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1.
Lancet Oncol ; 22(1): 118-131, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33387490

RESUMO

BACKGROUND: Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. METHODS: This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. FINDINGS: Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0-19·4; cohort B1 22·0 months [21·4-22·7], cohort B2 18·2 months [17·0-18·9], cohort C1 17·9 months [14·3-19·7], cohort C2 15·9 months [12·7-16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3-4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). INTERPRETATION: APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. FUNDING: Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.


Assuntos
Adenocarcinoma/tratamento farmacológico , Albuminas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígenos CD40/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Nivolumabe/administração & dosagem , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/secundário , Idoso , Albuminas/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Antígenos CD40/imunologia , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nivolumabe/efeitos adversos , Paclitaxel/efeitos adversos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Fatores de Tempo , Resultado do Tratamento , Estados Unidos
2.
Sci Adv ; 6(24): eaay9691, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32577507

RESUMO

Tumor heterogeneity and evolution drive treatment resistance in metastatic colorectal cancer (mCRC). Patient-derived xenografts (PDXs) can model mCRC biology; however, their ability to accurately mimic human tumor heterogeneity is unclear. Current genomic studies in mCRC have limited scope and lack matched PDXs. Therefore, the landscape of tumor heterogeneity and its impact on the evolution of metastasis and PDXs remain undefined. We performed whole-genome, deep exome, and targeted validation sequencing of multiple primary regions, matched distant metastases, and PDXs from 11 patients with mCRC. We observed intricate clonal heterogeneity and evolution affecting metastasis dissemination and PDX clonal selection. Metastasis formation followed both monoclonal and polyclonal seeding models. In four cases, metastasis-seeding clones were not identified in any primary region, consistent with a metastasis-seeding-metastasis model. PDXs underrepresented the subclonal heterogeneity of parental tumors. These suggest that single sample tumor sequencing and current PDX models may be insufficient to guide precision medicine.

3.
Nat Commun ; 11(1): 2156, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358485

RESUMO

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional , DNA Topoisomerases Tipo II/metabolismo , Progressão da Doença , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Células HT29 , Humanos , Ligases/metabolismo , Camundongos , Proteínas do Grupo Polycomb/metabolismo , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Inibidores da Topoisomerase/farmacologia
4.
BMC Pulm Med ; 19(1): 5, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30616547

RESUMO

BACKGROUND: Inhibition of interleukin (IL)-13, a Type 2 inflammatory mediator in asthma, improves lung function and reduces exacerbations; however, more effective therapies are needed. A subset of asthma patients also exhibits elevated IL-17, which is associated with greater disease severity, neutrophilic inflammation, and steroid resistance. BITS7201A is a novel, humanized bispecific antibody that binds and neutralizes both IL-13 and IL-17. METHODS: Safety, pharmacokinetics, and immunogenicity of BITS7201A were evaluated in a phase 1 study. Part A was a single ascending-dose design with 5 cohorts: 30-, 90-, and 300-mg subcutaneous (SC), and 300- and 750-mg intravenous (IV). Part B was a multiple ascending-dose design with 3 cohorts: 150-, 300-, and 600-mg SC every 4 weeks × 3 doses. Both parts enrolled approximately 8 healthy volunteers into each cohort (6 active: 2 placebo). Part B included an additional cohort of patients with mild asthma (600-mg SC). RESULTS: Forty-one subjects (31 active, 10 placebo) and 26 subjects (20 active, 6 placebo) were enrolled into Parts A and B, respectively. The cohort with mild asthma patients was terminated after enrollment of a single patient. No deaths, serious adverse events, or dose-limiting adverse events occurred. In Part A, 12 active (39%) and 5 placebo subjects (50%), and in Part B, 6 active (30%) and 3 placebo subjects (50%) experienced at least 1 treatment-emergent adverse event (TEAE). The most common AEs were fatigue (n = 3) and influenza-like illness (n = 2). One injection-site reaction was reported. Two subjects with elevated blood eosinophil counts at baseline had transient elevations in blood eosinophils (≥Grade 2, > 1500 cells/µL). In Parts A and B, 16 of 30 (53%) and 16 of 17 (94%) active subjects, respectively, tested positive for anti-drug antibodies (ADAs). No anaphylaxis or hypersensitivity events occurred. BITS7201A exhibited single- and multiple-dose pharmacokinetic characteristics consistent with an IgG monoclonal antibody; exposure generally increased dose-proportionally. Postdose elevations of the serum pharmacodynamic biomarkers, IL-17AA and IL-17FF, occurred, confirming target engagement. CONCLUSIONS: BITS7201A was well tolerated, but was associated with a high incidence of ADA formation. TRIAL REGISTRATION: ClinicalTrials.gov , NCT02748642; registered April 6, 2016 (retrospectively registered).


