← Product Code [PQP](/productcode/PQP) · P170019S015 # FoundationOne CDx (P170019S015) _Foundation Medicine, Inc. · PQP · May 19, 2020 · Pathology · APPR_ **Canonical URL:** https://fda-staging.innolitics.com/device/P170019S015 ## Device Facts - **Applicant:** Foundation Medicine, Inc. - **Product Code:** [PQP](/productcode/PQP.md) - **Decision Date:** May 19, 2020 - **Decision:** APPR - **Device Class:** Class 3 - **Review Panel:** Pathology ## Indications for Use FoundationOne® CDx is a next generation sequencing (NGS) based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations (indels), and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. The test is intended as a companion diagnostic (CDx) to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Additionally, FICDx 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. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product. ## Device Story FoundationOne®CDx (F1CDx) is an NGS-based IVD for tumor mutation profiling and companion diagnostic (CDx) use. Input: DNA from FFPE tumor tissue. Process: Whole-genome shotgun library construction; hybrid-capture of 324 cancer-related genes; sequencing on Illumina HiSeq 4000. Analysis: Proprietary pipeline detects base substitutions, indels, CNAs, and rearrangements; calculates MSI, TMB, and HRD status. Output: Clinical report identifying genomic alterations and CDx-relevant therapy eligibility. Used in clinical laboratories (Cambridge, MA; Morrisville, NC) by trained personnel. Healthcare providers use output to select targeted therapies (e.g., olaparib for mCRPC patients with HRR mutations). Benefits: Enables precision oncology by matching patients to FDA-approved therapies based on tumor genomic profile. ## Clinical Evidence Clinical evidence from the PROfound Phase III trial (N=387). Primary endpoint: radiological progression-free survival (rPFS) by BICR. Results: 66% reduction in risk of progression/death in Cohort A (HR=0.34; p<0.0001); 51% reduction in Cohort A+B (HR=0.49; p<0.0001). Analytical validation included 230 specimens (120 HRR positive, 110 negative) showing high concordance (PPA 93.96%, NPA 99.96%) with an orthogonal NGS method. ## Technological Characteristics NGS-based hybrid-capture assay targeting 324 genes. DNA input: 50-1000 ng from FFPE. Sequencing: Illumina HiSeq 4000 (SBS chemistry). Analysis: Proprietary bioinformatics pipeline (BWA, Picard, GATK, custom variant callers). Connectivity: Networked laboratory environment. Sterilization: N/A (reagents qualified). Software: Custom analysis pipeline (locked). ## Regulatory Identification A next generation sequencing (NGS) oncology panel is a device used for the qualitative detection of germline or somatic variants in one or more cancer-related genes. The device is intended to be used on DNA or RNA isolated from human clinical specimens. ## Predicate Devices - FoundationFocus CDxBRCA ([P160018](/device/P160018.md)) ## Reference Devices - PathVysion HER-2 DNA Probe Kit - PATHWAY Anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody - InSite HER-2/neu Kit - SPOT-Light HER2 CISH Kit - Bond Oracle HER2 IHC System - HER2 CISH pharmDx Kit - HercepTest - HER2 FISH pharmDx Kit - THxID BRAF Kit - cobas 4800 BRAF V600 Mutation Test - Oncomine Dx Target Test - therascreen BRAF V600E RGQ PCR Kit - Praxis Extended RAS Panel - cobas KRAS Mutation Test - therascreen KRAS RGQ PCR Kit - Vysis ALK Break Apart FISH Probe Kit - ALK (D5F3) CDx Assay - cobas EGFR Mutation Test v2 - therascreen EGFR RGQ PCR Kit - therascreen PIK3CA RGQ PCR Kit ## Submission Summary (Full Text) > This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com. > > Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a PMA](https://innolitics.com/services/regulatory/), [a 510(k)](https://innolitics.com/services/510ks/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/). {0} # SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) ## I. GENERAL INFORMATION Device Generic Name: Next Gen Sequencing oncology panel, somatic or germline variant detection system Device Trade Name: FoundationOne®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: P170019/S015 Date of FDA Notice of Approval: May 19, 2020 The original PMA (P170019) for FoundationOne®CDx (F1CDx) was approved on November 30, 2017 for the detection of genetic alterations in patients who may benefit from one of fifteen FDA-approved therapies for non-small cell lung cancer (NSCLC), melanoma, breast cancer, colorectal cancer, and ovarian cancer. Subsequently, seven PMA supplements were approved for expanding the indications for use of F1CDx since its original approval. PMA supplement (P170019/S005) for adding genomic loss of heterozygosity (LOH) was approved on April 10, 2019. PMA supplement (P170019/S004) for adding an indication for Lynparza® (olaparib) in ovarian cancer patients with BRCA1/2 alterations was approved on July 1, 2019. PMA supplement (P170019/S008) for adding an indication for Tagrisso® (osimertinib) in NSCLC patients with EGFR exon 19 deletions and EGFR exon 21 L858R alterations was approved on July 1, 2019. PMA supplement P170019/S006 for adding an indication for PIQRAY® (alpelisib) in breast cancer patients with PIK3CA alterations was approved on December 3, 2019. PMA supplement (P170019/S010) for adding a second site in Morrisville, NC, where the F1CDx assay will be performed was approved on December 16, 2019. PMA supplement (P170019/S013) for adding an indication for PEMZYRE® (pemigatinib) in cholangiocarcinoma patients with FGFR2 fusions was approved on April 17, 2020. PMA supplement (P170019/S011) for adding an indication for TABRECTA® (capmatinib) in NSCLC patients with MET single nucleotide variants (SNVs) and indels that lead to MET exon 14 skipping was approved on May 6, 2020. The current supplement was submitted to expand the intended use of F1CDx to include a companion diagnostic indication for homologous recombination repair (HRR) gene PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 1 {1} alterations in patients with metastatic castrate resistant prostate cancer (mCRPC) who may benefit from treatment with Lynparza® (olaparib). ## II. INDICATIONS FOR USE FoundationOne® CDx is a next generation sequencing (NGS) based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations (indels), and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. The test is intended as a companion diagnostic (CDx) to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Additionally, FICDx 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. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product. Table 1: Companion Diagnostic Indications | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Non-small cell lung cancer (NSCLC) | EGFR exon 19 deletions and EGFR exon 21 L858R alterations | Gilotrif® (afatinib), Iressa® (gefitinib), Tagrisso® (osimertinib), or Tarceva® (erlotinib) | | | EGFR exon 20 T790M alterations | Tagrisso® (osimertinib) | | | ALK rearrangements | Alecensa® (alectinib), Xalkori® (crizotinib), or Zykadia® (ceritinib) | | | BRAF V600E | Tafinlar® (dabrafenib) in combination with Mekinist® (trametinib) | | | MET single nucleotide variants (SNVs) and indels that lead to MET exon 14 skipping | Tabrecta™ (capmatinib) | | Melanoma | BRAF V600E | Tafinlar® (dabrafenib) or Zelboraf® (vemurafenib) | | | BRAF V600E and V600K | Mekinist® (trametinib) or Cotellic® (cobimetinib) in combination with Zelboraf® (vemurafenib) | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 2 {2} | Breast cancer | ERBB2 (HER2) amplification | Herceptin® (trastuzumab), Kadcyla® (ado-trastuzumab-emtansine), or Perjeta® (pertuzumab) | | --- | --- | --- | | | PIK3CA C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, H1047Y alterations | Piqray® (alpelisib) | | Colorectal cancer | KRAS wild-type (absence of mutations in codons 12 and 13) | Erbitux® (cetuximab) | | | KRAS wild-type (absence of mutations in exons 2, 3, and 4) and NRAS wild type (absence of mutations in exons 2, 3, and 4) | Vectibix® (panitumumab) | | Ovarian cancer | BRCA1/2 alterations | Lynparza® (olaparib) or Rubraca® (rucaparib) | | Cholangiocarcinoma | FGFR2 fusions and select rearrangements | Pemazyre™ (pemigatinib) | | Prostate cancer | Homologous Recombination Repair (HRR) gene (BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D and RAD54L) alterations | Lynparza® (olaparib) | The test is also used for detection of genomic loss of heterozygosity (LOH) from formalin-fixed, paraffin-embedded (FFPE) ovarian tumor tissue. Positive homologous recombination deficiency (HRD) status (defined as tBRCA-positive and/or LOH high) in ovarian cancer patients is associated with improved progression-free survival (PFS) from Rubraca (rucaparib) maintenance therapy in accordance with the RUBRACA product label. The F1CDx assay will be performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings/precautions and limitations are included in the FoundationOne CDx assay labeling. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 3 {3} # V. DEVICE DESCRIPTION FoundationOne®CDx (F1CDx) is performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. The assay includes reagents, software, instruments and procedures for testing DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples. The assay employs a single DNA extraction method from routine FFPE biopsy or surgical resection specimens, 50-1000 ng of which undergoes whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, 1 promoter region, 1 non-coding RNA (ncRNA), and select intronic regions from 34 commonly rearranged genes, 21 of which also include the coding exons (refer to Table 2 and Table 3, below, for the complete list of genes included in F1CDx). In total, the assay therefore detects alterations in a total of 324 genes. Using the Illumina® HiSeq 4000 platform, hybrid-capture selected libraries will be sequenced to high uniform depth (targeting > 500X median coverage with > 99% of exons at coverage > 100X). Sequence data is processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number alterations (amplifications and homozygous deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic signatures including microsatellite instability (MSI), tumor mutational burden (TMB), and positive homologous recombination deficiency (HRD) status (tBRCA-positive and/or LOH high) will be reported. Table 2: Genes with full coding exonic regions included in F1CDx for the detection of substitutions, insertion-deletions (indels), and copy number alterations (CNAs) | ABL1 | BRAF | CDKN1A | EPHA3 | FGFR4 | IKZF1 | MCL1 | NKX2-1 | PMS2 | RNF43 | TET2 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ACVR1B | BRCA1 | CDKN1B | EPHB1 | FH | INPP4B | MDM2 | NOTCH1 | POLD1 | ROS1 | TGFBR2 | | AKT1 | BRCA2 | CDKN2A | EPHB4 | FLCN | IRF2 | MDM4 | NOTCH2 | POLE | RPTOR | TIPARP | | AKT2 | BRD4 | CDKN2B | ERBB2 | FLT1 | IRF4 | MED12 | NOTCH3 | PPARG | SDHA | TNFAIP3 | | AKT3 | BRIP1 | CDKN2C | ERBB3 | FLT3 | IRS2 | MEF2B | NPM1 | PPP2R1A | SDHB | TNFRSF14 | | ALK | BTG1 | CEBPA | ERBB4 | FOXL2 | JAK1 | MEN1 | NRAS | PPP2R2A | SDHC | TP53 | | ALOX12B | BTG2 | CHEK1 | ERCC4 | FUBP1 | JAK2 | MERTK | NT5C2 | PRDM1 | SDHD | TSC1 | | AMER1 | BTK | CHEK2 | ERG | GABRA6 | JAK3 | MET | NTRK1 | PRKAR1A | SETD2 | TSC2 | | APC | C11orf30 | CIC | ERRF11 | GATA3 | JUN | MITF | NTRK2 | PRKCI | SF3B1 | TYRO3 | | AR | CALR | CREBBP | ESR1 | GATA4 | KDM5A | MKNK1 | NTRK3 | PTCH1 | SGK1 | U2AF1 | | ARAF | CARD11 | CRKL | EZH2 | GATA6 | KDM5C | MLH1 | P2RY8 | PTEN | SMAD2 | VEGFA | | ARFRP1 | CASP8 | CSF1R | FAM46C | GID4 (C17orf39) | KDM6A | MPL | PALB2 | PTPN11 | SMAD4 | VHL | | ARID1A | CBFB | CSF3R | FANCA | GNA11 | KDR | MRE11A | PARK2 | PTPRO | SMAR CA4 | WHSC1 | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 4 {4} | ASXL1 | CBL | CTCF | FANCC | GNA13 | KEAP1 | MSH2 | PARP1 | QKI | SMAR CB1 | WHSC1L1 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ATM | CCND1 | CTNNA1 | FANCG | GNAQ | KEL | MSH3 | PARP2 | RAC1 | SMO | WT1 | | ATR | CCND2 | CTNNB1 | FANCL | GNAS | KIT | MSH6 | PARP3 | RAD21 | SNCAIP | XPO1 | | ATRX | CCND3 | CUL3 | FAS | GRM3 | KLHL6 | MST1R | PAX5 | RAD51 | SOCS1 | XRCC2 | | AURKA | CCNE1 | CUL4A | FBXW7 | GSK3B | KMT2A (MLL) | MTAP | PBRM1 | RAD51B | SOX2 | ZNF217 | | AURKB | CD22 | CXCR4 | FGF10 | H3F3A | KMT2D (MLL2) | MTOR | PDCD1 | RAD51C | SOX9 | ZNF703 | | AXIN1 | CD274 | CYP17A1 | FGF12 | HDAC1 | KRAS | MUTYH | PDCD1G2 | RAD51D | SPEN | | | AXL | CD70 | DAXX | FGF14 | HGF | LTK | MYC | PDGFRA | RAD52 | SPOP | | | BAP1 | CD79A | DDR1 | FGF19 | HNF1A | LYN | MYCL | PDGFRB | RAD54L | SRC | | | BARD1 | CD79B | DDR2 | FGF23 | HRAS | MAF | MYCN | PDK1 | RAF1 | STAG2 | | | BCL2 | CDC73 | DIS3 | FGF3 | HSD3B1 | MAP2K1 | MYD88 | PIK3C2B | RARA | STAT3 | | | BCL2L1 | CDH1 | DNMT3A | FGF4 | ID3 | MAP2K2 | NBN | PIK3C2G | RB1 | STK11 | | | BCL2L2 | CDK12 | DOT1L | FGF6 | IDH1 | MAP2K4 | NF1 | PIK3CA | RBM10 | SUFU | | | BCL6 | CDK4 | EED | FGFR1 | IDH2 | MAP3K1 | NF2 | PIK3CB | REL | SYK | | | BCOR | CDK6 | EGFR | FGFR2 | IGF1R | MAP3K1 3 | NFE2L2 | PIK3R1 | RET | TBX3 | | | BCORL1 | CDK8 | EP300 | FGFR3 | IKBKE | MAPK1 | NFKBIA | PIM1 | RICTOR | TEK | | Table 3: Genes with Select Intronic Regions for the Detection of Gene Rearrangements, a Promoter Region and a ncRNA gene. | ALK | BRCA1 | ETV4 | EZR | KIT | MYC | NUTM1 | RET | DLC34A2 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | introns 18, 19 | introns 2, 7, 8, 12, 16, 19, 20 | introns 5, 6 | introns 9-11 | intron 16 | intron 1 | intron 1 | introns 7-11 | intron 4 | | BCL2 3'UTR | BRCA2 intron 2 | ETV5 introns 6, 7 | FGFR1 intron 1, 5, 17 | KMT2A (MLL) introns 6-11 | NOTCH2 intron 26 | PDGFRA introns 7, 9, 11 | ROS1 introns 31-35 | TERC ncRNA | | BCR introns 8, 13, 14 | CD74 introns 6-8 | ETV6 introns 5, 6 | FGFR2 intron 1, 17 | MSH2 intron 5 | NTRK1 introns 8-10 | RAF1 introns 4-8 | RSPO2 intron 1 | TERT Promoter | | BRAF introns 7-10 | EGFR introns 7, 15, 24-27 | EWSR1 introns 7-13 | FGFR3 intron 17 | MYB intron 14 | NTRK2 Intron 12 | RARA intron 2 | SDC4 intron 2 | TMPRSS 2 introns 1-3 | ### Test Output The test output includes: Category 1: CDx Claims noted in Table 1 of the Intended Use PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 5 {5} Category 2: Cancer Mutations with Evidence of Clinical Significance Category 3: Cancer Mutations with Potential Clinical Significance Genomic findings other than those listed in Table 1 of the intended use statement (i.e., Categories 2 and 3) are not prescriptive or conclusive for labeled use of any specific therapeutic product. ### Test Kit Contents The test includes a sample shipping kit, which is sent to ordering laboratories. The shipping kit contains the following components: - Specimen Preparation Instructions - Shipping Instructions - Return Shipping Label ### Instruments The F1CDx assay is intended to be performed with serial number-controlled instruments as indicated in Table 4, below. All instruments are qualified by Foundation Medicine, Inc. (FMI) under FMI's Quality System. Table 4: Instruments for use with the F1CDx assay | Instrument | | --- | | Beckman Biomek NXP Span-8 Liquid Handler | | Thermo Fisher Scientific KingFisher™ Flex with 96 Deep-well Head | | Illumina® cBot System | | Illumina® HiSeq 4000 System | ### Test Process All assay reagents included in the F1CDx assay process are qualified by FMI and are compliant with the medical device Quality Systems Regulation (QSR). ### A. Specimen Collection and Preparation Formalin-fixed, paraffin-embedded (FFPE) tumor specimens are collected and prepared following standard pathology practices. FFPE specimens may be received either as unstained slides or as an FFPE block. Prior to starting the assay, a Hematoxylin and Eosin (H&E) stained slide is prepared, and then reviewed by a board-certified pathologist to confirm disease ontology and to ensure that adequate tissue (0.6 mm³), tumor content (≥ 20% tumor) and sufficient nucleated cells are present to proceed with the assay. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 6 {6} ## B. DNA Extraction Specimens passing pathology review are queued for DNA extraction which begins with lysis of cells from FFPE tissue by digestion with a proteinase K buffer followed by automated purification using the 96-well KingFisher™ FLEX Magnetic Particle Processor. After completion of DNA extraction, double-stranded DNA (dsDNA) is quantified by the Quant-iT™ PicoGreen® fluorescence assay using the provided lambda DNA standards (Invitrogen) prior to Library Construction (LC). The sample must yield a minimum of 55 ng of genomic DNA to ensure sufficient DNA for quality control (QC) and to proceed with LC. ## C. Library Construction Library Construction (LC) begins with the normalization of DNA to 50-1000 ng. The normalized DNA samples are randomly sheared (fragmented) to ~200 bp by adaptive focused acoustic sonication using a Covaris LE220 before purification using a 1.8X volume of AMPure® XP Beads (Agencourt®). Solid-phase reversible immobilization (SPRI) purification and subsequent library construction with the NEBNext® reagents (custom-filled kits by NEB), including mixes for end repair, dA addition and ligation, are performed in 96-well plates (Eppendorf) on a Bravo Benchbot (Agilent) using the “with-bead” protocol¹ to maximize reproducibility and library yield. Indexed (6 bp barcodes) sequencing libraries are PCR amplified with HiFi™ (Kapa) for 10 cycles, and subsequently 1.8X SPRI purified. Purification and dilution for QC are performed. Following LC, a QC procedure is performed by quantifying single-stranded DNA (ssDNA) from purified libraries using the Quant-iT™ OliGreen® ssDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Libraries yielding insufficient sequencing library are failed. ## D. Hybrid Capture Hybrid Capture (HC) begins with normalization of each library to 500-2000 ng. Normalized samples then undergo solution hybridization which is performed using a > 50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 bp oligonucleotides. The baits target ~1.8 Mb of the human genome including all coding exons of 309 cancer-related genes, introns or non-coding regions of 35 genes, plus > 3,500 single nucleotide polymorphisms (SNPs) located throughout the genome. Baits are designed by tiling 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; SNP targets are allocated one bait each. Intronic baits are filtered for repetitive elements² as defined by the UCSC Genome RepeatMasker track. After hybridization, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen) and off-target material is removed by PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 7 {7} washing one time with 1X SSC at 25°C and four times with 0.25X SSC at 55°C. The PCR master mix is added to directly amplify (12 cycles) the captured library from the washed beads.³ After 12 cycles of amplification, the samples are 1.8X SPRI purified. Purification and dilution for QC are performed. Quality Control for Hybrid Capture is performed by measuring dsDNA yield using a Quant-i™ PicoGreen® dsDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Captured libraries yielding less than 140 ng of sequencing library are failed. ### E. Sequencing Sequencing is performed using off-board clustering on the Illumina cBot with patterned flow cell technology to generate monoclonal clusters from a single DNA template followed by sequencing using sequencing by synthesis (SBS) chemistry on the Illumina HiSeq 4000. Fluorescently labeled 3'-blocked dNTP's along with a polymerase are incorporated through the flow cell to create a growing nucleotide chain that is excited by a laser. A camera captures the emission color of the incorporated base and then is cleaved off. The terminator is then removed to allow the nucleotide to revert to its natural form and to allow the polymerase to add another base to the growing chain. A new pool of fluorescently labeled 3'-blocked dNTPs are added with each new sequencing cycle. The color changes for each new cycle as a new base is added to the growing chain. This method allows for millions of discrete clusters of clonal copies of DNA to be sequenced in parallel. ### F. Sequence Analysis Sequence data is analyzed using proprietary software developed by FMI. Sequence data is mapped to the human genome (hg19) using Burrows-Wheeler Aligner (BWA) v0.5.9.