FoundationOne Liquid CDx

{0} # 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 Device Procode: PQP Applicant's Name and Address: Foundation Medicine, Inc. 400 Summer Street Boston, MA 02210 Date(s) of Panel Recommendation: None Premarket Approval Application (PMA) Number: P190032/S029 Date of FDA Notice of Approval: May 27, 2026 The original Premarket Approval (PMA) (P190032) for FoundationOne® Liquid CDx (F1LCDx) 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). Subsequently, additional PMA supplements were approved for expanding the indications for use of F1LCDx since the original approval. See Section VII for more details. The current supplement was submitted to expand the indication for the F1LCDx test as a companion diagnostic for the indication listed in the table below. New Indication Being Sought in this PMA supplement submission. | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Prostate cancer | Homologous Recombination Repair (HRR) gene (*BRCA1, BRCA2, ATM, ATR, CDK12, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C*) | TALZENNA® (talazoparib) in combination with XTANDI® (enzalutamide) | ---PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 1 of 52 {1} ## 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 to analyze 324 genes and report genomic alterations in 311 genes. These include substitutions, insertions, and deletions (indels) in 311 genes, rearrangements in 8 genes and copy number alterations in 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 | | --- | --- | --- | | Breast Cancer | PIK3CA mutations | ITOVEBI^{TM} (inavolisib) in combination with palbociclib and fulvestrant | | | PIK3CA mutations C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y | PIQRAY^{®} (alpelisib) | | Colorectal cancer (CRC) | BRAF V600E | BRAFTOVI^{®} (encorafenib) in combination with cetuximab | | Non-small cell lung cancer (NSCLC) | ALK Rearrangements | ALECENSA^{®} (alectinib) | | | BRAF V600E | BRAFTOVI^{®} (encorafenib) in combination with MEKTOVI^{®} (binimetinib) | | | EGFR Exon 19 deletions and EGFR Exon 21 L858R substitution | EGFR tyrosine kinase inhibitors approved by FDA* | | | MET single nucleotide variants (SNVs) and indels that lead to MET exon 14 skipping | TABRECTA^{®} (capmatinib) | | | | TEPMETKO^{®} (tepotinib)** | | | ROS1 fusions** | ROZLYTREK^{®} (entrectinib) | | Prostate cancer | BRCA1, BRCA2, ATM alterations | LYNPARZA^{®} (olaparib) | | | BRCA1, BRCA2 alterations | AKEEGA^{®} (niraparib + abiraterone acetate) | | | | LYNPARZA^{®} (olaparib) in combination with abiraterone | | | | RUBRACA^{®} (rucaparib) | | | Homologous Recombination Repair | TALZENNA^{®} | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 2 of 52 {2} | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | | (HRR) gene (*BRCA1, BRCA2, ATM, ATR, CDK12, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C*) alterations | (talazoparib) in combination with XTANDI® (enzalutamide) | | Solid Tumors | *NTRK1/2/3* fusions** | ROZLYTREK® (entrectinib) | *For the most current information about the therapeutic products in this group, go to: https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools#Group_Labeling ** When considering eligibility for ROZLYTREK® based on the detection of NTRK1/2/3 and ROS1 fusions, or for TEPMETKO® based on the detection of MET SNVs and indels that lead to MET exon 14 skipping, testing using plasma specimens is only appropriate for patients for whom tumor tissue is not available for testing. 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 with the tumor types above who are negative for the mutations listed in Table 1 (see ** footnote under Table 1 for NTRK1/2/3 and ROS1 fusions for ROZLYTREK® and MET SNVs and indels that lead to MET exon 14 skipping for TEPMETKO®) 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 are not prescriptive or conclusive for labeled use of any specific therapeutic product. ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found in the F1LCDx labeling. ### V. DEVICE DESCRIPTION The F1LCDx assay is performed 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 isolated 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 fifteen of these genes (refer to Table 2 for the complete list of genes reported by F1LCDx). 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 in 311 genes. These include base substitutions and indels in 311 genes, copy number variants in three genes, and PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 3 of 52 {3} genomic rearrangements in eight genes. A subset of targeted regions in 75 genes is baited for enhanced sensitivity. Table 2: Complete List of Genes Interrogated by FoundationOne Liquid CDx¹ | 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 | | EPHA3 | EPHB1 | EPHB4 | ERBB2 | ERBB3 [Exons 3, 6, 7, 8, 10, 12, 20, 21, 23, 24, 25] | ERBB4 | ERCC4 | ERG | ERRFI1 | 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, Intron17] | 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] | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 4 of 52 {4} | 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 | | PRKCI | PTCH1 | **PTEN** | **PTPN11** | PTPRO | QKI | RAC1 | RAD21 | RAD51 | RAD51B | | RAD51C | RAD51D | RAD52 | RAD54L | **RAF1** [Exons 3, 4, 6, 7, 10, 14, 15, 17, Introns 4-8] | RARA [Intron 2] | **RB1** | RBM10 | REL | **RET** [Introns 7, 8, Exons 11, 13-16, Introns 9-11] | | RICTOR | RNF43 | **ROS1** [Exons 31, 36-38, 40, Introns 31- 35] | RPTOR | RSPO2* [Intron 1] | SDC4* [Intron 2] | SDHA | SDHB | SDHC | SDHD | | SETD2 | SF3B1 | SGK1 | SLC34A2* [Intron 4] | SMAD2 | SMAD4 | SMARCA4 | SMARCB1 | **SMO** | SNCAIP | | SOCS1 | SOX2 | SOX9 | SPEN | SPOP | SRC | STAG2 | STAT3 | **STK11** (LKB1) | SUFU | | SYK | TBX3 | TEK | TERC* [ncRNA] | **TERT*** [Promoter] | TET2 | TGFBR2 | TIPARP | TMPRSS2* [Introns 1-3] | TNFAIP3 | | TNFRSF14 | **TP53** | TSC1 | TSC2 | TYRO3 | U2AF1 | **VEGFA** | VHL | WHSC1 | WTI | | XPO1 | XRCC2 | ZNF217 | ZNF703 | | | | | | | $^{1}$While the F1LCDx assay interrogates 324 genes, including 309 genes with complete exonic (coding) coverage and 15 genes with only select non-coding coverage (indicated with an *), F1LCDx reports alterations only in 311 genes (309 genes with coding coverage and 2 genes with non-coding coverage). F1LCDx also reports Rearrangements in 13 genes from non-coding region, only when the partner gene is approved for reporting, e.g., CD74 in a ROS1-CD74 fusion. Select genes and select exons (indicated in bold) are baited for increased sensitivity. The reporting of rearrangements and copy number alterations as part of the tumor mutation profiling claim are restricted to those genes included in Table 3, below. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 5 of 52 {5} **Table 3: Genes for which copy number alterations and rearrangements are reported for tumor profiling by F1LCDx** | Alteration Type | Genes | | --- | --- | | Copy Number Alterations | *BRCA1, BRCA2, ERBB2* | | Rearrangements | *ALK, BRCA1, BRCA2, NTRK1, NTRK2, NTRK3* | The test report includes variants reported in the following levels: # **Level 1: Companion Diagnostics (CDx)** Clinical evidence should be presented from a prospectively designed clinical trial. Results can also be presented from a retrospective clinical bridging study demonstrating that the clinical endpoints are preserved using plasma samples in trials where enrollment was based on tissue test results. For follow-on markers, a clinical concordance study demonstrating non-inferiority to the original FDA-approved cfDNA-based companion diagnostic device is required. In addition to the clinical validation, analytical validation for each specific Level 1 CDx biomarker should be presented. # **Level 2: cfDNA Biomarkers with Strong Evidence of Clinical Significance in cfDNA** For a Level 2 claim of cfDNA biomarkers with strong evidence of clinical significance, clinical validation needs to be from evidence presented with FDA-approved liquid biopsy companion diagnostic biomarkers for the specific