FoundationOne Liquid CDx

{0} PMA P200016: FDA Summary of Safety and Effectiveness Data 1 of 49 # SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) ## I. GENERAL INFORMATION Device Generic Name: Next generation sequencing oncology panel, somatic or germline variant detection system Device Trade Name: FoundationOne® Liquid CDx (F1 Liquid CDx) Device Procode: PQP Applicant's Name and Address: Foundation Medicine, Inc. 150 Second Street Cambridge, MA 02141 Date(s) of Panel Recommendation: None Premarket Approval Application (PMA) Number: P200016 Date of FDA Notice of Approval: November 6, 2020 The FoundationOne® Liquid CDx was approved on August 26, 2020 as a companion diagnostic for BRCA1 and BRCA2 alterations in metastatic castration-resistant prostate cancer (mCRPC) patients who may benefit from treatment with RUBRACA® (rucaparib) and EGFR activating mutations (Exon 19 deletions and L858R substitution mutation) in patients with advanced and metastatic non-small cell lung cancer (NSCLC) who may benefit from treatment with IRESSA® (gefitinib), TAGRISSO® (osimertinib), and TARCEVA® (erlotinib). On October 26, 2020 the FoundationOne® Liquid CDx test was approved as a companion diagnostic for BRCA1 and BRCA2 alterations in epithelial ovarian cancer for patients who may benefit from treatment with RUBRACA® (rucaparib), ALK rearrangements in non-small cell lung cancer for patients who may benefit from treatment with ALECENSA® (alectinib), and PIK3CA mutations patients with breast cancer who may benefit from treatment with PIQRAY® (alpelisib). The current PMA was submitted to include the intended use of FoundationOne® Liquid CDx as a companion diagnostic for the indications listed in the table below: New Indication Being Sought in this PMA submission. | Biomarker(s) Detected | Therapy | Tumor Type | | --- | --- | --- | | BRCA1, BRCA2, and ATM alterations | Lynparza® (olaparib) | Prostate Cancer | ## II. INDICATIONS FOR USE FoundationOne® Liquid CDx is a qualitative next generation sequencing based in vitro diagnostic test that uses targeted high throughput hybridization-based capture technology {1} to detect and report substitutions, insertions and deletions (indels) in 311 genes, including rearrangements in four (4) genes, and copy number alterations in three (3) genes. FoundationOne® Liquid CDx utilizes circulating cell-free DNA (cfDNA) isolated from plasma derived from anti-coagulated peripheral whole blood of cancer patients collected in FoundationOne® Liquid CDx cfDNA blood collection tubes included in the FoundationOne® Liquid CDx Blood Sample Collection Kit. The test is intended to be used as a companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Table 1: Companion diagnostic indications | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Non-small cell lung cancer (NSCLC) | ALK Rearrangements | ALECENSA® (alectinib) | | | EGFR Exon 19 deletions and EGFR Exon 21 L858R alteration | IRESSA® (gefitinib) TAGRISSO® (osimertinib) TARCEVA® (erlotinib) | | Prostate cancer | BRCA1, BRCA2, and ATM alterations | LYNPARZA® (olaparib) | | | BRCA1, BRCA2 alterations | RUBRACA® (rucaparib) | | Ovarian Cancer | BRCA1, BRCA2 alterations | RUBRACA® (rucaparib) | | Breast Cancer | PIK3CA mutations C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y | PIQRAY® (alpelisib) | Additionally, FoundationOne® Liquid CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. A negative result from a plasma specimen does not mean that the patient's tumor is negative for genomic findings. Patients who are negative for the mutations listed in Table 1 should be reflexed to routine biopsy and their tumor mutation status confirmed using an FDA-approved tumor tissue test, if feasible. Genomic findings other than those listed in Table 1 of the intended use statement are not prescriptive or conclusive for labeled use of any specific therapeutic product. FoundationOne® Liquid CDx is a single-site assay performed at Foundation Medicine, Inc. in Cambridge, MA. ## III. CONTRAINDICATIONS There are no known contraindications. ## IV. WARNINGS AND PRECAUTIONS - Alterations reported may include somatic (not inherited) or germline (inherited) alterations; however, the test does not distinguish between germline and somatic PMA P200016: FDA Summary of Safety and Effectiveness Data {2} alterations. If a reported alteration is suspected to be germline, confirmatory testing should be considered in the appropriate clinical context. - The test is not intended to replace germline testing or to provide information about cancer predisposition. - Patients for whom no companion diagnostic alterations are detected should be considered for confirmation with an FDA-approved tumor tissue test, if possible. # V. DEVICE DESCRIPTION The FoundationOne® Liquid CDx assay is performed exclusively as a laboratory service using circulating cell-free DNA (cfDNA) isolated from plasma derived from anti-coagulated peripheral whole blood from patients with solid malignant neoplasms. The assay employs a single DNA extraction method to obtain cfDNA from plasma from whole blood. Extracted cfDNA undergoes whole-genome shotgun library construction and hybridization-based capture of 324 cancer-related genes. All coding exons of 309 genes are targeted; select intronic or non-coding regions are targeted in three genes (refer to Table 2 for the complete list of genes reported by FoundationOne® Liquid CDx). Hybrid-capture selected libraries are sequenced with deep coverage using the NovaSeq 6000 platform. Sequence data are processed using a custom analysis pipeline designed to detect genomic alterations, including base substitutions and indels in 311 genes, copy number variants in three genes, and genomic rearrangements in four genes. A subset of targeted regions in 75 genes is baited for increased sensitivity. Table 2: Genomic Regions in which Variants are Reported by FoundationOne® Liquid | ABL1 [Exons 4-9] | ACVR1B | AKT1 [Exon 3] | AKT2 | AKT3 | ALK [Exons 20-29, Introns 18,19] | ALOX12B | AMER1 (FAM123B) | APC | AR | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ARAF [Exons 4, 5, 7, 11, 13, 15, 16] | ARFRP1 | ARID1A | ASXL1 | ATM | ATR | ATRX | AURKA | AURKB | AXIN1 | | AXL | BAP1 | BARD1 | BCL2 | BCL2L1 | BCL2L2 | BCL6 | BCOR | BCORL1 | BCR* [Introns 8, 13, 14] | | BRAF [Exons 11-18, Introns 7-10] | BRCA1 [Introns 2, 7, 8, 12, 16, 19, 20] | BRCA2 [Intron 2] | BRD4 | BRIP1 | BTG1 | BTG2 | BTK [Exons 2, 15] | C11orf30 (EMSY) | C17orf39 (GID4) | | CALR | CARD11 | CASP8 | CBFB | CBL | CCND1 | CCND2 | CCND3 | CCNE1 | CD22 | | CD70 | CD74* [Introns 6-8] | CD79A | CD79B | CD274 (PD-L1) | CDC73 | CDH1 | CDK12 | CDK4 | CDK6 | | CDK8 | CDKN1A | CDKN1B | CDKN2A | CDKN2B | CDKN2C | CEBPA | CHEK1 | CHEK2 | CIC | | CREBBP | CRKL | CSF1R | CSF3R | CTCF | CTNNA1 | CTNNB1 [Exon 3] | CUL3 | CUL4A | CXCR4 | | CYP17A1 | DAXX | DDR1 | DDR2 [Exons 5, 17, 18] | DIS3 | DNMT3A | DOT1L | EED | EGFR [Introns 7, 15, 24-27] | EP300 | PMA P200016: FDA Summary of Safety and Effectiveness Data {3} PMA P200016: FDA Summary of Safety and Effectiveness Data 4 of 49 | EPHA3 | EPHB1 | EPHB4 | ERBB2 | ERBB3 [Exons 3, 6, 7, 8, 10, 12, 20, 21, 23, 24, 25] | ERBB4 | ERCC4 | ERG | ERRF11 | ESR1 [Exons 4-8] | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ETV4* [Intron 8] | ETV5* [Introns 6, 7] | ETV6* [Introns 5, 6] | EWSR1* [Introns 7-13] | EZH2 [Exons 4, 16, 17, 18] | EZR* [Introns 9-11] | FAM46C | FANCA | FANCC | FANCG | | FANCL | FAS | FBXW7 | FGF10 | FGF12 | FGF14 | FGF19 | FGF23 | FGF3 | FGF4 | | FGF6 | FGFR1 [Introns 1, 5, Intron 17] | FGFR2 [Intron 1, Intron 17] | FGFR3 [Exons 7, 9 (alternative designation exon 10), 14, 18, Intron 17] | FGFR4 | FH | FLCN | FLT1 | FLT3 [Exons 14, 15, 20] | FOXL2 | | FUBP1 | GABRA6 | GATA3 | GATA4 | GATA6 | GNA11 [Exons 4, 5] | GNA13 | GNAQ [Exons 4, 5] | GNAS [Exons 1, 8] | GRM3 | | GSK3B | H3F3A | HDAC1 | HGF | HNF1A | HRAS [Exons 2, 3] | HSD3B1 | ID3 | IDH1 [Exon 4] | IDH2 [Exon 4] | | IGF1R | IKBKE | IKZF1 | INPP4B | IRF2 | IRF4 | IRS2 | JAK1 | JAK2 [Exon 14] | JAK3 [Exons 5, 11, 12, 13, 15, 16] | | JUN | KDM5A | KDM5C | KDM6A | KDR | KEAP1 | KEL | KIT [Exons 8,9,11,12, 13, 17, Intron 16] | KLHL6 | KMT2A (MLL) [Introns 6, 8-11, Intron 7] | | KMT2D (MLL2) | KRAS | LTK | LYN | MAF | MAP2K1 (MEK1) [Exons 2, 3] | MAP2K2 (MEK2) [Exons 2-4, 6, 7] | MAP2K4 | MAP3K1 | MAP3K13 | | MAPK1 | MCL1 | MDM2 | MDM4 | MED12 | MEF2B | MEN1 | MERTK | MET | MITF | | MKNK1 | MLH1 | MPL [Exon 10] | MRE11A | MSH2 [Intron 5] | MSH3 | MSH6 | MST1R | MTAP | MTOR [Exons 19, 30, 39 40, 43-45, 47, 48, 53, 56] | | MUTYH | MYB* [Intron 14] | MYC [Intron 1] | MYCL (MYCL1) | MYCN | MYD88 [Exon 4] | NBN | NF1 | NF2 | NFE2L2 | | NFKBIA | NKX2-1 (TTF-1) | NOTCH1 | NOTCH2 [Intron 26] | NOTCH3 | NPM1 [Exons 4-6, 8, 10] | NRAS [Exons 2, 3] | NSD3 (WHSC1L1) | NT5C2 | NTRK1 [Exons 14, 15, Introns 8-11] | | NTRK2 [Intron 12] | NTRK3 [Exons 16, 17] | NUTM1* [Intron 1] | P2RY8 | PALB2 | PARK2 | PARP1 | PARP2 | PARP3 | PAX5 | | PBRM1 | PDCD1 (PD-1) | PDCD1L G2 (PD-L2) | PDGFRA [Exons 12, 18, Introns 7, 9, 11] | PDGFRB [Exons 12-21, 23] | PDK1 | PIK3C2B | PIK3C2G | PIK3CA [Exons 2, 3, 5-8, 10, 14, 19, 21 (Coding Exons 1, 2, 4-7, 9, 13, 18, 20)] | PIK3CB | | PIK3R1 | PIM1 | PMS2 | POLD1 | POLE | PPARG | PPP2R1A | PPP2R2A | PRDM1 | PRKAR1A | {4} The reporting of rearrangements and copy number alterations are restricted to those genes included in Table 3, below. Table 3: Genes for which copy number alterations and rearrangements are reported for tumor profiling by FoundationOne® Liquid CDx | Alteration Type | Genes | | --- | --- | | Copy Number Alterations | BRCA1, BRCA2, ERBB2 | | Rearrangements | ALK, BRCA1, BRCA2 | The test