⁴ PCR duplicate read removal and sequence metric collection is performed using Picard 1.47 (http://picard.sourceforge.net) and SAMtools 0.1.12a.⁵ Local alignment optimization is performed using Genome Analysis Toolkit (GATK) 1.0.4705.⁶ Variant calling is performed only in genomic regions targeted by the test. Base substitution detection is performed using a Bayesian methodology, which allows for the detection of novel somatic alterations at low mutant allele frequency (MAF) and increased sensitivity for alterations at hotspot sites through the incorporation of tissue-specific prior expectations.⁷ Reads with low mapping (mapping quality < 25) or base calling quality (base calls with quality ≤ 2) are discarded. Final calls are made at MAF ≥ 5% (MAF ≥ 1% at hotspots). To detect indels, de novo local assembly in each targeted exon is performed using the de-Bruijn approach.⁸ Key steps are: - Collecting all read-pairs for which at least one read maps to the target region. - Decomposing each read into constituent k-mers and constructing an enumerable graph representation (de-Bruijn) of all candidate non-reference haplotypes present. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 8 {8} - Evaluating the support of each alternate haplotype with respect to the raw read data to generate mutational candidates. All reads are compared to each of the candidate haplotypes via ungapped alignment, and a read 'vote' for each read is assigned to the candidate with best match. Ties between candidates are resolved by splitting the read vote, weighted by the number of reads already supporting each haplotype. This process is iterated until a 'winning' haplotype is selected. - Aligning candidates against the reference genome to report alteration calls. Filtering of indel candidates is carried out similarly to base substitutions, with an empirically increased allele frequency threshold at repeats and adjacent sequence quality metrics as implemented in GATK: % of neighboring bases mismatches < 25%, average neighboring base quality > 25, average number of supporting read mismatches ≤ 2. Final calls are made at MAF ≥ 5% (MAF ≥ 3% at hotspots). Copy number alterations (CNAs, for HD, CNA is zero) are detected using a comparative genomic hybridization (CGH)-like method. First, a log-ratio profile of the sample is acquired by normalizing the sequence coverage obtained at all exons and genome-wide SNPs (~3,500) against a process-matched normal control. This profile is segmented and interpreted using allele frequencies of sequenced SNPs to estimate tumor purity and copy number at each segment. Amplifications are called at segments with ≥ 6 copies (or ≥ 7 for triploid/≥ 8 for tetraploid tumors) and homozygous deletions at 0 copies, in samples with tumor purity ≥ 20%. Amplifications in ERBB2 are called positive at segments with ≥ 5 copies for diploid tumors. Genomic rearrangements are identified by analyzing chimeric read pairs. Chimeric read pairs are defined as read pairs for which reads map to separate chromosomes, or at a distance of over 10 megabase (Mb). Pairs are clustered by genomic coordinate of the pairs, and clusters containing at least five (5) chimeric pairs [three (3) for known fusions] are identified as rearrangement candidates. Filtering of candidates is performed by mapping quality (average read mapping quality in the cluster must be 30 or above) and distribution of alignment positions. Rearrangements are annotated for predicted function (e.g., creation of fusion gene). To determine microsatellite instability (MSI) status, 95 intronic homopolymer repeat loci (10-20 bp long in the human reference genome) with adequate coverage on F1CDx Assay are analyzed for length variability and compiled into an overall MSI score via principal components analysis. Using the 95 loci, for each sample the repeat length is calculated in each read that spans the locus. The means and variances of repeat lengths is recorded. Principal components analysis (PCA) is used to project the 190-dimension data onto a single dimension (the first principal component) that maximizes the data separation, producing an MSI score. Each sample is assigned a qualitative status of MSI-High (MSI-H) or MSI-Stable (MSS); ranges of the MSI score are assigned MSI-H or MSS by manual unsupervised clustering. Samples with low coverage (< 250X median) are assigned a status of MSI-unknown. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 9 {9} Tumor mutational burden (TMB) is measured by counting all synonymous and non-synonymous variants present at 5% allele frequency or greater, and filtering out potential germline variants according to published databases of known germline polymorphisms including Single Nucleotide Polymorphism database (dbSNP) and Exome Aggregation Consortium (ExAC). Additional germline alterations still present after database querying are assessed for potential germline status and filtered out using a somatic-germline/zygosity (SGZ) algorithm. Furthermore, known and likely driver mutations are filtered out to exclude bias of the data set. The resulting mutation number is then divided by the coding region corresponding to the number of total variants counted, or 793 kb. The resulting number is communicated as mutations per Mb unit (mut/Mb). After completion of the Analysis Pipeline, variant data is displayed in the FMI custom-developed CATi software applications with sequence quality control metrics. As part of data analysis QC for every sample, the FICDx assay assesses cross-contamination through the use of a SNP profile algorithm reducing the risk of false-positive calls that could occur as a result of an unexpected contamination event. Sequence data is reviewed by trained bioinformatics personnel. Samples failing any QC metrics are automatically held and not released. ### 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 FMI as a professional service prior to approval and release by the laboratory director or designee. ### H. Internal Process Controls Related to the System #### Positive Control Each assay run includes a control sample run in duplicate. The control sample contains a pool of ten HapMap cell lines and is used as a positive mutation detection control. One hundred (100) different germline SNPs present across the entire targeted region are required to be detected by the analysis pipeline. If SNPs are not detected as expected, this results in a QC failure as it indicates a potential processing error. #### Sensitivity Control The HapMap control pool used as the positive control is prepared to contain variants at 5%-10% MAF which must be detected by the analysis pipeline to ensure expected sensitivity for each run. #### Negative Control Samples are barcoded molecularly at the 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 PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 10 {10} identify potential contamination that may have occurred prior to molecular barcoding, and can detect contamination lower than 1%. # I. Variant Classification A clinical report is provided to the ordering physician for each FICDx test performed at Foundation Medicine, Inc. Each report is generated and reviewed by an internal team consisting of clinical bioinformatics analysts, scientists, curators, and pathologists with mutations positive for therapy identified. Each sample is assessed for mutations in the 14 HRR genes ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, and RAD54L (Table 5). For these genes, both deleterious and suspected deleterious mutations in short variant, copy number alteration, and rearrangement variant classes are determined by an in-house software pipeline. Alterations listed in the COSMIC database and homozygous deletions are considered deleterious. Suspected deleterious mutations include truncating events i.e., splice, frameshift, and nonsense alterations, as well as large rearrangements that disrupt the coding sequence. The COSMIC check is a second layer of check for HRR positive suspected deleterious alterations. All splice, nonsense, frameshift alterations in HRR genes are considered biomarker positive and would be considered as suspected deleterious mutations (or “likely” status in FMI reporting rules). If these mutations are additionally reported in COSMIC, they would be listed as deleterious mutations (or “known” status in FMI reporting). The FICDx assay is intended as an aid in selecting prostate cancer patients with deleterious or suspected deleterious HRR variants, identified by the rules below, and who may be eligible for treatment with Lynparza™ (olaparib). Table 5: Mutation types identified in the HRR genes | Variant Class | Alteration type | Description* | | --- | --- | --- | | Short Variant | Nonsense, frameshift, or splice site | Any deleterious nonsense, frameshift, or splicing event that spans or occurs within ±2 bases of the intron/exon junction. | | | Missense or non-frameshift | Any of the mutations listed in Table 6 for ATM, BRCA1, and BRCA2 | | Copy Number Alteration | Homozygous copy number loss | Deleterious homozygous copy number loss of one or more exons. | | Rearrangement | Rearrangement | Any rearrangement that disrupts protein function | *For BRCA2, truncating mutations must occur upstream of bases encoding amino acid 3326. Additionally, the frameshift mutation T367fs*13 is FANCL is ineligible. All short variants must occur in the canonical transcript. The specific deleterious mutation (DM) and suspected deleterious mutation (SDM) missense mutations or non-frameshift mutations for BRCA1, BRCA2 and ATM are PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 11 {11} shown in Table 6 below. However, any missense or non-frameshift mutations in the other 12 genes would not be considered HRR positive. Table 6: Eligible deleterious mutation in the ATM, BRCA1 and BRCA2 genes | ATM | BRCA1 | BRCA2 | | --- | --- | --- | | M1T | M1V | M1R | | R2032K | M1I | M1I | | R2227C | C61G | V159M | | R2547_S2549del | C64Y | V211L | | G2765S | R71G | V211I | | R2832C | R71K | R2336P | | S2855_V2856delinsRI (annotated as S2855_V2856>RI) | R1495M | R2336H | | R3008C | E1559K | | | R3008H | D1692N | | | 8418+5_8418+8delGTGA or 8418+1_8418+4delGTGA | D1692H | | | | R1699W | | | | A1708E | | | | G1788V | | ### VI. ALTERNATIVE PRACTICES AND PROCEDURES There are FDA-approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using FFPE tumor specimens, as listed in Table 1 of the F1CDx intended use statement. The approved CDx tests are listed in Table 7, below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Each alternative has its own advantages and disadvantages. Physicians should consider the best method that suits their patients and that best meets their expectations. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 12 {12} Table 7 List of FDA approved CDx assays for genes targeted by F1CDx | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | HER2-Amplification | PathVysion HER-2 DNA Probe Kit | Abbott Molecular, Inc. | FISH | HERCEPTIN (trastuzumab) | Breast cancer | | | PATHWAY Anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody | Ventana Medical Systems, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | InSite HER-2/neu Kit | Biogenex Laboratories, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | SPOT-Light HER2 CISH Kit | Life Technologies, Inc. | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | Bond Oracle HER2 IHC System | Leica Biosystems | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | HER2 CISH pharmDx Kit | Dako Denmark A/S | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | INFORM HER2 Dual ISH DNA Probe Cocktail | Ventana Medical Systems, Inc. | Dual ISH | HERCEPTIN (trastuzumab) | Breast cancer | | | HercepTest | Dako Denmark A/S | IHC | HERCEPTIN (trastuzumab) PERJETA (pertuzumab) KADCYLA (ado-trastuzumab emtansine) | Breast cancer Gastric or Gastroesophageal junction adenocarcinoma | | | HER2 FISH pharmDx Kit | Dako Denmark A/S | FISH | HERCEPTIN (trastuzumab) PERJETA (pertuzumab) KADCYLA (ado-trastuzumab emtansine) | Breast cancer Gastric or Gastroesophageal junction adenocarcinoma | | BRAF-V600 | THxID BRAF Kit | bioMerieux | PCR | MEKINIST (tramatenib) | Melanoma | | | cobas 4800 BRAF V600 Mutation Test | Roche Molecular Systems, Inc. | PCR | ZELBORAF (vemurafenib) | Melanoma | | BRAF-600E | THxID BRAF Kit | bioMerieux | PCR | TAFINLAR (dabrafenib) | Melanoma | | | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | TAFINLAR (dabrafenib) MEKINIST (trametinib) | NSCLC | | | therascreen BRAF V600E RGQ PCR Kit | QIAGEN | PCR | BRAFTOVI (encorafenib) Erbitux (cetuximab) | Colorectal cancer | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 13 {13} | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | NRAS | Praxis Extended RAS Panel | Illumina, Inc. | NGS | VECTIBIX (panitumumab) | Colorectal cancer | | KRAS | cobas KRAS Mutation Test | Roche Molecular Systems, Inc. | PCR | ERBITUX (cetuximab) VECTIBIX (panitumumab) | Colorectal cancer | | | therascreen KRAS RGQ PCR Kit | QIAGEN | PCR | ERBITUX (cetuximab) VECTIBIX (panitumumab) | Colorectal cancer | | | Praxis Extended RAS Panel | Illumina, Inc. | NGS | VECTIBIX (panitumumab) | Colorectal cancer | | ALK - fusion | Vysis ALK Break Apart FISH Probe Kit | Abbott Molecular, Inc. | FISH | XALKORI (crizotinib) | NSCLC | | | ALK (D5F3) CDx Assay | Ventana Medical Systems, Inc. | IHC | XALKORI (crizotinib) | NSCLC | | EGFR – Exon 19 deletions & L858R | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TARCEVA (erlotinib) TAGRISSO (osimertinib) IRESSA (gefitinib) | NSCLC | | | therascreen EGFR RGQ PCR Kit | QIAGEN | PCR | GILOTRIF (afatinib) IRESSA (gefitinib) | NSCLC | | | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | IRESSA (gefitinib) | NSCLC | | EGFR T790M | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TAGRISSO (osimertinib) | NSCLC | | BRCA1/2 | FoundationFocus CDxBRCA | Foundation Medicine, Inc. | NGS | RUBRACA (rucaparib) | Advanced ovarian cancer | | PIK3CA | therascreen PIK3CA RGQ PCR Kit | QIAGEN | PCR | PIQRAY (alpelisib) | Breast cancer | Abbreviations: FISH – fluorescence in situ hybridization; IHC – immunohistochemistry; CISH – chromogenic in situ hybridization; ISH – in situ hybridization; PCR – polymerase chain reaction; NGS – next generation sequencing. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 14 {14} ## VII. MARKETING HISTORY Foundation Medicine, Inc. initially designed and developed the FoundationOne® laboratory developed test (F1 LDT), and the first commercial sample was tested in 2012. The F1 LDT has been used to detect the presence of genomic alterations in FFPE tumor tissue specimens. The F1 LDT is not FDA-cleared or -approved. The F1CDx Premarket Approval (PMA) was originally approved on November 30, 2017 by FDA (P170019) and is commercially available in US since March 30, 2018. The following PMA supplements were approved by FDA for including additional indications for use to the originally approved Intended Use: - • P170019/S005 was approved on April 10, 2019 - • P170019/S004 and P170019/S008 were approved on July 1, 2019 - • P170019/S009 approved on August 21, 2019 - • P170019/S006 approved on December 3, 2019 - • P170019/S010 approved on December 16, 2019 - • P170019/S013 approved on April 17, 2020 - • P170019/S011 approved on May 5, 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 test results, and subsequently, inappropriate patient management decisions. Patients with false positive results may undergo treatment with one of the therapies listed in the above 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 therapy. There is also a risk of delayed results, which may lead to delay of treatment with indicated therapy. For the specific adverse events related to the approved therapeutics, please see approved drug product labels. ## IX. SUMMARY OF NON-CLINICAL STUDIES ### A. Laboratory Studies Performance characteristics were established using DNA derived from FFPE tissue from both bone metastasis and soft tissue types from prostate cancer patients. The study included a broad range of alteration types (substitution, insertion-deletion, copy number alterations, rearrangements) across a number of genes. For various analytical validation studies 175 unique samples [(121 HRR positive samples (151 variants) for analytical concordance, 7 samples (17 variants) for limit of detection and 47 samples (99 variants)] for precision/reproducibility studies were used. Total number of variants observed across all 175 samples were 267. The tables below (Table 8 and 9) include a list of genes/variant types that were represented in all the PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 15 {15} analytical validation studies to support performance of the assay for detection of all the variant types in the HRR genes. **Table: 8: Summary of samples across analytical validation studies** | Gene | Total | HD | RE | ID | SUB | | --- | --- | --- | --- | --- | --- | | *BRCA1* | 12 | 1 | 6 | 4 | 1 | | *BRCA2* | 77 | 17 | 6 | 38 | 16 | | *ATM* | 46 | 6 | 8 | 16 | 16 | | *BARD1* | 10 | 0 | 2 | 3 | 5 | | *BRIP1* | 14 | 1 | 2 | 4 | 7 | | *CDK12* | 48 | 3 | 4 | 29 | 12 | | *CHEK1* | 6 | 1 | 1 | 2 | 2 | | *CHEK2* | 12 | 2 | 2 | 8 | 0 | | *FANCL* | 4 | 0 | 0 | 4 | 0 | | *PALB2* | 16 | 1 | 2 | 1 | 12 | | *RAD51B* | 3 | 0 | 3 | 0 | 0 | | *RAD51C* | 5 | 2 | 1 | 2 | 0 | | *RAD51D* | 5 | 0 | 1 | 1 | 3 | | *RAD54L* | 2 | 0 | 1 | 0 | 1 | | *Total* | 267 | 37 | 40 | 115 | 75 | SUB: Base Substitutions. Patients who have other variant types (non-sense, missense, splice site, etc.) ID: Insertion/Deletion. Patients who have small insertions/deletions which lead to a frameshift HD: Homozygous deletion (copy number alteration, CN=0). Patients who have homozygous gene loss; RE: Large Rearrangement. Patients who have exonic or multi-exon insertions or deletion **Table 9: Variants Used is Three Key Analytical Validation Studies** | | LoD | | | | Precision | | | | Accuracy | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | HD | RE | ID | SUB | HD | RE | ID | SUB | HD | RE | ID | SUB | | *BRCA1* | 0 | 1 | 0 | 0 | 1 | 2 | 1 | 1 | 0 | 2 | 3 | 0 | | *BRCA2* | 1 | 0 | 0 | 0 | 1 | 3 | 11 | 7 | 15 | 3 | 27 | 9 | | *ATM* | 0 | 1 | 0 | 1 | 4 | 4 | 4 | 3 | 2 | 3 | 12 | 12 | | *BARD1* | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 4 | 0 | 1 | 2 | 1 | | *BRIP1* | 0 | 0 | 0 | 1 | 1 | 2 | 2 | 4 | 0 | 0 | 2 | 2 | | *CDK12* | 0 | 1 | 1 | 0 | 0 | 0 | 7 | 3 | 3 | 3 | 21 | 9 | | *CHEK1* | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 2 | 0 | 0 | 2 | 0 | | *CHEK2* | 0 | 1 | 0 | 0 | 1 | 0 | 2 | 0 | 1 | 1 | 6 | 0 | | **FANCL* | 0 | 0 | 1 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | | *PALB2* | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 9 | 1 | 0 | 0 | 2 | | *RAD51B* | 0 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | | **RAD51C* | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | | *RAD51D* | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 1 | 1 | 1 | | *RAD54L* | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | *Not enrolled in the clinical trial PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 16 {16} # 1. Analytical Accuracy/Concordance ## a. Comparison to an Orthogonal Method An analytical accuracy study was performed to demonstrate the concordance