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Introduction: Increase in targeted therapies has resulted in the need for a single assay capable of detecting diverse biomarkers indicated for these agents. Comprehensive genomic profiling (CGP) provides such a solution, but due to the complexity and number of assays available today, standardization of validation has become critically important. We present FoundationOne CDx, the first NGS-based comprehensive companion diagnostics (CDx) platform developed and performed in compliance with FDA 21 CFR part 820. The assay interrogates 324 genes, and has CDx indications in five tumor types associated with 17 targeted therapies (Table 1). The versatile assay design will facilitate streamlined development of future CDx indications. Methods: DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB). Results: Clinical validity was established such that the concordance between CGP and approved CDx were statistically non-inferior to that of two runs of approved CDx. For analytical validity, limit of detection (LoD) was at allele frequency 4% for known substitutions and indels. LoD was 16% tumor content for copy number amplifications, 30% for homozygous deletions, 11% for rearrangements, 12% for MSI, and 20% for TMB. Concordance with an orthogonal NGS platform was 94.6% for substitutions and indels. Within-assay reproducibility had PPA 99.4%. Conclusion: Rapid expansion of targeted therapies and CDx has necessitated a new approach and urgency to defining performance standards. We developed a comprehensive CDx assay and demonstrated clinical and analytical validity to support and accelerate using CGP for routine clinical care. Table 1. Companion Diagnostic IndicationsIndicationBiomarkerTherapyNon-small cell lung cancer (NSCLC)EGFR exon 19 deletions and EGFR exon 21 L858R alterationsafatinib, gefitinib, or erlotinibEGFR exon 20 T790M alterationsosimertinibALK rearrangementsalectinib, crizotinib, or ceritinibBRAF V600Edabrafenib in combination with trametinibMelanomaBRAF V600Edabrafenib, vemurafenibBRAF V600E and V600Ktrametinib, cobimetinib, in combination with vemurafenibBreast cancerERBB2 (HER2) amplificationtrastuzumab, ado-trastuzumab-emtansine, or pertuzumabColorectal cancerKRAS wild-type (absence of mutations in codons 12 and 13)cetuximabKRAS and NRAS wild-type (absence of mutations in exons 2, 3, and 4)panitumumabOvarian cancerBRCA1/2 alterationsrucaparib Citation Format: James X. Sun, Yali Li, Coren Milbury, Joel Skoletsky, Christine Burns, Wai-ki Yip, Jun Luo, Ninad Dewal, Adrienne Johnson, Kyle Gowen, Jing Tong, Yuting He, Jie He, Pei Ma, Jared White, Steve Roels, John Truesdell, Eric Peters, Houston Gilbert, Charlie Wu, Erica Schleifman, Johannes Noe, Carl Barrett, Kenneth Thress, Suzanne Jenkins, Julia Elvin, Geoff Otto, Doron Lipson, Jeffrey Ross, Vincent Miller, Philip Stephens, Michael Doherty, Christine Vietz. A clinically validated comprehensive companion diagnostic platform for care of patients with advanced cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4757.

Introduction: Patients with HER2-positive breast cancer may benefit from targeted therapies including trastuzumab and pertuzumab. While FDA has approved companion diagnostics (CDx) using FISH or IHC for identifying ERBB2 amplifications, molecular diagnostic testing in breast cancer is rapidly evolving towards comprehensive genomic profiling (CGP) due to an increasing number of biomarkers. However, clinical validity of ERBB2 testing in CGP has not yet been demonstrated in an FDA-approved manner. We present the first ERBB2 test using NGS as a follow-on CDx, which is part of FoundationOne CDx (F1CDx). Methods: Clinical validity was established against the FDA-approved HER2 FISH test, using retrospective breast cancer patient samples. For each sample, two tests using the FISH assay were performed, and clinical validity was established such that the F1CDx result is statistically non-inferior to the performance between two runs of FISH. Concordance was calculated on samples that agree between the two FISH tests. Results: Results are shown in Table 1. An exploratory analysis including low-level ERBB2 amplifications (CN=4; ploidy+2) detected by F1CDx demonstrated an improved PPA of 93.8%. A non-inferiority test demonstrated a margin of 8.0%. Further analysis showed that the discordance is most significant among samples with a low FISH ratio. In samples that are FISH-positive but F1CDx-negative, a second test of FISH only yields a FISH-FISH concordance of only 55%, suggesting significantly decreased FISH-FISH reproducibility. Conclusion: We presented a follow-on CDx for ERBB2. The F1CDx platform provides a single assay that identifies cancer patients who may be eligible to receive FDA-approved targeted therapeutics, conserves tissue by avoiding serial testing and can serve as clinical trial assay for studies requiring a molecular biomarker for eligibility. The data demonstrating clinical and analytical validity of ERBB2 may accelerate the use of CGP for routine clinical use. Table 1. The ERBB2 companion diagnostic in FoundationOne CDxCompanion diagnosticERBB2 amplificationIndicated useTrastuzumab, ado-trastuzumab-emtansine, or pertuzumab in breast cancerApproved CDx comparatorHER2 FISH PharmDx® Kit (Dako Denmark A/S)Positive percent agreement (PPA)89.4% (101/113)Negative percent agreement (NPA)98.4% (180/183)Median unique sequence coverage>500XPrecision100%Limit of Detection19.7% tumor content Citation Format: Wai-ki Yip, Joel Skoletsky, Pei Ma, Jun Luo, Coren Milbury, Christine Burns, John Truesdell, Julia Elvin, Geoff Otto, Doron Lipson, Jeffrey Ross, Vincent Miller, Philip Stephens, Michael Doherty, Christine Vietz, James X. Sun, Yali Li. An ERBB2 follow-on companion diagnostic for clinical care of patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1607.