report includes variants reported in the following categories; see Table 4: Table 4. Category Definitions | Category | FoundationOne® Liquid CDx | | | | Comments | | --- | --- | --- | --- | --- | --- | | | Prescriptive use for a Therapeutic Product | Clinical Performance | Analytical Performance | | | | Category 1: Companion Diagnostic (CDx) | Yes | Yes | Yes | ctDNA biomarkers linked to the safe and effective use of the corresponding therapeutic product, for which FoundationOne® Liquid CDx has demonstrated clinical performance shown to support therapeutic efficacy and strong analytical performance for the biomarker. | | | Category 2: | No | No | Yes | ctDNA biomarkers with strong evidence of clinical significance | | | Category 3: | No | No | Yes | ctDNA biomarkers with strong evidence of clinical significance | | | Category 4: | No | No | Yes | ctDNA biomarkers with strong evidence of clinical significance | | PMA P200016: FDA Summary of Safety and Effectiveness Data {5} | Category | FoundationOne® Liquid CDx | | | Comments | | --- | --- | --- | --- | --- | | | Prescriptive use for a Therapeutic Product | Clinical Performance | Analytical Performance | | | ctDNA Biomarkers with Strong Evidence of Clinical Significance in ctDNA | | | | presented by other FDA-approved liquid biopsy companion diagnostics for which FoundationOne® Liquid CDx has demonstrated analytical reliability but not clinical performance. | | Category 3A: Biomarkers with Evidence of Clinical Significance in tissue supported by strong analytical validation using ctDNA | No | No | Yes | ctDNA biomarkers with evidence of clinical significance presented by tissue-based FDA-approved companion diagnostics for which FoundationOne® Liquid CDx has demonstrated analytical performance including analytical accuracy, and concordance of blood-based testing to tissue-based testing for the biomarker. | | Category 3B: Biomarkers with Evidence of Clinical Significance in tissue supported by analytical validation using ctDNA | No | No | Yes | ctDNA biomarkers with evidence of clinical significance presented by tissue-based FDA-approved companion diagnostics for which FoundationOne® Liquid has demonstrated minimum analytical performance including analytical accuracy. | | Category 4: Other Biomarkers with Potential Clinical Significance | No | No | Yes | ctDNA biomarkers with emergent evidence based on peer-reviewed publications for genes/variants in tissue, variant information from well-curated public databases, or in-vitro pre-clinical models, for which FoundationOne® Liquid CDx has demonstrated minimum analytical performance. | ## FoundationOne® Liquid cfDNA CDx Blood Specimen Collection Kit Contents The test includes a blood specimen collection kit, which is sent to ordering laboratories. The shipping kit contains the following components: - Specimen preparation and shipping instructions - Two FoundationOne® Liquid CDx cfDNA Blood Collection Tubes (8.5 mL nominal fill volume per tube) - Return shipping label ## Instruments The FoundationOne® Liquid CDx assay is intended to be performed with the serial number-controlled instruments indicated in Table 5, below. All instruments are qualified by Foundation Medicine, Inc. (Foundation Medicine or FMI) under Foundation Medicine's Quality System. PMA P200016: FDA Summary of Safety and Effectiveness Data {6} Table 5: Instruments for use with the FoundationOne® Liquid CDx assay | Instrument | | --- | | Illumina NovaSeq 6000 | | Beckman Biomek NXP Span-8 Liquid Handler | | Thermo Scientific Kingfisher Flex DW 96 | | Bravo Benchbot | | Hamilton STARlet STAR Liquid Handling Workstation | ## Test Process All assay reagents included in the FoundationOne® Liquid CDx assay process are qualified by Foundation Medicine and are compliant with the medical device Quality System Regulation (QSR). ## A. Specimen Collection and Preparation Whole blood specimens are collected in FoundationOne® Liquid CDx cfDNA Blood Collection Tubes (BCT) provided as a component of the FoundationOne® Liquid CDx specimen collection kit. Prior to cfDNA isolation, the plasma is separated from whole blood by centrifugation, which separates the plasma from the buffy coat (white blood cells) and red blood cells. The plasma layer is removed from the buffy coat to avoid contamination of cellular DNA into the plasma sample. A residual volume of plasma remains in the tube to avoid disturbing the buffy coat. A second spin of the separated plasma at high speed further pellets cell debris and protein. ## B. DNA Extraction Following the separation of plasma from whole blood, cfDNA is isolated from plasma using the KingFisher™ Flex Magnetic Particle Processor, which uses an efficient and automated method to purify cfDNA. The KingFisher™ Instrument uses magnetic rods to move nucleic acid through purification phases of binding, washing, and elution to yield high purity cfDNA. After isolating cfDNA, the Agilent 4200 TapeStation is used to quantify cfDNA. ## C. Library Construction Library Construction (LC) begins with the normalization of cfDNA. The samples are purified, using AMPure XP Beads (Agencourt). Solid-phase reversible immobilization (SPRI) purification is used subsequent to library construction with the NEBNext kits (NEB), including mixes for end repair with blunt-end and 5'-phosphorylate the cfDNA fragments using T4 Polynucleotide Kinase and T4 DNA Polymerase. This step prepares the 3'-end for dA-addition while also preparing the 5'-end of the DNA fragment for ligation. Second, dA-addition will incorporate a single dAMP to the 3'-end of the End-Repaired material. After dA-addition, a universal Y-adaptor is ligated onto each end of the DNA fragment using a DNA ligase. These steps are performed in 96-well plates (Eppendorf) on a Bravo Benchbot (Agilent) using the "with-bead" protocol to maximize reproducibility and library yield. Indexed (Foundation Medicine customized six base pair barcodes) sequencing libraries are PCR amplified with a high-fidelity DNA polymerase (HiFi™, Kapa) for ten cycles, SPRI purified and quantified by PicoGreen fluorescence assay PMA P200016: FDA Summary of Safety and Effectiveness Data {7} (Invitrogen). Process matched control (PMC) is prepared and added to the plate with other cfDNA samples at the beginning of LC. ## D. Hybrid Capture Hybrid Capture begins with the normalization of each library from 500 ng to 2000 ng. Solution hybridization is performed using a &gt;50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 base pair oligonucleotides (Integrated DNA Technology) for baits. The baits target regions from 324 cancer-related genes including all coding exons of 309 genes and only select introns or non-coding regions in 15 genes. Baits were designed by appointing overlapping 120 bp DNA sequence intervals covering target exons (60 bp overlap) and introns (20 bp overlap), with a minimum of three baits per target; single nucleotide polymorphism (SNP) targets were allocated one bait each. Intronic baits were filtered for repetitive elements as defined by the University of California at Santa Cruz (UCSC) Genome Repeat Masker track. Hybrid selection of targets demonstrating reproducibly low coverage was boosted by increasing the number of baits for these targets. Upon completion of the pre-capture normalization, blocking DNA (adaptor block, Cot, Salmon Sperm DNA) is added to the sequencing library and the mixture is lyophilized in a 96-well plate. The library is then re-suspended in nuclease-free water, heat denatured at 95°C for 5 minutes, temperature ramps from 95°C to 68°C to anneal blocking DNA, and then the samples are incubated at 68°C for a minimum of 5 minutes before the addition of the bait set reagent. After a 20-24-hour incubation, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen) and off-target library is removed by washing one time with Saline Sodium Citrate (SSC) at 25°C and four times with SSC at 55°C. The PCR master mix is added to directly amplify the captured library from the washed beads. After amplification, the samples are SPRI purified and quantified by PicoGreen. ## E. Sequencing Sequencing on the Illumina NovaSeq 6000 platform employs on-board cluster generation (OBCG) using patterned flow cell (FC) technology to generate monoclonal clusters via ExAmp from a single DNA template. The clusters are then sequenced using sequencing by synthesis (SBS) chemistry. The NovaSeq system is capable of sequencing up to two flow cells at a time. During OBCG, a single DNA template is introduced into each of the primer substrate layered nanowells of the flow cell, where the template is immediately and rapidly amplified by ExAmp. This rapid amplification prevents other DNA templates from binding, ensuring a monoclonal cluster is formed in each nanowell. The procedure allows for fixed size and spacing of the clusters which results in improved and more accurate resolution. A growing nucleotide chain is created on the flow cell by incorporating fluorescently labeled, 3'-blocked dNTPs. After excitation by a laser, the camera captures the emission color of the incorporated, fluorescently labeled nucleotide. The 3'-block is then removed, reverting the nucleotide to its natural form, which allows the polymerase to add another base to the growing double strand of DNA. With each PMA P200016: FDA Summary of Safety and Effectiveness Data 8 of 49 {8} successive SBS cycle, a new fluorescently labeled 3'-blocked dNTP is added. SBS allows for millions of discrete clusters of clonal copies of DNA to be sequenced in parallel. ## F. Sequence Analysis Sequence data are analyzed using mainly proprietary software developed by Foundation Medicine. External tools