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1.
Proc Natl Acad Sci U S A ; 109(8): 3041-6, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22323597

RESUMO

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity, which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications, clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however, the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs, most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification, revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well.


Assuntos
Receptores ErbB/genética , Amplificação de Genes , Heterogeneidade Genética , Glioblastoma/enzimologia , Glioblastoma/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Proliferação de Células , Segregação de Cromossomos/genética , Simulação por Computador , Genoma Humano/genética , Glioblastoma/patologia , Humanos , Hibridização in Situ Fluorescente
2.
Histopathology ; 63(2): 279-86, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23701505

RESUMO

AIM: To study prostate cancer zonal differences in TMPRSS2-ERG gene rearrangement. METHODS AND RESULTS: We examined 136 well-characterized dominant anterior prostatic tumours, including 61 transition zone (TZ) and 75 anterior peripheral zone (PZ) lesions, defined using strict anatomical considerations. TMPRSS2-ERG FISH and ERG protein immunohistochemistry were performed on tissue microarrays. FISH results, available for 56 TZ and 71 anterior PZ samples, were correlated with ERG staining and TZ-associated 'clear cell' histology. Fewer TZ cancers (four of 56; 7%) were rearranged than anterior PZ cancers (18 of 71; 25%) (P = 0.009); deletion was the sole mechanism of TZ cancer rearrangement. ERG protein overexpression was present in 4% (two of 56; both FISH+) and 30% (21 of 71; 17 FISH+) of TZ and anterior PZ tumours, respectively. 'Clear cell' histology was present in 21 of 56 (38%) TZ and eight of 71 (11%) anterior PZ tumours. Seven per cent of cancers with and 21% without this histology had rearrangement, regardless of zonal origin. CONCLUSIONS: TMPRSS2-ERG rearrangement occurs in dominant TZ and anterior PZ prostate cancers, with all rearranged TZ cancers in this cohort showing deletion. ERG immunohistochemistry demonstrated excellent sensitivity (86%) and specificity (96%) for TMPRSS2-ERG rearrangement. TMPRSS2-ERG fusion is rare in TZ tumours and present at a low frequency in tumours displaying 'clear cell' histology.


Assuntos
Rearranjo Gênico , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/genética , Transativadores/genética , Transativadores/metabolismo , Estudos de Coortes , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Neoplasias da Próstata/patologia , Análise Serial de Tecidos , Regulador Transcricional ERG
3.
Proc Natl Acad Sci U S A ; 106(6): 1886-91, 2009 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-19181860

RESUMO

We have identified new genomic alterations in the breast cancer cell line HCC1954, using high-throughput transcriptome sequencing. With 120 Mb of cDNA sequences, we were able to identify genomic rearrangement events leading to fusions or truncations of genes including MRE11 and NSD1, genes already implicated in oncogenesis, and 7 rearrangements involving other additional genes. This approach demonstrates that high-throughput transcriptome sequencing is an effective strategy for the characterization of genomic rearrangements in cancers.


Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Rearranjo Gênico , Genoma Humano/genética , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular Tumoral , DNA Complementar , Proteínas de Ligação a DNA/genética , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Proteína Homóloga a MRE11 , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética
4.
Mod Pathol ; 23(10): 1325-33, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20562851