Assuntos
Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/farmacocinética , Asma/terapia , Interleucina-13/imunologia , Interleucina-17/imunologia , Adolescente , Adulto , Biomarcadores/sangue , Eosinófilos/citologia , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina G/sangue , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Método Simples-Cego , Adulto Jovem
5.
Lancet Respir Med ; 6(8): 615-626, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30072107

RESUMO

BACKGROUND: Heterogeneity in the progression of idiopathic pulmonary fibrosis (IPF) might reflect diversity in underlying pathobiology, and represents a major challenge in the prediction of clinical progression and treatment benefit. Previous studies have found peripheral blood concentrations of several protein biomarkers to be prognostic for overall survival duration in patients with IPF, but these findings have generally not been directly compared and replicated between cohorts. We aimed to use the pivotal trials for pirfenidone to evaluate prognostic and predictive properties of biomarkers across multiple endpoints, and whether they are modulated by pirfenidone treatment. METHODS: We did post-hoc analyses of test and replication cohorts from CAPACITY 004 (NCT00287716), CAPACITY 006 (NCT00287729), and ASCEND (NCT01366209) trials for the plasma proteins CCL13, CCL17, CCL18, CXCL13, CXCL14, COMP, interleukin 13, MMP3, MMP7, osteopontin, periostin, and YKL40. Eligible participants had IPF and received pirfenidone 2403 mg/day or placebo in CAPACITY (test cohort) or ASCEND (replication cohort), were aged 40-80 years, and without missing biomarker data at baseline. To identify biomarkers that were consistently prognostic for clinical outcome measures, the primary analysis was the association between biomarker concentrations at baseline and absolute change in percentage of predicted forced vital capacity (FVC%pred) at 12 months (CAPACITY week 48, ASCEND week 52) in the placebo group. Biomarkers within the test cohort that met predefined success criteria of a prognostic p value less than 0·10 from multivariate analysis were further assessed in the replication cohort. Furthermore, the predictive effect size (ie, biomarkers that were predictive for benefit from pirfenidone) was calculated as the difference in FVC%pred treatment effect (pirfenidone in relation to placebo) between high versus low biomarker subgroups at week 48 (test cohort) or week 52 (replication cohort). FINDINGS: Several baseline biomarkers (CCL13, CCL18, COMP, CXCL13, CXCL14, periostin, and YKL40) were prognostic for progression outcomes in the placebo groups of the test cohort. However, only CCL18 was consistently prognostic for absolute change in percentage of FVC%pred in both the test (p=0·032) and replication (p=0·004) cohorts. Pirfenidone treatment benefit was consistent regardless of baseline biomarker concentration. INTERPRETATION: Blood CCL18 concentrations were the most consistent predictor of disease progression across IPF cohorts with potential to inform new target discovery and clinical trial design. Future validation of these findings in prospective studies is warranted. FUNDING: Genentech Inc.


Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Ensaios Clínicos como Assunto/normas , Fibrose Pulmonar Idiopática/tratamento farmacológico , Piridonas/administração & dosagem , Idoso , Biomarcadores/sangue , Progressão da Doença , Feminino , Seguimentos , Humanos , Fibrose Pulmonar Idiopática/genética , Masculino , Modelos de Riscos Proporcionais
9.
Respir Res ; 17: 29, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26993628

