FoundationOne Liquid CDx (F1LCDx)

{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. Date(s) of Panel Recommendation: None Premarket Approval Application (PMA) Number: P190032/S011 Date of FDA Notice of Approval: October 11, 2023 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 its 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 | | --- | --- | --- | | Non-small cell lung cancer (NSCLC) | *BRAF* V600E | BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) | ## 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 detect 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 PMA P190032/S011: FDA Summary of Safety and Effectiveness Data 1 of 34 {1} companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. **Table 1: Companion diagnostic indications** | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Non-small cell lung cancer (NSCLC) | *ALK* Rearrangements | ALECENSA® (alectinib) | | | *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* | | | *EGFR* Exon 20 insertions | EXKIVITY® (mobocertinib) | | | *MET* single nucleotide variants (SNVs) and indels that lead to *MET* exon 14 skipping | TABRECTA® (capmatinib) | | | *ROS1* fusions** | ROZLYTREK® (entrectinib) | | Prostate cancer | *BRCA1*, *BRCA2*, and *ATM* alterations | LYNPARZA® (olaparib) | | | *BRCA1*, *BRCA2* alterations | RUBRACA® (rucaparib) | | Breast Cancer | *PIK3CA* mutations C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y | PIQRAY® (alpelisib) | | Solid Tumors | *NTRK1/2/3* fusions** | ROZLYTREK® (entrectinib) | | Colorectal cancer (CRC) | *BRAF* V600E | BRAFTOVI® (encorafenib) in combination with cetuximab | *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 Additionally, FoundationOne Liquid CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. A negative result from a plasma specimen does not mean that the patient’s tumor is negative for genomic findings. Patients who are negative for the mutations listed in Table 1 (see **Note for PMA P190032/S011: FDA Summary of Safety and Effectiveness Data 2 of 34 {2} NTRK1/2/3 and ROS1 fusions) should be reflexed to routine biopsy and their tumor mutation status confirmed using an FDA-approved tumor tissue test, if feasible. **Note: when considering eligibility for ROZLYTREK® based on the detection of NTRK1/2/3 and ROS1 fusions, testing using plasma specimens is only appropriate for patients for whom tumor tissue is not available for testing. Genomic findings other than those listed in Table 1 of the intended use statement are not prescriptive or conclusive for labeled use of any specific therapeutic product. FoundationOne Liquid CDx is a single-site assay performed at Foundation Medicine, Inc. in Cambridge, MA. ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found in the F1LCDx labeling. ### V. DEVICE DESCRIPTION The F1LCDx assay is performed exclusively as a laboratory service using circulating cell-free DNA (cfDNA) isolated from plasma derived from anti-coagulated peripheral whole blood from patients with solid malignant neoplasms. The assay employs a single DNA extraction method to obtain cfDNA from plasma from whole blood. Extracted cfDNA undergoes whole-genome shotgun library construction and hybridization-based capture of 324 cancer-related genes. All coding exons of 309 genes are targeted; select intronic or non-coding regions are targeted in three genes (refer to Table 2 for the complete list of genes targeted 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 3 genes, and genomic rearrangements in 8 genes. A subset of targeted regions in 75 genes is baited for increased sensitivity. Table 2: Genomic Regions in which Variants are Targeted by F1LCDx¹ | 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- | BRCA1 [Introns 2, 7, | BRCA2 [Intron 2] | BRD4 | BRIP1 | BTG1 | BTG2 | BTK [Exons 2, 15] | C11orf30 (EMSY) | C17orf39 (GID4) | PMA P190032/S011: FDA Summary of Safety and Effectiveness Data 3 of 34 {3} | 18, Introns 7-10] | 8, 12, 16, 19, 20] | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 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 | ERRF11 | ESR1 [Exons 4-8] | | ETV4* [Intron 8] | ETV5* [Introns 6, 7] | ETV6* [Introns 5, 6] | EWSR1* [Introns 7-13] | EZH2 [Exons 4, 16, 17, 18] | EZR* [Introns 9-11] | FAM46C | FANCA | FANCC | FANCG | | FANCL | FAS | FBXW7 | FGF10 | FGF12 | FGF14 | FGF19 | FGF23 | FGF3 | FGF4 | | FGF6 | FGFR1 [Introns 1, 5, Intron 17] | FGFR2 [Intron 1, Intron 17] | FGFR3 [Exons 7, 9 (alternative designation exon 10), 14, 18, Intron 17] | FGFR4 | FH | FLCN | FLT1 | FLT3 [Exons 14, 15, 20] | FOXL2 | | FUBP1 | GABRA6 | GATA3 | GATA4 | GATA6 | GNA11 [Exons 4, 5] | GNA13 | GNAQ [Exons 4, 5] | GNAS [Exons 1, 8] | GRM3 | | GSK3B | H3F3A | HDAC1 | HGF | HNF1A | HRAS [Exons 2, 3] | HSD3B1 | ID3 | IDH1 [Exon 4] | IDH2 [Exon 4] | | IGF1R | IKBKE | IKZF1 | INPP4B | IRF2 | IRF4 | IRS2 | JAK1 | JAK2 [Exon 14] | JAK3 [Exons 5, 11, 12, 13, 15, 16] | | JUN | KDM5A | KDM5C | KDM6A | KDR | KEAP1 | KEL | KIT [Exons 8, 9, 11, 12, 13, 17, Intron 16] | KLHL6 | KMT2A (MLL) [Introns 6, 8-11, Intron 7] | | KMT2D (MLL2) | KRAS | LTK | LYN | MAF | MAP2K1 (MEK1) [Exons 2, 3] | MAP2K2 (MEK2) [Exons 2-4, 6, 7] | MAP2K4 | MAP3K1 | MAP3K13 | | MAPK1 | MCL1 | MDM2 | MDM4 | MED12 | MEF2B | MEN1 | MERTK | MET | MITF | | MKNK1 | MLH1 | MPL [Exon 10] | MRE11A | MSH2 [Intron 5] | MSH3 | MSH6 | MST1R | MTAP | MTOR [Exons 19, 30, 39, 40, 43-45, 47, 48, 53, 56] | | MUTYH | MYB* [Intron 14] | MYC [Intron 1] | MYCL (MYCL1) | MYCN | MYD88 [Exon 4] | NBN | NF1 | NF2 | NFE2L2 | | NFKBIA | NKX2-1 (TTF-1) | NOTCH1 | NOTCH2 [Intron 26] | NOTCH3 | NPM1 [Exons 4-6, 8, 10] | NRAS [Exons 2, 3] | NSD3 (WHSC1L1) | NT5C2 | NTRK1 [Exons 14, 15, Introns 8-11] | | NTRK2 [Intron 12] | NTRK3 [Exons 16, 17] | NUTM1* [Intron 1] | P2RY8 | PALB2 | PARK2 | PARP1 | PARP2 | PARP3 | PAX5 | PMA P190032/S011: FDA Summary of Safety and Effectiveness Data 4 of 34 {4} | *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 are restricted to those genes included in Table 3, below. **Table 3: Genes for which copy number alterations and rearrangements are reported for tumor profiling by 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 PMA P190032/S011: FDA Summary of Safety and Effectiveness Data 5 of 34 {5} 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 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 PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 6 of 34 {6} # 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 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 binding, washing, and elution to yield high purity cfDNA. After isolating cfDNA, the Agilent 4200 TapeStation is used to quantify cfDNA. # C. Library Construction Library Construction (LC) begins with the normalization of cfDNA. The samples are purified, using AMPure XP Beads (Agencourt). Solid-phase reversible immobilization (SPRI) purification is used subsequent to library construction with the NEBNext kits (NEB), including mixes for end repair with blunt-end and 5'- phosphorylate the cfDNA fragments using T4 Polynucleotide Kinase and T4 DNA Polymerase. This step prepares the 3'-end for dA-addition while also preparing the 5'-end of the DNA fragment for ligation. Second, dA-addition will incorporate a single dAMP to the 3'-end of the End-Repaired material. After dA-addition, a universal Y-adaptor is ligated PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 7 of 34 {7} 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, SPRI purified and quantified by PicoGreen fluorescence assay (Invitrogen). Process matched control (PMC) is prepared and added to the plate with other cfDNA samples at the beginning of LC. On May 25, 2022, F1LCDx was approved for a change in the design of the primers used for LC. Specifically, a change was made to the regions of the primer that hybridize to the universal Y-adaptors during the PCR amplification process, which resulted in increased amplification efficiency and reduced the minimum recommended cfDNA input level to 20ng. ### D. Hybrid Capture Hybrid Capture begins with the normalization of each library from 500 ng to 2000 ng. 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. Upon completion of the pre-capture normalization, blocking DNA (adaptor block, Cot, Salmon Sperm DNA) is added to the sequencing library and the mixture is lyophilized in a 96-well plate. The library is then re-suspended in nuclease-free water, heat denatured at 95°C for 5 minutes, temperature ramps from 95°C to 68°C to anneal blocking DNA, and then the samples are incubated at 68°C for a minimum of 5 minutes before the addition of the bait set reagent. After a 20-24-hour incubation, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen) and off-target library is removed by washing one time with Saline Sodium Citrate (SSC) at 25°C and four times with SSC at 55°C. The PCR master mix is added to directly amplify the captured library from the washed beads. After amplification, the samples are SPRI purified and quantified by PicoGreen. PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 8 of 34 {8} ## E. Sequencing Sequencing on the Illumina NovaSeq 6000 platform employs on-board cluster generation (OBCG) using patterned flow cell (FC) technology to generate monoclonal clusters via ExAmp from a single DNA template. The clusters are then sequenced using sequencing by synthesis (SBS) chemistry. The NovaSeq system is capable of sequencing up to two flow cells at a time. During OBCG, a single DNA template is introduced into each of the primer substrate layered nanowells of the flow cell, where the template is immediately and rapidly amplified by ExAmp. This rapid amplification prevents other DNA templates from binding, ensuring a monoclonal cluster is formed in each nanowell. The procedure allows for fixed size and spacing of the clusters which results in improved and more accurate resolution. A growing nucleotide chain is created on the flow cell by incorporating fluorescently labeled, 3'-blocked deoxynucleoside 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). 