Foundationone cdx assay

CGP was performed using the FDA-approved FoundationOne CDx assay (Foundation Medicine, Cambridge, MA) in a Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited laboratory using previously described methods.^{22 (link)} Prior to nucleic acid extraction, hematoxylin and eosin-stained slides were reviewed to confirm the presence of tumor. DNA extracted from formalin-fixed paraffin-embedded tissues underwent hybrid-capture based next generation sequencing using the FoundationOne platform which interrogates all coding exons of 324 cancer-related genes and introns from 31 genes commonly rearranged in cancer. Data were analyzed for all types of genomic alterations, including base substitutions, insertions/deletions, copy number alterations, and gene rearrangements. In addition, variant-level loss of heterozygosity (LOH), tumor mutational burden (TMB), and microsatellite instability (MSI) were determined. TMB was evaluated on up to 1.1 Mb of sequenced DNA, and MSI was assessed from DNA sequencing across 95 loci as previously described.^{23 (link),24 (link)} TMB ≥ 20 mutations/Mb was considered High (TMB-High), >10 mutations/Mb considered intermediate (TMB-Int), and 0-9 mutations/Mb to be TMB low (TMB-low).

A collection of tumor samples, including both resections (archival) and post-anti-PD-1/PD-L1 progression biopsies (new), was assembled from patients during screening. Each sample was sequenced using the Foundation Medicine NGS FoundationOneCDx assay^{68 (link)}. The assay detects mutations and copy number variations in 324 cancer-related genes and selected rearrangements. The assay also provides information on microsatellite instability and TMB. Detailed information on the assay and the variant calling pipeline are available at ref. ⁷³ . TMB is reported as low (<10 mutations Mb⁻¹) or high (≥10 mutations Mb⁻¹). Mutation profiles included co-occurring and mutually exclusive gene alterations. For each gene, substitutions, short insertions and deletions, rearrangements and copy number changes of known or likely functional relevance detected using the assay were included. Additionally, tumor PD-L1 expression was assessed locally using PD-L1 immunohistochemistry assays. PD-L1 expression was evaluated in TCs and considered positive when expressed in ≥1% of neoplastic cells.