RESUMO

BACKGROUND: Quilizumab, a humanized IgG1 monoclonal antibody, targets the M1-prime segment of membrane-expressed IgE, leading to depletion of IgE-switched and memory B cells. In patients with mild asthma, quilizumab reduced serum IgE and attenuated the early and late asthmatic reaction following whole lung allergen challenge. This study evaluated the efficacy and safety of quilizumab in adults with allergic asthma, inadequately controlled despite high-dose inhaled corticosteroids (ICS) and a second controller. METHODS: Five hundred seventy-eight patients were randomized to monthly or quarterly dosing regimens of subcutaneous quilizumab or placebo for 36 weeks, with a 48-week safety follow-up. Quilizumab was evaluated for effects on the rate of asthma exacerbations, lung function, patient symptoms, serum IgE, and pharmacokinetics. Exploratory analyses were conducted on biomarker subgroups (periostin, blood eosinophils, serum IgE, and exhaled nitric oxide). RESULTS: Quilizumab was well tolerated and reduced serum total and allergen-specific IgE by 30-40 %, but had no impact on asthma exacerbations, lung function, or patient-reported symptom measures. At Week 36, the 300 mg monthly quilizumab group showed a 19.6 % reduction (p = 0.38) in the asthma exacerbation rate relative to placebo, but this was neither statistically nor clinically significant. Biomarker subgroups did not reveal meaningful efficacy benefits following quilizumab treatment. CONCLUSIONS: Quilizumab had an acceptable safety profile and reduced serum IgE. However, targeting the IgE pathway via depletion of IgE-switched and memory B cells was not sufficient for a clinically meaningful benefit for adults with allergic asthma uncontrolled by standard therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT01582503.


Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Asma/tratamento farmacológico , Asma/imunologia , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Administração por Inalação , Adolescente , Corticosteroides/administração & dosagem , Adulto , Idoso , Antiasmáticos/administração & dosagem , Antiasmáticos/efeitos adversos , Asma/diagnóstico , Terapia Combinada , Relação Dose-Resposta a Droga , Feminino , Humanos , Hipersensibilidade/diagnóstico , Masculino , Pessoa de Meia-Idade , Prevenção Secundária/métodos , Resultado do Tratamento , Adulto Jovem
10.
RNA Biol ; 12(6): 628-42, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25864709

RESUMO

A growing number of gene-centric studies have highlighted the emerging significance of lncRNAs in cancer. However, these studies primarily focus on a single cancer type. Therefore, we conducted a pan-cancer analysis of lncRNAs comparing tumor and matched normal expression levels using RNA-Seq data from ∼ 3,000 patients in 8 solid tumor types. While the majority of differentially expressed lncRNAs display tissue-specific expression we discovered 229 lncRNAs with outlier or differential expression across multiple cancers, which we refer to as 'onco-lncRNAs'. Due to their consistent altered expression, we hypothesize that these onco-lncRNAs may have conserved oncogenic and tumor suppressive functions across cancers. To address this, we associated the onco-lncRNAs in biological processes based on their co-expressed protein coding genes. To validate our predictions, we experimentally confirmed cell growth dependence of 2 novel oncogenic lncRNAs, onco-lncRNA-3 and onco-lncRNA-12, and a previously identified lncRNA CCAT1. Overall, we discovered lncRNAs that may have broad oncogenic and tumor suppressor roles that could significantly advance our understanding of cancer lncRNA biology.


Assuntos
Perfilação da Expressão Gênica , Neoplasias/metabolismo , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , Bases de Dados de Ácidos Nucleicos , Humanos
11.
Genome Biol ; 15(8): 429, 2014 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25116943

RESUMO

BACKGROUND: Long intergenic non-coding RNAs (lncRNAs) represent an emerging and under-studied class of transcripts that play a significant role in human cancers. Due to the tissue- and cancer-specific expression patterns observed for many lncRNAs it is believed that they could serve as ideal diagnostic biomarkers. However, until each tumor type is examined more closely, many of these lncRNAs will remain elusive. RESULTS: Here we characterize the lncRNA landscape in lung cancer using publicly available transcriptome sequencing data from a cohort of 567 adenocarcinoma and squamous cell carcinoma tumors. Through this compendium we identify over 3,000 unannotated intergenic transcripts representing novel lncRNAs. Through comparison of both adenocarcinoma and squamous cell carcinomas with matched controls we discover 111 differentially expressed lncRNAs, which we term lung cancer-associated lncRNAs (LCALs). A pan-cancer analysis of 324 additional tumor and adjacent normal pairs enable us to identify a subset of lncRNAs that display enriched expression specific to lung cancer as well as a subset that appear to be broadly deregulated across human cancers. Integration of exome sequencing data reveals that expression levels of many LCALs have significant associations with the mutational status of key oncogenes in lung cancer. Functional validation, using both knockdown and overexpression, shows that the most differentially expressed lncRNA, LCAL1, plays a role in cellular proliferation. CONCLUSIONS: Our systematic characterization of publicly available transcriptome data provides the foundation for future efforts to understand the role of LCALs, develop novel biomarkers, and improve knowledge of lung tumor biology.