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 PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 9 of 34 {9} originate and terminate in reference nodes from the locus. Increased k-mer sizes may be used to account for ambiguities, cycles, and other problematic regions within the graph. The result of the graph traversal is a set of candidate variants. For each variant, there is a set of k-mers supporting the variant and a set of k-mers that would support the reference or another variant at the location. Each candidate variant is then scanned against reads in the locus to identify which reads support either the candidate variant or a different variant or reference at the location. The cluster membership of the supporting reads is then assessed to determine which clusters show unambiguous support for the variant and which have conflicting assignments, indicating that the variant may have arisen as an error in sequencing or library preparation. The final variant calls are made based on a model that takes into account the coverage at the location, the number of supporting read clusters and their redundancy level, and the number of error-containing clusters. ### G. Report Generation Approved results are annotated by automated software with CDx relevant information and are merged with patient demographic information and any additional information provided by Foundation Medicine as a professional service prior to approval and release by the laboratory director or designee. ### 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. ---PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 10 of 34 {10} ## I. Classification Criteria for CDx Biomarkers Detected by F1LCDx 1. *BRCA1 and BRCA2 alterations to identify patients eligible for rucaparib in prostate cancer:* The CDx classification criteria and the list of *BRCA1/BRCA2* missense mutations for rucaparib, based on the trial pre-specifications are described in Table 5 and Table 6; however, not all the missense mutations listed below were observed in the TRITON2, and PROfound clinical studies. **Table 5: Classification Criteria for Deleterious Tumor *BRCA* Variants** | Qualification Criteria | Sequence Classification | Methodology | | --- | --- | --- | | A *BRCA1* or *BRCA2* alteration that includes any of the sequence classifications | Protein truncating mutations | Sequence analysis identifies premature stop codons anywhere in the gene coding region, except, 3' of and including *BRCA2* K3326* | | | Splice site mutations | Sequence analysis identifies variant splice sequences at intron/exon junctions -/+ 2bp of exon starts/ends | | | Homozygous deletions | Sequence analysis identifies deletions in both gene alleles of ≥1 exon in size | | | Large protein truncating rearrangements | Sequence analysis identifies protein truncating rearrangements | | | Deleterious missense mutations | Curated list | **Table 6: Deleterious *BRCA* Missense Alterations in rucaparib trial** | *BRCA1* Alterations (Protein Change) | | | | | *BRCA2* Alterations (Protein Change) | | | | --- | --- | --- | --- | --- | --- | --- | --- | | M1V | C44Y | R71T | R1699W | G1770V | M1V | R2336P | T2722R | | M1T | C44F | R71M | R1699Q | M1775K | M1T | R2336L | D2723H | | M1R | C47S | S770L | G1706R | M1775R | M1R | R2336H | D2723G | | M1I | C47Y | R1495T | G1706E | C1787S | M1I | T2412I | G2724W | | M18T | C47F | R1495M | A1708E | G1788V | D23N | R2602T | G2748D | | L22S | C61S | R1495K | S1715R | P1812A | D23Y | W2626C | A2911E | | I26N | C61G | E1559K | S1722F | A1823T | S142N | I2627F | E3002K | | T37K | C61Y | E1559Q | V1736A | V1833M | S142I | R2659T | R3052W | | C39R | C64R | T1685A | G1738R | W1837R | V159M | R2659K | D3095G | | C39G | C64G | T1685I | G1738E | V1838E | V211I | E2663V | D3095E | | C39Y | C64Y | D1692N | K1759N | | V211L | S2670L | N3124I | | C39W | C64W | M1689R | L1764P | | Y600C | I2675V | N3187K | | H41R | R71G | D1692H | I1766N | | K1530N | T2722K | | | C44S | R71K | D1692Y | I1766S | | | | | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 11 of 34 {11} 2. *ATM, BRCA1 and BRCA2 alterations to identify patients eligible for olaparib in mCRPC:* **Table 7: Rules Applied to the Aforementioned Genes:** | Qualification Criteria | Sequence Classification | Methodology | Comments | | --- | --- | --- | --- | | A gene alteration that includes any of the sequence classifications | Protein truncating mutations | Sequence analysis identifies premature stop codons anywhere in the gene coding region, except 3' of and including *BRCA2* K3326* | Does not include VUS. Includes mutations on the canonical transcript only for genes *ATM, BRCA1, and BRCA2*. | | | Splice site mutations | Sequence analysis identifies variant splice sequences at intron/exon junctions -/+ 2bp of exon starts/ends | Does not include VUS. Includes indels that extend through ± 2bp from the intron/exon junction. Includes mutations on the canonical transcript only for genes *ATM, BRCA1, and BRCA2*. | | | Homozygous deletions | Sequence analysis identifies deletions in both gene alleles of ≥1 exon in size | Does not include VUS Only reported for *BRCA1* and *BRCA2*. Not reported for *ATM*. | | | Large protein truncating rearrangements | Sequence analysis identifies protein truncating rearrangements | Does not include VUS | | | Deleterious missense mutations | Curated list | Protein effects from list of missense mutations on the canonical transcript only for genes *ATM, BRCA1, and BRCA2*. | Alterations reported are limited to those within the alteration-calling capabilities of FMI as of March 2, 2020. ATM missense mutations were identified from the ClinVar database. Should the calling capabilities expand, additional alterations that meet the above criteria may also be reported, per FDA approval. **Table 8. List of Deleterious Missense Mutations by Protein Effect, Implemented on the Respective Canonical Transcript.