Clinical next‐generation sequencing in patients with non–small cell lung cancer

Abstract

BACKGROUND: A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC). METHODS: Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. RESULTS: Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 13183. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. CONCLUSIONS: NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing. Cancer 2015;121:631-9.

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

“…Novel research techniques, such as next generation sequencing, have been developed (1,2) and the usage of DNA from stored formalin-fixed and paraffin-embedded (FFPE) tissues libraries has increased. For example, comprehensive analysis of DNA alteration with next generation sequencing enables us to identify the novel genomic alterations in patients with cancer recurrence, which may provide more therapeutic targets.…”

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

“…Yet studies have also demonstrated the risk of artifacts which can cause false-positive results from challenging cancer biopsies such as FFPE specimens [2][3][4][5]12,14 . Additionally, recent publications have highlighted high failure rates for NGS using oncology specimens 16 and false-positive calls with commercial targeted NGS panels that are aggravated by the use of low-input DNA 12 . As a result, some laboratories have either modified or added additional checks and balances to commercially available targeted NGS technologies to improve performance, often to ensure accuracy or confirm the findings from the bioinformatic analyses 5,10,11 .…”

Sample types applied for molecular diagnosis of therapeutic management of advanced non-small cell lung cancer in the precision medicine

“…Currently, the expansion of numbers of these gene alterations seems to pose some problems. One of these problems that need to be surmounted is the necessarily longer time required to interpret data on whole gene sequences [144]. Another problem pertains to generating a readable report of the alterations that are identified For tissue biopsies and cell blocks, serial sections should be cut, and HE-stained sections need to be analyzed by experienced pathologists to formulate definitive histologic diagnoses and evaluate the tumor cellularity.…”