Assuntos
Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , RNA Longo não Codificante/genética , Transcriptoma , Bases de Dados Genéticas , Exoma , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Análise de Sequência de DNA , Análise de Sequência de RNA/métodos
12.
Nucleic Acids Res ; 42(14): e113, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25030904

RESUMO

High-throughput sequencing technologies, including RNA-seq, have made it possible to move beyond gene expression analysis to study transcriptional events including alternative splicing and gene fusions. Furthermore, recent studies in cancer have suggested the importance of identifying transcriptionally altered loci as biomarkers for improved prognosis and therapy. While many statistical methods have been proposed for identifying novel transcriptional events with RNA-seq, nearly all rely on contrasting known classes of samples, such as tumor and normal. Few tools exist for the unsupervised discovery of such events without class labels. In this paper, we present SigFuge for identifying genomic loci exhibiting differential transcription patterns across many RNA-seq samples. SigFuge combines clustering with hypothesis testing to identify genes exhibiting alternative splicing, or differences in isoform expression. We apply SigFuge to RNA-seq cohorts of 177 lung and 279 head and neck squamous cell carcinoma samples from the Cancer Genome Atlas, and identify several cases of differential isoform usage including CDKN2A, a tumor suppressor gene known to be inactivated in a majority of lung squamous cell tumors. By not restricting attention to known sample stratifications, SigFuge offers a novel approach to unsupervised screening of genetic loci across RNA-seq cohorts. SigFuge is available as an R package through Bioconductor.


Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Isoformas de RNA/metabolismo , Análise de Sequência de RNA/métodos , Software , Processamento Alternativo , Carcinoma de Células Escamosas/genética , Proteínas de Transporte/genética , Análise por Conglomerados , Éxons , Genes p16 , Loci Gênicos , Neoplasias de Cabeça e Pescoço/genética , Peptídeos e Proteínas de Sinalização Intracelular , Calicreínas/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares , Carcinoma de Células Escamosas de Cabeça e Pescoço
13.
Nucleic Acids Res ; 42(13): e107, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24970867

RESUMO

Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however, this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method, called UNCeqR, that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation, the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models, including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort, UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA, ERBB2 and FGFR2). In summary, integrating RNA-seq with DNA-WES increases mutation detection performance, especially for low purity tumors.


Assuntos
Análise Mutacional de DNA/métodos , Neoplasias/genética , Análise de Sequência de RNA/métodos , Neoplasias da Mama/genética , Feminino , Genes Neoplásicos , Humanos , Neoplasias Pulmonares/genética , Taxa de Mutação
14.
J Mol Diagn ; 16(4): 440-51, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24814956

RESUMO

The use of massively parallel sequencing for studying RNA expression has greatly enhanced our understanding of the transcriptome through the myriad ways these data can be characterized. In particular, clinical samples provide important insights about RNA expression in health and disease, yet these studies can be complicated by RNA degradation that results from the use of formalin as a clinical preservative and by the limited amounts of RNA often available from these precious samples. In this study we describe the combined use of RNA sequencing with an exome capture selection step to enhance the yield of on-exon sequencing read data when compared with RNA sequencing alone. In particular, the exome capture step preserves the dynamic range of expression, permitting differential comparisons and validation of expressed mutations from limited and FFPE preserved samples, while reducing the data generation requirement. We conclude that cDNA hybrid capture has the potential to significantly improve transcriptome analysis from low-yield FFPE material.


Assuntos
Adenocarcinoma/genética , DNA Complementar/genética , Exoma , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/genética , RNA/genética , Análise de Sequência de RNA/métodos , Adenocarcinoma de Pulmão , Fusão Gênica , Humanos
15.
Nucleic Acids Res ; 41(19): e178, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23935067

