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

Scott Morris, Janakiraman Subramanian, Esma Gel, George Runger, Eric Thompson, David Mallery, Glen Weiss

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background Next 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. Methods 2,723 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. Results 6.4% 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. Conclusions Specimen 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.

Original languageEnglish (US)
Article numbere0196556
JournalPLoS One
Volume13
Issue number4
DOIs
StatePublished - Apr 1 2018

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Tumors
neoplasms
Neoplasms
sampling
testing
drugs
Fine Needle Biopsy
Tumor Burden
Quality Control
Pharmaceutical Preparations
Needles
quality control
Quality control
Tissue
Mutation
mutation
Biopsy
Testing
Libraries
Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Performance of next-generation sequencing on small tumor specimens and/or low tumor content samples using a commercially available platform. / Morris, Scott; Subramanian, Janakiraman; Gel, Esma; Runger, George; Thompson, Eric; Mallery, David; Weiss, Glen.

In: PLoS One, Vol. 13, No. 4, e0196556, 01.04.2018.

Research output: Contribution to journalArticle

Morris, Scott ; Subramanian, Janakiraman ; Gel, Esma ; Runger, George ; Thompson, Eric ; Mallery, David ; Weiss, Glen. / Performance of next-generation sequencing on small tumor specimens and/or low tumor content samples using a commercially available platform. In: PLoS One. 2018 ; Vol. 13, No. 4.
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abstract = "Background Next 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. Methods 2,723 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. Results 6.4{\%} 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. Conclusions Specimen 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.",
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