involving the simultaneous generation of tens to hundreds of genomic rearrangements, called chromothripsis. To understand the molecular consequences of these events, we sequenced the genomes and transcriptomes of two prostate tumors exhibiting evidence of chromothripsis. We identified several complex fusion transcripts, each containing sequence from three different genes, originating from different parts of the genome. One such poly-gene fusion transcript appeared to be expressed from a chain of small genomic fragments. Furthermore, we detected poly-gene fusion transcripts in the prostate cancer cell line LNCaP, suggesting they may represent a common phenomenon. Finally in one tumor with chromothripsis, we identified multiple mutations in the p53 signaling pathway, expanding on recent work associating aberrant DNA damage response mechanisms with chromothripsis. Overall, our data show that chromothripsis can manifest as massively rearranged transcriptomes. The implication that multigenic changes can give rise to poly-gene fusion transcripts is potentially of great significance to cancer genetics.
Assuntos
Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Fusão Gênica , Humanos , Masculino , Mutação , Neoplasias da Próstata/patologiaRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor central in the regulation of key cellular processes including cell metabolism, tissue differentiation, and regulation of the immune system. PPARγ is required for normal differentiation of the urothelium and is thought to be an essential driver of the luminal subtype of bladder cancer. However, the molecular components that regulate PPARG gene expression in bladder cancer remain unclear. Here, we developed an endogenous PPARG reporter system in luminal bladder cancer cells and performed genome-wide CRISPR knockout screening to identify bona fide regulators of PPARG gene expression. Functional validation of the dataset confirmed GATA3, SPT6, and the cohesin complex components SMC1A, and RAD21, as permissive upstream positive regulators of PPARG gene expression in luminal bladder cancer. In summary, this work provides a resource and biological insights to aid our understanding of PPARG regulation in bladder cancer.
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BACKGROUND: Androgen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10-100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription. RESULTS: To characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity. CONCLUSIONS: Using a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.
Assuntos
Elementos Facilitadores Genéticos , Receptores Androgênicos/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Humanos , Masculino , Anotação de Sequência Molecular , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Próstata/genética , Reprodutibilidade dos TestesRESUMO
Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.
Assuntos
Biomarcadores Farmacológicos/análise , Cistadenocarcinoma Seroso/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genômica/métodos , Humanos , Metabolômica/métodos , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteômica/métodos , Integração de SistemasRESUMO
Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial proliferation that is most commonly encountered as an incidental finding in the peritoneal cavity. There is controversy in the literature about whether WDPM is a neoplasm or a reactive process and, if neoplastic, whether it is a variant or precursor of epithelial malignant mesothelioma or is a different entity. Using whole exome sequencing of five WDPMs of the peritoneum, we have identified distinct mutations in EHD1, ATM, FBXO10, SH2D2A, CDH5, MAGED1, and TP73 shared by WDPM cases but not reported in malignant mesotheliomas. Furthermore, we show that WDPM is strongly enriched with C > A transversion substitution mutations, a pattern that is also not found in malignant mesotheliomas. The WDPMs lacked the alterations involving BAP1, SETD2, NF2, CDKN2A/B, LASTS1/2, PBRM1, and SMARCC1 that are frequently found in malignant mesotheliomas. We conclude that WDPMs are neoplasms that are genetically distinct from malignant mesotheliomas and, based on observed mutations, do not appear to be precursors of malignant mesotheliomas.
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BACKGROUND: Malignant peritoneal mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. METHODS: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multi-omics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular, we examined BAP1 mutation and copy number status and its relationship to immune checkpoint inhibitor activation. RESULTS: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. CONCLUSIONS: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phases I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.
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Biomarcadores Tumorais/genética , Haploinsuficiência , Mesotelioma/genética , Neoplasias Peritoneais/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Biomarcadores Tumorais/metabolismo , Humanos , Imunoterapia , Mesotelioma/classificação , Mesotelioma/terapia , Mutação , Neoplasias Peritoneais/classificação , Neoplasias Peritoneais/terapia , Microambiente Tumoral , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
IMPORTANCE: The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics. OBJECTIVE: To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment. DESIGN, SETTING, AND PARTICIPANTS: Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes. EXPOSURE: Enzalutamide, 160 mg, daily orally. MAIN OUTCOMES AND MEASURES: Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels). RESULTS: The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations. CONCLUSIONS AND RELEVANCE: Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.