RESUMO

Identifying variants using high-throughput sequencing data is currently a challenge because true biological variants can be indistinguishable from technical artifacts. One source of technical artifact results from incorrectly aligning experimentally observed sequences to their true genomic origin ('mismapping') and inferring differences in mismapped sequences to be true variants. We developed BlackOPs, an open-source tool that simulates experimental RNA-seq and DNA whole exome sequences derived from the reference genome, aligns these sequences by custom parameters, detects variants and outputs a blacklist of positions and alleles caused by mismapping. Blacklists contain thousands of artifact variants that are indistinguishable from true variants and, for a given sample, are expected to be almost completely false positives. We show that these blacklist positions are specific to the alignment algorithm and read length used, and BlackOPs allows users to generate a blacklist specific to their experimental setup. We queried the dbSNP and COSMIC variant databases and found numerous variants indistinguishable from mapping errors. We demonstrate how filtering against blacklist positions reduces the number of potential false variants using an RNA-seq glioblastoma cell line data set. In summary, accounting for mapping-caused variants tuned to experimental setups reduces false positives and, therefore, improves genome characterization by high-throughput sequencing.


Assuntos
Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Alinhamento de Sequência/métodos , Software , Artefatos , Linhagem Celular Tumoral , Mapeamento Cromossômico , Bases de Dados de Ácidos Nucleicos , Exoma , Humanos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos
16.
PLoS One ; 8(2): e56823, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23451093

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Idoso , Aberrações Cromossômicas , Classe I de Fosfatidilinositol 3-Quinases , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Receptores ErbB/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Masculino , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/genética , Fosfatidilinositol 3-Quinases/genética , Fatores de Transcrição SOXB1/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
17.
Arch Pathol Lab Med ; 137(1): 32-40, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22583114

RESUMO

CONTEXT: Precise subtype diagnosis of non-small cell lung carcinoma is increasingly relevant, based on the availability of subtype-specific therapies, such as bevacizumab and pemetrexed, and based on the subtype-specific prevalence of activating epidermal growth factor receptor mutations. OBJECTIVES: To establish a baseline measure of interobserver reproducibility for non-small cell lung carcinoma diagnoses with hematoxylin-eosin for the current 2004 World Health Organization classification, to estimate interobserver reproducibility for the therapeutically relevant squamous/nonsquamous subsets, and to examine characteristics that improve interobserver reproducibility. DESIGN: Primary, resected lung cancer specimens were converted to digital (virtual) slides. Based on a single hematoxylin-eosin virtual slide, pathologists were asked to assign a diagnosis using the 2004 World Health Organization classification. Kappa statistics were calculated for each pathologist-pair for each slide and were summarized by classification scheme, pulmonary pathology expertise, diagnostic confidence, and neoplastic grade. RESULTS: The 12 pulmonary pathology experts and the 12 community pathologists each independently diagnosed 48 to 96 single hematoxylin-eosin digital slides derived from 96 cases of non-small cell lung carcinoma resection. Overall agreement improved with simplification from the comprehensive 44 World Health Organization diagnoses (κ  =  0.25) to their 10 major header subtypes (κ  =  0.48) and improved again with simplification into the therapeutically relevant squamous/nonsquamous dichotomy (κ  =  0.55). Multivariate analysis showed that higher diagnostic agreement was associated with better differentiation, better slide quality, higher diagnostic confidence, similar years of pathology experience, and pulmonary pathology expertise. CONCLUSIONS: These data define the baseline diagnostic agreement for hematoxylin-eosin diagnosis of non-small cell lung carcinoma, allowing future studies to test for improved diagnostic agreement with reflex ancillary tests.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/classificação , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/diagnóstico , Coleta de Dados , Amarelo de Eosina-(YS) , Feminino , Hematoxilina , Humanos , Internet , Masculino , Variações Dependentes do Observador , Patologia Cirúrgica , Coloração e Rotulagem , Organização Mundial da Saúde
18.
BMC Bioinformatics ; 13: 221, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22946927

RESUMO

BACKGROUND: Next-generation sequencing technologies have become important tools for genome-wide studies. However, the quality scores that are assigned to each base have been shown to be inaccurate. If the quality scores are used in downstream analyses, these inaccuracies can have a significant impact on the results. RESULTS: Here we present ReQON, a tool that recalibrates the base quality scores from an input BAM file of aligned sequencing data using logistic regression. ReQON also generates diagnostic plots showing the effectiveness of the recalibration. We show that ReQON produces quality scores that are both more accurate, in the sense that they more closely correspond to the probability of a sequencing error, and do a better job of discriminating between sequencing errors and non-errors than the original quality scores. We also compare ReQON to other available recalibration tools and show that ReQON is less biased and performs favorably in terms of quality score accuracy. CONCLUSION: ReQON is an open source software package, written in R and available through Bioconductor, for recalibrating base quality scores for next-generation sequencing data. ReQON produces a new BAM file with more accurate quality scores, which can improve the results of downstream analysis, and produces several diagnostic plots showing the effectiveness of the recalibration.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/normas , Software , Calibragem , Genoma , Modelos Logísticos , Alinhamento de Sequência
19.
PLoS One ; 7(5): e36530, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22590557

