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MOTIVATION: Tumours are composed of distinct cancer cell populations (clones), which continuously adapt to their local micro-environment. Standard methods for clonal deconvolution seek to identify groups of mutations and estimate the prevalence of each group in the tumour, while considering its purity and copy number profile. These methods have been applied on cross-sectional data and on longitudinal data after discarding information on the timing of sample collection. Two key questions are how can we incorporate such information in our analyses and is there any benefit in doing so? RESULTS: We developed a clonal deconvolution method, which incorporates explicitly the temporal spacing of longitudinally sampled tumours. By merging a Dirichlet Process Mixture Model with Gaussian Process priors and using as input a sequence of several sparsely collected samples, our method can reconstruct the temporal profile of the abundance of any mutation cluster supported by the data as a continuous function of time. We benchmarked our method on whole genome, whole exome and targeted sequencing data from patients with chronic lymphocytic leukaemia, on liquid biopsy data from a patient with melanoma and on synthetic data and we found that incorporating information on the timing of tissue collection improves model performance, as long as data of sufficient volume and complexity are available for estimating free model parameters. Thus, our approach is particularly useful when collecting a relatively long sequence of tumour samples is feasible, as in liquid cancers (e.g. leukaemia) and liquid biopsies. AVAILABILITY AND IMPLEMENTATION: The statistical methodology presented in this paper is freely available at github.com/dvav/clonosGP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias , Estudos Transversais , Exoma , Humanos , Mutação , Neoplasias/genética , Software , Microambiente Tumoral , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Aiming to improve treatment options for BRAF wild-type melanoma, we previously conducted the DOC-MEK study of docetaxel with MEK inhibitor (MEKi) selumetinib or placebo, revealing trends to prolongation of progression-free survival (hazard ratio 0.75, P = 0.130), and improved response rates (32% vs 14%, P = 0.059) with docetaxel plus selumetinib. NRAS status did not associate with outcome. Here, the aim was to identify novel biomarkers of response to MEKi. METHODS: A MEK 6 gene signature was quantified using NanoString and correlated with clinical outcomes. Two components of the gene signature were investigated by gene silencing in BRAF/NRAS wild-type melanoma cells. RESULTS: In melanomas of patients on the selumetinib but not the placebo arm, two gene signature components, dual-specificity protein phosphatase 4 (DUSP4) and ETS translocation variant 4 (ETV4), were expressed more highly in responders than non-responders. In vitro, ETV4 depletion inhibited cell survival but did not influence sensitivity to MEKi selumetinib or trametinib. In contrast, DUSP4-depleted cells showed enhanced cell survival and increased resistance to both selumetinib and trametinib. CONCLUSIONS: ETV4 and DUSP4 associated with clinical response to docetaxel plus selumetinib. DUSP4 depletion induced MEKi resistance, suggesting that DUSP4 is not only a biomarker but also a mediator of MEKi sensitivity. CLINICAL TRIAL REGISTRATION: DOC-MEK (EudraCT no: 2009-018153-23).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Fosfatases de Especificidade Dupla/genética , Melanoma/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Proteínas Proto-Oncogênicas c-ets/genética , Benzimidazóis/administração & dosagem , Docetaxel/administração & dosagem , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , TranscriptomaRESUMO
BACKGROUND: Single gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing. METHODS AND FINDINGS: We validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care. Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US$449) per patient, the panel was less expensive locally than performing more than two or three single gene tests. Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of "real world" data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds. CONCLUSIONS: This study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.
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Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Colorretais/diagnóstico , Genômica , Melanoma/diagnóstico , Patologia/métodos , Patologia/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/economia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Criança , Tomada de Decisão Clínica , Neoplasias Colorretais/economia , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Melanoma/economia , Melanoma/genética , Melanoma/terapia , Pessoa de Meia-Idade , Programas Nacionais de Saúde , Estudos Prospectivos , Estudos Retrospectivos , Reino Unido , Adulto JovemRESUMO
The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia.
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Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Sequenciamento Completo do Genoma , Mutação , Genômica , PrognósticoRESUMO
Cancer is characterised by complex somatically acquired genetic aberrations that manifest as intra-tumour and inter-tumour genetic heterogeneity and can lead to treatment resistance. In this case study, we characterise the genome-wide somatic mutation dynamics in a metastatic melanoma patient during therapy using low-input (50 ng) PCR-free whole genome sequencing of cell-free DNA from pre-treatment and post-relapse blood samples. We identify de novo tumour-specific somatic mutations from cell-free DNA, while the sequence context of single nucleotide variants showed the characteristic UV-damage mutation signature of melanoma. To investigate the behaviour of individual somatic mutations during proto-oncogene B-Raf -targeted and immune checkpoint inhibition, amplicon-based deep sequencing was used to verify and track frequencies of 212 single nucleotide variants at 10 distinct time points over 13 months of treatment. Under checkpoint inhibition therapy, we observed an increase in mutant allele frequencies indicating progression on therapy 88 days before clinical determination of non-response positron emission tomogrophy-computed tomography. We also revealed mutations from whole genome sequencing of cell-free DNA that were not present in the tissue biopsy, but that later contributed to relapse. Our findings have potential clinical applications where high quality tumour-tissue derived DNA is not available.
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Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient.
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DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Neoplasias/genética , Biópsia , DNA de Neoplasias/sangue , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Melanoma/diagnóstico , Melanoma/genética , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/patologia , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
TGFß signals through serine/threonine kinase receptors and intracellular Smad transcription factors. An important regulatory step involves ubiquitination of Smads and/or TGFß receptors by specific ubiquitin ligases, in a process that can be reversed by the deubiquitinating enzyme UCH37. Here, to explore the physiological role of UCH37 in TGFß signalling we have generated stable and inducible HaCAT keratinocyte and Colo-357 pancreatic carcinoma cell lines mis-expressing UCH37. We show that UCH37 knockdown significantly inhibits the activity of a TGFß-dependent gene reporter and selectively decreases levels of some TGFß-dependent target genes, notably p21 and PAI-1, but only during the early phase of TGFß receptor activation. Interestingly, UCH37 knockdown in Colo-357 cells had no effect on TGFß-dependent cell proliferation and epithelial-mesenchymal transition, yet significantly impaired cell migration. Collectively, our data indicate that UCH37 sustains early TGFß pathway activation kinetics that determines threshold-specific gene expression patterns, and that opposing actions of ubiquitin ligases and deubiquitinases influences distinct biological TGFß-dependent biological responses. Moreover, we suggest that UCH37 could represent a viable target for novel and selective cancer therapeutics.