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1.
Blood ; 132(23): 2456-2464, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30249784

RESUMO

The introduction of novel agents has led to major improvements in clinical outcomes for patients with multiple myeloma. To shorten evaluation times for new treatments, health agencies are currently examining minimal residual disease (MRD) as a surrogate end point in clinical trials. We assessed the prognostic value of MRD, measured during maintenance therapy by next-generation sequencing (NGS). MRD negativity was defined as the absence of tumor plasma cell within 1 000 000 bone marrow cells (<10-6). Data were analyzed from a recent clinical trial that evaluated the role of transplantation in newly diagnosed myeloma patients treated with lenalidomide, bortezomib, and dexamethasone (RVD). MRD negativity was achieved at least once during maintenance in 127 patients (25%). At the start of maintenance therapy, MRD was a strong prognostic factor for both progression-free survival (adjusted hazard ratio, 0.22; 95% confidence interval, 0.15-0.34; P < .001) and overall survival (adjusted hazard ratio, 0.24; 95% confidence interval, 0.11-0.54; P = .001). Patients who were MRD negative had a higher probability of prolonged progression-free survival than patients with detectable residual disease, regardless of treatment group (RVD vs transplant), cytogenetic risk profile, or International Staging System disease stage at diagnosis. These results were similar after completion of maintenance therapy. Our findings confirm the value of MRD status, as determined by NGS, as a prognostic biomarker in multiple myeloma, and suggest that this approach could be used to adapt treatment strategies in future clinical trials.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Sequenciamento de Nucleotídeos em Larga Escala , Mieloma Múltiplo/metabolismo , Idoso , Medula Óssea/metabolismo , Medula Óssea/patologia , Bortezomib/administração & dosagem , Dexametasona/administração & dosagem , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Lenalidomida/administração & dosagem , Quimioterapia de Manutenção , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Neoplasia Residual , Plasmócitos/metabolismo , Plasmócitos/patologia , Taxa de Sobrevida
4.
Nature ; 543(7647): 723-727, 2017 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-28329770

RESUMO

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Região Variável de Imunoglobulina/imunologia , Linfoma de Célula do Manto/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfócitos T CD4-Positivos/imunologia , Citotoxicidade Imunológica , Análise Mutacional de DNA , Epitopos de Linfócito T/imunologia , Exoma/genética , Genômica , Antígenos HLA-D/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Imunoterapia/tendências , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Linfoma de Célula do Manto/terapia , Mutação , Proteômica
5.
Arthritis Rheumatol ; 69(4): 774-784, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28002888

RESUMO

OBJECTIVE: Ankylosing spondylitis (AS), a chronic inflammatory disorder, has a notable association with HLA-B27. One hypothesis suggests that a common antigen that binds to HLA-B27 is important for AS disease pathogenesis. This study was undertaken to determine sequences and motifs that are shared among HLA-B27-positive AS patients, using T cell repertoire next-generation sequencing. METHODS: To identify motifs enriched among B27-positive AS patients, we performed T cell receptor ß (TCRß) repertoire sequencing on samples from 191 B27-positive AS patients, 43 B27-negative AS patients, and 227 controls, and we obtained >77 million TCRß clonotype sequences. First, we assessed whether any of 50 previously published sequences were enriched in B27-positive AS patients. We then used training and test cohorts to identify discovered motifs that were enriched in B27-positive AS patients versus controls. RESULTS: Six previously published and 11 discovered motifs were enriched in the B27-positive AS samples as compared to controls. After combining motifs related by sequence, we identified a total of 15 independent motifs. Both the full set of 15 motifs and a set of 6 published motifs were enriched in the B27-positive AS patients as compared to B27-positive healthy individuals (P = 0.049 and P = 0.001, respectively). Using an independent cohort, we validated that at least some of these motifs were associated with AS, and not simply with B27-positive status. CONCLUSION: We identified TCRß motifs that are enriched in B27-positive AS patients as compared to B27-positive healthy controls. This suggests that a common antigen, presented by HLA-B27 and detected by CD8+ T cells, may be associated with AS disease pathogenesis.


Assuntos
Antígeno HLA-B27/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Espondilite Anquilosante/imunologia , Adolescente , Adulto , Motivos de Aminoácidos , Sequência de Aminoácidos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência , Adulto Jovem
6.
PLoS One ; 10(10): e0141561, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26509579

RESUMO

Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We developed a novel multiplex assay that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously. We multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from 5 individuals with frequencies as low as 1 per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with ELISPOT. Applying this technology we have shown that the vast majority of shared antigen-specific clonotypes identified in different individuals display the same specificity. We also showed that shared antigen-specific clonotypes are simpler sequences and are present at higher frequencies compared to non-shared clonotypes specific to the same antigen. In conclusion this technology enables sensitive and quantitative monitoring of T cells specific for hundreds or thousands of antigens simultaneously allowing the study of T cell responses with an unprecedented resolution and scale.


