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1.
BMC Cancer ; 13: 206, 2013 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-23617802

RESUMO

BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.


Assuntos
Análise Mutacional de DNA/métodos , Janus Quinase 2/genética , Leucemia Eritroblástica Aguda/genética , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Alelos , Primers do DNA , Éxons , Reações Falso-Positivas , Células HL-60 , Humanos , Neoplasia Residual
2.
J Thorac Oncol ; 13(10): 1588-1594, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30056163

RESUMO

INTRODUCTION: Malignant pleural mesothelioma is an aggressive malignancy with limited systemic therapy options. Promising results have been reported with use of anti-programmed cell death 1 therapy; however, its benefits appear to be confined to a subgroup of patients. Microsatellite instability (MSI) results from the inactivation of DNA mismatch repair genes and results in a high tumor mutational burden, a phenomenon that has not been seen with mesothelioma. MSI and protein absence have been shown to correlate in colorectal cancer, such that most centers have adopted immunohistochemistry (IHC) to screen for MSI-high colorectal cancers. We profiled a large cohort of patients with mesothelioma to determine the rate of negative IHC staining results the four common mismatch repair proteins. DESIGN: A tissue microarray comprising 335 patients with malignant pleural mesothelioma were used. IHC for the four common mismatch repair proteins (mutL homolog 1; PMS1 homolog 2, mismatch repair system component; mutS homolog 2; and mutS homolog 6) was performed. Programmed death ligand 1 IHC staining with the E1L3N clone was also performed. DNA was isolated from IHC equivocal samples and analyzed for microsatellite instability by using the Promega MSI Analysis System (version 1.2, Promega, Madison, WI). RESULTS: Of the patients profiled, 329 had intact mismatch repair proteins by IHC. Six samples with IHC testing results indicating absent mismatch repair protein were analyzed for MSI and confirmed to be negative. Of the six IHC-negative samples, five were negative for programmed death ligand 1 staining and one sample had more than 5% staining. CONCLUSION: In this large retrospective series, we were unable to identify any patients with malignant pleural mesothelioma with microsatellite instability. Response to anti-programmed cell death 1-based immunotherapy may be driven by other mechanisms.


Assuntos
Imuno-Histoquímica/métodos , Neoplasias Pulmonares/genética , Mesotelioma/genética , Instabilidade de Microssatélites/efeitos dos fármacos , Neoplasias Pleurais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/patologia , Mesotelioma Maligno , Pessoa de Meia-Idade , Neoplasias Pleurais/patologia , Estudos Retrospectivos , Adulto Jovem
3.
Nat Commun ; 9(1): 3970, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266954

RESUMO

Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.


Assuntos
Proteína BRCA1/genética , Metilação de DNA , Indóis/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Dosagem de Genes , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
4.
Clin Cancer Res ; 24(3): 569-580, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29061645

RESUMO

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths.Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing.Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival.Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569-80. ©2017 AACRSee related commentary by Peng and Mills, p. 508.


Assuntos
Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Reparo de DNA por Recombinação , Proteína do Retinoblastoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Cistadenocarcinoma Seroso/diagnóstico , Feminino , Recombinação Homóloga , Humanos , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/metabolismo , Prognóstico , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Análise de Sobrevida , Avaliação de Sintomas
5.
Oncotarget ; 5(23): 12016-26, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25427447

RESUMO

Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer (GC) but is poorly described in terms of molecular changes. Here, we explored the role of TP53, a commonly mutated gene in GC, to determine if p53 protein expression and/or the presence of somatic mutations in TP53 can be used as a predictive marker for patients at risk of progressing to GC from IM. Immunohistochemistry and high resolution melting were used to determine p53 protein expression and TP53 mutation status respectively in normal gastric mucosa, IM without concurrent GC (IM-GC), IM with concurrent GC (IM+GC) and GC. This comparative study revealed an incremental increase in p53 expression levels with progression of disease from normal mucosa, via an IM intermediate to GC. TP53 mutations however, were not detected in IM but occurred frequently in GC. Further, we identified increased protein expression of Mdm2/x, both powerful regulators of p53, in 100% of the IM+GC cohort with these samples also exhibiting high levels of wild-type p53 protein. Our data suggests that TP53 mutations occur late in gastric carcinogenesis contributing to the final transition to cancer. We also demonstrated involvement of Mdmx in GC.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteínas de Ciclo Celular , Análise Mutacional de DNA , Progressão da Doença , Humanos , Imuno-Histoquímica , Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/genética , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/patologia
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