RESUMO

TMPRSS2-ERG gene rearrangement is seen in about half of clinically localized prostate cancers, yet controversy exists with regard to its prognostic implications. Similarly, the relationship of TMPRSS2-ERG fusion to Gleason score and morphology remains uncertain. We assigned Gleason scores and recorded morphological features for 521 clinically localized prostate cancers sampled in triplicate and arrayed in eight tissue microarray blocks. Fluorescence in situ hybridization was performed to delineate TMPRSS2-ERG aberrations. Using maximum Gleason score, based on three core evaluation, and overall Gleason score, based on prostatectomy sections, Fisher's exact test was performed for tumors with TMPRSS2-ERG translocation/deletion, copy number increase (≥ 3) of the TMPRSS2-ERG region without translocation/deletion, and copy number increase and concomitant translocation/deletion. In all, 217 (42%) translocation/deletion and 30 (5.9%) copy number increase-alone cases were detected. Among 217 translocation/deletion cases, 32 had translocation/deletion with copy number increase. In all, 237, 200, and 75 cancers had maximum core-specific Gleason score of 6, 7, and 8-10, respectively. Tumors with translocation/deletion tended toward lower Gleason scores than those without (P=0.002) with similar results for overall Gleason score (P=0.02); copy number increase cases tended toward higher Gleason scores than those without (P<0.001). Gleason score of 8-10 tumors demonstrated lower odds of translocation/deletion (odds ratio (OR) 0.38; 95% CI 0.21-0.68) and higher odds of copy number increase alone (OR 7.33; 95% CI 2.65-20.31) or copy number increase+translocation/deletion (OR 3.03; 95% CI 1.12-8.15) relative to Gleason score of <7 tumors. No significant difference in TMPRSS2-ERG incidence was observed between patients with and without cribriform glands, glomerulations, signet-ring cells, or intraductal cancer (P=0.821, 0.095, 0.132, 0.375). TMPRSS2-ERG gene fusion is associated with lower core-specific and overall Gleason scores and not with high-grade morphologies. Conversely, TMPRSS2-ERG copy number increase, with or without rearrangement, is associated with higher Gleason score. These findings indicate that translocation/deletion of TMPRSS2-ERG is not associated with histological features of aggressive prostate cancer.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Dosagem de Genes , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Masculino , Análise Serial de Tecidos
5.
Clin Cancer Res ; 15(6): 2091-7, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19276271

RESUMO

PURPOSE: To assess the feasibility of characterizing gene copy number alteration by fluorescence in situ hybridization (FISH) of circulating tumor cells (CTC) isolated using the CellSearch system in patients with progressive castration-resistant metastatic prostate cancer. EXPERIMENTAL DESIGN: We used probe combinations that included the androgen receptor (AR) and MYC genes for FISH analysis of CTC samples collected from 77 men with castration-resistant metastatic prostate cancer. RESULTS: High-level chromosomal amplification of AR was detected in 38% and relative gain of MYC in 56% of samples analyzed. No such abnormalities were detected in samples with CTC counts of <10, reflecting ascertainment difficulty in these lower count samples. CONCLUSION: The CTC isolated from our patient cohort present a very similar molecular cytogenetic profile to that reported for late-stage tumors and show that FISH analysis of CTC can be a valuable, noninvasive surrogate for routine tumor profiling. That as many as 50% of these patients have substantial amplification of the AR locus indicates that androgen signaling continues to play an important role in late-stage prostate cancer.


Assuntos
Hibridização in Situ Fluorescente/métodos , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/patologia , Dosagem de Genes , Genes myc , Humanos , Masculino , Neoplasias da Próstata/genética , Receptores Androgênicos/genética
6.
Clin Cancer Res ; 13(7): 2023-9, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17404082

RESUMO

PURPOSE: To better direct targeted therapies to the patients with tumors that express the target, there is an urgent need for blood-based assays that provide expression information on a consistent basis in real time with minimal patient discomfort. We aimed to use immunomagnetic-capture technology to isolate and analyze circulating tumor cells (CTC) from small volumes of peripheral blood of patients with advanced prostate cancer. EXPERIMENTAL DESIGN: Blood was collected from 63 patients with metastatic prostate cancer. CTCs were isolated by the Cell Search system, which uses antibodies to epithelial cell adhesion marker and immunomagnetic capture. CTCs were defined as nucleated cells positive for cytokeratins and negative for CD45. Captured cells were analyzed by immunofluorescence, Papanicolau staining, and fluorescence in situ hybridization. RESULTS: Most patients (65%) had 5 or more CTCs per 7.5 mL blood sample. Cell counts were consistent between laboratories (c = 0.99) and did not change significantly over 72 or 96 h of storage before processing (c = 0.99). Their identity as prostate cancer cells was confirmed by conventional cytologic analysis. Molecular profiling, including analysis of epidermal growth factor receptor (EGFR) expression, chromosome ploidy, and androgen receptor (AR) gene amplification, was possible for all prostate cancer patients with >or=5 CTCs. CONCLUSIONS: The analysis of cancer-related alterations at the DNA and protein level from CTCs is feasible in a hospital-based clinical laboratory. The alterations observed in EGFR and AR suggest that the methodology may have a role in clinical decision making.