used include: 1) BWA (Burrows-Wheeler Aligner) v0.7.17, for aligning sequence reads to the genomic reference, 2) Samtools v1.6 for utility operations, 3) Picard tools v1.56 for metrics calculations, and 4) Biopython for the pairwise2 sequence alignment module. Reads from each Illumina flow cell are demultiplexed (sorted into sets of reads deriving from distinct samples), and their fragment barcodes (FBCs) are extracted and encoded into the read names. For each sample, read pairs with matching, valid FBCs are aligned and processed together to: 1) identify clusters of reads originating from the same original fragment; 2) merge overlapping read pairs into single reads, where possible; and 3) generate consensus reads representing all information in the set of reads for each cluster, encoding positions with mismatches (errors) with base quality 20. The consensus reads are then aligned to the reference genome to generate the 'consensus' BAM. For the detection of short variants (e.g., substitutions and small indels) in each target region of interest, a de novo assembly is performed. This is done using proprietary software to generate a de Bruijn graph including all k-mers in reads mapping to a particular locus. The graph is parsed to identify paths that originate and terminate in reference nodes from the locus. Increased k-mer sizes may be used to account for ambiguities, cycles, and other problematic regions within the graph. The result of the graph traversal is a set of candidate variants. For each variant, there is a set of k-mers supporting the variant and a set of k-mers that would support the reference or another variant at the location. Each candidate variant is then scanned against reads in the locus to identify which reads support either the candidate variant or a different variant or reference at the location. The cluster membership of the supporting reads is then assessed to determine which clusters show unambiguous support for the variant and which have conflicting assignments, indicating that the variant may have arisen as an error in sequencing or library preparation. The final variant calls are made based on a model that takes into account the coverage at the location, the number of supporting read clusters and their redundancy level, and the number of error-containing clusters. ## G. Report Generation Approved results are annotated by automated software with CDx relevant information and are merged with patient demographic information and any additional information provided by Foundation Medicine as a professional service prior to approval and release by the laboratory director or designee. PMA P200016: FDA Summary of Safety and Effectiveness Data 9 of 49 {9} # H. Internal Process Controls # Positive Control Each assay run includes a control sample run in duplicate. The control sample contains a pool of eleven HapMap cell lines and is used as a positive mutation detection control. 100 different germline SNPs present across the entire targeted region are required to be detected by the analysis pipeline. # Sensitivity Control The HapMap control pool used as the positive control is prepared to contain variants at $0.1\%$ , $10\%$ mutant allele frequency (MAF) which must be detected by the analysis pipeline to ensure expected sensitivity for each run. # Negative Control Samples are barcoded molecularly at the library construction (LC) stage. Only reads with a perfect molecular barcode sequence are incorporated into the analysis. The Analysis Pipeline includes an algorithm that analyzes the SNP profile of each specimen to identify potential contamination that may have occurred prior to molecular barcoding. # I. CDx Classification Criteria 1. BRCA1 and BRCA2 alterations to identify patients eligible for rucaparib in prostate and ovarian cancer: The CDx classification criteria and the list of BRCA1/BRCA2 missense mutations for rucaparib, based on the trial prespecifications are described in Table 6 and Table 7; however, not all the missense mutations listed below were observed in the TRITON2, ARIEL2, and PROfound clinical studies. Table 6: Classification Criteria for Deleterious Tumor BRCA Variants | Qualification Criteria | Sequence Classification | Methodology | | --- | --- | --- | | A BRCA1 or BRCA2 alteration that includes any of the sequence classifications | Protein truncating mutations | Sequence analysis identifies premature stop codons anywhere in the gene coding region, except: 3’ of and including BRCA2 K3326* | | | Splice site mutations | Sequence analysis identifies variant splice sequences at intron/exon junctions -/+ 2bp of exon starts/ends | | | Homozygous deletions | Sequence analysis identifies deletions in both gene alleles of ≥1 exon in size | | | Large protein truncating rearrangements | Sequence analysis identifies protein truncating rearrangements | | | Deleterious missense mutations | Curated list | PMA P200016: FDA Summary of Safety and Effectiveness Data {10} Table 7: Deleterious BRCA Missense Alterations in rucaparib | BRCA1 Alterations (Protein Change) | | | | | BRCA2 Alterations (Protein Change) | | | | --- | --- | --- | --- | --- | --- | --- | --- | | M1V | C44Y | R71T | R1699W | G1770V | M1V | R2336P | T2722R | | M1T | C44F | R71M | R1699Q | M1775K | M1T | R2336L | D2723H | | M1R | C47S | S770L | G1706R | M1775R | M1R | R2336H | D2723G | | M1I | C47Y | R1495T | G1706E | C1787S | M1I | T2412I | G2724W | | M18T | C47F | R1495M | A1708E | G1788V | D23N | R2602T | G2748D | | L22S | C61S | R1495K | S1715R | P1812A | D23Y | W2626C | A2911E | | I26N | C61G | E1559K | S1722F | A1823T | S142N | I2627F | E3002K | | T37K | C61Y | E1559Q | V1736A | V1833M | S142I | R2659T | R3052W | | C39R | C64R | T1685A | G1738R | W1837R | V159M | R2659K | D3095G | | C39G | C64G | T1685I | G1738E | V1838E | V211I | E2663V | D3095E | | C39Y | C64Y | D1692N | K1759N | | V211L | S2670L | N3124I | | C39W | C64W | M1689R | L1764P | | Y600C | I2675V | N3187K | | H41R | R71G | D1692H | I1766N | | K1530N | T2722K | | | C44S | R71K | D1692Y | I1766S | | | | | 2. ATM, BRCA1 and BRCA2 alterations to identify patients eligible for olaparib in mCRPC: Table 8: Rules Applied to the Aforementioned Genes: | Qualification Criteria | Sequence Classification | Methodology | Comments | | --- | --- | --- | --- | | A gene alteration that includes any of the sequence classifications | Protein truncating mutations | Sequence analysis identifies premature stop codons anywhere in the gene coding region, except: 3' of and including BRCA2 K3326* | Does not include VUS.Includes mutations on the canonical transcript only for genes ATM, BRCA1, and BRCA2. | | | Splice site mutations | Sequence analysis identifies variant splice sequences at intron/exon junctions -/+ 2bp of exon starts/ends | Does not include VUS. Includes indels that extend through ±2bp from the intron/exon junction.Includes mutations on the canonical transcript only for genes ATM, BRCA1, and BRCA2. | | | Homozygous deletions | Sequence analysis identifies deletions in both gene alleles of ≥1 exon in size | Does not include VUS Only reported for BRCA1&2.Not reported for ATM. | | | Large protein truncating rearrangements | Sequence analysis identifies protein truncating rearrangements | Does not include VUS | | | Deleterious missense mutations | Curated list | Protein effects from list of missense mutations on the canonical transcript only for genes ATM, BRCA1, and BRCA2. | PMA P200016: FDA Summary of Safety and Effectiveness Data {11} Alterations reported are limited to those within the alteration-calling capabilities of FMI as of March 2, 2020. ATM missense mutations were identified from the ClinVar database. Should the calling capabilities expand, additional alterations that meet the above criteria may also be reported, per FDA approval. Table 9. List of Deleterious Missense Mutations by Protein Effect, Implemented on the Respective Canonical Transcript. | BRCA1 | | BRCA2 | | ATM | | | --- | --- | --- | --- | --- | --- | | Protein Effect (PE) | FMI Annotated PE | Protein Effect (PE) | FMI Annotated PE | Protein Effect (PE) | FMI Annotated PE | | MIV | MIV | MIR | MIR | MIT | MIT | | MII | MII | MII | MII | R2032K | R2032K | | C6IG | C6IG | VI59M | VI59M | R2227C | R2227C | | C64Y | C64Y | V211L | V211L | R2547 S2549del | R2547 S2549del | | R7IG | R7IG | V211I | V211I | G2765S | G2765S | | R7IK | R7IK | R2336P | R2336P | R2832C | R2832C | | RI495M | RI495M | R2336H | R2336H | S2855 V2856delinsR1 | S2855 V2856delinsR1 S2855 V2856>R1 | | EI559K | EI559K | | | R3008C | R3008C | | DI692N | DI692N | | | R3008H | R3008H | | DI692H | DI692H | | | [VUS from Jan 2016 HRR* List to be Excluded] | | | RI699W | RI699W | | | V2424G | V2424G | | AI708E | AI708E | | | [Excluded from Jan 2016 HRR List] | | | G1788V | GI788V | | | K750K | splice site 2250G>A | HRR = Homologous Recombination Repair genes Intronic Variants | Gene | Chr | Position | Ref | Alt | dbSNP | FMI Protein Effect | | --- | --- | --- | --- | --- | --- | --- | | ATM | chr11 | 108128198 | T | G | rs730881346 | [Variant Not Called by FMI] | | ATM | chr11 | 108214102 | AGTGA | A | rs730881295 | splice site 8418+5_8418+8delGTGA or splice site 8418+1_8418+4delGTGA | 3. CDx classification criteria for EGFR alterations: - Base substitutions resulting in EGFR L858R - In-frame deletions occurring within EGFR Exon 19 4. ALK rearrangements to identify patients eligible for treatment with ALECENSA® (alectinib): CDx positivity for an ALK rearrangement is based on the following variant classification criteria: - The ALK rearrangement must have pathogenic driver status (FMI driver status of "known" or "likely") - AND the disease type must be NSCLC - AND one of the following two conditions must hold: 1. The partner gene is EML4, or 2. The ALK breakpoint occurs within ALK intron 19 PMA P200016: FDA Summary of Safety and Effectiveness Data {12} VI. ALTERNATIVE PRACTICES AND PROCEDURES There are FDA-approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using cfDNA isolated from plasma samples, as listed in Table 1 of the FoundationOne® Liquid CDx intended use statement. The approved CDx tests are listed in Table 10, below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/media/119249/download. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle. Table 10: FDA-approved companion diagnostic (CDx) alternatives | Biomarker(s) Detected | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | EGFR Exon 19 deletions and L858R Substitution Mutation | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | Polymerase Chain Reaction (PCR) | TARCEVA® (erlotinib), TAGRISSO® (osimertinib), and IRESSA® (gefitinib) | NSCLC | | | Guardant360 CDx | Guardant Health, Inc. | NGS | | | | | | | | TAGRISSO® (osimertinib) | | | PIK3CA: C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y | therascreen PIK3CA RGQ PCR test | QIAGEN, Inc. | PCR | PIQRAY® (alpelisib) | Breast Cancer | There are no FDA-approved CDx alternatives using cfDNA isolated from plasma for the detection of genomic alterations of BRCA1, BRCA2, and ATM for the identification of mCRPC patients eligible for treatment with LYNPARZA® (olaparib). VII. MARKETING HISTORY Foundation Medicine designed and developed FoundationOne® Liquid CDx based on previous versions of the assay, including the FoundationACT (FACT) and FoundationOne® Liquid laboratory developed test (LDT), a revised version of FACT. The first commercial sample was tested in 2016. The FACT and FoundationOne® Liquid LDTs have been used to detect the presence of genomic alterations in blood and plasma specimens. Neither the FACT nor FoundationOne® Liquid LDTs were FDA-cleared or - approved. The FoundationOne® Liquid CDx test was approved on August 26, 2020 for the detection of genomic alterations of BRCA1 or BRCA2 for the identification of mCRPC patients eligible for treatment with RUBRACA® (rucaparib) and the detection of EGFR Exon 19 deletions (Exon 19del) and L858R substitutions in plasma obtained from patients with advanced and metastatic NSCLC for treatment with TARCEVA® (erlotinib), TAGRISSO® (osimertinib), and IRESSA® (gefitinib). The FoundationOne® Liquid CDx PMA P200016: FDA Summary of Safety and Effectiveness Data 13 of 49 {13} assay was also approved for tumor mutation profiling for substitutions and indels to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. The FoundationOne® Liquid CDx test was approved on October 26, 2020 as a companion diagnostic for BRCA1 and BRCA2 alterations in epithelial ovarian cancer for patients who may benefit from treatment with RUBRACA® (rucaparib), ALK rearrangements in NSCLC for patients who may benefit from treatment with ALECENSA® (alectinib), and PIK3CA mutations in patients with breast cancer who may benefit from treatment with PIQRAY® (alpelisib). This approval also included the addition of rearrangements in three (3) genes, and copy number alterations in three (3) genes for tumor profiling. The FoundationOne® Liquid CDx assay has been marketed in the United States, the European Union, and in several other foreign countries since August 2020. ## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect FoundationOne® Liquid CDx test results, and subsequently, inappropriate patient management decisions. Patients with false positive CDx biomarker results may undergo treatment with one of the therapies listed in the intended use statement without clinical benefit and may experience adverse reactions associated with the therapy. Patients with false negative results may not be considered for treatment with the indicated targeted therapy. There is also a risk of delayed results, which may lead to delay of treatment with the indicated therapy. For the specific adverse events related to the approved therapeutics, please see approved drug product labels. For the specific adverse events that occurred in the clinical study, please see the FDA approved package inserts for LYNPARZA® (olaparib) which is available at Drugs@FDA. ## IX. SUMMARY OF NONCLINICAL STUDIES ### A. Laboratory Studies Performance characteristics were established using circulating cfDNA derived from blood specimens extracted from a wide range of tumor types and performed as described in the Summary of Safety and Effectiveness Data for P190032 and P200006. Table 11 and Table 12 below provides a summary of the number of tumor types and relevant ATM, BRCA1, and BRCA2 variants or variant types included in each study described below or referenced. As summarized in the table below, each study included a broad range of representative alteration types (substitutions, insertion-deletions, copy number alterations, rearrangements) in various genomic contexts across several genes. Due to the lack of sufficient volume of clinical specimens, some of the studies used contrived samples, which consisted of enzymatically sheared cell line DNA spiked into human plasma and diluted with cfDNA isolated from healthy donor plasma. A PMA P200016: FDA Summary of Safety and Effectiveness Data 14 of 49 {14} contrived sample functional characterization (CSFC) study (Section IX.A.1) was conducted to demonstrate comparable performance of sheared cell line DNA samples as compared to cfDNA isolated from plasma specimens obtained from cancer positive patient specimens. Clinical specimens were used to assess analytical accuracy, precision and confirmation of the estimated limit of detection (LoD), and evaluate sample stability. The validation studies included &gt;7,000 sample replicates, &gt;31,000 unique variants, &gt;30 tumor types, representing all 311 genes targeted by the assay. Please refer to the Summary of Safety and Effectiveness Data for P190032 and P200006 for the representation of tumor types and variants included in the previous device approvals. Table 11: Representation of tumor types and variants* across validation studies | Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # Unique | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | Targeted Genes | Subs | Indels | Rearrang. | Copy Number Losses | | Contrived Sample Functional Characterization (CSFC) Study | Breast cancer Colorectal Cancer (CRC) Lung cancer Contrived samples | 13 | 1843 | 228 | 563 | 81 | 11 | 1 | | Orthogonal Concordance | 23 cancer types Contrived samples | 278 | 0 | 64 | 541 | 12 | 11 | 0 | | LoD Estimation | Prostate Contrived samples | 10 | 877 | 286 | 1490 | 247 | 32 | 3 | | LoB | Healthy Donors | 28 | 79 | 322 | 26134 | 4482 | 911 | 42 | | FoundationOne® Liquid CDx to Validated Tumor Tissue Test Concordance: BRCA1 and BRCA2 Variants | Prostate cancer Ovarian cancer | 279 | 0 | 2 | 100 | 87 | 9 | 2 | | Potentially Interfering Substances | Contrived samples | 9 | 336 | 18 | 16 | 11 | 11 | 2 | | Hybrid Capture Bait Specificity | 25 cancer types Contrived samples | 3546 | 0 | 324 | 0 | 0 | 0 | 0 | | Reagent Stability | Contrived samples | 8 | 142 | 279 | 1090 | 215 | 32 | 2 | | Reagent Interchangeability | Contrived samples | 8 | 192 | 20 | 15 | 11 | 11 | 1 | | Precision study 1 | Breast cancer CRC Lung cancer Ovarian cancer Prostate cancer Skin cancer Contrived samples | 47 | 1121 | 280 | 900 | 229 | 63 | 5 | PMA P200016: FDA Summary of Safety and Effectiveness Data {15} | Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # Unique | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | Targeted Genes | Subs | Indels | Rearrang. | Copy Number Losses | | Precision study 2 | Lung cancer Prostate cancer Stomach cancer CRC Bile duct cancer Breast cancer | 10 | 230 | 6 | 6 | 4 | 0 | 0 | | DNA Extraction | CRC Prostate cancer Breast cancer Lung cancer Skin cancer | 6 | 72 | 161 | 265 | 53 | 2 | 0 | | Whole Blood Sample Stability | Lung cancer CRC Prostate cancer Breast cancer | 11 | 22 | 66 | 75 | 15 | 1 | 0 | | Inverted Tube Whole Blood Sample Stability | Lung cancer CRC Breast cancer Ovarian cancer Prostate cancer | 130 | 260 | 237 | 594 | 91 | 5 | 0 | | Cross Contamination | Contrived samples | 5 | 376 | 39 | 9 | 5 | 4 | 1 | | Guard Banding | Contrived samples | 10 | 375 | 20 | 17 | 12 | 12 | 1 | | Clinical validation for detection of EGFR Exon 19 deletions and L858R alterations: non-inferiority study | Lung cancer | 177 | 0 | 1 | 5 | 7 | 0 | 0 | | Clinical validation study for detection of deleterious alterations in BRCA1 and BRCA2 in prostate cancer | Prostate cancer | 199 | 0 | 2 | 44 | 55 | 8 | 1 | | Blood Collection Tube Equivalence | Ovarian cancer Breast cancer CRC Prostate cancer Lung cancer Skin cancer Stomach cancer | 60 | 192 | 116 | 135 | 39 | 13 | 0 | | Automation Line Equivalence | Contrived samples | 8 | 187 | 303 | 1926 | 337 | 63 | 4 | | Variant Report Curation | Breast cancer CRC Lung cancer | 19 | 57 | 183 | 300 | 104 | 15 | 2 | PMA P200016: FDA Summary of Safety and Effectiveness Data {16} | Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # Unique | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | Targeted Genes | Subs | Indels | Rearrang. | Copy Number Losses | | | Prostate cancer Skin cancer | | | | | | | | | Pan-tumor performance (includes historical analysis) | 20 cancer types | 19868 | 0 | 0 | 0 | 0 | 0 | 0 | | Molecular Index Barcode Performance | 25 cancer types Contrived samples | 7637 | 0 | 324 | 0 | 0 | 0 | 0 | | FoundationOne® Liquid LDT to FoundationOne® Liquid CDx Concordance | 25 cancer types | 927 | 0 | 73 | 1815 | 376 | 109 | 0 | *Variant result totals may