** | *BRCA1* | | *BRCA2* | | *ATM* | | | --- | --- | --- | --- | --- | --- | | Protein Effect (PE) | FMI Annotated PE | Protein Effect (PE) | FMI Annotated PE | Protein Effect (PE) | FMI Annotated PE | | M1V | M1V | M1R | M1R | M1T | M1T | | MII | MII | MII | MII | R2032K | R2032K | | C61G | C61G | V159M | V159M | R2227C | R2227C | | C64Y | C64Y | V211L | V211L | R2547- S2549del | R2547- S2549del | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 12 of 34 {12} | R71G | R71G | V211I | V211I | G2765S | G2765S | | --- | --- | --- | --- | --- | --- | | R71K | R71K | R2336P | R2336P | R2832C | R2832C | | R1495M | R1495M | R2336H | R2336H | S2855-V2856delinsR1 | S2855-V2856delinsR1 S2855-V2856>R1 | | E1559K | E1559K | | | R3008C | R3008C | | D1692N | D1692N | | | R3008H | R3008H | | D1692H | D1692H | | | [VUS from Jan 2016 HRR* List to be Excluded] | | | R1699W | R1699W | | | V2424G | V2424G | | A1708E | A1708E | | | [Excluded from Jan 2016 HRR List] | | | G1788V | G1788V | | | K750K | splice site 2250G>A | HRR = Homologous Recombination Repair genes ### Intronic Variants | Gene | Chr | Position | Ref | Alt | dbSNP | FMI Protein Effect | | --- | --- | --- | --- | --- | --- | --- | | *ATM* | *chr11* | *108128198* | *T* | *G* | *rs730881346* | **[Variant Not Called by FMI]** | | *ATM* | *chr11* | *108214102* | AGTGA | *A* | *rs730881295* | splice site 8418+5_8418+8delGTGA or splice site 8418+1_8418+4delGTGA | 3. *CDx classification criteria for EGFR alterations:* 1. Base substitutions resulting in *EGFR* L858R 2. In-frame deletions occurring within *EGFR* Exon 19 4. *ALK rearrangements to identify patients eligible for treatment with ALECENSA® (alectinib):* CDx positivity for an *ALK* rearrangement is based on the following variant classification criteria: - The *ALK* rearrangement must have pathogenic driver status (FMI driver status of "known" or "likely") - AND the disease type must be NSCLC - AND one of the following two conditions must hold: 1. The partner gene is *EML4*, or 2. The *ALK* breakpoint occurs within *ALK* intron 19 5. *SNVs and indels that lead to MET exon 14 skipping to identify patients eligible for treatment with TABRECTA® (capmatinib):* 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. PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 13 of 34 {13} # 6. *Biomarker Rules for Rearrangements that Lead to NTRK1, NTRK2, or NTRK3 Fusions:* 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. # 7. *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. # 8. *EGFR exon 20 insertions to identify NSCLC patients eligible for treatment with EXKIVITY:* CDx positivity for *EGFR* exon 20 insertions is determined if the following criteria were met: - Any in-frame insertions affecting amino acids 762 – 775 (inclusive) in *EGFR* exon 20 # 9. *BRAF V600E to identify metastatic CRC patients eligible for treatment with BRAFTOV1 (encorafenib) in combination with cetuximab and* ---PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 14 of 34 {14} 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 BRAF V600E to identify patients with NSCLC eligible for treatment with BRAFTOV1 (encorafenib) in combination with MEKTOV1 (binimetinib). However, FoundationOne CDx (F1CDx) test from Foundation Medicine, Inc. (FMI) is an FDA-approved CDx (P170019/S039) 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. The approved PMA supplements that affected the intended use are listed in Table 9. Table 9. Marketing History | Submission No. | Date of Approval | Biomarker/Update | Indication | Drug | | --- | --- | --- | --- | --- | | P200006 | October 26, 2020 | ALK Rearrangements | NSCLC | ALECENSA® (alectinib) | | | | PIK3CA 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 | NSCLC | TABRECTA® (capmatinib) | | P190032/S004 | December 22, | NTRK1/2/3 fusions | Solid Tumors | ROZLYTREK® | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 15 of 34 {15} | Submission No. | Date of Approval | Biomarker/Update | Indication | Drug | | --- | --- | --- | --- | --- | | | 2022 | *ROS1* fusions | NSCLC | (entrectinib) | | P190032/S008 | December 19, 2022 | *EGFR* Exon 19 deletions and *EGFR* Exon 21 L858R alteration | NSCLC | *EGFR* tyrosine kinase inhibitors approved by FDA | | P190032/S005 | May 3, 2023 | *EGFR* Exon 20 insertions | NSCLC | EXKIVITY® (mobocertinib) | | P190032/S010 | June 8, 2023 | *BRAF* V600E alteration | CRC | BRAFTOVI® (encorafenib) in combination with cetuximab | ### 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 BRAFTOVI (encorafenib) and MEKTOVI (binimetinib) which is available at Drugs@FDA. ### IX. SUMMARY OF NONCLINICAL STUDIES #### A. Laboratory Studies The evidence in support of the analytical performance of F1LCDx in detecting *BRAF* V600E is presented in this section. Analytical accuracy/concordance and precision near the limit of