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Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses
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Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

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Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular

Vincent Plagnol1, Samuel Woodhouse1, Karen Howarth1

  • 1Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom.

Plos One
|March 16, 2018
PubMed

Insights

The InVisionFirst assay accurately detects low-frequency mutations in circulating tumor DNA (ctDNA) for cancer care. This highly sensitive and specific assay is validated for clinical use in profiling patients and guiding treatment decisions.

Area of Science:

  • Oncology
  • Genomics
  • Molecular Diagnostics

Background:

  • Circulating tumor DNA (ctDNA) analysis is crucial for cancer patient profiling and guiding targeted therapy decisions.
  • Detecting low-frequency mutations (below 0.5% VAF) in ctDNA requires highly sensitive analytical methods for clinical utility.
  • Objective assessment of assay performance necessitates rigorous analytical validation.

Purpose of the Study:

  • To develop and analytically validate the InVisionFirst™ assay for profiling actionable genomic alterations in cell-free DNA.
  • To assess the assay's sensitivity, specificity, and robustness in detecting various mutation types across a range of variant allele fractions (VAFs).
  • To confirm the assay's suitability for clinical applications in cancer care.

Main Methods:

  • Development of the InVisionFirst™ assay utilizing enhanced tagged amplicon sequencing (eTAm-Seq™) technology.
  • Profiling of 36 cancer-associated genes for point mutations, indels, amplifications, and gene fusions in cell-free DNA.
  • Analytical validation using patient samples (non-small cell lung cancer patients and healthy donors) and contrived samples with known VAFs, compared against digital PCR (dPCR).

Main Results:

  • The InVisionFirst assay demonstrated high sensitivity: 99.48% for SNVs at 0.25%-0.33% VAF and 92.46% for indels at 0.25% VAF.
  • Exceptional specificity (99.9997% per base) was maintained, with high detection rates at low VAFs.
  • The assay successfully detected gene fusions (ALK, ROS1) and amplifications (ERBB2, FGFR1, MET, EGFR) with high sensitivity and specificity, showing strong concordance with dPCR.

Conclusions:

  • The InVisionFirst assay is a highly sensitive, specific, and robust method for detecting actionable genomic alterations in ctDNA.
  • The assay meets the analytical requirements for clinical applications, supporting its use in cancer patient profiling and treatment guidance.
  • This validated assay enables reliable detection of low-frequency mutations, advancing precision oncology.

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