Assuntos
Técnicas Citológicas , Células Neoplásicas Circulantes , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Idoso , Idoso de 80 Anos ou mais , Castração , Receptores ErbB/biossíntese , Citometria de Fluxo , Imunofluorescência , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Reprodutibilidade dos Testes
7.
Clin Cancer Res ; 11(11): 4182-90, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15930355

RESUMO

Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mutação , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Western Blotting , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Genótipo , Humanos , Mesilato de Imatinib , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo
8.
Nat Genet ; 45(7): 791-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23685749

RESUMO

Adenoid cystic carcinomas (ACCs) are among the most enigmatic of human malignancies. These aggressive salivary gland cancers frequently recur and metastasize despite definitive treatment, with no known effective chemotherapy regimen. Here we determined the ACC mutational landscape and report the exome or whole-genome sequences of 60 ACC tumor-normal pairs. These analyses identified a low exonic somatic mutation rate (0.31 non-silent events per megabase) and wide mutational diversity. Notably, we found mutations in genes encoding chromatin-state regulators, such as SMARCA2, CREBBP and KDM6A, suggesting that there is aberrant epigenetic regulation in ACC oncogenesis. Mutations in genes central to the DNA damage response and protein kinase A signaling also implicate these processes. We observed MYB-NFIB translocations and somatic mutations in MYB-associated genes, solidifying the role of these aberrations as critical events in ACC. Lastly, we identified recurrent mutations in the FGF-IGF-PI3K pathway (30% of tumors) that might represent new avenues for therapy. Collectively, our observations establish a molecular foundation for understanding and exploring new treatments for ACC.


Assuntos
Carcinoma Adenoide Cístico/genética , Transformação Celular Neoplásica/genética , Mutação , Neoplasias das Glândulas Salivares/genética , Animais , Células COS , Carcinoma Adenoide Cístico/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Chlorocebus aethiops , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Estudos de Associação Genética , Humanos , Modelos Biológicos , Mutação/fisiologia , Neoplasias das Glândulas Salivares/metabolismo , Transdução de Sinais/genética , Análise Serial de Tecidos
9.
Eur Urol ; 60(5): 897-904, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21802835

RESUMO

BACKGROUND: Abiraterone acetate (AA) is an androgen biosynthesis inhibitor shown to prolong life in patients with castration-resistant prostate cancer (CRPC) already treated with chemotherapy. AA treatment results in dramatic declines in prostate-specific antigen (PSA) in some patients and no declines in others, suggesting the presence of molecular determinants of sensitivity in tumors. OBJECTIVE: To study the role of transmembrane protease, serine 2 (TMPRSS2)-v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion, an androgen-dependent growth factor, in circulating tumor cells (CTCs) as a biomarker of sensitivity to AA. DESIGN, SETTING, AND PARTICIPANTS: The predictive value of TMPRSS2-ERG status was studied in 41 of 48 men with postchemotherapy-treated CRPC enrolled in sequential phase 2 AA trials. INTERVENTION: Patients received AA 1000 mg daily and continuously. MEASUREMENTS: TMPRSS2-ERG status was characterized by a sensitive, analytically valid reverse transcription polymerase chain reaction assay in CTCs enriched from ethylene-diaminetetraacetic acid anticoagulated blood obtained prior to AA treatment. Outcomes were measured by PSA Working Group 1 criteria. RESULTS AND LIMITATIONS: Standard procedures for specimen acquisition, processing, and testing using the validated TMPRSS2-ERG assay on a multiplex platform gave intra-assay and interassay coefficients of variation <7%. TMPRSS2-ERG fusion was present in 15 of 41 patients (37%), who had a median baseline CTC count of 17 (interquartile range: 7-103 cells per 7.5 ml). A PSA decline ≥50% was observed in 7 of 15 patients (47%) with the fusion and in 10 of 26 patients (38%) without the fusion. Although limited by the low number of patients, a posttherapy CTC count of less than five per 7.5 ml was prognostic for longer survival relative to a CTC count five or more. TMPRSS2-ERG status did not predict a decline in PSA or other clinical outcomes. CONCLUSIONS: Molecular profiles of CTCs with an analytically valid assay identified the presence of the prostate cancer-specific TMPRSS2-ERG fusion but did not predict for response to AA treatment. This finding demonstrates the role of CTCs as surrogate tissue that can be obtained in a routine practice setting. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00474383 (COU-AA-003), NCT00485303 (COU-AA-004).