include variants classified as VUS or benign. Clinical oncology blood specimens can be constrained by factors such as limitations in blood draw volumes and cfDNA concentration. For studies where clinical samples carrying CDx biomarkers/alteration types were not evaluated due to limitations in sample availability, a post-market study (See Section XIII) is planned to confirm the performance of the FoundationOne® Liquid CDx test using intended use clinical specimens. In some studies when use of clinical specimens was not feasible due to volume limitations, contrived samples were used which consisted of enzymatically sheared cell line DNA spiked into human plasma from healthy donors, extracted according to the assay's standard procedure, and the isolated cfDNA was then diluted with cfDNA. To support such use, a contrived sample functional characterization (CSFC) study was conducted to demonstrate comparable performance of sheared cell line DNA samples as compared to cfDNA isolated from plasma. Highly actionable alterations were identified in the 39 contrived samples representing 17 genes and included 17 substitutions, 6 indels, 6 copy number losses, and 9 rearrangements that were used across validation studies. The 39 contrived samples included 1 ATM substitutions, 2 ATM indels, 2 BRCA1 (positive for 2 indels and 1 substitutions), and 3 BRCA2 samples (positive for 5 indels). These samples were used to supplement the samples used to support the performance of the ATM, BRCA1 and BRCA2 CDx indications listed in Table 1. 1. Contrived Sample Functional Characterization (CSFC) Study: See Summary of Safety and Effectiveness Data for P190032 and P200006 and Section XIII. 2. Analytical Accuracy/Concordance with an Orthogonal Method: See Summary of Safety and Effectiveness Data for P190032 and P200006. PMA P200016: FDA Summary of Safety and Effectiveness Data {17} PMA P200016: FDA Summary of Safety and Effectiveness Data 18 of 49 # 3. Analytical Sensitivity: a. Limit of Blank (LoB): See Summary of Safety and Effectiveness Data for P190032. b. Limit of Detection (LoD): The LoD for each variant type was established by processing a total of 1,069 sample replicates across ten contrived (enzymatically fragmented cell-line gDNA) samples representing short variants, rearrangements, and copy number alterations (homozygous deletions). For this study the initial cfDNA input was set at 45 ng for contrived samples; however, the final cfDNA input varied depending upon the starting VAF and/or tumor fraction and the targeted VAF and/or tumor fraction dilution levels. The LoD was determined using the conservative hit rate approach for the majority of variants. LoD by hit rate was defined as the mean VAF value (for short variants and rearrangements) or mean tumor fraction value (for copy number alterations) at the lowest dilution level tested with at least 95% detection across replicates. The hit rate was computed as the number of replicates with positive variant calls per the total number of replicates tested at each level. Short variants with hit rates of at least 95% at all dilution levels or hit rates below 95% for all dilution levels were excluded from analysis as LoD could not be reliably estimated. The median estimated LoD for CDx alterations are presented in Table 12. The median LoD for targeted short variant, rearrangement, and copy number alterations were consistent with the platform LoD. Table 12: LoD estimation for CDx alterations | Gene | Alteration Subtype | # Samples Evaluated | Median LoD^{1} | | --- | --- | --- | --- | | ATM | Indel | 1 | 0.51% | | | ATM-EXPH5 Truncation^{2} | 1 | 1.13% | | BRCA1 | Substitutions | 8 | 0.34% VAF | | | Indels | 1 | 0.38% VAF | | | Rearrangement^{2} | 1 | 0.87% VAF | | BRCA2 | Substitutions | 17 | 0.37% VAF | | | Indels | 2 | 0.36% VAF | | | BRCA2-EDA Truncation^{2} | 1 | 0.48% VAF | | | Copy Number Loss^{1} | 1 | 48.1% TF | The Estimated LoDs for one ATM and several BRCA1 and BRCA2 short variants were confirmed at values higher than the LoDs estimated for the non-CDx alterations. (see Precision: Reproducibility and Reproducibility section below, Tables 14 and 15 for confirmed LoD values). 1 The accuracy of %VAF/%TF have not been analytically validated. 2 The LoD for these alterations was determined using clinical specimens. # 4. Analytical Specificity: a. Potentially Interfering Substances: {18} See Summary of Safety and Effectiveness Data for P190032. b. Hybrid Capture Bait Specificity: See Summary of Safety and Effectiveness Data for P190032. 5. Carryover/Cross-Contamination: See Summary of Safety and Effectiveness Data for P190032. 6. Precision: Repeatability and Reproducibility at LoD Precision was evaluated for alterations associated with CDx claims, as well as tumor mutation profiling variants. Repeatability including intra-run performance (run on the same plate under the same conditions) and reproducibility including inter-run performance (run on different plates under different conditions) were assessed and compared across three reagent lots, two sequencers, and two processing runs. a. Results for a subset of highly-actionable alterations A set of 39 unique samples were used to evaluate precision of FoundationOne® Liquid CDx for detecting a set of highly-actionable variants, including 8 contrived samples representing various targeted alterations and 31 clinical samples. The samples representing CDx alterations are summarized in Table 13. The 31 clinical samples consisted of 7 different cancers (10 lung, 6 prostate, 3 colon, 2 melanoma, 4 ovarian, 5 breast, and 1 unknown). The samples included 30 actionable gene alterations including 7 BRCA1 or BRCA2 alterations and 4 ATM alterations. The remaining samples included multiple other actionable genes and variant types. Target alterations were assessed near LoD and/or 2x - 3x LoD. Each sample was divided into 24 aliquots, with 12 duplicates being processed on the same plate under the same conditions. The cfDNA input for the library construction step was set at 45 ng for the contrived samples, and ranged from 24ng - 45ng for the clinical samples, with preference towards the lower, more challenging cfDNA input amounts. Across 47 samples (31 clinical specimens at one dilution level and 8 contrived samples across two dilution levels), a total of 57 unique alterations were evaluated. Table 13: CDx sample set assessed for precision | Targeted Alteration | Disease Ontology of Patient from which Sample was Derived | | --- | --- | | ATM K1773fs*3 (5318delA) | Contrived | | ATM splice site 8850+1G>A | Prostate cancer | PMA P200016: FDA Summary of Safety and Effectiveness Data 19 of 49 {19} | Targeted Alteration | Disease Ontology of Patient from which Sample was Derived | | --- | --- | | ATM I2012fs*4 | Prostate cancer | | ATM-EXPHS Truncation | Prostate cancer | | BRCA1, BRCA2 alterations | 6 contrived samples | | BRCA1 E23fs*17 | Ovary cancer | | BRCA1 Q780* | Ovary high grade serous carcinoma | | BRCA1 Rearrangement | Unknown primary malignant neoplasm | | BRCA2 G267* | Ovary serous carcinoma | | BRCA2 S2988fs*12 | Ovary cancer | | BRCA2-EDA Truncation | Prostate cancer | The repeatability of CDx alterations is summarized in Table 14 and the reproducibility of CDx alterations is summarized in Table 15. Table 14: Repeatability of CDx alterations targeted in precision study at $\geq 1\mathrm{x}{\mathrm{{LoD}}}^{1}$ | Variant Type | Alteration | Concordant Pairs | Repeatability (%) | 95% CIs (%) | VAF/TF2Level Tested | X LoD Tested | | --- | --- | --- | --- | --- | --- | --- | | Short variant | ATM I2012fs*4 | 12/12 | 100 | (73.54, 100) | 0.86% | 1.7 | | Short variant | ATM splice site 8850+1G>A | 12/12 | 100 | (73.54, 100) | 0.56% | 1.1 | | Short variant | ATM K1773fs*3 | 12/12 | 100 | (73.54, 100) | 0.77% | 1.5 | | Short variant | ATM K1773fs*3 | 12/12 | 100 | (73.54, 100) | 1.04% | 2.0 | | Rearrangement | ATM-EXPH5 truncation | 12/12 | 100 | (73.54, 100) | 1.13% | \(1.0^3\) | | Short variant | BRCA1 2338C>T | 12/12 | 100 | (73.54, 100) | 1.11% | 3.3 | | Short variant | BRCA1 2475delC | 12/12 | 100 | (73.54, 100) | 0.61% | 1.6 | | Short variant | BRCA1 2475delC | 12/12 | 100 | (73.54, 100) | 0.93% | 3.3 | | Short variant | BRCA1 2612C>TT | 11/11 | 100 | (71.51, 100) | 0.51% | 1.3 | | Short variant | BRCA1 68_69delAG | 12/12 | 100 | (73.54, 100) | 0.66% | 1.7 | | Short variant | BRCA1 P871fs*32 | 12/12 | 100 | (73.54, 100) | 1.08% | 2.8 | | Rearrangement | BRCA1-BRCA1 | 12/12 | 100 | (73.54, 100) | 0.87% | 1.0 | | Short variant | BRCA2 3599_3600delGT | 12/12 | 100 | (73.54, 100) | 0.58% | 1.6 | | Short variant | BRCA2 3599_3600delGT | 12/12 | 100 | (73.54, 100) | 0.92% | 2.6 | | Short variant | BRCA2 4284_4285insT | 12/12 | 100 | (73.54, 100) | 0.94% | 2.6 | | Short variant | BRCA2 4284_4285insT | 11/11 | 100 | (71.51, 100) | 1.26% | 3.5 | | Short variant | BRCA2 5351delA | 12/12 | 100 | (73.54, 100) | 1.22% | 3.2 | | Short variant | BRCA2 5351delA | 12/12 | 100 | (73.54, 100) | 1.85% | 4.9 | | Short variant | BRCA2 5351delA | 11/11 | 100 | (71.51, 100) | 1.07% | 2.8 | | Short variant | BRCA2 5351delA | 12/12 | 100 | (73.54, 100) | 2.24% | 5.9 | | Short variant | BRCA2 5465_5466insA | 12/12 | 100 | (73.54, 100) | 0.92% | 2.4 | | Short variant | BRCA2 5465_5466insA | 11/11 | 100 | (71.51, 100) | 1.19% | 3.1 | | Short variant | BRCA2 8961_8964delGAGT | 12/12 | 100 | (73.54, 100) | 1.07% | 2.8 | | Short variant | BRCA2 799G>T | 10/12 | 83.33 | (51.59, 97.91) | 0.5% | 1.5 | | Short variant | BRCA2 9097_9098insA | 6/11 | 54.55 | (23.38, 83.25) | 0.71% | 1.9 | | Short variant | BRCA2 9097_9098insA | 10/12 | 83.33 | (51.59, 97.91) | 1.03% | 2.7 | | Copy Number Loss | BRCA2 loss | 11/12 | 91.67 | (61.52, 99.79) | 39.43% | 0.8 | PMA P200016: FDA Summary of Safety and Effectiveness Data {20} PMA P200016: FDA Summary of Safety and Effectiveness Data 21 of 49 | Variant Type | Alteration | Concordant Pairs | Repeatability (%) | 95% CIs (%) | VAF/TF² Level Tested | X LoD Tested | | --- | --- | --- | --- | --- | --- | --- | | Rearrangement | BRCA2-EDA | 11/11 | 100 | (71.51, 100) | 0.48% | 0.6 | 1 Clinical samples were mostly tested at 2x – 3x LoD rather than 1x – 1.5x LoD 2 The accuracy of %VAF/%TF have not been analytically validated. 