RESUMO

BACKGROUND: Lung adenocarcinoma (LAD) has extreme genetic variation among patients, which is currently not well understood, limiting progress in therapy development and research. LAD intrinsic molecular subtypes are a validated stratification of naturally-occurring gene expression patterns and encompass different functional pathways and patient outcomes. Patients may have incurred different mutations and alterations that led to the different subtypes. We hypothesized that the LAD molecular subtypes co-occur with distinct mutations and alterations in patient tumors. METHODOLOGY/PRINCIPAL FINDINGS: The LAD molecular subtypes (Bronchioid, Magnoid, and Squamoid) were tested for association with gene mutations and DNA copy number alterations using statistical methods and published cohorts (n = 504). A novel validation (n = 116) cohort was assayed and interrogated to confirm subtype-alteration associations. Gene mutation rates (EGFR, KRAS, STK11, TP53), chromosomal instability, regional copy number, and genomewide DNA methylation were significantly different among tumors of the molecular subtypes. Secondary analyses compared subtypes by integrated alterations and patient outcomes. Tumors having integrated alterations in the same gene associated with the subtypes, e.g. mutation, deletion and underexpression of STK11 with Magnoid, and mutation, amplification, and overexpression of EGFR with Bronchioid. The subtypes also associated with tumors having concurrent mutant genes, such as KRAS-STK11 with Magnoid. Patient overall survival, cisplatin plus vinorelbine therapy response and predicted gefitinib sensitivity were significantly different among the subtypes. CONCLUSIONS/ SIGNIFICANCE: The lung adenocarcinoma intrinsic molecular subtypes co-occur with grossly distinct genomic alterations and with patient therapy response. These results advance the understanding of lung adenocarcinoma etiology and nominate patient subgroups for future evaluation of treatment response.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Instabilidade Cromossômica , Metilação de DNA , DNA de Neoplasias , Dosagem de Genes , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Idoso , Estudos de Coortes , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Taxa de Sobrevida
20.
Clin Cancer Res ; 16(19): 4864-75, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20643781

RESUMO

PURPOSE: Lung squamous cell carcinoma (SCC) is clinically and genetically heterogeneous, and current diagnostic practices do not adequately substratify this heterogeneity. A robust, biologically based SCC subclassification may describe this variability and lead to more precise patient prognosis and management. We sought to determine if SCC mRNA expression subtypes exist, are reproducible across multiple patient cohorts, and are clinically relevant. EXPERIMENTAL DESIGN: Subtypes were detected by unsupervised consensus clustering in five published discovery cohorts of mRNA microarrays, totaling 382 SCC patients. An independent validation cohort of 56 SCC patients was collected and assayed by microarrays. A nearest-centroid subtype predictor was built using discovery cohorts. Validation cohort subtypes were predicted and evaluated for confirmation. Subtype survival outcome, clinical covariates, and biological processes were compared by statistical and bioinformatic methods. RESULTS: Four lung SCC mRNA expression subtypes, named primitive, classical, secretory, and basal, were detected and independently validated (P < 0.001). The primitive subtype had the worst survival outcome (P < 0.05) and is an independent predictor of survival (P < 0.05). Tumor differentiation and patient sex were associated with subtype. The expression profiles of the subtypes contained distinct biological processes (primitive: proliferation; classical: xenobiotic metabolism; secretory: immune response; basal: cell adhesion) and suggested distinct pharmacologic interventions. Comparison with lung model systems revealed distinct subtype to cell type correspondence. CONCLUSIONS: Lung SCC consists of four mRNA expression subtypes that have different survival outcomes, patient populations, and biological processes. The subtypes stratify patients for more precise prognosis and targeted research.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , RNA Mensageiro/genética , Idoso , Carcinoma de Células Escamosas/classificação , Carcinoma de Células Escamosas/diagnóstico , Estudos de Coortes , Feminino , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/diagnóstico , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Análise de Sobrevida , Resultado do Tratamento
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