RESUMEN
BACKGROUND: Several targeted therapies have been approved for treatment of solid tumors. Identification of gene mutations that indicate response to these therapies is rapidly progressing. A 34-gene next-generation sequencing (NGS) panel, developed and validated by us, was evaluated to detect additional mutations in community-based cancer specimens initially sent to our reference laboratory for routine molecular testing. METHODS: Consecutive de-identified clinical specimens (n = 121) from melanoma cases (n = 31), lung cancer cases (n = 27), colorectal cancer cases (n = 33), and breast cancer cases (n = 30) were profiled by NGS, and the results were compared with routine molecular testing. RESULTS: Upon initial mutation testing, 20 % (24/121) were positive. NGS detected ≥1 additional mutation not identified by routine testing in 74 % of specimens (90/121). Of the specimens with additional mutations, 16 harbored mutations in National Comprehensive Cancer Network guideline genes. These various additional mutations were in gene regions not routinely covered, in genes not routinely tested, and/or present at low allele frequencies. Moreover, NGS yielded no false negatives. Overall, NGS detected mutations in 59 % of the genes (20/34) included in the panel, 75 % of which (15/20) were detected in multiple tumor types. Mutations in TP53 were found in 51 % of tumors tested (62/121). Mutations in at least one other (non-TP53) gene present in the panel were detected in 64 % of cases (77/121). CONCLUSION: This assay provides improved breadth and sensitivity for profiling clinically relevant genes in these prevalent solid tumor types.
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Biomarcadores de Tumor , Mutación , Neoplasias/diagnóstico , Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Biopsia , Femenino , Frecuencia de los Genes , Pruebas Genéticas/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Detection of the BRAF V600E mutation is required for use of the BRAF inhibitor, vemurafenib, in patients with metastatic melanoma. Although the Roche Cobas 4800 BRAF V600 Mutation Test is approved, it detects primarily the single-nucleotide V600E mutation and could miss other potentially relevant V600 mutations. To assess the detection rate of the cobas assay for V600 mutations in clinical specimens, we compared the results of this assay with Sanger sequencing in 295 melanoma FFPE samples. Twenty samples were excluded because of invalid results on the cobas (n = 3), sequencing (n = 15), or both (n = 2). V600 mutations were detected by the cobas test in 96 (34.9%) of 275 samples and by Sanger sequencing in 118 (42.9%) of 275 samples. Thus, relative to Sanger sequencing, the cobas test exhibited 80.5% sensitivity (95% CI, 72.4% to 86.6%) and 99.4% specificity (95% CI, 96.5% to 99.9%). Of 23 samples with positive sequencing results but negative cobas results, 21 harbored dinucleotide mutations (V600E in 6, V600K in 10, and V600R in 5); the other two involved single-nucleotide mutations (V600E and V600G). These findings indicate that the cobas assay may miss many V600 mutations in clinical specimens. In our study, the addition of Sanger sequencing for samples with negative cobas results increased the detection rate to 42.9%. This approach could help maximize the number of patients who benefit from BRAF inhibitor treatment.
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Análisis Mutacional de ADN , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Humanos , Melanoma/secundario , Persona de Mediana Edad , Mutación Missense , Sensibilidad y Especificidad , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: The natural history and the possible changes of celiac disease (CD) prevalence over time are still unclear. OBJECTIVES: 1) To establish whether loss of tolerance to gluten may occur at any age; 2) to investigate possible changes of CD prevalence over time; and 3) to investigate CD-related co-morbidities. METHODS: We analyzed 3,511 subjects with matched samples from 1974 (CLUE I) and 1989 (CLUE II). To avoid a selection bias regarding survival, we also screened 840 CLUE I participants who deceased after the 1974 survey. Outcome measure. CD autoimmunity (positivity to auto-antibodies) over time. RESULTS: CD autoimmunity was detected in seven subjects in 1974 (prevalence 1:501) and in an additional nine subjects in 1989 (prevalence 1:219). Two cases of CD autoimmunity were found among the 840 subjects deceased after CLUE I. Compared to controls, untreated CD subjects showed increased incidence of osteoporosis and associated autoimmune disorders, but they did not reach statistical significance. CONCLUSIONS: During a 15-year period CD prevalence increased 2-fold in the CLUE cohort and 5-fold overall in the US since 1974. The CLUE study demonstrated that this increase was due to an increasing number of subjects that lost the immunological tolerance to gluten in their adulthood.
