RESUMEN
Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. cfDNA from 31 patients with diverse advanced cancers and known tumor mutation status was deep sequenced using targeted hotspot panels. Forty-five of 52 expected mutations and two additional mutations (KRAS p.Q61H and EZH2 p.Y646F) were detected in plasma through a custom bioinformatics pipeline. We observed comparable cfDNA concentration/integrity between collection tubes within 16 hours of plasma separation and equal analytical performance among NGS platforms, with 1% detection sensitivity for cfDNA genotyping.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/sangre , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Femenino , Genómica/métodos , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neoplasias/sangreRESUMEN
Clinical next-generation sequencing (NGS) assay choice requires careful consideration of panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, compatibility with low quality and quantity of nucleic acids, and work flow feasibility. Herein, in a high-volume clinical molecular diagnostic laboratory, we have validated a targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay) coupled with high-throughput sequencing using Ion Proton sequencer for routine screening of solid tumors. The panel screens 143 genes using low amounts of formalin-fixed, paraffin-embedded DNA (20 ng) and RNA (10 ng). A large cohort of 121 tumor samples representing 13 tumor types and 6 cancer cell lines was used to assess the capability of the panel to detect 148 single-nucleotide variants, 49 insertions or deletions, 40 copy number aberrations, and a subset of gene fusions. High levels of analytic sensitivity and reproducibility and robust detection sensitivity were observed. Furthermore, we demonstrated the critical utility of sequencing paired normal tissues to improve the accuracy of detecting somatic mutations in a background of germline variants. We also validated use of the Ion Chef automated bead templating and chip loading system, which represents a major work flow improvement. In summary, we present data establishing the OncoMine Comprehensive Assay-Ion Proton platform to be well suited for implementation as a routine clinical NGS test for solid tumors.
Asunto(s)
Biomarcadores de Tumor , Amplificación de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Neoplasias/diagnóstico , Neoplasias/genética , Fusión de Oncogenes , Línea Celular Tumoral , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Flujo de TrabajoRESUMEN
Microsatellites are short tandem repeats of deoxyribonucleic acid (DNA) sequences which are distributed throughout the genome. Tumors in patients with Lynch syndrome tend to accumulate mutations in microsatellites at a much higher rate than other sequences in the genome resulting in microsatellite instability (MSI). This is due to germline mutations in mismatch repair (MMR) genes. Using small pool-polymerase chain reaction (SP-PCR), previous studies have shown that mutant alleles can be detected in microsatellites of DNA from peripheral blood lymphocytes (PBLs) of Lynch syndrome patients at frequencies that were low, but significantly higher than frequencies in PBLs of age-matched non-Lynch syndrome controls. In the present study, SP-PCR detection of frequency of mutant MSI alleles (FMMA) was performed on PBLs and saliva samples from four sets of families. Each family set consisted of a mutation carrying affected proband (initial tumor bearer), a germline mutation-carrier sibling without tumors, and an age-matched normal control, either related (for 3 family sets) without mutation carrier status or unrelated (for 1 family set) without mutation carrier status. FMMAs of saliva and PBL DNA were compared between each proband, sibling and control for each family set, and between family sets. In all five statistically significant saliva comparisons identified between germline mutation carriers (FMMA: 0.080-0.261) and normal controls (FMMA: 0.003-0.087), the measured FMMAs were always higher in the carriers (p < 0.05). A logistic regression model of the data showed a significant increase in FMMAs in saliva DNA from siblings with MMR mutation compared to the normal controls (p < 0.001). These results indicated that the increased FMMAs observed in the saliva DNA as well as PBL DNA of MMR gene mutation carriers compared to normal controls are real and repeatable. Furthermore, the logistic regression also indicated that the FMMAs seen in saliva were nearly double those seen in PBLs (p < 0.001). Saliva testing, a less-invasive procedure than PBL testing, is more sensitive and appears to be a viable alternative for identifying MSI in carriers with MMR mutations.