detection (LoD) studies were conducted to support the indication for *BRAF* V600E using clinical samples. For F1LCDx platform-level validation (P190032), due to the challenges with obtaining sufficient volume of clinical specimens, analytical performance characteristics were established for some of the studies using contrived samples, which consisted of enzymatically sheared cell line DNA spiked into human plasma from healthy donors, extracted according to the assay’s standard procedure, and diluted with cfDNA isolated from healthy donor plasma. A contrived sample functional characterization (CSFC) study was conducted to demonstrate comparable performance of sheared cell line DNA samples spiked into plasma as compared to cfDNA isolated from plasma specimens obtained from cancer positive intended use patient specimens. These contrived samples were used to establish the LoD for insertion variants evaluated in the platform-level LoD PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 16 of 34 {16} establishment study. For information regarding the platform-level validation, refer to Section IX.A. in P190032 Summary of Safety and Effectiveness Data. # 1. Analytical Accuracy/Concordance An analytical accuracy/concordance study was performed to demonstrate the concordance between F1LCDx and an externally validated NGS assay (evNGS) for the detection of *BRAF* V600E. For this study, a total of 318 biomarker-positive and biomarker-negative samples from residual banked cfDNA derived from clinical colorectal cancer (CRC, n=203) and NSCLC (n=115) samples, as well as clinical samples from the BEACON trial (CRC samples) were identified for testing. Of these samples, 14 CRC samples were excluded due to F1LCDx or evNGS quality control (QC) failures. Analytical concordance of F1LCDx for detecting *BRAF* V600E was determined with 304 samples tested by F1LCDx assay (Table 10). Since specimens were selected based on F1LCDx and confirmed by the evNGS assay, positive predictive value (PPV) and negative predictive value (NPV) are estimated conditional on F1LCDx. PPV was estimated as 100% (91/91) with two-sided 95% confidence interval (CI) (95.95%, 100%), and NPV as 99.53% (212/213) with two-sided 95% CI (97.39%, 99.92%), as shown in Table 10, below. For informational purposes, unadjusted positive percent agreement (PPA) and negative percent agreement (NPA) are also displayed. Analytical concordance of F1LCDx for detecting *BRAF* V600E in NSCLC is displayed in Table 11. **Table 10. Concordance summary for *BRAF* V600E by F1LCDx and the evNGS in CRC and NSCLC** | | evNGS | | | PPV/NPV (95% CI^{1}) | | | --- | --- | --- | --- | --- | --- | | | | *BRAF* V600E positive | *BRAF* V600E negative | | Total | | **F1LCDx** | ***BRAF* V600E positive** | 91 | 0 | 91 | PPV: 100% (95.95%, 100%) | | | ***BRAF* V600E negative** | 1 | 212 | 213 | NPV: 99.53% (97.39%, 99.92%) | | | **Total** | 92 | 212 | 304 | | | | **PPA/NPA (Unadjusted) (95% CI^{1})** | PPA: 98.91% (94.1%, 99.81%) | NPA: 100% (98.22%, 100%) | | | $^{1}$Calculated with Wilson 2-sided 95% CI In the one (1) discordant sample that was F1LCDx-negative/evNGS-positive, there were low supporting reads (4) and a percent variant allele frequency (VAF) of 0.5% for F1LCDx. This discordance may be due to the low percent VAF, which did not pass F1LCDx calling threshold for *BRAF* V600E (refer to Section IX.A.2.b. below) PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 17 of 34 {17} Table 11. Concordance summary for BRAF V600E by F1LCDx and the evNGS in NSCLC | | evNGS | | | | | | --- | --- | --- | --- | --- | --- | | | | BRAF V600E positive | BRAF V600E negative | Total | PPV/NPV (95% CI^{1}) | | F1LCDx | BRAF V600E positive | 10 | 0 | 10 | PPV: 100% (72.5%, 100%) | | | BRAF V600E negative | 0 | 105 | 105 | NPV: 100% (96.47%, 100%) | | | Total | 10 | 105 | 115 | | | | PPA/NPA (Unadjusted) (95% CI^{1}) | PPA: 100% (72.25%, 100%) | NPA: 100% (96.47%, 100%) | | | $^{1}$Calculated with Wilson 2-sided 95% CI The lowest VAF for BRAF V600E positive samples from patients with NSCLC used in the analytical accuracy study was 1.0%. However, the lowest VAF of the sample in the clinical trial (refer to section X below) evaluated in the clinical bridging study was 0.099%, with 14/48 (29%) BRAF V600E positive samples having a VAF < 0.5%. To demonstrate accuracy for BRAF V600E variants below 1%, FMI provided supplementary evidence in the form of positive concordance data from the F1LCDx orthogonal platform concordance study with BRAF V600E samples with VAFs between 0.11% and 0.43% by F1LCDx from patients with other cancers (n=5). Four out of 5 samples tested were concordant, i.e., determined to be positive by F1LCDx and the NGS comparator method. ## 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). A supplemental LoB study was performed for F1LCDx to support the updated LC input range (20-60 ng) by collecting whole blood samples from 44 healthy donors and preparing two plasma cfDNA replicates per donor for a total of 88 cfDNA sample replicates. Additionally, one matched gDNA replicate per donor was isolated from buffy coat and mechanically fragmented for F1LCDx testing to obtain non-tumor variant (e.g., germline) information to support the LoB analysis. One cfDNA replicate was excluded from the analysis due to failure at the DNA extraction step. All variants were determined to have an LoB of 0, based on the detection rate not significantly exceeding 0.5%. Further BRAF V600E was not observed in any of the replicates. ### b. Limit of Detection (LoD) The LoD for BRAF V600E was estimated panel-wide (for short variants) as 0.4% VAF as part of the LoD study for PMA P190032 (refer to Section IX.A.3.b in the Summary of Safety and Effectiveness Data for P190032). To confirm the LoD, FMI performed a LoD confirmation study using two (2) clinical specimens PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 18 of 34 {18} [one (1) from a patient with CRC and one (1) from patient with NSCLC] positive for *BRAF* V600E. These two samples were tested near the platform estimated LoD of 0.4% VAF for short variants to assess the VAF necessary for the accurate detection and sensitivity of *BRAF* V600E. The LoDs were confirmed using 24 replicates for CRC sample and 22 replicates for NSCLC sample. The LoD for *BRAF* V600E is confirmed to be 0.86% VAF in patients with NSCLC as the lowest VAF level tested in the confirmation of LoD study for which 100% hit rate was observed across the two samples, as summarized in Table 13, below. This LoD is similar to the LoD observed when testing patient samples from CRC patients, 0.70% VAF as shown in Table 12. **Table 12: Confirmation of LoD Results for Targeted *BRAF* V600E** | Disease Ontology | Target Alteration | # Agree | # Valid Total | Observed Average VAF (%) | Hit Rate (%) 95% CI | | --- | --- | --- | --- | --- | --- | | CRC | 1799T>A | 24 | 24 | 0.70 | 100 [86.2, 100] | | NSCLC | 1799T>A | 22 | 22 | 0.86 | 100 [85.13, 100] | ### 3. Precision and Reproducibility Within-Laboratory (Intermediate) Precision A precision study was conducted using two clinical samples harboring *BRAF* V600E, one (1) sample from a patient with metastatic CRC, and one (1) sample from a patient with NSCLC. Reproducibility including inter-run performance (run on different plates under different conditions) and repeatability including intra-run performance (run on the same plate under the same conditions) were assessed and compared in duplicates across two different sequencers and three different reagent lots, across multiple days of performance by multiple operators. The results for the precision study for the clinical samples near LoD are summarized in Table 13 and Table 14 below. **Table 13. Reproducibility results for *BRAF* V600E variant** | Disease Ontology | Targeted Alteration | # Agree | # Valid Total | Reproducibility (%) [95% CI] | Observed Average VAF (%) | Fold LoD^{1} | | --- | --- | --- | --- | --- | --- | --- | | CRC | 1799T>A | 24 | 24 | 100 [86.2, 100] | 0.7 | 1.76x | | NSCLC | 1799T>A | 22 | 22 | 100 [85.1, 100] | 0.86 | 2.17x | $^{1}$Fold LoD value = observed average %VAF / 1x LoD (%VAF) **Table 14. Repeatability results for *BRAF* V600E variant** | Disease Ontology | Targeted Alteration | # Agree | # Valid Total | Reproducibility (%) [95% CI] | | --- | --- | --- | --- | --- | | CRC | 1799T>A | 12 | 12 | 100 [75.75, 100] | | NSCLC | 1799T>A | 10 | 10 | 100 [72.25, 100] | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 19 of 34 {19} Inter-run reproducibility was evaluated across 24 CRC and 22 NSCLC sample replicates and intra-run repeatability was evaluated across 12 (CRC) and 10 (NSCLC) duplicates per plate. No discordances were observed. Overall, both reproducibility and repeatability were 100%. The corresponding two- sided Wilson score 95% CIs are provided for the reproducibility and repeatability. # **B. Animal Studies** No animal studies were conducted using the F1LCDx assay. # **X. SUMMARY OF PRIMARY CLINICAL STUDY** The reasonable assurance of safety and effectiveness for F1LCDx for detection of BRAF V600E in patients with NSCLC who may benefit from treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib) was established through retrospective analysis of banked plasma samples from patients enrolled in the clinical trial, the PHAROS (ARRAY-818-202) trial, as well as BRAF V600E-negative tissue-matched plasma samples from commercial sources and plasma samples from the FMI archives. Data from this clinical study were the basis for the PMA supplement approval decision. A summary of the clinical study is presented below. # **A. Study Design** # 1. PHAROS Study Design The PHAROS trial is an open-label, multicenter, single-arm study in patients with BRAF V600E-positive metastatic NSCLC. Eligible patients were either treatment-naïve or had received treatment with chemotherapy and/or immunotherapy (previously treated). Prior use of BRAF inhibitors or MEK inhibitors was not permitted. The major efficacy outcome measure was confirmed objective response rate (ORR) by an independent review committee (IRC) per Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria to determine whether treatment with BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) is effective. A key secondary efficacy outcome was duration of response (DoR) as assessed by an IRC. The PHAROS clinical study population