Assuntos
Androstadienos/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/genética , Inibidores Enzimáticos/uso terapêutico , Células Neoplásicas Circulantes/metabolismo , Proteínas de Fusão Oncogênica/genética , Orquiectomia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Acetato de Abiraterona , Quimioterapia Adjuvante , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Células Neoplásicas Circulantes/patologia , Cidade de Nova Iorque , Seleção de Pacientes , Valor Preditivo dos Testes , Prostatectomia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Radioterapia Adjuvante , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 17-alfa-Hidroxilase/metabolismo , Taxa de Sobrevida , Fatores de Tempo , Resultado do Tratamento
10.
Cancer Res ; 69(4): 1400-6, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19190343

RESUMO

A significant number of prostate cancers have been shown to have recurrent chromosomal rearrangements resulting in the fusion of the androgen-regulated TMPRSS2 promoter to a member of the ETS transcription factor family, most commonly ERG. This results in ERG overexpression, which may have a direct causal role in prostate tumorigenesis or progression. However, the clinical significance of the rearrangement is unclear, and in particular, relationship to outcome has been inconsistent in recent reports. We analyzed TMPRSS2-ERG gene rearrangement status by fluorescence in situ hybridization in 521 cases of clinically localized surgically treated prostate cancer with 95 months of median follow-up and also in 40 unmatched metastases. Forty-two percent of primary tumors and 40% of metastases had rearrangements. Eleven percent had copy number increase (CNI) of the TMPRRS2-ERG region. Rearrangement alone was associated with lower grade, but not with stage, biochemical recurrence, metastases, or death. CNI with and without rearrangement was associated with high grade and advanced stage. Further, a subgroup of cancers with CNI and rearrangement by deletion, with two or more copies of the deleted locus, tended to be more clinically aggressive. DNA index assessment revealed that the majority of tumors with CNI of TMPRSS2-ERG had generalized aneuploidy/tetraploidy in contrast to tumors without TMPRSS2-ERG CNI, which were predominantly diploid. We therefore conclude that translocation of TMPRSS2-ERG is not associated with outcome, and the aggressive clinical features associated with CNI of chromosome 21 reflect generalized aneuploidy and are not due to CNI specifically of rearranged TMPRSS2-ERG.


Assuntos
Fusão Gênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Serina Endopeptidases/genética , Transativadores/genética , Translocação Genética , Aberrações Cromossômicas , Citometria de Fluxo , Deleção de Genes , Rearranjo Gênico , Humanos , Masculino , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prostatectomia , Neoplasias da Próstata/patologia , Regulador Transcricional ERG , Resultado do Tratamento
11.
Cancer Inform ; 6: 59-75, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19259404

RESUMO

We report a method, Expression-Microarray Copy Number Analysis (ECNA) for the detection of copy number changes using Affymetrix Human Genome U133 Plus 2.0 arrays, starting with as little as 5 ng input genomic DNA. An analytical approach was developed using DNA isolated from cell lines containing various X-chromosome numbers, and validated with DNA from cell lines with defined deletions and amplifications in other chromosomal locations. We applied this method to examine the copy number changes in DNA from 5 frozen gastrointestinal stromal tumors (GIST). We detected known copy number aberrations consistent with previously published results using conventional or BAC-array CGH, as well as novel changes in GIST tumors. These changes were concordant with results from Affymetrix 100K human SNP mapping arrays. Gene expression data for these GIST samples had previously been generated on U133A arrays, allowing us to explore correlations between chromosomal copy number and RNA expression levels. One of the novel aberrations identified in the GIST samples, a previously unreported gain on 1q21.1 containing the PEX11B gene, was confirmed in this study by FISH and was also shown to have significant differences in expression pattern when compared to a control sample. In summary, we have demonstrated the use of gene expression microarrays for the detection of genomic copy number aberrations in tumor samples. This method may be used to study copy number changes in other species for which RNA expression arrays are available, e.g. other mammals, plants, etc., and for which SNPs have not yet been mapped.

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