3 LoD was not directly established for this variant. The %VAF tested is considered the LoD for ATM rearrangements. Table 15: Reproducibility of CDx alterations targeted in precision study at ≥1x LoD¹ | Variant Type | Alteration | Concordant Replicates | Reproducibility (%) | 95% CIs (%) | VAF/TF² Level Tested | X LoD Tested | | --- | --- | --- | --- | --- | --- | --- | | Short variant | ATM I2012fs*4 | 24/24 | 100 | (85.75, 100) | 0.86% | 1.7 | | Short variant | ATM splice site 8850+1G>A | 24/24 | 100 | (85.75, 100) | 0.56% | 1.1 | | Short variant | ATM K1773fs*3 | 24/24 | 100 | (85.75, 100) | 0.77% | 1.5 | | Short variant | ATM K1773fs*3 | 24/24 | 100 | (85.75, 100) | 1.04% | 2.0 | | Rearrangement | ATM-EXPH5 truncation | 24/24 | 100 | (85.75, 100) | 1.13% | 1.0³ | | Short variant | BRCA1 2338C>T | 24/24 | 100 | (85.75, 100) | 1.11% | 3.3 | | Short variant | BRCA1 2475delC | 24/24 | 100 | (85.75, 100) | 0.61% | 1.6 | | Short variant | BRCA1 2475delC | 24/24 | 100 | (85.75, 100) | 0.93% | 2.4 | | Short variant | BRCA1 2612C>TT | 23/23 | 100 | (85.18, 100) | 0.51% | 1.3 | | Short variant | BRCA1 68_69delAG | 24/24 | 100 | (85.75, 100) | 0.66% | 1.7 | | Short variant | BRCA1 P871fs*32 | 24/24 | 100 | (85.75, 100) | 1.08% | 2.8 | | Rearrangement | BRCA1-BRCA1 | 24/24 | 100 | (85.75, 100) | 0.87% | 1.0 | | Short variant | BRCA2 3599_3600delGT | 24/24 | 100 | (85.75, 100) | 0.58% | 1.6 | | Short variant | BRCA2 3599_3600delGT | 24/24 | 100 | (85.75, 100) | 0.92% | 2.6 | | Short variant | BRCA2 4284_4285insT | 24/24 | 100 | (85.75, 100) | 0.94% | 2.6 | | Short variant | BRCA2 4284_4285insT | 23/23 | 100 | (85.18, 100) | 1.26% | 3.5 | | Short variant | BRCA2 5351delA | 24/24 | 100 | (85.75, 100) | 1.22% | 3.4 | | Short variant | BRCA2 5351delA | 24/24 | 100 | (85.75, 100) | 1.85% | 5.1 | | Short variant | BRCA2 5351delA | 23/23 | 100 | (85.18, 100) | 1.07% | 3.0 | | Short variant | BRCA2 5351delA | 24/24 | 100 | (85.75, 100) | 2.24% | 6.2 | | Short variant | BRCA2 5465_5466insA | 24/24 | 100 | (85.75, 100) | 0.92% | 2.6 | | Short variant | BRCA2 5465_5466insA | 23/23 | 100 | (85.18, 100) | 1.19% | 3.3 | | Short variant | BRCA2 799G>T | 22/24 | 91.67 | (73.0, 98.97) | 0.5% | 1.4 | | Short variant | BRCA2 8961_8964delGAGT | 24/24 | 100 | (85.75, 100) | 1.07% | 3.0 | | Short variant | BRCA2 9097_9098insA | 22/24 | 91.67 | (73.0, 98.97) | 1.03% | 2.9 | | Short variant | BRCA2 799G>T | 22/24 | 91.67 | (73.0, 98.97) | 0.5% | 1.4 | | Short variant | BRCA2 9097_9098insA | 5/23 | 21.74 | (7.46, 43.7) | 0.71% | 2.0 | | Short variant | BRCA2 9097_9098insA | 22/24 | 91.67 | (73.0, 98.97) | 1.03% | 2.9 | | Copy Number Loss | BRCA2 loss | 21/24 | 87.5 | (67.64, 97.34) | 39.43% | 0.8 | | Rearrangement | BRCA2-EDA | 23/23 | 100 | (85.18, 100) | 0.48% | 1.0 | 1 Clinical samples were mostly tested at 2x – 3x LoD rather than 1x – 1.5x LoD 2 The accuracy of %VAF/%TF have not been analytically validated. 3 LoD was not directly established for this variant. The %VAF tested is considered the LoD for ATM rearrangements. For repeatability, 28 samples with 19 unique targeted alterations were evaluated. Of the 19 unique alterations that were targeted in 24 of the 28 {21} samples, 16 alterations (84.2%) demonstrated 100% repeatability. Four BRCA2 samples demonstrated repeatability below 95% (54.6% - 91.7%). The BRCA2 loss was tested at an 39.4% TF below the estimated LoD of 48.1% TF and used a cfDNA input below the recommended cfDNA input of 30 ng. Of the remaining 3 poorly performing samples, only one was at a % VAF (0.5% VAF) near the estimated LoD (0.37% VAF), while the remaining 2 were tested at levels higher than the estimated LoDs for each sample. Therefore, the reason for the observed performance was not determined. Reproducibility of 100% was observed in 16 of 19 (84.2%) unique alterations in 24 of the 30 samples included in the study. Six BRCA2 samples demonstrated reproducibility below 95% (21.7 - 91.7). The BRCA2 loss was tested at an %TF below the estimated LoD and used a cfDNA input below the recommended cfDNA input of 30 ng. Of the remaining 5 poorly performing samples, only one was at a %VAF (0.5% VAF) near the estimated LoD (0.37% VAF), while the remaining 4 were tested at levels higher than the estimated LoDs for each sample. Therefore, the reason for the observed performance was not determined. Samples included in the precision/reproducibility study BRCA1 and BRCA2 alterations included prostate and non-prostate specimens. Data from a postmarket study will be provided using clinical samples from patients with prostate cancer to demonstrate performance in the intended specimen type. Also see the Summary of Safety and Effectiveness Data for P190032 and P200006 and Section XIII. b. Confirmation of LoD and Precision in Clinical Specimens: The combined confirmation of LoD and precision study was performed as described in the Summary of Safety and Effectiveness for P190032. In this study, 29 clinical cfDNA samples targeting variants at 1-1.5x LoD were evaluated to confirm LoD and precision in clinical specimens. Of the samples included in this study 9 clincial samples were associated with the olaparib indication listed in Table 1. All 9 specimens had 100% reproducibility at the levels tested. A summary of the Confirmation of LoD and precision results for a subset of highly-actionable alterations are provided in Table 16. See the Summary of Safety and Effectiveness Data for P190032 and P200006 and Section XIII. Table 16: Confirmation of LoD* and precision in clinical specimens for CDx alterations | Target Alteration | LoD | Mean Level Tested | Reproducibility (95% CI) | | --- | --- | --- | --- | | ATM I2012fs*4 | 0.51% MAF | 0.86% | 100% (85.8, 100.0) | | ATM splice site 8850+1G>A | 0.51% MAF | 0.56% | 100% (85.8, 100.0) | | ATM-EXPH5 truncation | Not Determined | 1.13 %VAF | 100% (85.8, 100.0) | | BRCA1 E23fs*17 | 0.38% VAF | 0.66% VAF | 100% (85.8, 100.0) | | BRCA1 Q780* | 0.34% VAF | 1.11%VAF | 100% (85.8, 100.0) | | BRCA1 Rearrangement | 0.26%-.47% VAF^{1} | 0.87% VAF | 100% (85.8, 100.0) | PMA P200016: FDA Summary of Safety and Effectiveness Data 22 of 49 {22} | BRCA2 S2988fs*12 | 0.36% VAF | 1.07% VAF | 100% (85.8, 100.0) | | --- | --- | --- | --- | | BRCA2- EDA Truncation | 0.26%-.47% VAF¹ | 0.48% VAF | 100% (85.2, 100.0) | In general, most of the targeted variants were tested at levels higher than estimated near LoD (~1x); therefore, the tested LoD level values (%VAF/%TF) are considered to be the confirmed LoD. The LoD for ATM rearrangements was not previously determined; however, as indicated previously, the variant was confirmed at 1.13% VAF which is considered to be the confirmed LoD. A post-market study is planned to demonstrate precision using samples at near the estimated LoD for those tested above or below the estimated LoD and in plasma specimens obtained from patients with prostate cancer (See Section XIII). c. Tumor Mutation Profiling Variants: See Summary of Safety and Effectiveness Data for P190032 and P200006 and Section XIII. d. Reagent Lot-to-Lot Reproducibility: See Summary of Safety and Effectiveness Data for P190032. e. Instrument-to-Instrument Reproducibility: See Summary of Safety and Effectiveness Data for P190032. f. Reagent Lot Interchangeability: See Summary of Safety and Effectiveness Data for P190032. g. Curator Precision: See Summary of Safety and Effectiveness Data for P190032. 7. Comparability Across Cancer Types: See Summary of Safety and Effectiveness Data for P190032 and P200006. 8. Stability: a. Reagent Stability: See Summary of Safety and Effectiveness Data for P190032. b. Stability of cfDNA and Plasma Samples: See Summary of Safety and Effectiveness Data for P190032. PMA P200016: FDA Summary of Safety and Effectiveness Data 23 of 49 {23} c. Whole Blood Specimen Stability and Inverted Tube Stability: See Summary of Safety and Effectiveness Data for P190032 and Section XIII. 9. Guard-banding and Robustness: a. DNA Extraction: See Summary of Safety and Effectiveness Data for P190032. b. cfDNA Input: See Summary of Safety and Effectiveness Data for P190032 and P200006 and Section XIII. c. Molecular Index Barcode Performance: See Summary of Safety and Effectiveness Data for P190032. d. Automation Line Equivalence: See Summary of Safety and Effectiveness Data for P190032. B. Animal Studies Not Applicable. C. Additional Studies The following studies in this section were performed in support of the clinical validation studies. 