tumor type at the biomarker or variant level. Such claims should also be supported by analytical performance for each biomarker from at least limit of detection (LoD), precision/ reproducibility, and accuracy studies. # **Level 3A: Biomarkers with Evidence of Clinical Significance in Tissue Supported by Strong Analytical Validation Using cfDNA and Concordance Between cfDNA and Tissue** Clinical evidence can be provided from tissue-based companion diagnostics. This should also be supported by analytical validation (LoD, precision, analytical accuracy, and concordance study to a tissue-based test) for the specific tumor type at the biomarker or variant level, using a representative approach for SNVs and indels. Evidence evaluating concordance between cfDNA- and tissue-samples for FDA-approved tissue markers should be demonstrated using an FDA-approved tissue test or a validated tissue test. # **Level 3B: Biomarkers with Evidence of Clinical Significance in Tissue Supported by Analytical Validation Using cfDNA** Clinical evidence can be provided from tissue-based companion diagnostics, with analytical validation supported by a representative approach for SNVs and indels from key analytical studies (such as LoD, accuracy, and precision). # **Level 4: Other Biomarkers with Potential Clinical Significance** Biomarkers not categorized into Levels 1, 2, or 3 can be included under Level 4 for informational purposes or to be used to direct patients toward clinical trials for which they may be eligible. Such claims can be supported by clinical rationale for inclusion in PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 6 of 52 {6} the panel. Such rationale could also include peer-reviewed publications for genes/ variants in tissue, variant information from well curated public databases, or in vitro pre-clinical models. Analytical validation should be supported by a representative approach for SNVs and indels from key analytical studies (such as LoD, accuracy, and precision). ### FoundationOne® Liquid CDx cfDNA 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 F1LCDx assay is intended to be performed with the serial number-controlled instruments indicated in Table 4, below. All instruments are qualified by Foundation Medicine, Inc. (Foundation Medicine or FMI) under Foundation Medicine's Quality System. Table 4: Instruments for use with the F1LCDx assay | Instrument | | --- | | Illumina NovaSeq 6000 | | Thermo Fisher Scientific Kingfisher Flex DW and 96 | | Hamilton STARlet STAR Liquid Handling Workstation | ### Test Process All assay reagents including blood collection tubes included in the F1LCDx 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 F1LCDx cfDNA Blood Collection Tubes (BCT) provided as a component of the F1LCDx specimen collection kit. Prior to cfDNA isolation, the plasma is collected 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 PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 7 of 52 {7} 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 after 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 liquid handling workstation (Hamilton STAR) using the "with-bead" protocol to maximize reproducibility and library yield. Dual-indexed (Foundation Medicine customized six base pair barcodes) sequencing libraries are PCR amplified with a high-fidelity DNA polymerase (HiFi™, Kapa) for ten cycles, and SPRI purified. Process matched control (PMC) is prepared and added to the plate with other cfDNA samples at the beginning of LC. ### D. Hybrid Capture Solution hybridization is performed using a >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. A 25 μl fixed volume of library from the LC step, blocking DNA (adaptor block, Cot, Salmon Sperm DNA) and the bait reagents are combined and the mixture is lyophilized in a 96-well plate. The library is then re-suspended in buffer and the samples are incubated at 95°C for 5 minutes to denature dsDNA libraries to ssDNA libraries. The incubation temperature is then reduced to 65°C for 2-4 hours to facilitate the hybridization of ssDNA libraries to the bait set. After the 65°C incubation, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen) and off-target library is removed by washing two times with Wash Buffer 1 at 65°C and three times with Wash Buffer 2 at 48°C. The PCR master mix is added to PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 8 of 52 {8} 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 technology to generate monoclonal clusters via exclusion amplification (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 nano-wells 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 nano-well. 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 deoxynucleotide triphosphates (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 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' binary alignment map (BAM). PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 9 of 52 {9} # - • **Short Variant (Base Substitutions and Indels) Detection** 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. After identifying the supporting reads for each candidate short variant, the AP analyzes the short variant candidates and calculates metrics used to evaluate the quality of the variant call. The final variant calls are made based on a series of quality control filters, which will reject a call based on the intrinsic sample noise, the expected noise level for the particular variant, and other known error modes (e.g., sequence homology). # - • **Rearrangement Detection** The same assembly procedure used to detect short variants is also leveraged to detect rearrangements. Paths in the de Bruijn graph described above that originate in reference nodes of the locus but do not return to that locus are further analyzed as rearrangement candidates. Such paths correspond to sequences that are anchored in the locus of interest, but that diverge to sequences not normally found in that locus. When this exogenous sequence can be mapped unambiguously to another location in the genome, the rearrangement candidate is further evaluated using quality control filters related to sequence repetitiveness, mapping confidence, and the quality and amount of read support. Rearrangement candidates passing all filters are further characterized for functional impact and called as rearrangements. # - • **Copy Number Alteration Detection** ---PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 10 of 52 {10} Copy number alterations (CNAs) are detected using a comparative panel of normals (PoN)-like method. Normalized target coverage obtained by comparison with a historical set of reference samples (Panel of Normals, or PoN), along with the allele frequencies of targeted SNPs, are used to generate a copy-number model of the targeted genomic regions. This model consists of an overall purity estimate and a copy number state for each distinct genomic segment. Candidates are annotated with regards to functional effect and cancer relevance status. Amplifications are called at segments with ≥ 6 copies (or ≥ 7 for triploid/≥ 8 for tetraploid tumors) and homozygous deletions at 0 copies. Amplifications in ERBB2 are called positive at segments with ≥ 5 copies for diploid tumors. Additional requirements are placed on these calls, including QC metrics for the overall quality and quantity of data supporting the modeled copy-number level. For example, amplifications are required to cover more than 80% of the exons in a gene, and all calls are required to involve at least two consecutive coverage targets. ### G. Report Generation Approved results are annotated by automated software with CDx relevant information and are merged with patient demographic information prior to approval and release by the laboratory director or designee. ### H. Internal Process Controls #### Process 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. Classification Criteria for CDx Biomarkers Detected by F1LCDx PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 11 of 52 {11} # **1. CDx classification criteria for Homologous Recombination Repair (HRR) genes for patients with prostate cancer** # **1.1. HRR alterations (BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, and RAD54L) to identify patients eligible for TALZENNA® (talazoparib) in combination with XTANDI® (enzalutamide).