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Anticuerpos/sangre , Enfermedades Autoinmunes/epidemiología , Autoinmunidad , Enfermedad Celíaca/epidemiología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Osteoporosis/epidemiología , Transglutaminasas/inmunología , Adolescente , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Comorbilidad , Glútenes/inmunología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Prevalencia , Proteína Glutamina Gamma Glutamiltransferasa 2 , Sensibilidad y Especificidad , Estados Unidos , Adulto JovenRESUMEN
BACKGROUND & AIMS: Biomarkers are being developed as alternatives to liver biopsy for predicting liver fibrosis in patients with chronic hepatitis C. Hepascore uses noninvasive serum markers and has been validated in Australian and European populations for predicting different degrees of fibrosis. This study validated this test in a U.S. population. METHODS: Patients with chronic hepatitis C virus infection were assigned to training (n = 203) or validation (n = 188) sets. Liver fibrosis was staged according to the METAVIR scoring system. The Hepascore algorithm uses data on age, sex, as well as total bilirubin, gamma-glutamyl transferase, alpha2-macroglobulin, and hyaluronic acid levels. RESULTS: The ability of Hepascore to predict significant fibrosis (F2-4) as determined by the area under the receiver operating curve was similar in training (0.83) and validation sets (0.81) and was comparable to results seen in previous studies. A cutoff score of > or =0.55 was best for predicting significant fibrosis, with a sensitivity and specificity of 82% and 65% and positive and negative predictive values of 70% and 78%. When compared with 2 simple indices, FIB-4 (age, platelets, AST, and ALT) and APRI (AST/platelet ratio index), Hepascore performed better at excluding advanced fibrosis by using a low cutoff score but worse at predicting fibrosis by using a high cutoff score. An algorithm with Hepascore followed by FIB-4 or APRI spared 103 of 391 individuals a liver biopsy and missed advanced fibrosis in only 1 patient. CONCLUSIONS: Hepascore accurately predicted likelihood of developing fibrosis and could alleviate the need for liver biopsy in a subset of patients.
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Hepatitis C Crónica/complicaciones , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Índice de Severidad de la Enfermedad , Adulto , Factores de Edad , Bilirrubina/sangre , Biomarcadores , Femenino , Humanos , Ácido Hialurónico/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Suero/química , Factores Sexuales , Estados Unidos , alfa-Macroglobulinas/análisis , gamma-Glutamiltransferasa/sangreRESUMEN
The hepatic isoform 1A1 of uridine diphosphate glucuronosyltransferase is responsible for glucuronidation and detoxification of SN-38, the active metabolite of irinotecan. The presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter leads to a significant decrease in SN-38 glucuronidation. Patients with the UGT1A1 (TA)7 allele are more likely to experience severe neutropenia and diarrhea following irinotecan chemotherapy. We assessed the distribution of the UGT1A1 (TA)n polymorphism in healthy male and female US residents of European and Asian descent. We used a fluorescent polymerase chain reaction-based assay to detect UGT1A1 (TA)n polymorphisms in 138 healthy volunteers (56 Caucasians, 37 Chinese, 37 Filipino and eight Japanese) between the ages of 18 and 65 years. The chi-test was used to assess between-group differences in the distribution of UGT1A1 (TA)n genotypes. The UGT1A1 (TA)6/6 genotype was significantly more common in Asians than in Caucasians (76 vs. 46%), whereas the (TA)6/7 (39 vs. 20%) and (TA)7/7 (13 vs. 5%) genotypes were more common in Caucasians than in Asians. Genotype distributions did not differ significantly between men and women in either group. The UGT1A1 (TA)5/5 genotype was detected in one Caucasian woman. In conclusion, consistent with previous reports, the UGT1A1 (TA)7/7 genotype was significantly more common in Caucasians than in Asians. UGT1A1 (TA)n/n genotype distribution did not vary with sex in individuals of European or Asian descent.