1. Blood Collection Tube Equivalence: See Summary of Safety and Effectiveness Data for P190032 and Section XIII. 2. Concordance with a Validated Tumor-tissue NGS by Gene and Variant Type The concordance between the FoundationOne® Liquid CDx and a validated tumor-tissue NGS orthogonal assay was performed to establish the ability of the FoundationOne® Liquid CDx test to detect ATM, BRCA1, and BRCA2 alterations in plasma samples obtained from patients whose prostate tumors were identified as positive by a validated tumor-tissue based NGS orthogonal assay. Agreements (PPA and NPA) were calculated by gene and by variant type using the comparator method as the reference. The PPA estimate across all 3 genes was 74.29%, it was highest in the BRCA2 gene (PPA=76.47%). PPA estimates for ATM and BRCA1 PMA P200016: FDA Summary of Safety and Effectiveness Data 24 of 49 {24} were $70.83\%$ and $70.43\%$ respectively. NPA point estimates in all cases were greater than $99\%$ (Table 17). Concordances per variant type in each gene were calculated based on the results of 325 samples from the clinical bridging study. Copy number losses in ATM are not reported by FoundationOne® Liquid CDx, and therefore were not assessed within this analysis. All data analysis was based on variant level data rather than at the sample level, i.e., a sample can have multiple positive (or negative) calls in the same gene of the same variant type, and as such the numbers reported in the tables below are based on targeted variant locations across all 3 genes for each sample. For short variants, the set of all observed variants in the data were considered as being either positively or negatively called in each sample. For rearrangements, all possible rearrangement types (truncation, deletion, and duplication) that could be called as a biomarker were examined. For copy number alterations, only homozygous deletions were measured, consistent with the CDx biomarker definition. Results were as follows in Table 17 and Table 18. Table 17: Concordance by Gene and Overall Variant Type | Gene/Variant Type | F1LCDx+/ Orth+ | F1LCDx- / Orth + | F1LCDx+/ Orth- | F1LCDx- / Orth- | PPA (%) (95% CI*) | NPA (%) (95% CIs) | | --- | --- | --- | --- | --- | --- | --- | | ATM | 34 | 14 | 37 | 22990 | 70.83% (56.82%, 81.76%) | 99.84% (99.78%, 99.88%) | | BRCA1 | 5 | 2 | 3 | 2590 | 71.43% (35.89%, 91.78%) | 99.88% (99.66%, 99.96%) | | BRCA2 | 65 | 20 | 11 | 20054 | 76.47% (66.43%, 84.22%) | 99.95% (99.90%, 99.97%) | | Short Variant | 90 | 17 | 44 | 42099 | 84.11% (76.02%, 89.84%) | 99.90% (99.86%, 99.92%) | | Rearrangement | 10 | 8 | 6 | 2901 | 55.56% (33.72%, 75.44%) | 99.79% (99.55%, 99.90%) | | Copy Number Alteration (gain) | 4 | 11 | 1 | 634 | 26.67% (10.90%, 51.95%) | 99.84% (99.11%, 99.99%) | | Total | 104 | 36 | 51 | 45634 | 74.29% (66.47%, 80.81%) | 99.89% (99.85%, 99.92%) | Abbreviations: F1LCDx = FoundationOne® Liquid CDx; Orth = orthogonal method Score method was used to estimate $95\%$ two-sided CI Additional analysis was performed for each of the variant types by gene. Table 18: Concordance by Variant Type by Gene | Gene | Variant Type | F1LCDx+/ Orth+ | F1LCDx- / Orth + | F1LCDx+/ Orth- | F1LCDx- / Orth- | PPA (%) (95% CI1) | NPA (%) (95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | | ATM | SV | 31 | 12 | 35 | 22022 | 72.09% (57.31%, 83.25%) | 99.84% 99.78%, 99.89%) | | | RE | 3 | 2 | 2 | 318 | 60.00% (23.07%, 88.24%) | 99.38% (97.75%, 99.83%) | | BRCA1 | SV | 3 | 0 | 1 | 1296 | 100% (43.85%, 100%) | 99.92% (99.56%, 100%)2 | PMA P200016: FDA Summary of Safety and Effectiveness Data {25} | Gene | Variant Type | F1LCDx+/Orth+ | F1LCDx-/Orth - | F1LCDx+/Orth- | F1LCDx-/Orth- | PPA (%) (95% CI^{1}) | NPA (%) (95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | | | RE | 2 | 2 | 2 | 969 | 50.00% (15.00%, 85.00%) | 99.79% (98.25%, 99.94%) | | | CNA | 0 | 0 | 0 | 325 | N/A^{3} | 100.00% (98.83%, 100%) | | BRCA2 | SV | 56 | 5 | 8 | 18781 | 91.80% (82.21%, 96.45%) | 99.96% (99.92%, 99.98%) | | | RE | 5 | 4 | 2 | 964 | 55.56% (26.65%, 81.12%) | 99.79% (98.25%, 99.94%) | | | CNA | 4 | 11 | 1 | 309 | 26.67% (10.90%, 51.95%) | 99.68% (98.20%, 99.98%) | Abbreviations: F1LCDx = FoundationOne® Liquid CDx; Orth = orthogonal method 1 Score method was used to estimate 95% two-sided CI 2 Actual upper bound is 99.996% 3 PPA was not evaluable, as the denominator was 0. In general, the point estimates of NPA were very high, indicating that if an alteration is not detected by the orthogonal method, it likely will not be detected in FoundationOne® Liquid CDx, providing a high level of confidence in variants reported by FoundationOne® Liquid CDx. The F1L CDx-/Orth+ discordances observed are likely due to biological differences between tumor tissue and plasma, and random chance due to small sample size. Differences in tumor fraction and %VAF values between the variants present in the tissue and liquid samples due to shedding of cfDNA into the circulation, as well as tumor heterogeneity in the tumor tissue specimens could contribute to discordant results. Another potentially explanatory trend was sample quality. Many of the discordant samples (40 of 64 total samples with at least one discordance) had at least one in-process QC metric that was not met, resulting in qualified results. For the orthogonal method, 22 of 38 (57.89%) samples with a F1L CDx+/Orth- discordance had a QC flag in the corresponding Orthogonal method sample. Of the other samples with an FoundationOne® Liquid CDx variant detected, only 9 of 87 (10.34%) had a QC flag in the corresponding orthogonal method sample. This difference is highly significant (p &lt; 1×10⁻¹³). This suggests that poor quality of some of the tissue samples explains some of the discordances seen. This trend was seen in liquid samples as well, although the difference was not statistically significant. For samples with an Orth+/F1L CDx- discordance, 11 of 35 (31.43%) had a flag in the liquid samples, compared to 24 of 96 (25%) in other samples with a tumor tissue result. Moreover, the number of samples is small enough that results can be skewed due to random variance. Overall, low VAF and TF in either assay as well as poor sample quality explain most of the discordances observed. The data provided in this study also supports the recommendation for samples yielding a negative result by the FoundationOne® Liquid CDx test should be reflected to a FDA-approved tumor-tissue based CDx test. PMA P200016: FDA Summary of Safety and Effectiveness Data 26 of 49 {26} D. Animal Studies Not Applicable X. SUMMARY OF PRIMARY CLINICAL STUDY Foundation Medicine performed a clinical bridging study to establish a reasonable assurance of safety and effectiveness of the FoundationOne® Liquid CDx for the new CDx indications being sought. Data from this clinical study was the basis for the PMA approval decision. A summary of the clinical study is presented below. The safety and effectiveness of FoundationOne® Liquid CDx for detecting BRCA1, BRCA2 and/or ATM alterations in prostate cancer patients who may benefit from treatment with olaparib versus enzalutamide or abiraterone acetate was demonstrated in a prospectively defined retrospective analysis of specimens from patients enrolled in Cohort A of the study D081DC00007 (PROfound). PROfound is a prospective, multicenter, randomized, open-label, phase III trial evaluating the efficacy and safety of olaparib versus enzalutamide or abiraterone acetate in subjects with mCRPC who have failed prior treatment with a new hormonal agent (NHA) who have qualifying mutations in one of 15 genes directly or indirectly involved in homologous recombination repair, as determined by a tumor tissue test. All patients were required to have a qualifying homologous recombination repair (HRR) mutation assessed using the FoundationOne LDT tumor tissue-based clinical trial assay (F1 LDT CTA) to be randomized. Qualifying HRR gene alterations included alterations in BRCA1, BRCA2 and ATM for Cohort A, and BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and RAD54L for Cohort B. This indication for FoundationOne Liquid CDx was based on plasma samples available from patients enrolled into Cohort A only. Cohort A of PROfound included patients who had a qualifying tumor mutation in either BRCA1, BRCA2, or ATM genes. A bridging study was conducted to assess: 1) the concordance between ATM, BRCA1, and BRCA2 status (alteration positive or negative) by the F1 LDT CTA used for enrollment and the FoundationOne® Liquid CDx and 2) the effectiveness of olaparib in patients identified to harbor ATM, BRCA1, or BRCA2 alterations by the FoundationOne® Liquid CDx test. Throughout this document, the term ATM/BRCA1/BRCA2 is used to denote patients who carry mutations in BRCA1, BRCA2 or ATM, unless specified. A. Study Design The PROfound study is a prospective, multi-center, randomized, open-label, Phase III trial evaluating the efficacy and safety of olaparib versus enzalutamide or abiraterone acetate in patients with mCRPC who have failed prior treatment with a NHA and have a qualifying tumor mutation in one of 15 genes involved in the homologous recombination repair pathway. Patients were divided into two cohorts (Cohort A or Cohort B) based on their HRR gene mutation status. As stated above, patients randomized into Cohort A, included patients whose tumor tissue specimens were PMA P200016: FDA Summary of Safety and Effectiveness Data 27 of 49 {27} identified as harboring alterations in BRCA1, BRCA2 or ATM, while those randomized into Cohort B included those patients whose tumor tissue specimens were identified as harboring alterations in one of the remaining 11 HRR genes (i.e., BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and RAD54L). In the device bridging study, all available plasma samples, meeting the inclusion/exclusion criteria below, from Cohort A or screen failed patients collected at baseline prior to randomization into the AstraZeneca PROfound clinical trial were tested with FoundationOne® Liquid CDx. 1. Clinical Bridging Study Inclusion and Exclusion Criteria a. Sample inclusion criteria: - Patient provided appropriate consent for sample testing and diagnostic development - Specimens in frozen plasma. - Samples must meet minimum criteria for FoundationOne® Liquid CDx operational testing requirements - Samples must have a minimum plasma volume of 2.5mL. - For FoundationOne® Liquid CDx: Samples must have DNA content, as assessed by the TapeStation assay, &gt;30 ng of DNA for LC input for primary analysis - Samples obtained in accordance with FoundationOne® Liquid CDx sample criteria. b. Sample exclusion criteria: - Tissue, other liquid samples are excluded - Samples that do not meet minimum FoundationOne® Liquid CDx operational testing requirements - Samples with plasma volume &lt;2.5mL will be excluded. - Samples with &lt;30 ng of DNA content for LC input for primary analysis - Samples not obtained in accordance with FoundationOne® Liquid CDx sample criteria. 