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Biomarcadores Tumorais/sangue , DNA de Neoplasias/sangue , Neoplasias de Próstata Resistentes à Castração/sangue , Receptores Androgênicos/sangue , Proteínas de Ligação a Retinoblastoma/sangue , Ubiquitina-Proteína Ligases/sangue , Idoso , Idoso de 80 Anos ou mais , Antagonistas de Receptores de Andrógenos/administração & dosagem , Benzamidas , Variações do Número de Cópias de DNA , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Genômica , Humanos , Masculino , Nitrilas , Feniltioidantoína/administração & dosagem , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Resultado do Tratamento , beta Catenina/sangueRESUMO
PURPOSE: Although novel agents targeting the androgen-androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients. EXPERIMENTAL DESIGN: Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR. RESULTS: On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ(2)). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ(2)) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression). CONCLUSIONS: AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC.
Assuntos
Androstenos/farmacologia , Biomarcadores Tumorais/sangue , DNA de Neoplasias/sangue , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Taxoides/farmacologia , Idoso , Idoso de 80 Anos ou mais , Androstenos/uso terapêutico , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Intervalo Livre de Doença , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Metástase Neoplásica , Células Neoplásicas Circulantes , Modelos de Riscos Proporcionais , Neoplasias de Próstata Resistentes à Castração/sangue , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/mortalidade , Taxoides/uso terapêuticoRESUMO
RNA editing modifies the sequence of primary transcripts, potentially resulting in profound effects to RNA structure and protein-coding sequence. Recent analyses of RNA sequence data are beginning to provide insights into the distribution of RNA editing across the entire transcriptome, but there are few published matched whole genome and transcriptome sequence datasets, and designing accurate bioinformatics methodology has proven highly challenging. To further characterize the RNA editome, we analyzed 16 paired DNA-RNA sequence libraries from prostate tumor specimens, employing a comprehensive strategy to rescue low coverage sites and minimize false positives. We identified over a hundred thousand putative RNA editing events, a third of which were recurrent in two or more samples, and systematically characterized their type and distribution across the genome. Within genes the majority of events affect non-coding regions such as introns and untranslated regions (UTRs), but 546 genes had RNA editing events predicted to result in deleterious amino acid alterations. Finally, we report a potential association between RNA editing of microRNA binding sites within 3' UTRs and increased transcript expression. These results provide a systematic characterization of the landscape of RNA editing in low coverage sequence data from prostate tumor specimens. We demonstrate further evidence for RNA editing as an important regulatory mechanism and suggest that the RNA editome should be further studied in cancer.
Assuntos
Biologia Computacional/métodos , Neoplasias da Próstata/genética , Edição de RNA , Sequência Conservada , DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , RNA/genéticaRESUMO
BACKGROUND: Genomic analyses of hundreds of prostate tumors have defined a diverse landscape of mutations and genome rearrangements, but the transcriptomic effect of this complexity is less well understood, particularly at the individual tumor level. We selected a cohort of 25 high-risk prostate tumors, representing the lethal phenotype, and applied deep RNA-sequencing and matched whole genome sequencing, followed by detailed molecular characterization. RESULTS: Ten tumors were exposed to neo-adjuvant hormone therapy and expressed marked evidence of therapy response in all except one extreme case, which demonstrated early resistance via apparent neuroendocrine transdifferentiation. We observe high inter-tumor heterogeneity, including unique sets of outlier transcripts in each tumor. Interestingly, outlier expression converged on druggable cellular pathways associated with cell cycle progression, translational control or immune regulation, suggesting distinct contemporary pathway affinity and a mechanism of tumor stratification. We characterize hundreds of novel fusion transcripts, including a high frequency of ETS fusions associated with complex genome rearrangements and the disruption of tumor suppressors. Remarkably, several tumors express unique but potentially-oncogenic non-ETS fusions, which may contribute to the phenotype of individual tumors, and have significance for disease progression. Finally, one ETS-negative tumor has a striking tandem duplication genotype which appears to be highly aggressive and present at low recurrence in ETS-negative prostate cancer, suggestive of a novel molecular subtype. CONCLUSIONS: The multitude of rare genomic and transcriptomic events detected in a high-risk tumor cohort offer novel opportunities for personalized oncology and their convergence on key pathways and functions has broad implications for precision medicine.