** Deleterious or suspected deleterious HRR gene (*BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, and RAD54L*) variants, identified by the rules below in Table 5. **Table 5. Biomarker definition for HRR gene (*BRCA1, BRCA2, ATM, ATR, CDK12, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C*) Alterations for Talazoparib in combination with Enzalutamide Treatment** | Gene | Variant Class | Description | | --- | --- | --- | | *BRCA1* *BRCA2* *ATM* *ATR* *CDK12* *CHEK2* *FANCA* *MLH1* *MRE11A* *NBN* *PALB2* *RAD51C* | Short Variant | Any nonsense, frameshift, or splice site alteration^{1} - Any splice alterations within the splice donor or acceptor site^{2} - For BRCA2, truncating mutations must occur upstream of bases encoding amino acid 3326 Any of the additional mutations listed in Tables 6 – 10 | | | Copy Number | Homozygous deletion of one or more exons, regardless of transcript. Only reported for *BRCA1* and *BRCA2*. | | | Rearrangement | Any inactivating rearrangement, regardless of transcript | $^{1}$ Missense mutations in the start codon (except for those in tables 6-10) and short variant deletions spanning from upstream of the start codon (annotated as M1?) are biomarker negative. $^{2}$ The splice site is defined as the first or last two bases of the intron (except for those in tables 6-10). **Table 6. List of short variants in *BRCA1\*** | M1I | C47S | T1685A | W1718C | P1812A | | --- | --- | --- | --- | --- | | M1R | C47Y | T1685I | S1722F | A1823T | | M1T | C61Y | H1686R | V1736A | V1833M | | M1V | C61G | V1688del | V1736G | W1837C | | M18K | C61S | M1689R | G1738E | W1837G | | M18T | C64G | T1691I | G1738R | W1837R | | L22S | C64R | T1691K | D1739G | V1838E | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 12 of 52 {12} | C24R | C64Y | D1692Y | D1739V | Y1853C | | --- | --- | --- | --- | --- | | I26N | C64W | D1692H | G1748D | splice site 212+3A>G | | T37K | R71G | D1692N | R1753T | splice site 213-11T>G | | T37R | R71K | A1693del | K1759N | splice site 213-12A>G | | C39R | R71M | C1697R | L1764P | splice site 302-3C>G | | C39Y | R71T | R1699Q | I1766S | splice site 4675+3A>T | | C39G | S770L | R1699W | I1766N | splice site 4986+3G>C | | C39W | R1495K | L1705P | G1770V | splice site 4986+5G>A | | H41R | R1495M | G1706E | M1775K | splice site 4986+6T>C | | C44F | R1495T | G1706R | M1775R | splice site 4986+6T>G | | C44S | E1559K | A1708E | L1780P | splice site 5074+3A>G | | C44Y | E1559Q | V1714G | C1787S | splice site 5194-12G>A | | C47F | A1623G | S1715N | C1787_G1788>SD | splice site 5406+4A>G | | C47R | S1655F | S1715R | G1788V | | **Table 7. List of short variants in *BRCA2\*** | M1R | K1530N | R2659G | D2723G | D3095E | | --- | --- | --- | --- | --- | | M1I | R2336H | R2659K | D2723H | D3095G | | M1V | R2336P | R2659T | D2723V | N3124I | | M1T | R2336L | Y2660D | G2724W | N3187K | | D23N | T2412I | E2663K | Y2726C | splice site 316+4delA | | D23Y | L2510P | E2663V | G2748D | splice site 316+5G>A | | S142I | R2602T | S2670L | R2784W | splice site 8487+3A>G | | S142N | H2623R | I2675V | G2793R | splice site 8754+3G>C | | V159M | W2626C | L2686P | Q2829R | splice site 8754+4A>G | | G173R | W2626R | L2688P | A2911E | splice site 8754+5G>A | | V211I | I2627F | T2722K | E3002K | splice site 8954-5A>G | | V211L | L2647P | T2722R | R3052W | | | Y600C | L2653P | D2723A | G3076V | | **Table 8. List of short variants in *ATM\*** | M1I | E2039K | A2622V | F2827C | splice site 331+5G>A | | --- | --- | --- | --- | --- | | M1L | A2067D | D2625_A2626>EP | R2832C | splice site 8418+5_8418+8delGTGA | | M1T | R2227C | V2662D | S2855_V2856>RI | | | P292L | Y2470D | D2708N | D2913Y | | | D2016G | A2524P | V2716A | R3008C | | | R2032K | R2547_S2549del | G2765S | R3008H | | **Table 9. List of Short Variants in *CHEK2*** | R117G | K373E | | --- | --- | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 13 of 52 {13} | G151S | A392V | | --- | --- | **Table 10. List of Short Variants in *FANCA*, *MLH1*, *RAD51C*** | Gene | Short Variant | | --- | --- | | *FANCA* | F320L, F1263del | | *MLH1* | R265C | | *RAD51C* | L219S | *Note: This biomarker definition includes ATM and BRCA1/2 inactivating mutation lists that may change over time as a result of periodic review of new evidence, in accordance with FDA-approved variant classification procedures.* # **1.2. HRR alterations (*BRCA1*, *BRCA2*, *ATM*) to identify patients eligible for *LYNPARZA*$^{®}$ (olaparib).** Deleterious or suspected deleterious HRR gene (*BRCA1*, *BRCA2*, *ATM*) variants, identified by the rules below in Table 11. **Table 11. Biomarker definition for HRR gene (*BRCA1*, *BRCA2*, *ATM*) Alterations for Olaparib treatment** | Gene | Variant Class | Description | | --- | --- | --- | | *BRCA1* *BRCA2* *ATM* | Short Variant | Any inactivating short variant alteration meeting the definition in table 5. Any of the *BRCA1*, *BRCA2*, or *ATM* additional mutations listed in Tables 6 – 8 | | | Copy Number | Homozygous deletion meeting the definition in table 5. | | | Rearrangement | Inactivating rearrangements meeting the definition in table 5. | # **1.3. HRR alterations (*BRCA1*, *BRCA2*) eligible for *AKEEGA*$^{®}$ (niaparib + abiraterone acetate), *LYNPARZA*$^{®}$ (olaparib) in combination with abiraterone, or *RUBRACA*$^{®}$ (rucaparib).** Deleterious or suspected deleterious HRR gene (*BRCA1*, *BRCA2*) variants, identified by the rules below in Table 12. **Table 12. Biomarker definition for HRR gene (*BRCA1*, *BRCA2*) Alterations for treatment with Niraparib+ abiraterone acetate, or Olaparib in combination with abiraterone, or Rucaparib** | Gene | Variant Class | Description | | --- | --- | --- | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 14 of 52 {14} | *BRCA1 BRCA2* | Short Variant | Any inactivating short variant alteration meeting the definition in table 5. Any of the *BRCA1* or *BRCA2* additional mutations listed in Tables 6 – 7 | | --- | --- | --- | | | Copy Number | Homozygous deletion meeting the definition in table 5. | | | Rearrangement | Inactivating rearrangements meeting the definition in table 5. | # **2. CDx classification criteria for EGFR alterations** - Base substitutions resulting in EGFR L858R - In-frame deletions occurring within EGFR Exon 19 # **3. 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 # **4. SNVs and indels that lead to MET exon 14 skipping to identify patients eligible for treatment with TABRECTA® (capmatinib) and TEPMETKO®:** A SNV or indel in *MET* shall be considered to result in skipping of exon 14 if one or more of the following criteria are met: 1. Deletions greater than or equal to 5 bp that affect positions -3 to -30 in the intronic region immediately adjacent to the splice acceptor site at the 5' boundary of *MET* exon 14. 2. Indels affecting positions -1 or -2 at the splice acceptor site of the 5' boundary of *MET* exon 14. 