Assuntos
ELISPOT , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos T/genética , Receptores Imunológicos/genética , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Evolução Clonal/genética , Evolução Clonal/imunologia , ELISPOT/métodos , ELISPOT/normas , Humanos , Reprodutibilidade dos Testes
7.
Lancet Oncol ; 16(5): 541-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25842160

RESUMO

BACKGROUND: Diffuse large-B-cell lymphoma is curable, but when treatment fails, outcome is poor. Although imaging can help to identify patients at risk of treatment failure, they are often imprecise, and radiation exposure is a potential health risk. We aimed to assess whether circulating tumour DNA encoding the clonal immunoglobulin gene sequence could be detected in the serum of patients with diffuse large-B-cell lymphoma and used to predict clinical disease recurrence after frontline treatment. METHODS: We used next-generation DNA sequencing to retrospectively analyse cell-free circulating tumour DNA in patients assigned to one of three treatment protocols between May 8, 1993, and June 6, 2013. Eligible patients had diffuse large-B-cell lymphoma, no evidence of indolent lymphoma, and were previously untreated. We obtained serial serum samples and concurrent CT scans at specified times during most treatment cycles and up to 5 years of follow-up. VDJ gene segments of the rearranged immunoglobulin receptor genes were amplified and sequenced from pretreatment specimens and serum circulating tumour DNA encoding the VDJ rearrangements was quantitated. FINDINGS: Tumour clonotypes were identified in pretreatment specimens from 126 patients who were followed up for a median of 11 years (IQR 6·8-14·2). Interim monitoring of circulating tumour DNA at the end of two treatment cycles in 108 patients showed a 5-year time to progression of 41·7% (95% CI 22·2-60·1) in patients with detectable circulating tumour DNA and 80·2% (69·6-87·3) in those without detectable circulating tumour DNA (p<0·0001). Detectable interim circulating tumour DNA had a positive predictive value of 62·5% (95% CI 40·6-81·2) and a negative predictive value of 79·8% (69·6-87·8). Surveillance monitoring of circulating tumour DNA was done in 107 patients who achieved complete remission. A Cox proportional hazards model showed that the hazard ratio for clinical disease progression was 228 (95% CI 51-1022) for patients who developed detectable circulating tumour DNA during surveillance compared with patients with undetectable circulating tumour DNA (p<0·0001). Surveillance circulating tumour DNA had a positive predictive value of 88·2% (95% CI 63·6-98·5) and a negative predictive value of 97·8% (92·2-99·7) and identified risk of recurrence at a median of 3·5 months (range 0-200) before evidence of clinical disease. INTERPRETATION: Surveillance circulating tumour DNA identifies patients at risk of recurrence before clinical evidence of disease in most patients and results in a reduced disease burden at relapse. Interim circulating tumour DNA is a promising biomarker to identify patients at high risk of treatment failure. FUNDING: National Cancer Institute and Adaptive Biotechnologies.


Assuntos
DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Criança , DNA de Neoplasias/isolamento & purificação , Feminino , Humanos , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes , Tomografia Computadorizada por Raios X
8.
Biol Blood Marrow Transplant ; 20(9): 1307-13, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24769317

RESUMO

Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10(-4) after induction predicts subsequent relapse. Likewise, MRD ≥ 10(-4) in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10(-6) or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10(-4) before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10(-6) had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse.


Assuntos
Genes Codificadores dos Receptores de Linfócitos T/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoglobulinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodos , Adolescente , Adulto , Idoso , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Adulto Jovem
9.
Blood ; 123(20): 3073-9, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24646471

RESUMO

We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively. Patients who were MRD(-) by sequencing had a significantly longer time to tumor progression (TTP) (median 80 vs 31 months; P < .0001) and overall survival (median not reached vs 81 months; P = .02), compared with patients who were MRD(+). When stratifying patients by different levels of MRD, the respective TTP medians were: MRD ≥10(-3) 27 months, MRD 10(-3) to 10(-5) 48 months, and MRD <10(-5) 80 months (P = .003 to .0001). Ninety-two percent of VGPR patients were MRD(+). In complete response patients, the TTP remained significantly longer for MRD(-) compared with MRD(+) patients (131 vs 35 months; P = .0009).


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Neoplasia Residual , Prognóstico
10.
PLoS One ; 8(9): e74231, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069285

RESUMO

In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10(-5) which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.


