Performance of next-generation sequencing on small tumor specimens and/or low tumor content samples using a commercially available platform

Abstract

BackgroundNext generation sequencing tests (NGS) are usually performed on relatively small core biopsy or fine needle aspiration (FNA) samples. Data is limited on what amount of tumor by volume or minimum number of FNA passes are needed to yield sufficient material for running NGS. We sought to identify the amount of tumor for running the PCDx NGS platform.Methods2,723 consecutive tumor tissues of all cancer types were queried and reviewed for inclusion. Information on tumor volume, success of performing NGS, and results of NGS were compiled. Assessment of sequence analysis, mutation calling and sensitivity, quality control, drug associations, and data aggregation and analysis were performed.Results6.4% of samples were rejected from all testing due to insufficient tumor quantity. The number of genes with insufficient sensitivity make definitive mutation calls increased as the percentage of tumor decreased, reaching statistical significance below 5% tumor content. The number of drug associations also decreased with a lower percentage of tumor, but this difference only became significant between 1–3%. The number of drug associations did decrease with smaller tissue size as expected. Neither specimen size or percentage of tumor affected the ability to pass mRNA quality control. A tumor area of 10 mm2 provides a good margin of error for specimens to yield adequate drug association results.ConclusionsSpecimen suitability remains a major obstacle to clinical NGS testing. We determined that PCR-based library creation methods allow the use of smaller specimens, and those with a lower percentage of tumor cells to be run on the PCDx NGS platform.

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis

“…Tissue samples taken for pathological diagnosis, including fresh, frozen and formalin-fixed tissues, are often used for comprehensive cancer genome analysis. Recent technical developments, especially high-throughput sequencing, enables researchers to subject formalin-fixed paraffin-embedded (FFPE) tissues to cancer genome analysis using next generation sequencing (NGS) [1–4]. A major advantage of FFPE tissues over frozen tissue is that pathologists can observe the cancer lesions in the same region of the samples that is subjected to genome analysis to directly and accurately compare the histological findings and genomic profiles [5].…”

Next-Generation Sequencing May Discriminate Extreme Long-term versus Short-term Survival in Patients with Metastatic Small Cell Lung Cancer (SCLC)

“…A comprehensive NGS test was performed to analyze actionable gene mutations, copy number variations (CNVs), and mRNA expression by using the Paradigm Cancer Diagnostic's (PCDx) NGS platform according to the previously described methodology [11]. The PCDx test is a clinical-grade targeted NGS test run in a Clinical Laboratory Improvement Amendments–certified and College of American Pathologists–accredited laboratory [12]. The platform measures genomic, transcriptomic, and proteomic aberrations linked with 86 unique therapies based on published patient research information for tumors all cancer types.…”

“…The platform measures genomic, transcriptomic, and proteomic aberrations linked with 86 unique therapies based on published patient research information for tumors all cancer types. Accordingly, the PCDx test helps to guide treatment especially for tumor types that have potential targeted therapy options, like breast cancer, colorectal cancer, and NSCLC, but also SCLC, mesothelioma, and gastric cancer [12]. The sequencing was performed by using the Ion 318 chip on the Ion PGM sequencer (Thermo Fisher Scientific, Waltham, MA, USA).…”

Tissue surface area and tumor cell count affect the success rate of the Oncomine Dx Target Test in the analysis of biopsy tissue samples

“…In October 2019, it was granted expanded approval for use in the detection of mutations of EGFR on exons 18 to 21 5 . However, NGS‐based companion diagnostics sometimes fail to analyze gene alterations because of the insufficient quantity or quality of specimens; failure caused by insufficient quantity has been previously reported in 6.4% to 33.8% of cases 6,7,8 . Although an improvement of the analysis success rate is needed in this setting, few studies have reported on the specimens that are suitable for NGS‐based companion diagnostics, including ODxTT.…”