comprises BRAF V600E-positive subjects from treatment naïve and previously treated patient Cohorts. Ninety-eight (98) subjects were enrolled based on the presence of BRAF V600E by local tissue testing or clinical trial assays (CTA). # 2. Clinical Bridging Study Design A clinical bridging study was conducted to evaluate: 1) the concordance between the F1LCDx assay and the CTAs for the detection of BRAF V600E, and 2) the clinical validity of F1LCDx in identifying patients with NSCLC with BRAF PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 20 of 34 {20} V600E who may be eligible for treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib). F1LCDx testing was performed on patients with available plasma samples from the PHAROS trial that tested positive for BRAF V600E by local tissue testing or clinical trial assays (CTA). The PHAROS clinical study did not include patients negative for BRAF V600E and therefore did not represent the F1LCDx (+), tissue-based CTA (-) [F1LCDx+ CTA-] subgroup of the F1LCDx intended use population. As such, supplemental matched tissue and plasma samples were obtained from subjects available through commercial sources and only plasma samples from the FMI archives, and a sensitivity analysis was performed to evaluate the potential impact of the F1LCDx(+)/CTA (-) population on the efficacy in the F1LCDx intended use population. ### 3. Clinical Inclusion and Exclusion Criteria The sample inclusion and exclusion criteria for the retrospective testing of the clinical bridging study were: Sample inclusion criteria: - Specimens in frozen plasma. - Samples meet minimum criteria for F1LCDx operational testing requirements - For F1LCDx: Samples must have cfDNA input, as assessed by the TapeStation assay, ≥ 20 ng, for primary analysis Sample exclusion criteria: - Tissue, other liquid samples are excluded - Samples that do not meet minimum F1LCDx operational testing requirements - For F1LCDx: Samples with <20 ng DNA as assessed by the TapeStation assay ### 4. Follow-up Schedule The F1LCDx clinical bridging study involved only retrospective testing of plasma samples; as such, no additional patient follow-up was conducted. ### 5. Clinical Endpoints The primary endpoint of the PHAROS trial is the ORR defined as the proportion of patients who have achieved a confirmed best ORR (CR or PR) as determined by IRC per RECIST v1.1 in both the treatment naïve and previously treated settings. ### B. Accountability of PMA Cohort The F1LCDx clinical bridging study included a total of 98 patient samples from PHAROS trial, which consists of 59 treatment-naïve and 39 previously treated patient samples. Of these, 48 subjects (49%) tested BRAF V600E positive by F1LCDx and were included in the primary objective analysis set, while 33 (34%) tested negative, and 17 (17%) subjects enrolled in the PHAROS trial were not evaluable due to extraction failures and lack of available plasma (Figure 1). PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 21 of 34 {21} For biomarker-negative (negative for BRAF V600E) samples, matched tissue and plasma samples were procured from commercial sources and plasma only samples from the FMI archives. The 120 negative samples consisted of 70 procured commercial tissue samples with matched plasma and 50 negative plasma samples from FMI archives. The samples procured from commercial vendors were tested using PCR and NGS based tests, which were representative of the CTAs used to enroll subjects into the PHAROS clinical study. Three procured samples did not have enough material for the external testing and were unevaluable for CTA. As a result, these 3 samples were not included in the analysis. Therefore, a total of 117 (120 less 3) CTA (-) samples were included in the analysis. Among the 70 procured plasma samples, 18 samples were unevaluable by F1LCDx (14 extraction failures and 4 QC failures). Figure 1: Sample Processing and Accountability Flow Chart  ### C. Study Population Demographics and Baseline Parameters The key demographic and clinical characteristics for F1LCDx-evaluable patients and F1LCDx-unevaluable patients are summarized in Table 15 (treatment naïve) and Table 16 (previously treated), below. An analysis comparing the two groups was performed as part of the sensitivity analysis in the clinical bridging study to identify those characteristics that were imbalanced. PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 22 of 34 {22} **Table 15. Demographics and Clinical Characteristics in the F1LCDx Evaluable-and F1LCDx-Unevaluable Subsets (treatment naïve Cohort)** | Covariate | F1LCDx Evaluable/CTA+ | F1LCDx Unevaluable/CTA+ | Difference in mean/proportion (95% CI) | p-value comparing the two subsets* | | --- | --- | --- | --- | --- | | Age | | | | 0.94 | | Mean | 66.62 | 66.00 | 0.62 (-8, 7) | | | Minimum | 51.00 | 47.00 | | | | Q1 | 60.25 | 60.00 | | | | Median | 68.00 | 70.00 | | | | Q3 | 73.00 | 72.00 | | | | Maximum | 83.00 | 82.00 | | | | SD | 8.15 | 10.82 | | | | Sex | | | | 0.49 | | Male | 21 (42%) | 5 (55.46%) | -13.56% (-42.07%, 18.45%) | | | Female | 29 (58%) | 4 (44.44%) | 13.56% (-18.45%, 42.07%) | | | ECOG status | | | | 1.00 | | 0 | 16 (32%) | 3 (33.33%) | -1.33% [-34.54%, 