Assuntos
Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoensaio/métodos , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Antígenos/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Citomegalovirus/imunologia , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas da Matriz Viral/imunologia
11.
Blood ; 120(26): 5173-80, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23074282

RESUMO

The persistence of minimal residual disease (MRD) during therapy is the strongest adverse prognostic factor in acute lymphoblastic leukemia (ALL). We developed a high-throughput sequencing method that universally amplifies antigen-receptor gene segments and identifies all clonal gene rearrangements (ie, leukemia-specific sequences) at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In the present study, the assay specifically detected 1 leukemic cell among greater than 1 million leukocytes in spike-in experiments. We compared this method with the gold-standard MRD assays multiparameter flow cytometry and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) using diagnostic and follow-up samples from 106 patients with ALL. Sequencing detected MRD in all 28 samples shown to be positive by flow cytometry and in 35 of the 36 shown to be positive by ASO-PCR and revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively. We conclude that this new method allows monitoring of treatment response in ALL and other lymphoid malignancies with great sensitivity and precision. The www.clinicaltrials.gov identifier number for the Total XV study is NCT00137111.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Sequência de Bases , Criança , Evolução Clonal/genética , Evolução Clonal/fisiologia , Genes de Cadeia Pesada de Imunoglobulina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Modelos Biológicos , Técnicas de Diagnóstico Molecular/métodos , Dados de Sequência Molecular , Neoplasia Residual , Reação em Cadeia da Polimerase/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , Sensibilidade e Especificidade
12.
Nature ; 466(7308): 869-73, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20668451

RESUMO

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.


Assuntos
Genes Neoplásicos/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , MAP Quinase Quinase 4/genética , Masculino , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/genética , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas Quinases/genética , Receptores Acoplados a Proteínas-G/genética
13.
Proc Natl Acad Sci U S A ; 107(28): 12587-92, 2010 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-20616066

RESUMO

A unique microarray-based method for determining the extent of DNA methylation has been developed. It relies on a selective enrichment of the regions to be assayed by target amplification by capture and ligation (mTACL). The assay is quantitatively accurate, relatively precise, and lends itself to high-throughput determination using nanogram amounts of DNA. The measurements using mTACLs are highly reproducible and in excellent agreement with those obtained by sequencing (r = 0.94). In the present work, the methylation status of >145,000 CpGs from 5,472 promoters in 221 samples was measured. The methylation levels of nearby CpGs are correlated, but the correlation falls off dramatically over several hundred base pairs. In some instances, nearby CpGs have very different levels of methylation. Comparison of normal and tumor samples indicates that in tumors, the promoter regions of genes involved in differentiation and signaling are preferentially hypermethylated, whereas those of housekeeping genes remain hypomethylated. mTACL is a platform for profiling the state of methylation of a large number of CpG in many samples in a cost-effective fashion, and is capable of scaling to much larger numbers of CpGs than those collected here.


Assuntos
Metilação de DNA , Diferenciação Celular/genética , DNA/genética , Fosfatos de Dinucleosídeos , Genoma , Humanos , Metilação
14.
BMC Bioinformatics ; 11: 305, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20525369

RESUMO

BACKGROUND: Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profiles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profiles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fixed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. RESULTS: Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically significant negative correlation between methylation profiles and gene expression in the panel of breast cancer cell lines. Subnetwork enrichment of these genes has identified 35 common regulators with 6 or more predicted markers. In addition to identifying epigenetically regulated genes, we show evidence of differentially expressed methylation patterns between the basal and luminal subtypes. CONCLUSIONS: Our results indicate that the proposed computational protocol is a viable platform for identifying epigenetically regulated genes. Our protocol has generated a list of predictors including COL1A2, TOP2A, TFF1, and VAV3, genes whose key roles in epigenetic regulation is documented in the literature. Subnetwork enrichment of these predicted markers further suggests that epigenetic regulation of individual genes occurs in a coordinated fashion and through common regulators.