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Asiático/genética , Frecuencia de los Genes , Glucuronosiltransferasa/genética , Polimorfismo Genético , Población Blanca/genética , Adolescente , Adulto , Anciano , Antineoplásicos Fitogénicos/farmacocinética , Camptotecina/análogos & derivados , Camptotecina/farmacocinética , China/etnología , Femenino , Genotipo , Humanos , Irinotecán , Japón/etnología , Masculino , Tasa de Depuración Metabólica/genética , Persona de Mediana Edad , Filipinas/etnología , Estados UnidosRESUMEN
BACKGROUND: Von Willebrand disease (VWD) diagnosis and classification usually require a combination of nonspecific and VW-factor (VWF)-specific assays. We evaluated the analytical performance of a commercially available collagen-binding assay (CBA) and its usefulness in conjunction with other assays for laboratory diagnosis of VWD. METHODS: We used a commercial CBA ELISA (Life Technologies) to evaluate 3085 plasma samples. We used standard procedures to perform other assays, including factor VIII activity (FVIII:C), VWF antigen (VWF:Ag), ristocetin cofactor activity, VWF collagen binding capacity (VWF:CB), and VWF multimeric analysis. RESULTS: CBA intra- and interassay CVs were <6% and <13%, respectively. Reference intervals were 45%-198% for VWF:CB and 0.75-1.32 for the VWF:CB/Ag ratio. Of 3085 samples tested, 235 (8%) had results commonly associated with VWD. Multimer analysis and phenotypic data in 156 samples identified VWD types as: 91 (58%) type 1, 62 (40%) type 2, and 3 (2%) type 3. Of the 91 type 1 samples, proportional decreases in functional activity were seen in 75 samples (82%) according to CBA and in 63 samples (69%) according to the ristocetin cofactor assay. Of the type 2 samples, 10 were further identified as probable type 2A, 26 as probable type 2B, 12 as probable type 2M, and 14 could not be subtyped. VWF:CBA/Ag ratios <0.5 occurred in 83% of VWD type 2A and 2B samples, indicating characteristic functional discordance. Mean (SD) VWF:CB values were significantly higher in individuals without group O blood [113 (45)] than in those with group O blood [83 (32)] (t-test, P = 0.007). CONCLUSIONS: The commercial CBA assay produces reliable results and is useful for laboratory diagnosis of VWD.
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Colágeno/metabolismo , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Fenotipo , Unión Proteica , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
5-Fluorouracil (5-FU) has been used for more than 40 years in the treatment of neoplastic disease, and remains the standard first-line treatment for colorectal cancer in combination with irinotecan and leucovorin. Previous studies indicated that measurement of dihydropyrimidine dehydrogenase (DPD) gene expression before treatment was valuable in determining the potential benefit of and toxicity to 5-FU treatment. In this study, we investigated the association between intratumoral DPD gene expression and the adjacent normal tissue DPD gene expression and DPD mRNA expression level in non-paired colon tumor and normal colon tissue specimens. In addition, we have compared the difference of DPD gene expression at three different RNA concentrations from the same specimen (180, 100 and 5 ng/reaction, respectively). DPD expression was measured by quantitative RT-PCR using a LightCycler instrument in a total of 31 specimens. Gene expression values were expressed as a ratio of target gene (DPD) to the internal reference gene (G6PDH). Our study revealed no statistically significant difference (p=0.23) between tumor tissues and matched normal tissue in DPD expression. In contrast, the data on DPD mRNA expression in non-paired colon tumor and normal tissue specimens revealed a significant difference (p=0.0004) between the tumor group and the normal group. In the three RNA concentration groups, there was no significant difference (p=0.55) in gene expression at the different RNA concentrations from the same donor. These results demonstrate that intratumoral gene expression levels of DPD do not correlate with tumor cell percentage or with RNA concentration. Thus, DPD mRNA expression appears to be a valid sensitivity test for 5-FU in spite of a varying density of tumor cells and RNA yield in specimens submitted for analysis.
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Antineoplásicos/uso terapéutico , Colon/enzimología , Neoplasias Colorrectales/enzimología , Dihidrouracilo Deshidrogenasa (NADP)/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Antineoplásicos/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Humanos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.