RESUMO
BACKGROUND: Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. METHODS: We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. RESULTS: Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. CONCLUSIONS: We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.
RESUMO
Intra-tumoral genomic heterogeneity is a well-established biologic property of human malignancies with emerging roles in cancer progression and therapy resistance. However, its impact on the clinical utility of genomic testing in patient management remains unclear. Furthermore, best practices to account for heterogeneity in the provision of highly accurate, clinically valid molecular testing have yet to be firmly established. Genomic biomarkers for the management of colorectal carcinoma are both well-established (ie, KRAS, NRAS) and emerging (BRAF, PIK3CA, and others) in respect to therapy selection and clinical trial eligibility. Critically, standard colorectal carcinoma management requires the exclusion of KRAS and NRAS mutations; thus optimal procedures to control for potential intra-tumoral heterogeneity are clinically important. Here, we assessed heterogeneity among three intra-tumoral sites within 99 colorectal carcinomas cases on a CLIA-validated oncology next generation sequencing assay and examined whether a pooling strategy overcame any discordant results. Overall, 11% of cases demonstrated discordant mutation results between sites; 2% of cases were discrepant for mutations within RAS genes while the remainder was discrepant in PIK3CA. Half of the discrepant cases were associated with borderline tumor cellularity assessment. Further, a sample pooling strategy across all three sites successfully detected the relevant mutation in all but one case. Our results indicate that intra-tumoral genomic heterogeneity of clinically relevant genes within colorectal carcinoma is a relatively infrequent occurrence and that a simple strategy to pool DNA from several tumor sites with adequate cellularity minimizes the risk of false negative results even further to ensure optimal patient management.