3. Base substitutions and indels affecting positions 0, +1, +2, or +3 at the splice donor site of the 3' boundary of *MET* exon 14. # **5. Biomarker Rules for Rearrangements that Lead to NTRK1, NTRK2, or NTRK3 Fusions:** PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 15 of 52 {15} Rearrangements in NTRK1, NTRK2, or NTRK3 shall be considered CDx biomarker positive, that is, to lead to a NTRK1, NTRK2, or NTRK3 RNA fusion, if the following criterion is met: - In-strand rearrangement events that may lead to an NTRK1, NTRK2 or NTRK3 RNA fusion with a previously reported or novel partner gene in which the kinase domain is not disrupted. This also includes rearrangement events that result in reciprocal fusions (NTRK may be on either the 5' or the 3' end of the detected fusion). In this regard out-of-strand events are considered as non-fusion rearrangements and are classified as CDx biomarker negative. Intragenic fusions in which genomic rearrangement events are wholly internal to the NTRK1, NTRK2, or NTRK3 genes (i.e., NTRK1-NTRK1, NTRK2-NTRK2, NTRK3-NTRK3 events) are also considered biomarker negative. Unidentified partners (encoded as N/A) or LINC non-coding partners are also considered CDx biomarker negative. ### 6. Biomarker Rules for Rearrangements that Lead to ROS1 Fusions: Rearrangements in ROS1 shall be considered CDx biomarker positive, i.e., to lead to ROS1 RNA fusion, if the following condition is met: - In-strand rearrangement events that may lead to a ROS1 RNA fusion with another protein coding gene in which the ROS1 kinase domain is not disrupted. ROS1 must be on the 3' end of the detected fusion. In this regard, out-of-strand events are considered as non-fusion rearrangements and are classified as CDx biomarker negative. Intragenic fusions in which genomic rearrangement events are wholly internal to the ROS1 (i.e., ROS1-ROS1 events) are also considered biomarker negative. Unidentified partners (encoded as N/A) or LINC non-coding partners are also considered CDx biomarker negative. ROS1 fusions with novel partners are required to be in frame. ### 7. CDx classification criteria for ITOVEBI™ (inavolisib) in combination with palbociclib and fulvestrant for patients with breast cancer Presence of a PIK3CA alteration listed in Table 13. Table 13. Eligible PIK3CA short variants | R88Q | N345D | E453G | E542V | Q546H | H1047L | G1049R | | --- | --- | --- | --- | --- | --- | --- | | G106A | N345H | E453K | E545A | Q546K | H1047N | G1049S | | G106D | N345I | E453Q | E545D | Q546L | H1047P | | | G106R | N345K | E453V | E545G | Q546P | H1047Q | | | G106S | N345S | E542A | E545K | Q546R | H1047R | | | G106V | N345T | E542D | E545L | M1043I | H1047T | | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 16 of 52 {16} | K111N | N345Y | E542G | E545Q | M1043T | H1047Y | | | --- | --- | --- | --- | --- | --- | --- | | K111R | C420R | E542K | E545R | M1043V | G1049A | | # **8. CDx classification criteria for PIQRAY® (alpelisib) for patients with breast cancer** Presence of a *PIK3CA* alteration resulting in H1047R, E545K, E542K, C420R, E545A, E545D [1635G>T only], E545G, Q546E, Q546R, H1047L, or H1047Y. # **9. BRAF V600E to identify metastatic CRC patients eligible for treatment with BRAFTOV1 (encorafenib) in combination with cetuximab and NSCLC patients for treatment with BRAFTOV1 (encorafenib) in combination with MEKTOV1 (binimetinib)** CDx classification criteria for BRAF substitutions: - Base substitutions resulting in *BRAF V600E* # **VI. ALTERNATIVE PRACTICES AND PROCEDURES** There are no FDA-approved CDx alternatives using cfDNA isolated from plasma for the detection of HRR gene alterations to identify patients with prostate cancer eligible for treatment with TALZENNA® (talazoparib) in combination with XTANDI® (enzalutamide). However, FoundationOne CDx (F1CDx) from Foundation Medicine, Inc. (FMI) is an FDA-approved CDx (P170019/S060) using formalin-fixed, paraffin-embedded (FFPE) tissue specimens for this indication. There are FDA-approved alternatives for the detection of select CDx and tumor profiling genetic alterations using either cfDNA isolated from plasma samples or FFPE tissue specimens. 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. # **VII. MARKETING HISTORY** The F1LCDx assay was FDA-approved on August 26, 2020, and subsequently commercialized in the United States. The F1LCDx assay has been marketed in the United States, the European Union, and in several other foreign countries since the approval. On September 21, 2022, the companion diagnostic indication for F1LCDx to identify patients with ovarian cancer harboring BRCA1 or BRCA2 alterations for treatment with RUBRACA (rucaparib) was removed. On May 17, 2024, the companion diagnostic PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 17 of 52 {17} indication for F1LCDx to identify patients with NSCLC harboring *EGFR* exon 20 insertions for treatment with EXKIVITY (mobocertinib) was removed. The approved PMA supplements that affected the intended use are listed in Table 14. **Table 14. Marketing History** | Submission No. | Date of Approval | Biomarker/Update | Patient Population | Drug | | --- | --- | --- | --- | --- | | P200006 | October 26, 2020 | *ALK* Rearrangements | NSCLC | ALECENSA® (alectinib) | | | | *PIK3 CA* alterations | Breast Cancer | PIQRAY® (alpelisib) | | P200016 | November 6, 2020 | *BRCA1*, *BRCA2*, and *ATM* alterations | Prostate Cancer | LYNPARZA® (olaparib) | | P190032/S001 | July 15, 2021 | *MET* single nucleotide variants (SNVs) and indels that lead to *MET* exon 14 skipping alterations | NSCLC | TABRECTA® (capmatinib) | | P190032/S008 | December 19, 2022 | *EGFR* Exon 19 deletions and Exon 21 L858R substitution mutation) | NSCLC | FDA-approved *EGFR* tyrosine kinase inhibitors. | | P190032/S004 | December 22, 2022 | *NTRK1/2/3* fusions | Solid tumors | ROZLYTREK® (entrectinib) | | | | *ROS1* | NSCLC | ROZLYTREK® (entrectinib) | | P190032/S010 | June 8, 2023 | *BRAF V600E* | Colorectal cancer (CRC) | BRAFTOVI® (encorafenib) in combination with cetuximab | | P190032/S011 | October 11, 2023 | *BRAF V600E* | NSCLC | BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) | | P190032/S014 | June 28, 2024 | *BRCA1* and *BRCA2* alterations | Prostate cancer | AKEEGA® (niraparib + abiraterone acetate). | | P190032/S016 | August 30, 2024 | *BRCA1* and *BRCA2* alterations | Prostate cancer | LYNPARZA® (olaparib) in combination with abiraterone | | P190032/S023 | October 10, 2024 | *PIK3CA*-mutations | Breast cancer | ITOVEBI® (inavolisib) in combination with palbociclib and fulvestrant | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 18 of 52 {18} | P190032/S015 | November 14, 2024 | MET exon 14 skipping alterations | NSCLC | TEPMETKO® (tepotinib) | | --- | --- | --- | --- | --- | ### 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 F1LCDx assay 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 insert for TALZENNA ® (talazoparib) and XTANDI® (enzalutamide) which are available at Drugs@FDA. ### IX. SUMMARY OF NON-CLINICAL STUDIES #### A. Laboratory Studies The primary evidence supporting the performance of F1LCDx in detecting HRR gene (*BRCA1*, *BRCA2*, *ATM*, *ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) alterations in metastatic castration-resistant prostate cancer patients was from data generated using intended use specimens across the validation studies. In addition to the platform-level validation results, as well as validation results for three (3) HRR genes (*ATM*, *BRCA1*, and *BRCA2*) in mCRPC patients (refer to section IX.A in P190032 and P200016 Summary of Safety and Effectiveness Data, respectively), additional evidence of the performance of F1LCDx in detecting HRR (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) gene alterations is provided through analytical concordance, precision and confirmation of the limit of detection (LoD) studies using clinical samples. Table 15 below includes a list of genes/variant types that were represented in the key analytical validation studies, in accordance with their prevalence in mCRPC, to support performance of the assay for detection of the variant types in the HRR genes (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) that were not evaluated in P200016. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 19 of 52 {19} **Table 15. Variants Used in the Key Analytical Validation Studies** | | LoD/Precision | | | Accuracy | | | | --- | --- | --- | --- | --- | --- | --- | | | RE | ID | SUB | RE | ID | SUB | | *ATR* | 1 | 1 | 0 | 0 | 12 | 4 | | *CDK12* | 1 | 1 | 1 | 0 | 39 | 11 | | *CHEK2* | 0 | 1 | 1 | 0 | 18 | 16 | | *FANCA* | 1 | 1 | 0 | 0 | 3 | 2 | | *MLH1* | 1 | 1 | 0 | 0 | 1 | 0 | | *MRE11A* | 1 | 1 | 0 | 0 | 3 | 2 | | *NBN* | 1 | 0 | 1 | 0 | 4 | 0 | | *PALB2* | 1 | 0 | 1 | 0 | 7 | 4 | | *RAD51C* | 1 | 1 | 0 | 0 | 1 | 1 | RE: rearrangement ID: insertion/deletion SUB: base substitutions The prevalence of RE alterations in these genes ranged from 0% to 0.28% in the TALAPRO-2 trial and an external publicly available prostate cancer database that uses another NGS-based technology. The prevalence of ID and SUB alterations in these genes ranged from 0.03% to 13.84% in the TALAPRO-2 trial and an external publicly available prostate cancer database that uses another NGS-based technology. Prostate cancer samples evaluated in the analytical validation studies were prepared using the updated workflow of the Library Construction (LC) and Hybrid Capture (HC) of the F1LCDx assay. ## 1. Analytical Accuracy/Concordance ### Comparison to an Orthogonal Method An analytical accuracy study was conducted previously as part of P200016 to demonstrate the concordance between F1LCDx and an externally validated, ctDNA-based, NGS assay (evNGS) for the detection of *BRCA1*, *BRCA2*, and *ATM* alterations. An additional analytical accuracy study was conducted in this sPMA to demonstrate the concordance between F1LCDx and an externally validated NGS assay (evNGS) for the detection of additional HRR gene (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) alterations. In total, 163 clinical samples from prostate cancer patients with valid results from both F1LCDx and the evNGS assay were included in the concordance analysis with representation of *ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C* base substitutions, insertions, and deletions. The study was conducted using 78 biomarker-negative and 85 biomarker positive samples as determined by F1LCDx results during the sample selection step and containing a total of 128 variants. Samples were residual cfDNA specimens from clinical prostate cancer samples from the FMI sample archives, the TALAPRO-2 trial, or processed under previous orthogonal studies. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 20 of 52 {20} The F1LCDx and evNGS results for the detection of HRR gene (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) alterations using evNGS as the comparator assay are provided in the contingency table in Table 16. To align with the product specifications of the evNGS assay, variants below the threshold of 0.5% VAF as detected by the evNGS assay are excluded from analysis. The Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) using the evNGS assay results as the reference were calculated at the sample level without adjusting for the distribution of samples selected using F1LCDx, positive predictive value (PPV) and negative predictive value (NPV) were also estimated conditional on F1LCDx. The statistical analysis in Table 16 showed a PPV of 81.61% with 95% CI (73.06%, 88.75%) and an NPV of 97.37% with 95% CI (90.90%, 99.28%), and a PPA of 97.26% with 95% CI (90.55%, 99.25%) and an NPA of 82.22% with 95% CI (73.06%, 88.75%) unadjusted for prevalence. **Table 16. Contingency Table Comparing the Sample-Level Detection of HRR gene (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) alterations by F1LCDx and the evNGS** | | evNGS+ | evNGS- | Total | | | --- | --- | --- | --- | --- | | **F1LCDx+** | 71 | 16 | 87 | PPV: 81.61% [95%CI*:72.19%, 88.35%] | | **F1LCDx-** | 2 | 74 | 76 | NPV:97.37%[95% CI*: 90.90%, 99.28%] | | **Total** | 73 | 90 | 163 | | | | PPA:97.26% [95%CI*:90.55%, 99.25%] | NPA:82.22% [95% CI*: 73.06%, 88.75%] | | | \*95% 2-sided confidence intervals (CI) were calculated using the Wilson score method The concordance results at the variant level are provided in the contingency table in Table 17. The statistical analysis in Table 17 showed a PPA of 97.89% with 95% CI (92.65%, 99.42%) and a NPA of 99.92% with 95% CI (99.88%, 99.95%) unadjusted for prevalence. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 21 of 52 {21} Table 17. Contingency Table Comparing the Variant-Level Detection of HRR gene (ATR, CDK12, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C) alterations by F1LCDx and the evNGS | | evNGS+ | evNGS- | Total | | --- | --- | --- | --- | | F1LCDx+ | 93 | 25 | 118 | | F1LCDx- | 2 | 31,013 | 31,0115 | | Total | 95 | 31,038 | 31,133 | | PPA:97.89% [95%CI*:92.65%, 99.42%] | | | | | NPA:99.92% [95% CI*: 99.88%, 99.95%] | | | | *95% 2-sided confidence intervals (CI) were calculated using the Wilson score method There were 27 discordant calls between F1LCDx and the evNGS test results from 18 discordant samples. Of the 18 samples, the following was observed: - Two samples expressed the two variants detected by the evNGS that were not detected by F1LCDx. These two variants detected by the evNGS had corresponding F1LCDx test results exhibiting low tumor fraction. - Sixteen samples exhibited the 25 variants detected by F1LCDx and not the evNGS. For these 25 alterations, 15 were variants occurring in a repetitive context representing a challenging variant condition for the evNGS assay and 9 were variants detected in F1LCDx that were below the LoD for the evNGS. The remaining alteration was a complex deletion-insertion event that may be impacted by variant calling sensitivity. ## 2. Analytical Sensitivity ### a. Limit of Blank (LoB) The LoB of F1LCDx was evaluated in the platform LoB study for PMA P190032 (refer to Section IX.A.3.a. in the Summary of Safety and Effectiveness Data for P190032). An additional LoB study was performed for P190032/S017 to support the updated LC/HC workflow of F1LCDx by collecting plasma cfDNA and corresponding donor-matched white blood cell (WBC) genomic DNA (gDNA) samples from 47 healthy donors with no known cancer diagnosis. A total of 32,273 unique variants were included in the study, including short variants (SV) for all HRR genes and rearrangements (RE) for all HRR genes except for RAD51C. Only one biomarker positive HRR variant was detected, CHEK2_1043T>A, in only one of the tested replicates, at 0.17% VAF, which was below the median LoD for short variants. The false positive rate for PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 22 of 52 {22} CHEK2_1043T>A was 1.0638%. No other CDx biomarker variants in other HRR genes (*i.e.*, *ATR*, *CDK12*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, and *RAD51C*) were detected. # **b. Limit of Detection (LoD)** The F1LCDx LoD for the detection of HRR gene (*BRCA1*, *BRCA2*, *ATM*, *ATR*, *CDK12*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, and *RAD51C*) alterations was established through CDx-specific and confirmation of platform-based LoD studies. The LoD for *BRCA1*, *BRCA2*, and *ATM* alterations was established through a previously conducted study for the approved PMA P200016. The LoD for the HRR genes (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) that were not evaluated in P200016 was confirmed through a precision study that used prostate cancer (PC) samples at or near 1x the median platform LoD for SVs (*i.e.*, gene substitutions and indels) and RE previously determined in P190032, as detailed in section 3 below. The LoD confirmation/Precision study also included one sample from colorectal cancer (CRC) harboring a *BRCA1* homozygous deletion (HD) tested at 2x LoD for HD. A summary of the confirmed LoD results is provided in Table 18 below. **Table 18. Confirmation of LoD results for HRR Gene Alterations** | Disease Ontology | Gene | Variant Type | Hit Rate (%) | Confirmed LoD | Median Platform LoD (%) | Fold LoD (x)* | | --- | --- | --- | --- | --- | --- | --- | | PC | *ATR* | RE | 94.44 | 0.52% VAF | 0.37 | 1.42 | | PC | *CDK12* | SV (substitution) | 100.00 | 0.57% VAF | 0.40 | 1.44 | | PC | *CDK12* | SV (insertion) | 100.00 | 0.54% VAF | 0.40 | 1.34 | | PC | *CDK12* | RE | 100.00 | 0.66% VAF | 0.37 | 1.79 | | PC | *CHEK2* | SV (substitution) | 94.44 | 0.49% VAF | 0.40 | 1.23 | | PC | *CHEK2* | SV (deletion) | 100.00 | 0.57% VAF | 0.40 | 1.43 | | PC | *PALB2* | RE | 100.00 | 0.70% VAF | 0.37 | 1.89 | | PC | *PALB2* | SV (substitution) | 96.88 | 0.55% VAF | 0.40 | 1.36 | | PC | *FANCA* | RE | 97.14 | 1.21% VAF | 0.90 | 1.35 | | PC | *FANCA* | SV (insertion) | 100.00 | 1.40% VAF | 0.82 | 1.70 | | PC | *MLH1* | SV (deletion) | 100.00 | 1.25% VAF | 0.82 | 1.53 | | PC | *MLH1* | RE | 100.00 | 1.16% VAF | 0.90 | 1.29 | | PC | *MRE11A* | SV (deletion) | 100.00 | 1.37% VAF | 0.82 | 1.67 | | PC | *NBN* | SV (substitution) | 100.00 | 1.30% VAF | 0.82 | 1.59 | | PC | *RAD51C* | SV (insertion) | 100.00 | 1.29% VAF | 0.82 | 1.57 | | PC | *RAD51C* | RE | 94.44 | 1.22% VAF | 0.90 | 1.35 | | CRC | *BRCA1* | HD | 100.00 | 61.80% TF | 30.40 | 2.03 | \* Fold LoD = [Confirmed LoD(%)] / [Median Platform LoD(%)] TF: tumor fraction PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 23 of 52 {23} ### 3. Precision and Reproducibility #### Site-to-Site Precision To evaluate the performance of HRR gene alteration detection, a site-to-site precision study was conducted using 18 prostate cancer specimens harboring HRR gene (*ATR*, *CDK12*, *CHEK2*, *FANCA*, *MLH1*, *MRE11A*, *NBN*, *PALB2*, *RAD51C*) alterations (biomarker-positive samples) at or near 1x the median platform LoD for short variants (substitutions, indels), and rearrangements. The study also included one clinical specimen from a CRC patient harboring a *BRCA1* HD tested at 2x LoD for HD. Table 19 below lists the biomarker-positive samples included in this study. **Table 19. List of samples and HRR alterations evaluated in the study** | Sample ID | Disease Ontology | Gene | Variant Type | Targeted Variant | | --- | --- | --- | --- | --- | | 1 | Prostate Cancer | *ATR* | SV (deletion) | p.I774fs*5 (2320delA) | | 2 | Prostate Cancer | *ATR* | RE | ATR- CMSS1 truncation | | 3 | Prostate Cancer | *CDK12* | SV (substitution) | p.Y279 * (837C>G) | | | | *CDK12* | SV (insertion) | p.Y913fs* 1 (2738_273 9insA) | | 4 | Prostate Cancer | *CDK12* | RE | CDK12- MICALCL truncation | | 5 | Prostate Cancer | *CHEK2* | SV (deletion) | p.T367fs* 15 (1100delC) | | 6 | Prostate Cancer | *CHEK2* | SV (substitution) | p.L354* (1061T>A) | | 7 | Prostate Cancer | *PALB2* | RE | PALB2- N/A truncation | | 8 | Prostate Cancer | *PALB2* | SV (substitution) | splice site 49- 1G>C | | 9^ | Prostate Cancer | *FANCA* | RE | FANCA- N/A truncation | | 10 | Prostate Cancer | *FANCA* | SV (insertion) | p.L432fs*53 (1290G>AT) | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 24 of 52 {24} | 11 | Prostate Cancer | *MLH1* | SV (deletion) | p.K618fs* 19 (1851delG) | | --- | --- | --- | --- | --- | | 12^ | Prostate Cancer | *MLH1* | RE | MLH1- TMPRSS2 truncation | | 13 | Prostate Cancer | *MRE11A* | SV (deletion) | p.E515fs* 8 (1543_154 6delGAAG) | | 14 | Prostate Cancer | *MRE11A* | RE | MRE11A- N/A truncation | | 15 | Prostate Cancer | *NBN* | RE | NBN-N/A truncation | | 16 | Prostate Cancer | *NBN* | SV (substitution) | p.E718* (2152G>T) | | 17 | Prostate Cancer | *RAD51C* | SV (insertion) | p.G149fs* 6 (444_445insT) | | 18^ | Prostate Cancer | *RAD51C* | RE | RAD51C- N/A truncation | | 19 | Colorectal Cancer# | *BRCA1* | HD | HD | # No PC samples with sufficient mass are available for this variant. ^ Samples selected from the talazoparib clinical program. An “-N/A” partner gene indicates that the partner gene was not identified/ represents intragenic breakpoints Samples were tested in duplicate across a total of 18 runs. Each run was performed using 2 reagent lots on 3 different Library Construction start days at 3 sites (2 replicates x 2 reagent lots x 3 LC start days x 3 sites) for a total of 36 replicates per sample. A total of 684 sample replicates (18 sample runs x 36 replicates per sample) were included in the study. Of the 684 replicates, 12 replicates failed to meet F1LCDx processing hybrid capture QC metrics. Thus, 672 valid replicates were evaluated. The 18 PC samples included in the precision study harbored a total of 19 HRR gene alterations targeted at ~1-1.5x LoD. Since there was no matched normal sample for BRCA1 HD, the CRC sample was processed without being diluted, at approximately 2x LoD. All 19 biomarker-positive samples were run at challenging DNA input (close to 20 ng). Reproducibility was evaluated by processing replicates from the same source sample, under conditions where one factor was changed at a time. The alterations had to be detected in each replicate of the source sample and meet the biomarker PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 25 of 52 {25} definition to be considered a positive call. The point estimates and 95% two-sided score CIs for reproducibility of each sample are detailed in Table 20. 19 out of the 20 targeted variants were above LoD and 1 variant was below LoD. Table 20. Reproducibility for All Targeted Variants | Alteration | Variant Type | Observed Average %VAF^{1}/%TF^{2} | Median Platform LoD | # of Positive Replicates | # of Valid Replicates | Reproducibility (%) | 95% Two-sided Score CI (%) | Fold LoD (x) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ATR-CMSS1 | RE | 0.52 | 0.37 | 34 | 36 | 94.44 | [81.86, 98.46] | 1.42 | | CDK12_837 C>G | SV (substitution) | 0.57 | 0.40 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.44 | | CDK12_273 8_2 739insA | SV (Insertion) | 0.54 | 0.40 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.34 | | CDK12-MICALCL | RE | 0.66 | 0.37 | 35 | 35 | 100.00 | [90.11, 100.00] | 1.79 | | CHEK2_110 0de IC | SV (Deletion) | 0.57 | 0.40 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.43 | | CHEK2_106 1T>A | SV (substitution) | 0.49 | 0.40 | 34 | 36 | 94.44 | [81.86, 98.46] | 1.23 | | PALB2-N/A | RE | 0.70 | 0.37 | 35 | 35 | 100.00 | [90.11, 100.00] | 1.89 | | PALB2_49- 1G>C | SV (substitution) | 0.55 | 0.40 | 31 | 32 | 96.88 | [84.26, 99.45] | 1.36 | | FANCA-N/A | RE | 1.21 | 0.90 | 34 | 35 | 97.14 | [85.47, 99.49] | 1.35 | | FANCA_129 0G>AT | SV (Insertion) | 1.40 | 0.82 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.70 | | MLH1_1851 delG | SV (Deletion) | 1.25 | 0.82 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.53 | | MLH1-TMPRSS2 | RE | 1.16 | 0.90 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.29 | | MRE11A_15 43_ 1546delGAA G | SV (Deletion) | 1.37 | 0.82 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.67 | | MRE11A- N/A | RE | 1.05 | 0.90 | 29 | 36 | 80.56 | [64.97, 90.25] | 1.17 | | NBN-N/A | RE | 1.16 | 0.90 | 31 | 36 | 86.11 | [71.34, 93.92] | 1.29 | | NBN_2152G >T | SV (substitution) | 1.30 | 0.82 | 35 | 35 | 100.00 | [90.11, 100.00] | 1.59 | | RAD51C_44 4_4 45insT | SV (insertion) | 1.29 | 0.82 | 36 | 36 | 100.00 | [90.36, 100.00] | 1.57 | | RAD51C-N/A | RE | 1.22 | 0.90 | 34 | 36 | 94.44 | [81.86, 98.46] | 1.35 | | BRCA1_loss | HD | 61.80 | 30.40 | 32 | 32 | 100.00 | [89.28, 100.00] | 2.03 | | ATR_2320del A | SV (Deletion) | 0.08 | 0.40 | 33 | 36 | 91.67% | [78.17, 97.13] | 0.19 | * Fold LoD = [Observed Average %VAF/TF] / [Median Platform LoD(%)] $^{1}$VAF is applicable for short variants (substitutions, insertions, deletions) and rearrangements $^{2}$Tumor fraction (TF) is applicable for homozygous deletions An “-N/A” partner gene indicates that the partner gene was not identified/ represents intragenic breakpoints PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 26 of 52 {26} One sample with a targeted MRE11A-N/A RE tested at 1.17x LoD demonstrated reproducibility of 80.56%. This sample had three replicates with evidence for the variant observed but filtered from reporting due to insufficient supporting read evidence and four replicates with a qualified QC status due to low coverage, which may impact variant calling sensitivity. One sample with a targeted NBN-N/A RE tested at 1.29x LoD demonstrated reproducibility of 86.11%. This sample had two replicates with no evidence of variant detected and three replicates with evidence for the variant observed but filtered from reporting due to insufficient supporting read evidence. One sample with a targeted ATR_2320delA demonstrated reproducibility <95%. This sample had observed reads below the median platform LoD. Repeatability was assessed by processing samples with two replicates from the same source sample and plate within each site, reagent lot, and LC start day. The result was considered to be in agreement if the duplicate replicates processed under identical conditions had the same detection status for the targeted variants. The repeatability results for each targeted variant are detailed in Table 21 below. **Table 21. Repeatability for All Targeted Variants** | Alteration | Variant Type | # of Agree Pairs | # of Valid Pairs | Repeatability (%) | 95% Two-sided Score CI (%) | | --- | --- | --- | --- | --- | --- | | *ATR-CMSS1* | RE | 16 | 18 | 88.89 | [67.20, 96.90] | | *CDK12_837 C>G* | SV (substitution) | 18 | 18 | 100.00 | [82.41, 100.00] | | *CDK12_273 8_2 739insA* | SV (Insertion) | 18 | 18 | 100.00 | [82.41, 100.00] | | *CDK12- MICALCL* | RE | 17 | 17 | 100.00 | [81.57, 100.00] | | *CHEK2_110 0de IC* | SV (Deletion) | 18 | 18 | 100.00 | [82.41, 100.00] | | *CHEK2_106 1T>A* | SV (substitution) | 16 | 18 | 88.89 | [67.20, 96.90] | | *PALB2-N/A* | RE | 17 | 17 | 100.00 | [81.57, 100.00] | | *PALB2_49- 1G>C* | SV (substitution) | 14 | 15 | 93.33 | [70.18, 98.81] | | *FANCA-N/A* | RE | 16 | 17 | 94.12 | [73.02, 98.95] | | *FANCA_129 0G>AT* | SV (Insertion) | 18 | 18 | 100.00 | [82.41, 100.00] | | *MLH1_1851 delG* | SV (Deletion) | 18 | 18 | 100.00 | [82.41, 100.00] | | *MLH1- TMPRSS2* | RE | 18 | 18 | 100.00 | [82.41, 100.00] | | *MRE11A_15 43 1546delGAA G* | SV (Deletion) | 18 | 18 | 100.00 | [82.41, 100.00] | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 27 of 52 {27} | Alteration | Variant Type | # of Agree Pairs | # of Valid Pairs | Repeatability (%) | 95% Two-sided Score CI (%) | | --- | --- | --- | --- | --- | --- | | MRE11A-N/A | RE | 13 | 18 | 72.22 | [49.13, 87.50] | | NBN-N/A | RE | 17 | 18 | 94.44 | [74.24, 99.01] | | NBN_2152G>T | SV (substitution) | 17 | 17 | 100.00 | [81.57, 100.00] | | RAD51C_444_45insT | SV (insertion) | 18 | 18 | 100.00 | [82.41, 100.00] | | RAD51C-N/A | RE | 16 | 18 | 88.89 | [67.20, 96.90] | | BRCA1_loss | HD | 15 | 15 | 100.00 | [79.61, 100.00] | | ATR_2320delA | SV (Deletion) | 17 | 18 | 94.44 | [74.24, 99.01] | ### B. Animal Studies No animal studies were conducted using the F1LCDx assay. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 28 of 52 {28} # X. SUMMARY OF PRIMARY CLINICAL STUDY The clinical performance of F1LCDx for identification of prostate cancer patients with HRR gene alterations who may benefit from treatment with TALZENNA® (talazoparib) in combination with XTANDI® (enzalutamide) was demonstrated based on results from the TALAPRO-2 clinical trial. A summary of the clinical study design is presented below: # A. TALAPRO-2 Study Design TALAPRO-2 is an international, double-blinded, placebo-controlled, randomized Phase 3 study of talazoparib in combination with enzalutamide compared with placebo in combination with enzalutamide in patients with mCRPC, where no systemic cancer treatments have been initiated after documentation of CRPC with the exception of androgen deprivation therapy (ADT) and first-generation anti-androgen agents. Part 1 was open-label and non-randomized and evaluated the safety, tolerability, and pharmacokinetics (PK) of talazoparib in combination with enzalutamide. The primary objective of Part 1 was to determine the starting dose of talazoparib in combination with enzalutamide to be used in Part 2. Part 2 was randomized, double-blinded, and placebo-controlled and evaluated the safety and efficacy of talazoparib in combination with enzalutamide compared with placebo in combination with enzalutamide. The major efficacy outcome measure of Part 2 was radiographic progression-free survival (rPFS) evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 and Prostate Cancer Working Group (PCWG3) (bone) criteria, assessed by Blinded Independent Central Review (BICR). An additional efficacy outcome measure was Overall Survival (OS). Part 2 of the study included 2 cohorts and were 1:1 randomized to talazoparib in combination with enzalutamide versus placebo in combination with enzalutamide. # Part 2 Cohort 1 The first cohort (Cohort 1) enrolled 805 mCRPC patients unselected for HRR status (referred to as an all-comers population). Although patients were unselected for HRR status, HRR deficiency assessment prior to randomization was required for stratification. # Part 2 Cohort 2 After enrollment for Cohort 1 was closed, enrollment into the study continued but was restricted to patients with HRR deficiencies (Cohort 2). Cohort 2 enrolled 230 patients harboring HRR gene alterations that were likely to sensitize the patient's tumor to poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition (HRR-deficient). Patients with HRR gene alterations were identified prior to randomization by central testing of tumor tissue or blood samples (liquid biopsy) using primarily the F1CDx PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 29 of 52 {29} during Cohort 1 of the study, and F1CDx and/or the F1LCDx assays during Cohort 2 of the study. Of the 805 mCRPC patients enrolled into Cohort 1, 169 patients were HRR-deficient and thus, there were 399 HRR-deficient patients from Cohort 1 and Cohort 2. ### **Clinical Validation Study Design** The clinical effectiveness of TALZENNA® in combination with XTANDI® among patients with HRR gene alterations as determined by F1LCDx was demonstrated through a clinical validation study using specimens from the TALAPRO-2 clinical trial. The clinical validation study was conducted to: (1) evaluate the efficacy of talazoparib in combination with enzalutamide in mCRPC patients with HRR gene alterations as determined by F1LCDx and (2) assess the robustness of the efficacy evaluated with sensitivity analyses that account for the uncertainty due to missing data for the F1LCDx biomarker status. The clinical validation for F1LCDx was based on 399 HRR-deficient patients from TALAPRO-2, which included clinical trial patients harboring HRR gene alterations from Cohorts 1 and 2. An additional analysis was conducted on the 805 patients in Cohort 1 of the TALAPRO-2 study unselected for HRR status. The clinical performance for F1LCDx was assessed through the primary efficacy analysis of HRR gene alteration positive patients as determined by F1LCDx in these populations. ### **1. Clinical Inclusion and Exclusion Criteria** #### **Inclusion criteria:** Enrollment in the TALAPRO-2 study was limited to patients who met the following key inclusion criteria: - At least 18 years of age. For Japan, at least 20 years of age. - Histologically or cytologically confirmed adenocarcinoma of the prostate without small cell or signet cell features. If the patient does not have a prior histological diagnosis, a baseline de novo biopsy must be used to confirm the diagnosis and to support biomarker analysis. - Asymptomatic or mildly symptomatic metastatic castration resistant prostate cancer (mCRPC). - For enrollment into Part 2 only (optional in Part 1): assessment of DNA damage repair (DDR) mutation status by prospective analysis of blood (liquid biopsy), or tissue (de novo or archival tissue), or historical analysis (with Sponsor pre-approval), of most recent tumor tissue per FoundationOne® testing. (Note: for patients enrolling in Part 1, DDR deficiency testing is optional). - For enrollment into Part 2 only (optional for Part 1): Unless prohibited by local regulations or ethics committee decision, consent to a saliva sample collection for retrospective sequencing of the same DDR genes tested on tumor tissue and blood (liquid biopsy), or a subset thereof, and to serve as a germline control in identifying tumor mutations. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 30 of 52 {30} - Surgically or medically castrated, with serum testosterone ≤50 ng/dL (≤1.73 nmol/L) at screening. Ongoing androgen deprivation therapy with a gonadotropin releasing hormone (GnRH) agonist or antagonist for patients who have not undergone bilateral orchiectomy must be initiated at least 4 weeks before Day 1 (Part 1) or randomization (Part 2) and must continue throughout the study. - Metastatic disease in bone documented on bone scan or in soft tissue documented on CT/MRI scan. - Progressive disease at study entry in the setting of medical or surgical castration. # Exclusion criteria: Patients were not permitted to enroll in the TALAPRO-2 study if they met any of the following key exclusion criteria: - Any prior systemic cancer treatment initiated in the non-metastatic CRPC or mCRPC disease state. (ADT and first-generation anti-androgens received in the CRPC disease state are NOT exclusionary). - Patients whose only evidence of metastasis is adenopathy below the aortic bifurcation. - Prior treatment with second-generation androgen receptor inhibitors (enzalutamide, apalutamide, and darolutamide), a PARP inhibitor, cyclophosphamide, or mitoxantrone for prostate cancer. - Prior treatment with platinum-based chemotherapy within 6 months (from the last dose) prior to Day 1 (Part 1) or randomization (Part 2), or any history of disease progression on platinum-based therapy within 6 months (from the last dose). - Treatment with cytotoxic chemotherapy which includes but is not limited to docetaxel, biologic therapy including sipuleucel-T, or radionuclide therapy received in the castration-sensitive prostate cancer is NOT exclusionary if discontinued in the 28 days prior to Day 1 (Part 1) or randomization (Part 2). Prior treatment with abiraterone in the castration-sensitive settings is not exclusionary if discontinued prior to randomization. Hormonal therapy (eg, bicalutamide, nilutamide, flutamide, estrogens) are not exclusionary if discontinued prior to randomization. Prednisone>10 mg/day (or equivalents) is exclusionary. - Treatment with any investigational agent within 4 weeks before Day 1 (Part 1) or randomization (Part 2). - Prior treatment with opioids for pain related to either primary prostate cancer or metastasis within 28 days prior to Day 1 (Part 1) or randomization (Part 2). - Current use of potent P-gp inhibitors within 7 days prior to Day 1 (Part 1) or randomization (Part 2). - Known or suspected brain metastasis or active leptomeningeal disease. - Symptomatic or impending spinal cord compression or cauda equina syndrome. PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 31 of 52 {31} ■ Any history of myelodysplastic syndrome, acute myeloid leukemia, or prior malignancy except for any of the following: ○ Carcinoma in situ or non melanoma skin cancer. ○ Any prior malignancies ≥3 years before randomization with no subsequent evidence of recurrence or progression regardless of the stage. ○ Stage 0 or Stage 1 cancer <3 years before randomization that has a remote probability of recurrence or progression in the opinion of the investigator. # 2. Follow-up Schedule Patients were followed-up in accordance with the TALAPRO-2 study safety follow-up and long-term follow-up schedules. The F1LCDx clinical validation study involved only testing and analysis of plasma cfDNA samples; as such, no additional patient subject follow-up was conducted with regard to the diagnostic study. # 3. Clinical Endpoints Clinical efficacy of talazoparib in combination with enzalutamide in mCRPC patients with HRR gene alterations by the F1LCDx assay was evaluated by the following primary efficacy endpoint: rPFS, defined as the time from the date of randomization to first objective evidence of radiographic progression as assessed in soft tissue per RECIST 1.1 or in bone (upon subsequent confirmation) per PCWG3 guidelines by BICR, or death, whichever occurred first. The primary efficacy analysis set included F1LCDx+ HRR-deficient patients, who were HRR gene alteration positive by the F1LCDx assay based on the F1LCDx biomarker definition for HRR gene alterations. # B. Accountability of PMA Cohort The clinical validation study was based on the combined HRR-deficient population in the TALAPRO-2 study as determined by the Clinical Trial Assays (CTA), i.e., HRRm CTA+ population, which consisted of 399 HRR-deficient patients (169 from Cohort 1 and 230 from Cohort 2). Of these 399 HRR-deficient patients, a total of 239 patients had valid results by F1LCDx prospective testing (i.e., on screening- phase samples) and 160 were not tested or did not have valid F1LCDx prospective results. An additional 115 patients had valid results by F1LCDx retrospective testing (i.e., on additional samples collected at screening and tested retrospectively). In total, of the 399 HRR-deficient patients in Cohort 1 and 2 of the study, 354 patients had valid results by either prospective or retrospective F1LCDx and 45 were not tested or did not have valid results by F1LCDx prospective or retrospective testing. Out of the 354 patients with F1LCDx valid results, 287 were HRR+ by F1LCDx and 67 were HRR- by F1LCDx (but positive by other enrolling assays) (Table 22). An additional analysis was conducted on the 805 patients in Cohort 1 of the TALAPRO-2 study unselected for HRR status (i.e., the all-comers population). Of these 805 patients unselected for HRR status, a total of 688 patients had valid results by either prospective PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 32 of 52 {32} or retrospective F1LCDx and 117 were not tested or did not have valid results by F1LCDx. Out of the 688 patients with valid F1LCDx results, 194 were HRR+ by F1LCDx and 494 were HRR- by F1LCDx (Table 22). Table 22. PMA sample accountability in the combined HRRm deficient population in Cohorts 1+2 and the all-comers population (Cohort 1) of the TALAPRO-2 study | Population | Subpopulation | Sample size (%) | | | --- | --- | --- | --- | | | | HRRm population from Cohorts 1+2 | All-comers population (Cohort 1) | | F1LCDx evaluable | Overall | 354 (88.72%) | 688 (85.47%) | | | • F1LCDx HRRm (F1LCDx+) | 287 (71.93%) | 194 (24.10%) | | | • By prospective testing | 201 (50.38%) | 20 (2.50%) | | | • By retrospective testing | 86 (21.55%) | 174 (20.50%) | | | • F1LCDx Non-HRRm (F1LCDx-) | 67 (16.79%) | 494 (61.37%) | | | • By prospective testing | 38 (9.52%) | 38 (4.73%) | | | • By retrospective testing | 29 (7.27%) | 456 (56.64%) | | F1LCDx unevaluable | Overall | 45 (11.28%) | 117 (14.53%) | | Overall | Overall | 399 (100.00%) | 805 (100.00%) | ### C. Study Population Demographics and Baseline Parameters The distributions of the selected demographic, patient and disease characteristics between the treatment and control arms were compared for the F1LCDx+ HRR-deficient (n=287) population. The results are shown in table 23. In general, most covariates are balanced between the treatment and control arms for the F1LCDx+ HRR-deficient patients. Table 23. Demographic and patient characteristics for F1LCDx HRR-deficient population | Covariate | Summary Statistics/Category | Treatment Group (N=142) | Placebo Group (N=145) | P value | | --- | --- | --- | --- | --- | | Previous treatment status | No Prior NHT or Taxane | 84 (59.15%) | 90 (62.07%) | 0.631 | | | Prior NHT or Taxane | 58 (40.85%) | 55 (37.93%) | | | | (Missing) | 0 (0.00%) | 0 (0.00%) | | | | Total | 142 (100.00%) | 145 (100.00%) | | | Age | min | 51.00 | 44.00 | 0.765 | | | Q1 | 64.00 | 64.00 | | | | median | 70.00 | 71.00 | | PMA P190032/S029: FDA Summary of Safety and Effectiveness Data 33 of 52 {33} | Covariate | Summary Statistics/Category | Treatment Group (N=142) | Placebo Group (N=145) | P value | | --- | --- | --- | --- | --- | | | mean | 69.94 | 69.99 | | | | Q3 | 76.00 | 76.00 | | | | max | 90.00 | 90.00 | | | | SD | 8.45 | 8.61 | | | | (Missing) | 0 (0.00%) | 0 (0.00%) | | | Age group | < 65 | 38 (26.76%) | 38 (26.21%) | 0.954 | | | >= 75 | 45 (31.69%) | 44 (30.34%) | | | | 65 to < 75 | 59 (41.55%) | 63 (43.45%) | | | | (Missing) | 0 (0.00%) | 0 (0.00%) | | | | Total | 142 (100.00%) | 145 (100.00%) | | | BMI | min | 18.60 | 18.00 | 0.562 | | | Q1 | 24.10 | 24.90 | | | | median | 27.30 | 27.80 | | | | mean | 28.03 | 28.45 | | | | Q3 | 30.60 | 30.90 | | | | max | 44.50 | 59.40 | | | | SD | 4.72 | 5.67 | | | | (Missing) | 1 (0.70%) | 2 (1.38%) | | | Baseline use of a bone protecting agent | No | 118 (83.10%) |…