Assuntos
Neoplasias da Mama/genética , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Neoplasias/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colágeno Tipo I/genética , Ilhas de CpG , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Estudo de Associação Genômica Ampla , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Ligação a Poli-ADP-Ribose , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-vav/genética , Fator Trefoil-1 , Proteínas Supressoras de Tumor/genética
15.
Proc Natl Acad Sci U S A ; 106(16): 6712-7, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19342489

RESUMO

Although genomewide association studies have successfully identified associations of many common single-nucleotide polymorphisms (SNPs) with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genomewide association studies. To identify these rare alleles we need high-throughput, high-accuracy resequencing technologies. Although array-based genotyping has allowed genomewide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for 2 main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing, and failure to achieve sufficient performance. We have recently solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data. The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA and ends with interrogation of purified DNA on resequencing arrays. We have used this pipeline to resequence approximately 5 Mb of DNA (on 3 arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study we obtained a false positive rate of approximately 1 in 500,000 bp and a false negative rate of approximately 10%.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA/métodos , Alelos , Automação , Pareamento Incorreto de Bases , Genoma Humano/genética , Humanos , Mutação/genética , Curva ROC , Análise de Sequência de DNA/normas
16.
BMC Med Genomics ; 2: 8, 2009 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-19228381

RESUMO

BACKGROUND: A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (approximately 40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. RESULTS: Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. CONCLUSION: MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

17.
Hum Mutat ; 29(3): 441-50, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18186519

RESUMO

Mismatch repair detection (MRD) was used to screen 93 matched tumor-normal sample pairs and 22 cell lines for somatic mutations in 30 cancer relevant genes. Using a starting amount of only 150 ng of genomic DNA, we screened 102 kb of sequence for somatic mutations in colon and breast cancer. A total of 152 somatic mutations were discovered, encompassing previously reported mutations, such as BRAF V600E and KRAS G12S, G12V, and G13D, as well as novel mutations, including some in genes in which somatic mutations have not previously been reported, such as MAP2K1 and MAP2K2. The distribution of mutations ranged widely within and across tumor types. The functional significance of many of these mutations is not understood, with patterns of selection only evident in KRAS and BRAF in colon cancer. These results present a novel approach to high-throughput mutation screening using small amounts of starting material and reveal a mutation spectrum across 30 genes in a large cohort of breast and colorectal cancers.


Assuntos
Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA/métodos , Mutação , Sequência de Bases , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Feminino , Humanos , Masculino
18.
Genome Biol ; 8(11): R246, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18028543

RESUMO

We have developed a new protocol for using molecular inversion probes to accurately and specifically measure allele copy number. The new protocol provides for significant improvements, including the reduction of input DNA (from 2 mug) by more than 25-fold (to 75 ng total genomic DNA), higher overall precision resulting in one order of magnitude lower false positive rate, and greater dynamic range with accurate absolute copy number up to 60 copies.


Assuntos
Alelos , Inversão Cromossômica , Sondas Moleculares , Linhagem Celular Tumoral , Aberrações Cromossômicas , Reações Falso-Positivas , Humanos , Perda de Heterozigosidade , Reação em Cadeia da Polimerase , Curva ROC
19.
Hum Genet ; 119(1-2): 75-83, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16391945

RESUMO

Single nucleotide polymorphisms (SNPs) that lead to non-synonymous changes in proteins may have functional effects and be subject to selection. Hence they are of particular interest in the study of genetic diseases. We have genotyped approximately 28,000 such SNPs in three ethnic populations (the HapMap plates) and ten primate species and analyzed these data for evidence of selection. We find SNPs predicted by PolyPhen to be damaging, have lower allele frequencies, and are particularly likely to be population-specific. We have also grouped SNPs by molecular function or biological process of the associated genes and find evidence that selection may be acting in concert on classes of genes.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Grupo com Ancestrais do Continente Asiático/genética , Bases de Dados Genéticas , Grupo com Ancestrais do Continente Europeu/genética , Polimorfismo de Nucleotídeo Único/genética , Animais , Frequência do Gene , Genética Populacional , Humanos , Primatas/genética , Seleção Genética
20.
Eur J Hum Genet ; 14(2): 207-15, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16306880

RESUMO

High-throughput genotyping technologies that enable large association studies are already available. Tools for genotype determination starting from raw signal intensities need to be automated, robust, and flexible to provide optimal genotype determination given the specific requirements of a study. The key metrics describing the performance of a custom genotyping study are assay conversion, call rate, and genotype accuracy. These three metrics can be traded off against each other. Using the highly multiplexed Molecular Inversion Probe technology as an example, we describe a methodology for identifying the optimal trade-off. The methodology comprises: a robust clustering algorithm and assessment of a large number of data filter sets. The clustering algorithm allows for automatic genotype determination. Many different sets of filters are then applied to the clustered data, and performance metrics resulting from each filter set are calculated. These performance metrics relate to the power of a study and provide a framework to choose the most suitable filter set to the particular study.


Assuntos
Algoritmos , Técnicas de Sonda Molecular , Polimorfismo de Nucleotídeo Único/genética , Projetos de Pesquisa , Análise por Conglomerados , Simulação por Computador , Marcadores Genéticos/genética , Genótipo , Reprodutibilidade dos Testes
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