RESUMEN
Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy.Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care.Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03).Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR.
Asunto(s)
Biomarcadores de Tumor , ADN Tumoral Circulante , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias/diagnóstico , Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neoplasias/mortalidad , Pronóstico , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR.
Asunto(s)
Análisis Mutacional de ADN/métodos , Melanoma/diagnóstico , Proteínas Proto-Oncogénicas B-raf/sangre , Neoplasias Cutáneas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Sistema Libre de Células , Detección Precoz del Cáncer , Femenino , Humanos , Masculino , Melanoma/genética , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas B-raf/genética , Sensibilidad y Especificidad , Neoplasias Cutáneas/genética , Análisis de Supervivencia , Adulto JovenRESUMEN
The Abstract is incorrect in PubMed. The corrected Abstract is provided here.
RESUMEN
Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were concordant for archival tissue and plasma cfDNA in 91% cases for BRAF mutations (kappa = 0.75, 95% confidence interval [CI] 0.63 - 0.88), in 99% cases for EGFR mutations (kappa = 0.90, 95% CI 0.71- 1.00), in 83% cases for KRAS mutations (kappa = 0.67, 95% CI 0.54 - 0.80) and in 91% cases for PIK3CA mutations (kappa = 0.65, 95% CI 0.46 - 0.85). Patients (n = 41) with > 1% of KRAS mutant cfDNA had a shorter median survival compared to 20 patients with = 1% of KRAS mutant DNA (4.8 vs. 7.3 months, p=0.008). Similarly, 67 patients with >1% of mutant cfDNA (BRAF, EGFR, KRAS, or PIK3CA) had a shorter median survival compared to 33 patients with = 1% of mutant cfDNA (5.5 vs. 9.8 months, p = 0.001), which was confirmed in multivariable analysis. [Corrected]
Asunto(s)
ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Fosfatidilinositol 3-Quinasa Clase I , Análisis Mutacional de ADN , Femenino , Genes erbB-1/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasias/mortalidad , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adulto JovenRESUMEN
UNLABELLED: Patients with Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) have a high frequency of BRAF(V600E) mutations and respond to RAF inhibitors. However, detection of mutations in tissue biopsies is particularly challenging in histiocytoses due to low tumor content and stromal contamination. We applied a droplet-digital PCR assay for quantitative detection of the BRAF(V600E) mutation in plasma and urine cell-free (cf) DNA and performed a prospective, blinded study in 30 patients with ECD/LCH. There was 100% concordance between tissue and urinary cfDNA genotype in treatment-naïve samples. cfDNA analysis facilitated identification of previously undescribed KRAS(G12S)-mutant ECD and dynamically tracked disease burden in patients treated with a variety of therapies. These results indicate that cfDNA BRAF(V600E) mutational analysis in plasma and urine provides a convenient and reliable method of detecting mutational status and can serve as a noninvasive biomarker to monitor response to therapy in LCH and ECD. SIGNIFICANCE: Patients with BRAF(V600E)-mutant histiocytic disorders have remarkable responses to RAF inhibition, but mutation detection in tissue in these disorders is challenging. Here, we identify that analysis of plasma and urinary cfDNA provides a reliable method to detect the BRAF(V600E) mutation and monitor response to therapy in these disorders.
Asunto(s)
Histiocitosis/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos , Biopsia , Niño , Codón , Estudios Transversales , Análisis Mutacional de ADN , Enfermedad de Erdheim-Chester/diagnóstico , Enfermedad de Erdheim-Chester/genética , Femenino , Frecuencia de los Genes , Genes ras , Genotipo , Histiocitosis/diagnóstico , Histiocitosis/tratamiento farmacológico , Histiocitosis de Células de Langerhans/diagnóstico , Histiocitosis de Células de Langerhans/genética , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Adulto JovenRESUMEN
Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis.