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Related Experiment Video

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Reference Samples to Compare Next-Generation Sequencing Test Performance for Oncology Therapeutics and Diagnostics.

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  • 1Department of Pathology, Washington University School of Medicine, St Louis, MO, USA.

American Journal of Clinical Pathology
|December 6, 2021
PubMed
Summary

This study evaluated next-generation sequencing (NGS) laboratory-developed tests (LDTs) using reference samples. Results showed variable accuracy in detecting genetic variants, highlighting the need for improved NGS LDT performance for targeted cancer therapies.

Keywords:
Companion diagnosticNext-generation sequencingPrecision medicineReference materialsTest performance

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Area of Science:

  • Molecular diagnostics
  • Genomics
  • Oncology

Background:

  • Laboratory-developed tests (LDTs) using next-generation sequencing (NGS) are crucial for selecting targeted therapies.
  • Concerns exist regarding the accuracy and standardization of these NGS LDTs across clinical laboratories.

Purpose of the Study:

  • To assess the performance of NGS LDTs in clinical laboratories.
  • To evaluate the accuracy of variant detection for targeted therapy selection.
  • To introduce traceable reference samples for NGS LDT validation.

Main Methods:

  • Engineered human cell lines (wet samples) and in silico mutagenized sequence files (dry samples) were used as reference materials.
  • CRISPR/Cas9 technology was employed for cell line engineering.
  • Single and multinucleotide variants in KRAS and NRAS genes were tested at varying allele fractions.

Main Results:

  • Twenty-one laboratories participated, testing both wet and dry samples.
  • Only 37% of laboratories correctly reported all variants.
  • The majority of errors were false negatives, indicating underdetection of genetic variants.

Conclusions:

  • Genetically engineered cell lines and in silico data serve as valuable complementary reference samples for NGS LDT evaluation.
  • Variable accuracy in variant detection among NGS LDTs can impact patient stratification for targeted therapies.
  • Standardized performance evaluation is essential for reliable NGS LDTs in clinical practice.