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Introduction: Patients whose tumors harbor NTRK gene fusions represent an extremely small proportion of the overall cancer population (i.e., ~0.32-1%). While adult and pediatric cancer patients with NTRK fusion-positive solid tumors are rare, they comprise a patient population with a targetable alteration for whom there is currently no accepted standard of care. Comprehensive genomic profiling assays are a critical component in the identification of this sub-population, and herein we describe the clinical validation and analytic performance of FoundationOne CDx™ (F1CDx) as the companion diagnostic for entrectinib, a potent inhibitor of ROS1 and TRK A/B/C kinases. F1CDx and entrectinib were approved in Japan for the respective diagnosis and treatment of adult and pediatric patients with NTRK fusion-positive advanced recurrent solid tumors. Methods: Analytical concordance was determined by comparing F1CDx to a validated RNA NGS fusion panel using 28 NTRK fusion-positive and 53 NTRK fusion-negative samples. Concordance was defined as the overall percent agreement (OPA) between the two assays. Positive and negative percent agreement (PPA, NPA) were calculated using the RNA assay as the reference. Analytical sensitivity was calculated from representative gene fusions based on the 95% probability of detection, and intermediate precision was assessed from NTRK-positive samples run at and slightly above the platform limit of detection. Clinical validation for F1CDx as a companion diagnostic for entrectinib in Japan was estimated based upon patients in the primary efficacy population who had samples remaining for F1CDx testing using multiple imputation and sensitivity analysis of the concordance of F1CDx to the various enrollment assays. Results: OPA between F1CDx and an orthogonally validated RNA NGS fusion panel was 92.6% (95% CI: 84.6-97.2%). PPA and NPA values were 85.7% (95% CI: 67.3-96.0%) and 96.2% (95% CI: 87.0-99.5%), respectively. Platform sensitivity for rearrangements was between 6.4-11.3% tumor purity. Repeatability for NTRK fusions was 100% (95% CI: 73.5-100.0%) and reproducibility was 100% (95% CI: 90.3-100.0%). OPA, PPA and NPA values comparing F1CDx to the multiple enrollment assays utilized for the STARTRK-2 trial were 95.7% (95% CI: 87.8-99.1%), 84.2% (95% CI: 60.4-96.6%), and 100.0% (95% CI: 92.9-100.0%), respectively. The previously reported overall response rate (ORR) for the primary efficacy population in the STARTRK-2 trial was 57.4% (95% CI: 42.3-70.7%)1. Bridging analysis estimated that the ORR for the F1CDx NTRK-positive population was 50.0% (95% CI: 24.7-75.4%), with acceptable balance between co-variates in the F1CDx evaluable and non-evaluable populations. Conclusions: The DNA-based F1CDx assay demonstrated strong concordance compared to an externally validated RNA NGS fusion panel (92.6% OPA) for the detection of NTRK fusions. Furthermore, an analysis of the clinical efficacy demonstrated comparable results between F1CDx NTRK-positive patients (ORR of 50%; 95% CI: 24.7-75.4%) and the overall NTRK fusion-positive population (ORR of 57%; 95% CI: 42.3-70.7%) from the STARTRK-2 clinical trial evaluating entrectinib in patients with solid tumors. 1. ESMO [https://www.esmo.org/Oncology-News/Entrectinib-NTRK-Fusion-Positive-Tumours-Demetri] Citation Format: David Fabrizio, Coren Milbury, Wai-Ki Yip, Bahar Yilmazel, Xiaobo Bai, Juan Liao, David Smith, Christine Burns, Christine Vietz. Clinical and analytic validation of FoundationOne CDx™ for NTRK fusion-positive solid tumors in patients treated with entrectinib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A028. doi:10.1158/1535-7163.TARG-19-A028

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.

Introduction: In patients with advanced-stage ovarian cancer, FDA has recently granted approval of treatment with PARP inhibitors (PARPi) in patients harboring deleterious BRCA mutations (~25% of population). However, there is clear evidence of “BRCA-like” patients who respond to PARPi without BRCA mutations. To identify such patients, it has been shown that deficiency in homologous recombination repair leads to a common phenotype of genome-wide loss of heterozygosity (LOH). Depending on the cutoff employed, genomic LOH could identify more than twice the number of ovarian cancer patients who could benefit from PARPi than measuring BRCA alone. We present here an NGS-based platform developed and performed in compliance with FDA 21 CFR part 820. The assay provides a tumor measurement of BRCA1/2 (FDA-approved as FoundationFocus CDx BRCA) as well as genomic LOH, and is on the same platform as the comprehensive FoundationOne CDx, which interrogates 324 genes. 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 (CNA), genomic rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB). A genome-wide LOH profile based on SNPs is measured as part of the CNA pipeline, and is summarized as the percentage of the tumor genome displaying LOH (scored from 0-100%), with ≥16% being considered LOH high based on clinical data derived from ARIEL2 Part 1, a phase II study of the PARPi rucaparib for the treatment of platinum-sensitive ovarian cancer (ARIEL2; NCT01891344) Results: For analytical validity, BRCA limit of detection (LoD) was at allele frequency 5.9% for substitutions and non-repetitive indels, and 30% tumor content for LOH. Overall percent agreement with comparator NGS assay was 97.3% for BRCA. No orthogonal platform concordance was established for LOH as no validated test exists. Within-assay reproducibility was measured with overall concordance of 100% for BRCA, and 98% for LOH. Conclusion: We developed a novel diagnostic assay (in compliance with FDA 21 CFR part 820) that can measure BRCA and genomic LOH simultaneously, and established robust analytical validation data. Citation Format: James X. Sun, Kevin Lin, Yali Li, Kyle Gowen, Yuting He, Coren Milbury, Christine Burns, Jun Luo, Steve Roels, Murtaza Mehdi, John Truesdell, Pei Ma, Lakshman Ramamurthy, Christine Vietz, Jeri Beltman, Thomas Harding, Doron Lipson, Jeffrey Ross, Vincent Miller, Philip Stephens, Michael Doherty, Julia Elvin. A validated diagnostic assay for identifying ovarian cancer patients with deleterious BRCA mutations and high genomic loss of heterozygosity (LOH) [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 4544.