Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies

Abstract

To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility. Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker. High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. .

Co-occurring alterations in the RAS-MAPK pathway limit response to MET inhibitor treatment inMETexon 14 skipping mutation positive lung cancer

“…This is the largest reported cohort to-date describing the genomic landscape of advanced-stage METex14-mutated NSCLC. We evaluated the frequency with which METex14 mutations co-occur with other cancer-associated mutations ( Supplemental Table 1) (11,15). Synonymous mutations and those with predicted neutral or unknown functional impact were excluded, as previously described (11), as were mutations previously associated with clonal hematopoiesis (16).…”

“…cfDNA was extracted from whole blood collected in 10-mL Streck tubes. After double centrifugation, 5-30 ng of cfDNA was isolated for digital sequencing of either a 70 or 73-gene panel ( Supplementary Table 6) as previously described (15). Only those genes in common to these two panels were included in subsequent analysis.…”

Genomic profile of urine has high diagnostic sensitivity compared to cytology in non‐invasive urothelial bladder cancer

“…Plasma is a well‐known liquid biopsy for analyzing the genetic status of tumors . However, the abundance of cell‐free tumor DNA in plasma is low, and therefore it is possible that mutations may not be detected in early‐stage cancer.…”

Identification of driver genes and somatic mutations in cell‐free DNA of patients with pulmonary lymphangioleiomyomatosis

“…NGS analysis of cfDNA from peripheral blood is effective to capture comprehensive genomic information of tumor cells when tissue genotyping is infeasible or insufficient in metastatic disease . Targeted cfDNA NGS testing enables detection of low‐frequency somatic mutations with high sensitivity due to low tumor shedding . Currently, cfDNA sequencing has been applied in many cancer types to identify oncogenic aberrations.…”

“…16 Targeted cfDNA NGS testing enables detection of low-frequency somatic mutations with high sensitivity due to low tumor shedding. 17 Currently, cfDNA sequencing has been applied in many cancer types to identify oncogenic aberrations. Despite dramatic changes in technology, a large knowledge deficit remains in the clinical value of fluid biopsy for LAM subjects.…”