between F1CDx and an externally validated NGS assay (evNGS) for the detection of HRR gene alterations. This study evaluated a set of 230 specimens including 120 HRR positive clinical specimens (151 total variants observed) and 110 HRR negative specimens. The positive variants cover a range of variant types including nonsense mutations, frameshift indels, mutations in the consensus splice donor and acceptor sequence, rearrangements, and homozygous deletions (copy number = 0). A summary of the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) and corresponding 95% two-sided exact confidence intervals (CIs) is provided in Table 10, below. **Table 10: Concordance Summary for HRR alterations** | Variant Types | F1CDx+ /evNGS+ | F1CDx- /evNGS+ | F1CDx+ /evNGS- | F1CDx- /evNGS- | PPA % [95% CI] | NPA [95% CI] | | --- | --- | --- | --- | --- | --- | --- | | **SUB** | 35 | 1 | 1 | 8243 | 97.22% [85.47%, 99.93%] | 99.99% [99.93%, 100.00%] | | **ID** | 75 | 6 | 2 | 17627 | 92.59% [84.57%, 97.23%] | 99.99% [99.96%, 100.00%] | | **RE** | 10 | 1 | 5 | 1824 | 90.91% [58.72%, 99.77%] | 99.73% [99.36%, 99.91%] | | **HD** | 20 | 1 | 3 | 1356 | 94.02% [88.06%, 97.56%] | 99.78% [99.36%, 99.95%] | | **Total** | 140 | 9 | 11 | 29050 | 93.96% [88.84, 97.20] | 99.96% [99.93%, 99.98%] | SUB: Base Substitutions. Patients who have other variant types (non-sense, missense, splice site, etc.); ID: Insertion/Deletion. Patients who have small insertions/deletions which lead to a frameshift; HD: Homozygous deletion (copy number alteration, CN=0). Patients who have homozygous gene loss; RE: Large Rearrangement. Patients who have exonic or multi-exon insertions or deletion. PPA/NPA may be biased due to differential sampling. Since the PPA and NPA were calculated without adjusting for the distribution of samples selected using F1CDx, positive predictive value (PPV) and negative predictive value (NPV) were also estimated conditional on F1CDx. The total number of alterations detected across all 120 samples by either F1CDx and/or evNGS was used to determine NPV. The observed PPV for short variants was 97.22% [95% confidence interval (CI): 85.83% - 99.86%], indels 97.40% [95% PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 17 {17} confidence interval (CI): 91.01%, 99.28%], rearrangements 66.67% [95% confidence interval (CI): 8.38%, 88.18%], and homozygous deletions 86.96% [95% confidence interval (CI): 66.41%, 97.22%]. The NPV for all variants was 99.97% [95% confidence interval (CI): 99.94%, 99.98%]. Differences in alteration calls between the two assays were noted. These differences were primarily due to differences in filtering employed by FICDx and evNGS. With FICDx as the screening assay, evNGS failed to detect 11 HRR variants that were detected by FICDx. Nine variants were detected by evNGS but not by FICDx. Seven of the nine were short variants (indels) that were just below the analytical detection threshold. The threshold for indel detection is higher for FICDx assay as compared to the evNGS assay. The FICDx variant calling pipeline imposes a filter (higher MAF threshold) for indels in homopolymer regions to reduce the likelihood of calling false positives resulting from artifacts introduced by the technology. As such, the difference observed was due to varying filter thresholds between the two platforms. In addition, three genes, *FANCL*, *RAD51B*, or *RAD54L*, were excluded from the analysis as these gene could not be evaluated by the evNGS. # **b. Comparison of variant concordance between soft tissue vs bone metastasis** Of the 230 patient samples, six were bone metastasis and 193 were soft tissue. A summary of the analysis is provided in Table 11, below. **Table 11: Soft tissue and bone metastasis concordance** | Soft Tissue | | | | | --- | --- | --- | --- | | | evNGS + | evNGS - | | | FICDx + | 114 | 9 | PPV: 92.68% (89.56%, 96.60%) | | FICDx - | 1 | 24383 | NPV: 99.98% (99.95%, 99.99%) | | | PPA: 95.80% (90.47%, 98.62%) | NPA: 99.96% (99.93%, 99.98%) | | | Bone Metastasis | | | | | | evNGS + | evNGS - | | | FICDx + | 6 | 0 | PPV: 100.00% (54.07%, 100.00%) | | FICDx - | 1 | 755 | NPV: 99.87% (99.27%, 100.00%) | | | PPA: 85.71% (42.13%, 99.64%) | NPA: 100.00% (99.51%, 100.00%) | | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 18 {18} ## 2. Analytical Sensitivity ### a. Limit of Detection (LoD) – Allele Fraction The LoD was evaluated in FFPE-derived DNA from seven prostate tumor specimens (both soft tissues and bone metastases) at lowest DNA input level (52 ng). The LoD for short variants of CDx biomarkers representing ATM (substitution), CDK12 (indel), PPP2R2A (7 bp indel), BRIP1 (substitution), PALB2 (substitution and indel), and FANCL (indel) are summarized in Table 12. Table 12: Summary of LoD for Alterations Associated with CDx Claims | Alteration | LoD* Allele Fraction (%) | | --- | --- | | HRR base substitutions | 5.44% - 6.33% | | HRR indels | 5.22% - 12.74% | *LoD calculations for the CDx variants were based on the 95% hit rate approach. Additional results for F1CDx estimating the LoD by allele fraction were included in PMA P170019 ### b. Limit of Blank (LoB) The limit of blank (LoB) of zero was confirmed by demonstrating that percentage of false-positive results did not exceed 5% (type I error risk α=0.05). Six HRR-biomarker negative samples were evaluated in 12 replicates each for the LoB assessment. Of the 72 aliquots, three failed prior to sequencing and 69 replicates were available for LoB analysis. The positive call rate is 0/69 (0%), confirming the LoB = zero. ### c. Analytical Sensitivity – Tumor Purity and Average Reads Using seven samples, LoD was determined for nine HRR rearrangements (two samples had multiple rearrangements) and one homozygous deletion based on tumor purity level. For each sample, five levels of tumor purity with 14 replicates for 20% tumor purity and 20 replicates for 15 – 2.5% tumor purity levels, were evaluated (total of 94 replicates per sample). LoD calculations were based on the empirical hit rate approach, and are summarized in Table 13. LoD values were determined to be 20.1% tumor purity for HRR gene rearrangements and 23.9% tumor purity for HRR gene homozygous deletions. Table 13: LoD by empirical hit rate based on tumor purity and average reads | Alteration | LoD Tumor Purity (%) | LoD Average Reads | | --- | --- | --- | | HRR rearrangements | 20.1% | 39.3 | | HRR homozygous deletions | 23.9% | N/A | Please refer to the Summary of Safety and Effectiveness Data P170019 (Section IX.A.2) for additional F1CDx platform-level F1CDx LoD data. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 19 {19} ### 3. Analytical Specificity #### a. Interfering Substances Please see the Summary of Safety and Effectiveness Data P160018 and P710019 for F1CDx platform validation of analytical specificity, including interfering substances and in silico hybrid capture bait specificity. ### 4. Carryover/Cross-Contamination Please see the Summary of Safety and Effectiveness Data P160018 and P170019 for F1CDx platform validation of carryover/cross-contamination. ### 5. Precision and Reproducibility An intermediate precision study was performed for alterations in the 14 HRR genes. This study evaluated two aspects: repeatability and reproducibility. Repeatability (intra-run) and reproducibility (inter-run) were assessed in replicates of two, per two runs (plates), with three sequencers and two reagent lots as full factorial. A total of 24 replicates per sample were processed. Samples were selected close to LoD and up to 2-3X LoD. A set of 47 unique FFPE samples from both soft tissue (38) and bone metastases (9) tumors carrying different variant types (62 alterations in HRR genes) were evaluated in the primary analysis. The primary analysis included 62 alterations at a range of MAF, chimeric read counts, and tumor purities. Several samples in the primary analysis included alterations that were very close to LoD and Variant Analysis Pipeline (VAP) thresholds. Some of these samples, the samples with rearrangements (RE) in particular, did not meet the acceptance criteria of ≥ 90%. Eight (8) of 15 RE samples were tested at 1x or just below 1x LoD. Five of those samples had up to 83% concordance for reproducibility and up to 80% concordance for repeatability. Three (3) other samples at 1x LoD and remaining seven (7) samples at 2–3x LoD had close to 100% concordance for both reproducibility and repeatability. Of the 11 samples with copy number alterations (CNA), 10 had homozygous deletion (HD, CNA =0) and one had copy number amplification. Of the 10 HD samples, five (5) samples were tested at 0.84 –1.8x LoD and four (4) samples were tested at 1.85 –2.7x LoD and one sample was tested at 3.5x LoD. Except for one sample (0.84x LoD), all samples had 100% concordance for both reproducibility and repeatability. A summary results and corresponding 95% two-sided exact confidence intervals (CIs) is provided below (Table 14). An additional secondary analysis was performed to assess all somatic alterations in HRR genes (99 alterations in total for secondary analysis) at levels just upstream of the variant analysis pipeline (VAP) thresholds. The results of the secondary exploratory analysis are enumerated in Table 15. Alterations and variant categories exceeded the acceptance criteria, demonstrating ≥ 90% reproducibility and ≥ 90% repeatability. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 20 {20} **Table 14: HRR Precision Primary Analysis Results** | Variant | N | N Reps | N detected | Repro % | L 95% CI | U 95% CI | N pair | N pairs agree | Rep % | L 95% CI | U 95% CI | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | HD | 11 | 259 | 242 | 93.4 | 89.7 | 96.1 | 129 | 122 | 94.6 | 89.1 | 97.8 | | Indel | 26 | 610 | 597 | 97.9 | 96.4 | 98.9 | 302 | 293 | 97.0 | 94.4 | 98.6 | | RE | 15 | 347 | 305 | 87.9 | 83.4 | 91.1 | 171 | 153 | 89.5 | 83.9 | 93.6 | | SUB | 10 | 238 | 226 | 95.0 | 91.4 | 97.4 | 118 | 108 | 91.5 | 85.0 | 95.9 | | **Total** | **62** | **1454** | **1370** | **94.2** | **92.9** | **95.4** | **720** | **676** | **93.9** | **91.9** | **95.5** | **Table 15: HRR Precision Secondary Analysis Results** | Variant | N | N Reps | N detected | Repro % | L 95% CI | U 95% CI | N pair | N pairs agree | Rep % | L 95% CI | U 95% CI | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | HD | 13 | 307 | 299 | 97.4 | 94.9 | 98.9 | 153 | 145 | 94.8 | 90.0 | 97.7 | | Indel | 34 | 793 | 790 | 99.6 | 98.9 | 99.9 | 392 | 389 | 99.2 | 97.8 | 99.8 | | RE | 16 | 371 | 366 | 98.7 | 96.9 | 99.6 | 183 | 178 | 97.3 | 93.7 | 99.1 | | SUB | 36 | 846 | 840 | 99.3 | 98.5 | 99.7 | 417 | 412 | 98.8 | 97.2 | 99.6 | | **Total** | **99** | **2317** | **2295** | **99.1** | **98.6** | **99.4** | **1145** | **1124** | **98.2** | **97.2** | **98.9** | HD: homologous recombination (CNA = 0), Indel: insertion and deletions, RE: rearrangements, SUB: base substitutions The variant component analysis was performed to assess the precision (mean, coefficient of variation, and standard deviation) using the underlying MAF for short variants (base subs and indels), TP (Tumor Purity) for HD/CNA and average reads for RE on the 62 alterations selected in the primary analysis. Of 62 alterations, three alterations (4.8%) were excluded from the variant component analysis due to reproducibility less than 50% (i.e., small number of positive call replicates for a reliable model). Based on the model, the mean, SD, and CV of repeatability and reproducibility were calculated for each alteration. The results of each short variant (base subs and indel), HD/CNA and RE assessed in the variant component analysis are summarized in Table 16, Table 17 and Table 18, respectively. **Table 16: Summary for base substitutions and indels component analysis results of each alteration** | Gene | Variant Type | Average MAF% | Repeatability | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | | | | | SD | CV | SD | CV | | *PALB2* | SUB | 35.1 | 0.03 | 0.08 | 0.02 | 0.08 | | *CHEK2* | ID | 45.9 | 0.03 | 0.06 | 0.02 | 0.06 | | *PALB2* | SUB | 6.9 | 0.01 | 0.18 | 0.01 | 0.24 | | *CDK12* | SUB | 17.2 | 0.01 | 0.07 | 0.02 | 0.12 | | *ATM* | ID | 13.5 | 0.01 | 0.09 | 0.01 | 0.11 | | *CDK12* | ID | 38.8 | 0.02 | 0.06 | 0.02 | 0.07 | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 21 {21} | Gene | Variant Type | Average MAF% | Repeatability | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | | | | | SD | CV | SD | CV | | BRCA2 | ID | 6.0 | 0.00 | 0.11 | 0.00 | 0.11 | | ATM | ID | 6.9 | 0.03 | 0.04 | 0.03 | 0.04 | | ATM | ID | 15.1 | 0.01 | 0.08 | 0.01 | 0.10 | | CDK12 | ID | 5.8 | 0.00 | 0.09 | 0.01 | 0.11 | | BARD1 | ID | 12.9 | 0.01 | 0.07 | 0.01 | 0.09 | | BRCA2 | ID | 10.8 | 0.01 | 0.09 | 0.01 | 0.11 | | CHEK1 | SUB | 31.6 | 0.02 | 0.07 | 0.02 | 0.07 | | BARD1 | SUB | 6.5 | 0.08 | 0.11 | 0.01 | 0.19 | | FANCL | ID | 43.6 | 0.03 | 0.07 | 0.03 | 0.08 | | RAD51C | ID | 57.3 | 0.01 | 0.03 | 0.01 | 0.03 | | BRCA1 | ID | 9.9 | 0.01 | 0.11 | 0.02 | 0.21 | | BRCA2 | ID | 59.9 | 0.02 | 0.04 | 0.02 | 0.04 | | ATM | ID | 49.9 | 0.02 | 0.05 | 0.02 | 0.05 | | BRCA2 | SUB | 36.3 | 0.03 | 0.07 | 0.03 | 0.09 | | BRCA2 | SUB | 52.7 | 0.03 | 0.05 | 0.03 | 0.07 | | BRCA2 | SUB | 66.3 | 0.03 | 0.04 | 0.03 | 0.05 | | FANCL | ID | 51.3 | 0.01 | 0.03 | 0.02 | 0.04 | | BRIP1 | ID | 16.1 | 0.02 | 0.09 | 0.01 | 0.10 | | CDK12 | ID | 15.4 | 0.01 | 0.07 | 0.01 | 0.12 | | CHEK2 | ID | 44.3 | 0.03 | 0.07 | 0.04 | 0.08 | | BRCA2 | ID | 48.3 | 0.02 | 0.05 | 0.03 | 0.05 | | BRCA2 | ID | 20.4 | 0.01 | 0.09 | 0.02 | 0.12 | | CDK12 | ID | 46.5 | 0.02 | 0.04 | 0.02 | 0.04 | | CDK12 | ID | 45.7 | 0.02 | 0.05 | 0.02 | 0.05 | | BRIP1 | ID | 46.0 | 0.01 | 0.03 | 0.04 | 0.09 | | RAD51D | SUB | 6.3 | 0.01 | 0.15 | 0.01 | 0.25 | | FANCL | ID | 42.9 | 0.02 | 0.05 | 0.02 | 0.06 | | BRIP1 | SUB | 19.1 | 0.01 | 0.07 | 0.01 | 0.08 | Table 17. Summary of HD variant component analysis results of each alteration | Gene | Variant Type | TP% | Repeatability | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | | | | | SD | CV | SD | CV | | CHEK1 | Amp | 79.9 | 0.02 | 0.02 | 0.03 | 0.03 | | ATM | loss | 82.2 | 0.06 | 0.07 | 0.07 | 0.08 | | BRCA1 | loss | 40.6 | 0.01 | 0.02 | 0.01 | 0.02 | | CHEK2 | loss | 55.9 | 0.03 | 0.06 | 0.05 | 0.09 | | RAD51C | loss | 55.0 | 0.01 | 0.02 | 0.01 | 0.02 | | ATM | loss | 65.4 | 0.16 | 0.25 | 0.16 | 0.25 | | ATM | loss | 49.9 | 0.09 | 0.18 | 0.09 | 0.18 | | ATM | loss | 43.6 | 0.09 | 0.20 | 0.09 | 0.22 | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 22 {22} Table 18: summary of RE variant component analysis results of each alteration | Gene | Variant Type | Average Reads | Repeatability | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | | | | | SD | CV | SD | CV | | ATM | RE | 153.1 | 17.26 | 0.11 | 27.64 | 0.18 | | BRCA1 | RE | 73.6 | 9.64 | 0.13 | 9.88 | 0.13 | | BRCA1 | RE | 12.7 | 2.90 | 0.22 | 2.92 | 0.22 | | BRCA2 | RE | 23.8 | 4.89 | 0.20 | 5.82 | 0.24 | | BRCA2 | RE | 14.7 | 2.58 | 0.17 | 3.44 | 0.23 | | BRCA2 | RE | 17.1 | 5.03 | 0.29 | 5.19 | 0.30 | | BRIP1 | RE | 169.7 | 16.54 | 0.09 | 20.89 | 0.12 | | PALB2 | RE | 38.6 | 6.60 | 0.17 | 7.82 | 0.20 | | RAD51B | RE | 236.9 | 20.84 | 0.08 | 32.68 | 0.13 | | ATM | RE | 75.8 | 13.27 | 0.17 | 14.71 | 0.19 | | RAD51B | RE | 12.5 | 3.06 | 0.24 | 3.06 | 0.24 | | CHEK1 | RE | 65.5 | 8.85 | 0.13 | 9.37 | 0.14 | | BRIP1 | RE | 68.2 | 10.3 | 0.15 | 14.70 | 0.21 | # Site-to-site reproducibility for the North Carolina site: A two-site reproducibility study including the second site in Morrisville, North Carolina was not conducted. Site-to-site reproducibility will be provided in a post-market study. # 6. Reagent Lot Interchangeability Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDxBRCA assay and F1CDx. For reagent lot interchangeability performance data, see the Summary of Safety and Effectiveness Data for P160018. # 7. Stability Studies # a. Reagent Stability Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDxBRCA Assay and F1CDx. For reagent stability performance data, see the Summary of Safety and Effectiveness Data for P160018. The claimed reagent stability is 4 months for the LC and HC kits, and 3 months for the sequencing kits at manufacturer's recommended storage condition. # b. DNA Stability Please refer to the Summary of Safety and Effectiveness Data for P160018 and P170019 for results on DNA stability. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 23 {23} # c. FFPE Slide Stability Please refer to the Summary of Safety and Effectiveness Data for P160018 and P170019 for data on the stability of FFPE slides. # 8. General Lab Equipment and Reagent Evaluation # a. DNA Amplification Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDxBRCA assay and F1CDx. For DNA amplification performance data, see the Summary of Safety and Effectiveness Data for P160018. # b. DNA Extraction An additional study was conducted to evaluate the quality of DNA extracted by F1CDx assay when processing prostate soft tissue and bone metastases samples. A total of 24 FFPE prostate bone metastasis specimens and 23 FFPE prostate soft tissue specimens were procured for evaluation in the study. Each of the 47 blocks were processed in duplicate from extraction through sequencing. It is known that prostate bone metastasis specimens are challenging for DNA extraction. Of the 94 (47x2) samples, 19 samples (1 bone met and 18 soft tissue) failed pathology review. Of the 75 samples (47 bone mets and 28 soft tissues), 22 bone mets (47%) and all 28 soft tissues (100%) passed the DNA extraction QC yielding ≥ 55 ng DNA. For the samples that passed the DNA extraction yield requirement, the success rate was 100% for LC, 96.0% for HC and 94% for sequencing. There is no significant difference between processing success rates for bone mets and soft tissue samples (Table 19). Table 19: Post-DNA extraction success rate | Process Steps | Overall % 95% CI | Bone mets % 95% CI | Soft Tissue % 95% CI | | --- | --- | --- | --- | | LC | 100% [92.9%, 100%] | 100% [84.6%, 100%] | 100% [87.7%, 100%] | | HC | 96% [86.3%, 99.5%] | 91% [70.8%, 98.9%] | 100% [87.7%, 100%] | | Sequencing | 94% [82.8%, 98.7%] | 95% [75.1%, 99.9%] | 93% [76.5%, 99.1%] | Additionally, the overall, positive and negative agreements between duplicates were determined to be 100% with corresponding exact 2-sided 95% CI of [98%, 100%], [72%, 100%] and [98%, 100%], respectively. In the 47 FFPE tissues, 11 HRR positive variants and 205 negative variants were identified. In the 11 HRR variants identified, the concordance was 100% for each variant type [substitutions (1), indels (7), rearrangements (1) and homozygous deletions (2)]. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 24 {24} Please refer to the Summary of Safety and Effectiveness Data for P160018 and P170019 for additional DNA extraction performance data. # 9. Guard banding/Robustness Please see the Summary of Safety and Effectiveness Data for P160018, P170019 and P160018/S001 for guardbanding /robustness results.. # 10. Tissue Comparability Please see the Summary of Safety and Effectiveness Data for P160018 and P170019 for tissue comparability study. # B. Animal Studies No animal studies were conducted using the F1CDx assay. # C. Additional Studies No additional studies were conducted using the F1CDx assay. # X. SUMMARY OF PRIMARY CLINICAL STUDY The PROfound study is a Phase III, randomized, open-label, multicenter trial to assess the efficacy and safety of olaparib monotherapy in patients with metastatic castration-resistant prostate cancer (mCRPC) that have qualifying homologous recombination repair (HRR) gene alterations that were predicted to be deleterious or suspected deleterious (known or predicted to be detrimental/lead to loss of function) and who have failed prior treatment with a new hormonal agent (NHA). The safety and effectiveness of F1CDx for detecting HRR gene alterations in mCRPC patients who may benefit from treatment with Lynparza® (olaparib) was demonstrated based on results from the PROfound trial. Patients were selected into the trial by FMI's Clinical Trial Improvement Amendments (CLIA) HRR clinical trial assay (CTA). The CLIA HRR CTA uses the same classification rules as the F1CDx test with an expanded curated mutation list determined for all 14 HRR genes including BRCA1 and BRCA2 prior to the start of the PROfound study. Since enrollment was based on FMI's F1CDx-based CLIA HRR assay, which is identical to the production F1CDx assay except some minor updates to the shared production analysis pipeline, the efficacy results are based on patients enrolled by the CLIA HRR assay (please see section D.2, below). The wet lab workflow (including reagents, equipment and QC) and the post-sequencing analysis pipeline are common between the CLIA HRR assay and production F1CDx assay. A summary of the clinical study is presented below. # A. Study Design The PROfound study was an international multicenter study conducted in 206 study centers in 20 countries (of these, 139 centres randomized patients): Argentina (6 sites), Australia (10 sites), Austria (5 sites), Brazil (14 sites), Canada (12 sites), PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 25 {25} Denmark (1 site), France (13 sites), Germany (15 sites), Israel (6 sites), Italy (10 sites), Japan (30 sites), Netherlands (6 sites), Norway (1 site), South Korea (9 sites), Spain (7 sites), Sweden (2 sites), Taiwan (9 sites), Turkey (8 sites), United Kingdom (5 sites) and United States (37 sites). First patient was enrolled in 6 February 2017 and the last was patient enrolled in 18 September 2018. The original protocol (v.1; 19 October, 2016) was amended 4 times (v.4; final version was dated 7 March, 2019) across all study sites to modify study procedures or patient eligibility criteria. All patients were required to have a qualifying HRR mutation assessed using the FMI CLIA HRR CTA to be randomized. Qualifying HRR gene alterations including 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. In addition, patients must have received a prior NHA (e.g., abiraterone acetate and/or enzalutamide) for the treatment of metastatic prostate cancer and/or castration-resistant prostate cancer (mCRPC) and, in the opinion of the investigator, progressed on this treatment. A total of 387 patients were enrolled based on the CTA. Patients were randomized in a 2:1 ratio to the treatments as specified below: - Investigators choice of NHA with either enzalutamide 160 mg orally once daily (od) or abiraterone acetate 1000 mg orally qd with prednisone 5 mg orally bd (prednisolone was permitted for use instead of prednisone, if necessary) The primary endpoint for the study was radiological progression-free survival (rPFS) of olaparib based on blinded independent central review (BICR) using RECIST 1.1 for soft tissue and prostate cancer working group 3 (PCWG3) for bone criteria in subjects with mCRPC with *BRCA1*, *BRCA2* or *ATM* qualifying mutations (Cohort A). The key secondary endpoints were objective response rate (ORR) by BICR assessment in subjects with measurable disease using RECIST 1.1 (soft tissue) and PCWG3 (bone) criteria (cohort A), rPFS by BICR using RECIST 1.1 (soft tissue) and PCWG3 (bone) criteria for (Cohort A+B) and overall survival (OS) for Cohort A. ### 1. Patient Information and Consent In the PROfound study, written informed consent was obtained from each patient before enrollment according to the regulatory and legal requirements of the participating countries. The patients must provide informed consent for the genetic sampling and analyses as part of study inclusion criteria. As part of this procedure, the Investigator explained orally and in writing the information about the nature, purpose, possible risk and benefit of the study, information about alternative treatment with non-investigational drugs. The investigator(s) is responsible for ensuring that consent is given freely and that the Subject understands that they are free to discontinue from the study at any time. The patient received all information that was required by regulatory authorities and International Conference on Harmonization guidelines. The Investigator PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 26 {26} provided the Sponsor with a copy of the IRB/IEC-approved Informed Consent Form (ICF) prior to the start of the study. The ICF was signed and dated; one copy was given to the patient, and the Investigator retained a copy as part of the clinical study records. The Investigator did not undertake any investigation specifically required for the clinical study until written consent had been obtained. ## 2. Key Clinical Inclusion and Exclusion Criteria ### Inclusion criteria: Enrollment in the PROfound study was limited to patients who met the following inclusion criteria: - Patients were to be male and at least 18 years of age. - Patients must have provided written informed consent. - Histologically confirmed diagnosis of prostate cancer. - Subjects must have progressed on prior NHA (e.g., abiraterone acetate and/or enzalutamide) for the treatment of metastatic prostate cancer and/or CRPC. - Radiographic progression at study entry while on androgen deprivation therapy (or after bilateral orchiectomy). - Qualifying HRR mutation in tumor tissue by the FMI CLIA HRR (Lynparza HRR) CTA Assay - Either archival or de novo biopsies are acceptable. - If subjects have a mutation in one of the 15 HRR genes based on prior prostate cancer tissue specimen testing by the commercially available FoundationOne assay, they must have the mutation confirmed as a qualifying mutation by FMI. Residual DNA (stored at FMI) from the original FoundationOne test will be used for confirmation. Subjects who do not have sufficient residual DNA from the original test will be analysed in-silico for qualifying HRR gene mutations based on their original FoundationOne test data, but these subjects must supply sufficient formalin fixed, paraffin embedded (FFPE) tumor sample to carry out retrospective central confirmation using the FMI CLIA HRR CTA Assay. - Patients must have had normal organ and bone marrow function measured within 28 days prior to administration of study treatment as defined below: - Haemoglobin (Hb) ≥10.0 g/dL with no blood transfusions in the past 28 days - Absolute neutrophil count ≥1.5 x 109/L - Platelet count ≥100 x 109/L - Total bilirubin ≤1.5 x institutional upper limit of normal (ULN) - Aspartate aminotransferase (serum glutamic oxaloacetic transaminase) / alanine aminotransferase serum glutamic pyruvate PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 27 {27} transaminase) ≤2.5 × institutional ULN unless liver metastases are present in which case they must be ≤5 × ULN - Patients must have creatinine clearance (CrCL) estimated of ≥51 mL/min using the Cockcroft-Gault equation for males or based on a 24 hour urine test. - Estimated CrCL = (140-age [years]) × weight (kg) serum creatinine (mg/dL) × 72 - ECOG PS 0-2. # **Exclusion criteria:** Patients were not permitted to enroll in the PROfound study if they met any of the following exclusion criteria: - Any previous treatment with PARP inhibitor, including olaparib - Patients who have any previous treatment with DNA-damaging cytotoxic chemotherapy, except if for non-prostate cancer indication and last dose >5 years prior to randomisation. For example, patients who received prior mitoxantrone or platinum-based chemotherapy for prostate cancer was excluded. - Prior estramustine was allowed. - Other malignancy (including myelodysplastic syndrome [MDS] and monoclonal gammopathy of undetermined significance) within the last 5 years except: adequately treated non-melanoma skin cancer or other solid tumours including lymphomas (without bone marrow involvement) curatively treated with no evidence of disease for ≥5 years - Patients with MDS/acute myeloid leukaemia (AML) or with features suggestive of MDS/AML - Patients who were receiving any systemic anti-cancer therapy (except radiotherapy) within 3 weeks prior to study treatment. - Persistent toxicities (>Grade 2, per the CTCAE) caused by previous cancer therapy, excluding alopecia or toxicities related to the use of LHRH agonist or antagonist - Patients with known brain metastases. A scan to confirm the absence of brain metastases was not required. - Patients invaluable for both bone and soft tissue progression as defined by meeting both of the following criteria: - A bone scan referred to as a superscan showing an intense symmetric activity in the bones. - No soft tissue lesion (measurable or non-measurable) that can be assessed by RECIST. ### 3. Follow-up schedule # Safety follow-up All randomised patients have a safety follow-up visit every 4 weeks post randomization until week 24. Following the week 24 visit, the safety follow-up PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 28 {28} schedule switches to a follow-up visit every 8 weeks. Safety follow-up visits continue using this schedule until the final study dose. After final study dose the patient receives a study discontinuation visit 7 days after the final study dose) and a safety follow-up visit 30 days (+/- 7 days) after the final study dose # Efficacy follow-up # a. From Enrolment to Radiographic Progression The baseline assessments of all imaging modalities should be performed as close as possible to the start of study treatment and no more than 4 weeks (-28 days) before randomization. Following the baseline assessment, subsequent assessments should be performed every 8 weeks (±7 days), relative to the date of randomization, until objective radiological disease progression has been confirmed by BICR or by Investigator Assessment, irrespective of treatment decisions or dose interruptions. # b. Post Radiographic Progression to 24 weeks following initiation of olaparib for patients who switch from NHA to olaparib post-progression Subjects who switch to olaparib post Radiographic Progression the visit schedule is every 4 weeks (±7 days) until 24 weeks following initiation of olaparib. Patients are then followed-up for efficacy every 12 weeks (±14 days) as part of survival follow-up. # Survival follow-up In survival follow-up, patients are followed up every 12 weeks (+/- 14 days) for 2nd progression and death. Patients who discontinue study treatment switch to survival follow-up. Patients on the Investigators Choice of NHA arm, who switch to olaparib post-progression enter survival follow-up after 24 weeks of treatment. Follow-up activities continue per the above schedules until the final data cut-off (DCO, last subject, last visit). Following final DCO, sites should continue to follow visits as per general practice. # B. Accountability of PMA Cohort # Cohort A A total of 4425 patients with mCRPC who had failed treatment with a prior NHA, were enrolled at 206 centers in 20 countries. Of these, 139 centres randomized patients. Of these, 139, 111 centers in 20 countries randomized patients into Cohort A. Patients with an available FFPE tumor sample were screened for qualifying HRR gene mutations using the FMI CLIA HRR CTA. Of the 245 patients with qualifying HRR gene mutations (BRCA1, and/or BRCA2 and/or ATM) that were randomized into Cohort A, 162 patients received olaparib and 83 patients received investigators choice of NHA. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 29 {29} Of the 245 patients randomized onto Cohort A, 224 patients had single mutations and 21 had co-occurring mutations. Of the 224 patients, 13 patients (5.8%) had a BRCA1 mutation, 127 patients (56.7%) had a BRCA2 mutation, and 84 patients (37.5%) had a ATM mutation alone. Of the 21 patients that had co-occurring mutations; 1 patient was BRCA1+ATM, 1 patient was BRCA1+RAD54L, 2 patients were BRCA2+ATM, 2 patients were BRCA2+BARD1, 4 patients were BRCA2+CDK12, 1 patient was BRCA2+CDK12+CHEK2, 2 patients were BRCA2+CHEK2, 1 patient was BRCA2+CHEK2+RAD51D, 3 patients were BRCA2+PPP2R2A, 1 patient was BRCA2+RAD51B, 1 patient was ATM+CHEK2, 1 patient ATM+PP2R2A, and 1 patient was ATM+RAD51B. Note: patients with co-occurring mutations (BRCA1, BRCA2, or ATM plus a Cohort B gene) were assigned to Cohort A. ## Cohort B Of these, 139 of these centers randomized patients with 85 centers in 18 countries randomizing patients into Cohort B. Patients with an available FFPE tumor sample were screened for qualifying HRR gene mutations using the FMI CLIA HRR CTA. Of the 142 patients with qualifying HRR gene mutations (BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and/or RAD54L) that were randomized into Cohort B, 94 patients received olaparib and 48 patients received investigators choice of NHA. Of the 142 patients randomized onto Cohort B, 135 patients had single mutation and 7 patients had co-occurring mutations. Of the 135 patients, 89 patients (65.9%) had a CDK12 mutations and 31.9% of patients had a single mutation in 1 of 9 HRR genes (BARD1, BRIP1, CHEK1, CHEK2, PALB2, PPP2R2A, RAD51B, RAD51D and RAD54L). No patients in Cohort B had a FANCL or RAD51C mutation alone and thus, not enrolled in the trial. For Cohort B, 7 patients had co-occurring mutations; 1 patient was BRCA2m+CDK12m, 1 patient was BARD1+CDK12, 1 patient was BRIP1+PALB2, 1 patient was CDK12+CHEK1, 2 patients were CDK12+PALB2 and 1 patient was PALB2+PPP2R2A. The majority (97.2%) of the PROfound trial patients (FAS; N=387) were confirmed as positive according to the testing criteria and mutation classification rules approved for the FICDx test (N=376). This subgroup is referred to as confirmed FMI FICDx subgroup (N=376). The efficacy of olaparib was studied in 376 patients with HRR mutations in mCRPC in the PROfound trial. A break-down by FAS (N=387) and confirmed FMI FICDx subgroup (N=376) is shown in Table 20. PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 30 {30} Table 20: Break-down of FAS and and confirmed FMI F1CDx subgroup by Cohort | | FAS | | | | | | Confirmed FMI F1CDx subgroup | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | Number of patients (N) | | Variant Status | | | | Number of patients (N) | | Variant Status | | | | | Gene** | Cohort A (245) | Cohort B (142) | HD | LR | ID | SUB | Cohort A (240) | Cohort B (136) | HD | LR | ID | SUB | | BRCA1 | 15 | 0 | 0 | 5 | 5 | 5 | 14 | 0 | 0 | 5 | 5 | 4 | | BRCA2 | 143 | 2 | 27 | 15 | 73 | 36 | 140 | 2 | 27 | 14 | 73 | 34 | | ATM | 90 | 2 | 10 | 8 | 39 | 41 | 88 | 2 | 10 | 8 | 38 | 40 | | BARD1 | 2 | 2 | 0 | 0 | 2 | 2 | 1 | 1 | 0 | 0 | 1 | 1 | | BRIP1 | 0 | 4 | 0 | 0 | 3 | 1 | 0 | 4 | 0 | 0 | 3 | 1 | | CDK12 | 5 | 94 | 5 | 5 | 68 | 39 | 5 | 89 | 5 | 5 | 65 | 36 | | CHEK1 | 0 | 3 | 0 | 0 | 2 | 1 | 0 | 3 | 0 | 0 | 2 | 1 | | CHEK2 | 5 | 12 | 1 | 0 | 15 | 1 | 5 | 12 | 1 | 0 | 15 | 1 | | FANCL* | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | | PALB2 | 0 | 8 | 0 | 0 | 6 | 3 | 0 | 7 | 0 | 0 | 6 | 2 | | RAD51B | 2 | 5 | 2 | 2 | 0 | 3 | 2 | 5 | 2 | 2 | 0 | 3 | | RAD51C* | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | | RAD51D | 1 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | | RAD54L | 1 | 5 | 0 | 1 | 1 | 4 | 1 | 5 | 0 | 1 | 1 | 4 | HD: Homozygous deletion. Patients who have homozygous gene loss LR: Large Rearrangement. Patients who have exonic or multi-exon insertions or deletion ID: Insertion/Deletion. Patients who have small insertions/deletions which lead to a frameshift SUB: Base Substitutions. Patients who have other variant types (non-sense, missense, splice site, etc.) *These two genes were not enrolled in the trial. **Patients harboring PPP2R2A were also enrolled in the trial. [FAS subgroup (PPP2R2A Cohort A 4, Cohort B 11); Confirmed FMI F1CDx subgroup (PPP2R2A Cohort A 4, Cohort B 10)]. ### C. Study Population Demographics and Baseline Parameters In the safety population, the median age was 65 years (range: 29 to 91 years), and 13.4% of patients were ≥75 years of age. Most patients were White (85.1%); 57.7% had an ECOG performance status of 0 at study entry, and 42.3% had an ECOG performance status of 1 at study entry. In the biomarker-defined population, the median age was 63 years (range: 39 to 91 years), and 10.2% of patients were ≥75 years of age. Most patients were White (81.6%); 59.2% had an ECOG performance status of 0 at study entry, and 40.8% had an ECOG performance status of 1 at study entry. Demographics for the overall safety population and for the biomarker-defined PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 31 {31} population are summarized in Table 21 and 22. Overall, 421 (90.9%) patients were treated at US sites and 42 (9.1%) patients were treated at Canadian sites. **Table 21: Demographic characteristics (FAS; Cohort A)** | | | Olaparib 300 mg bd (N=162) | Investigators choice of NHA (N=83) | Total (N=245) | | --- | --- | --- | --- | --- | | Age (years) | Mean | 68.0 | 68.1 | 68.1 | | | Standard deviation | 8.23 | 7.36 | 7.93 | | | Median | 68.0 | 67.0 | 68.0 | | | Min | 47 | 49 | 47 | | | Max | 86 | 86 | 86 | | Age group (years), n (%) | <65 | 54 | 23 | 77 | | | ≥65 | 108 | 60 | 168 | | Race, n (%) | White | 109 | 55 | 164 | | | Black or African | 2 | 1 | 3 | | | Asian | 43 | 19 | 62 | | | Other | 1 | 1 | 2 | | | Missing | 7 | 7 | 14 | | Ethnic group, n (%) | Hispanic or Latino | 12 | 9 | 21 | | | Not Hispanic or | 145 | 69 | 214 | | | Missing | 5 | 5 | 10 | **Table 22: Demographics Characteristics (FAS; Cohort B)** | | | Olaparib 300 mg bd (N=94) | Investigators choice of NHA (N=48) | Total (N=142) | | --- | --- | --- | --- | --- | | Age (years) | Mean | 69.2 | 70.3 | 69.6 | | | Standard deviation | 8.79 | 7.83 | 8.46 | | | Median | 69.0 | 69.5 | 69.0 | | | Min | 48 | 51 | 48 | | | Max | 91 | 87 | 91 | | Age group (years), n (%) | <65 | 28 (29.8) | 11 (22.9) | 39 (27.5) | | | ≥65 | 66 (70.2) | 37 (77.1) | 103 (72.5) | | Race, n (%) | White | 54 (57.4) | 30 (62.5) | 84 (59.2) | PMA P170019/S015: FDA Summary of Safety and Effectiveness Data Page 32 {32} | | | Olaparib 300 mg bd (N=94) | Investigators choice of NHA (N=48) | Total (N=142) | | --- | --- | --- | --- | --- | | | Black or African American | 5 (5.3) | 0 | 5 (3.5) | | | Asian | 26 (27.7) | 17 (35.4) | 43 (30.3) | | | Other | 1 (1.1) | 0 | 1 (0.7) | | | Missing | 8 (8.5) | 1 (2.1) | 9 (6.3) | | Ethnic group, n (%) | Hispanic or Latino | 5 (5.3) | 3 (6.3) | 8 (5.6) | | | Not Hispanic or Latino | 83 (88.3) | 43 (89.6) | 126 (88.7) | | | Missing | 6 (6.4) | 2 (4.2) | 8 (5.6) | Baseline disease characteristics, including site of the primary tumor, histologic subtype, and duration, are provided for the safety population and the biomarker-defined population in Table 23 and 24. Table 23: Baseline Disease Characteristics at baseline (FAS, cohort A) | | Number (%) of patients | | | | --- | --- | --- | --- | | | Olaparib 300 mg bd (N=162) | Investigators choice of NHA (N=83) | Total (N=245) | | Time from CRPC to randomisation (months) | | | | | Median | 24.2 | 23.7 | 24.1 | | Min, max | -6^{a}, 189 | 1, 175 | -6, 189 | | Time from mCRPC to randomisation (months) | | | | | Median | 23.3 | 22.5 | 23.1 | | Min, max | -6^{a}, 121 | 1, 105 | -6, 121 | | Histology type at diagnosis | | | | | Adenocarcinoma | 160 (98.8) | 80 (96.4) | 240 (98.0) | | Small cell carcinoma | 0 | 0 | 0 | | Other | 0 | 2 (2.4) | 2 (0.8) | | Missing | 2 (1.2) | 1 (1.2) | 3 (1.2) | | Total Gleason Score at diagnosis | | | | | 2 | 1 (0.6) | 0 | 1 (0.4) | | 3 | 0 | 0 | 0 | | 4 | 2 (1.2) | 0 | 2 (0.8) | | 5 | 2 (1.2) | 1 (1.2) | 3 (1.2) | | 6 | 6… --- **Source:** [https://fda-staging.innolitics.com/device/P170019S015](https://fda-staging.innolitics.com/device/P170019S015) **Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. 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