3. Clinical Endpoints The primary efficacy endpoint of Cohort A was radiographic progression-free survival (rPFS) as determined by Blinded independent central review (BICR) per RECIST version 1.1 and/or Prostate Cancer Clinical Trials Working Group 3 (PCWG3) (bone) criteria. Key secondary endpoints included confirmed objective response rate (ORR) (Cohort A), time to pain progression (Cohort A) and overall survival (Cohort A). The bridging study used the same primary endpoint for drug efficacy analysis. B. Accountability of PMA Cohort PMA P200016: FDA Summary of Safety and Effectiveness Data 28 of 49 {28} In total, 4,425 patients were screened and 387 (9.6%) were randomized into the PROfound study (Table 19). Of these 387 patients, 245 patients were randomized in Cohort A. 181 out of the 245 randomized patients in Cohort A both consented to the use of their sample for ctDNA CDx development and had a plasma sample available for testing. In total, 181/245 (73.9%) of the Cohort A patients were tested using the FoundationOne® Liquid CDx assay. Of these, 139 (76.8%) Cohort A patients had a successful FoundationOne® Liquid CDx test result and 42 Cohort A patients had a failed FoundationOne® Liquid CDx test result. This represents 56.7% (139/245) of total Cohort A patients. In addition, for the bridging study, 250 non-ATM/BRCA1/BRCA2 patient samples determined by F1 LDT CTA were randomly selected from the screen failure population and their matched plasma samples were tested by FoundationOne® Liquid CDx to determine the NPA between the FoundationOne® Liquid CDx test and the F1 LDT CTA. One hundred ninety-four (194) of 250 (77.6%) screen failed non-ATM/BRCA1/BRCA2 patients were successfully tested using the FoundationOne® Liquid CDx test. In total, 98/431 (22.7%) samples failed testing with the FoundationOne® Liquid CDx test. Eight (8) samples failed tests were due to insufficient DNA yield and 90 samples failed post DNA-extraction. Of the 139 successfully tested Cohort A patients by the FoundationOne® Liquid CDx, 111 patients were reported as BRCA1, BRCA2, and/or ATM mutation positive and 28 randomized patients were reported as non-ATM/BRCA1/BRCA2. Sample accountability for this clinical bridging study is summarized in Table 19 and Figure 1. Table 19: Sample accountability for olaparib clinical bridging study | Description | # patients | | --- | --- | | Patients randomized into PROfound | 387 | | BRCA1m, BRCA2m or ATMm (Cohort A) patients | 245 | | Cohort A patient samples tested with FoundationOne® Liquid CDx | 181 | | FoundationOne® Liquid CDx results available | 139 | | Cohort A patients ATM/BRCA1/BRCA2 positive by FoundationOne® Liquid CDx | 111 | PMA P200016: FDA Summary of Safety and Effectiveness Data 29 of 49 {29}  Figure 1 Overview of patients screened for the PROfound study a Includes 2013 patients who were non-ATM/BRCA1/BRCA2 using the F1 LDT CTA and 1 patient non-ATM/BRCA1/BRCA2 by a prior FoundationOne® test. In the figure above HRRm and non-HRRm refers to patients with or without ATM/BRCA1/BRCA2 alterations, respectively. b Includes patients who did not supply a sample for testing, patients who supplied an ineligible sample type and/or patient who supplied a sample after the testing window closed. # C. Study Population Demographics and Baseline Parameters Screened patients who were tissue positive for the pre-defined HRR genes were the primary population of interest. The demographics and baseline clinical characteristics of the Full Analysis Set (FAS)-confirmed F1 LDT CTA and the FoundationOne® Liquid CDx subgroups are summarized in Table 20 and Table 21. The demographics and baseline characteristics of the patients in the FoundationOne® Liquid CDx ATM/BRCA1/BRCA2 positive subgroup were generally balanced between treatment arms and consistent with those of the patients in the FAS. However, interpretation of the demographics and baseline characteristics of patients in the FoundationOne® Liquid CDx non-ATM/BRCA1/BRCA2 subgroup should be undertaken with caution due to the small numbers in this group. PMA P200016: FDA Summary of Safety and Effectiveness Data {30} Table 20: Demographic Characteristics for Cohort A Patients (FAS, Confirmed FoundationOne® Liquid CDx ATM/BRCA1/BRCA2 and FoundationOne® Liquid CDx non-ATM/BRCA1/BRCA2 patients) | | FAS | | Confirmed FoundationOne® Liquid CDx ATM/BRCA1/BRCA2 | | FoundationOne® Liquid CDx non-ATM/BRCA1/BRCA2 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Olaparib 300 mg bd (N=162) | Investigators choice of NHA (N=83) | Olaparib 300 mg bd (N=73) | Investigators choice of NHA (N=38) | Olaparib 300 mg bd (N=20) | Investigators choice of NHA (N=8) | | Age (years) | n | 162 | 83 | 73 | 38 | 20 | 8 | | | Mean | 68.0 | 68.1 | 65.9 | 68.4 | 71.8 | 65.6 | | | SD | 8.23 | 7.36 | 8.32 | 7.7 | 6.01 | 7.93 | | | Median | 68.0 | 67.0 | 65.0 | 68.5 | 72.5 | 65.5 | | | Min | 47 | 49 | 47 | 49 | 62 | 55 | | | Max | 86 | 86 | 81 | 86 | 86 | 75 | | Age group (years), n (%) | <65 | 54 (33.3) | 23 (27.7) | 32 (43.8) | 11 (28.9) | 3 (15.0) | 4 (50.0) | | | ≥65 | 108 (66.7) | 60 (72.3) | 41 (56.2) | 27 (71.1) | 17 (85,0) | 4 (50.0) | | Race, n (%) | White | 109 (67.3) | 55 (66.3) | 56 (76.7) | 30 (78.0) | 15 (75.0) | 5 (62.5) | | | Black or African American | 2 (1.2) | 1 (1.2) | 1 (1.4) | 0 | 0 | 0 | | | Asian | 43 (26.5) | 19 (22.9) | 10 (13.7) | 4 (10.5) | 5 (25.0) | 2 (25.0) | | | Other | 1 (0.6) | 1 (1.2) | 0 | 0 | 0 | 0 | | | Missing | 7 (4.3) | 7 (8.4) | 6 (8.2) | 4 (10.5) | 0 | 1 (12.5) | | Ethnic group, n (%) | Hispanic or Latino | 12 (7.4) | 9 (10.8) | 4 (5.5) | 2 (5.3) | 1 (5.0) | 2 (25.0) | | | Not Hispanic or Latino | 145 (89.5) | 69 (83.1) | 65 (89.0) | 33 (86.8) | 19 (95.0) | 5 (62.5) | | | Missing | 5 (3.1) | 5 (6.0) | 4 (5.5) | 3 (7.9) | 0 | 1 (12.5) | $\mathrm{SD} =$ standard deviation Table 21: Disease characteristics at baseline for Cohort A patients (FAS, confirmed F1 Liquid CDx ATM/BRCA1/BRCA2 and F1 Liquid CDx non-ATM/BRCA1/BRCA2 patients) | | FAS | | Confirmed F1 Liquid CDx ATM/BRCA1/BRCA2 | | F1 Liquid CDx non-ATM/BRCA1/BRCA2 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Olaparib 300 mg bd (N=162) | Investigators choice of NHA (N=83) | Olaparib 300 mg bd (N=73) | Investigators choice of NHA (N=38) | Olaparib 300 mg bd (N=20) | Investigators choice of NHA (N=8) | | Time from CRPC to randomization (months) | n | 160 | 82 | 73 | 37 | 19 | 8 | | | Median | 24.2 | 23.7 | 23.3 | 30.2 | 24.9 | 22.3 | | | Min, max | -6a, 189 | 1, 175 | -6,189 | 1,175 | 6,69 | 4,87 | | Time from mCRPC to randomization (months) | n | 160 | 82 | 73 | 37 | 19 | 8 | | | Median | 23.3 | 22.5 | 22.9 | 27.8 | 22.6 | 22.3 | | | Min, max | -6a, 121 | 1, 105 | -6,113 | 1,105 | 6,56 | 3,87 | | Histology type at diagnosis | Adenocarcinoma | 160 (98.8) | 80 (96.4) | 72 (98.6) | 36 (94.7) | 20 (100) | 8 (100) | | | Small cell carcinoma | 0 | 0 | 0 | 0 | 0 | 0 | | | Other | 0 | 2 (2.4) | 0 | 1 (2.6) | 0 | 0 | | | Missing | 2 (1.2) | 1 (1.2) | 1 (1.4) | 1 (2.6) | 0 | 0 | | Total Gleason | 1 | 0 | 0 | 0 | 0 | 0 | 0 | PMA P200016: FDA Summary of Safety and Effectiveness Data {31} | | FAS | Confirmed F1 Liquid CDx ATM/BRCA1/BRCA2 | F1 Liquid CDx non-ATM/BRCA1/BRCA2 | | --- | --- | --- | --- | | Olaparib 300 mg bd (N=162) | Investigators choice of NHA (N=83) | Olaparib 300 mg bd (N=73) | Investigators choice of NHA (N=38) | Olaparib 300 mg bd (N=20) | Investigators choice of NHA (N=8) | | Score at diagnosis | 2 | 1 (0.6) | 0 | 0 | 0 | 1 (5.0) | 0 | | 3 | 0 | 0 | 0 | 0 | 0 | 0 | | 4 | 2 (1.2) | 0 | 1 (1.4) | 0 | 0 | 0 | | 5 | 2 (1.2) | 1 (1.2) | 2 (2.7) | 0 | 0 | 1 (12.5) | | 6 | 6 (3.7) | 3 (3.6) | 3 (4.1) | 2 (5.3) | 1 (5.0) | 0 | | 7 | 41 (25.3) | 22 (26.5) | 20 (27.4) | 11 (28.9) | 7 (35.0) | 2 (25.0) | | 8 | 36 (22.2) | 12 (14.5) | 13 (17.8) | 6 (15.8) | 5 (25.0) | 2 (25.0) | | 9 | 59 (36.4) | 35 (42.2) | 26 (35.6) | 14 (36.8) | 6 (30.0) | 2 (25.0) | | 10 | 10 (6.2) | 7 (8.4) | 5 (6.8) | 3 (7.9) | 0 | 1 (12.5) | | Missing | 5 (3.1) | 3 (3.6) | 3 (4.1) | 2 (5.3) | 0 | 0 | | Sites of disease at baselineb | Total | 162 (100) | 83 (100) | 73 (100) | 38 (100) | 20 (100) | 8 (100) | | Prostate | 27 (16.7) | 12 (14.5) | 11 (15.1) | 3 (7.9) | 4 (20.0) | 2 (25.0) | | Loco-regional LNs | 35 (21.6) | 17 (20.5) | 15 (20.5) | 10 (26.3) | 10 (50.0) | 1 (12.5) | | Distant LNs | 59 (36.4) | 35 (42.2) | 31 (42.5) | 16 (42.1) | 8 (40.0) | 5 (62.5) | | Bone | 140 (86.4) | 73 (88.0) | 64 (87.7) | 34 (89.5) | 17 (85.0) | 7 (87.5) | | Respiratory | 30 (18.5) | 11 (13.3) | 18 (24.7) | 6 (15.8) | 2 (10.0) | 0 | | Liver | 18 (11.1) | 13 (15.7) | 8 (11.0) | 8 (21.1) | 1 (5.0) | 2 (25.0) | | Other distant sites | 34 (21.0) | 15 (18.1) | 17 (23.3) | 9 (23.7) | 3 (15.0) | 1 (12.5) | | Bone only | 42 (25.9) | 25 (30.1) | 16 (21.9) | 11 (28.9) | 3 (15.0) | 1 (12.5) | | LN only | 13 (8.0) | 5 (6.0) | 7 (9.6) | 3 (7.9) | 2 (10.0) | 0 | | Bone and LN only | 26 (16.0) | 14 (16.9) | 14 (19.2) | 5 (13.2) | 5 (25.0) | 2 (25.0) | | ECOG performance status at baseline | (0)…