24.03%] | | | 1 | 34 (68%) | 6 (66.67%) | 1.33% [-24.03%, 34.54%] | | | Race | | | | 1.00 | | Asian | 3 (6%) | 0 (0%) | 6% [-24.17%, 16.22%] | | | Black/African American | 1 (2%) | 0 (0%) | 2% [-27.96%, 10.5%] | | | White | 44 (88%) | 9 (100%) | -12% [-23.8%, 18.59%] | | | Unknown | 1 (2%) | 0 (0%) | 2% [-27.96%, 10.5%] | | | American Indian or Alaska Native | 1 (2%) | 0 (0%) | 2% [-27.96%, 10.5%] | | | Ethnicity | | | | 1.00 | | Not Hispanic or Latino | 49 (98%) | 9 (100%) | -2% [-10.5%, 27.96%] | | | Unknown | 1 (2%) | 0 (0%) | 2% [-27.96%, 10.5%] | | | Height BL^{#} | | | | 0.96 | | Mean | 167.19 | 166.60 | 0.59 [-8.1, 10] | | | Min | 143.00 | 149.90 | | | | Q1 | 159.25 | 160.75 | | | | Median | 165.10 | 166.50 | | | | Q3 | 175.23 | 172.73 | | | | Max | 210.50 | 182.00 | | | | SD | 11.90 | 11.44 | | | | N/A | 0 | 1 | | | | Weight BL^{#} | | | | 0.77 | | Mean | 74.01 | 78.12 | -4.11 [-17.65, 17.5] | | | Minimum | 39.80 | 50.10 | | | | Q1 | 55.67 | 60.70 | | | | Median | 71.34 | 70.00 | | | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 23 of 34 {23} | Covariate | F1LCDx Evaluable/CTA+ | F1LCDx Unevaluable/CTA+ | Difference in mean/proportion (95% CI) | p-value comparing the two subsets* | | --- | --- | --- | --- | --- | | Q3 | 94.00 | 91.00 | | | | Maximum | 125.20 | 143.70 | | | | SD | 22.14 | 29.33 | | | | TBSTAT^{a} | | | | 0.10 | | Current smoker | 5 (10%) | 3 (33.33%) | -23.33% [-55.09%, 0.78%] | | | Former smoker | 28 (56%) | 5 (55.56%) | 0.44% [-28.56%, 32.06%] | | | Never smoker | 17 (34%) | 1 (11.11%) | 22.89% [-11.5%, 39.47%] | | | Tissue Handling | | | | 0.84 | | Core Needle/Excisional Biopsy | 24 (48%) | 6 (66.67%) | -18.67% [-43.71%, 15.37%] | | | Fine Needle Aspiration (FNA) | 12 (24%) | 1 (11.11%) | 12.89% [-20.92%, 29.11%] | | | Other | 3 (6%) | 0 (0%) | 6% [-24.17%, 16.22%] | | | Resection | 9 (18%) | 2 (22.22%) | -4.22% [-37.77%, 16.19%] | | | N/A | 2 (4%) | 0 (0%) | 4% [-26.05%, 13.46%] | | | Sampling Site | | | | 0.66 | | Metastatic | 18 (36%) | 2 (22.22%) | 13.78% [-20.84%, 34.87%] | | | Primary | 19 (38%) | 5 (55.56%) | -17.56% [-45.86%, 14.48%] | | | Plasma | 13 (26%) | 2 (22.22%) | 3.78% [-30.28%, 24.67%] | | *p-value was from nonparametric Mann-Whitney Test for continuous measures, and Fisher-Freeman-Halton Test for categorical measures between the CDx-evaluable and CDx-unevaluable sets Q1: first quartile, Q3: third quartile, SD: standard deviation, ECOG: Eastern Cooperative Oncology Group$^{a}$ Height BL: Height baseline, Weight BL: Weight baseline, TBSTAT: Tobacco use status Table 16. Demographics and Clinical Characteristics in the F1LCDx Evaluable-and F1LCDx-Unevaluable Subsets (previously treated Cohort) | Covariate | F1LCDx Evaluable/CTA+ | F1LCDx Unevaluable/CTA+ | Difference in mean/proportion (95% CI) | p-value comparing the two subsets* | | --- | --- | --- | --- | --- | | Age | | | | 0.41 | | Mean | 69.00 | 72.38 | -3.38 [-12, 4] | | | Minimum | 53.00 | 55.00 | | | | Q1 | 62.00 | 69.00 | | | | Median | 71.00 | 72.00 | | | | Q3 | 76.00 | 80.75 | | | | Maximum | 86.00 | 83.00 | | | | SD | 8.99 | 9.83 | | | | Sex | | | | 0.04 | | Male | 13 (41.94%) | 7 (87.5%) | -45.56% [-64.17%, -6.89%] | | | Female | 18 (58.06%) | 1 (12.5%) | 45.56% [6.89%, 64.17%] | | | ECOG status | | | | 0.62 | | 0 | 5 (16.13%) | 2 (25%) | -8.87% [-44.12%, 15.44%] | | | 1 | 26 (83.87%) | 6 (75%) | 8.87% [-15.44%, 44.12%] | | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 24 of 34 {24} | Covariate | F1LCDx Evaluable/CTA+ | F1LCDx Unevaluable/CTA+ | Difference in mean/proportion (95% CI) | p-value comparing the two subsets* | | --- | --- | --- | --- | --- | | Race | | | | 0.72 | | Asian | 4 (12.9%) | 0 (0%) | 12.90% [-20.45%, 28.85%] | | | Black/African American | 2 (6.45%) | 0 (0%) | 6.45% [-26.32%, 20.72%] | | | White | 25 (80.65%) | 8 (100%) | -19.35% [-36.28%, 14.64%] | | | Unknown | 0 (0%) | 0 (0%) | 0% NA | | | American Indian or Alaska Native | 0 (0%) | 0 (0%) | 0% NA | | | Ethnicity | | | | NA* | | Not Hispanic or Latino | 31 (100%) | 8 (100%) | 0% [-11.03%, 32.44%] | | | Unknown | 0 (0%) | 0 (0%) | 0% NA | | | Height BL^{#} | | | | 0.06 | | Mean | 165.75 | 173.34 | -7.58 [-15, 0.3] | | | Min | 151.10 | 162.00 | | | | Q1 | 158.50 | 165.68 | | | | Median | 165.10 | 169.70 | | | | Q3 | 170.15 | 178.25 | | | | Max | 186.00 | 193.00 | | | | SD | 8.99 | 11.49 | | | | N/A | 1 | 0 | | | | Weight BL^{#} | | | | 0.28 | | Mean | 70.44 | 78.73 | -8.29 [-21.55, 6.45] | | | Minimum | 51.00 | 48.31 | | | | Q1 | 59.76 | 70.55 | | | | Median | 72.30 | 77.20 | | | | Q3 | 76.67 | 84.36 | | | | Maximum | 92.10 | 115.76 | | | | SD | 12.04 | 21.18 | | | | TBSTAT^{#} | | | | 0.02 | | Current smoker | 2 (6.45%) | 3 (37.50%) | -31.05% [-63.31%, -3.29%] | | | Former smoker | 18 (58.06%) | 5 (62.50%) | -4.44% [-33.87%, 31.06%] | | | Never smoker | 11 (35.48%) | 0 (0%) | 35.48% [0%, 53.05%] | | | Tissue Handling | | | | 1.00 | | Core Needle/Excisional Biopsy | 21 (67.74%) | 6 (75%) | -7.26% [-32.33%, 29.46%] | | | Fine Needle Aspiration (FNA) | 5 (16.13%) | 1 (12.5%) | 3.63% [-32.12%, 23.06%] | | | Other | 3 (9.68%) | 1 (12.5%) | -2.82% [-37.99%, 15.53%] | | | Resection | 2 (6.45%) | 0 (0%) | 6.45% [-26.32%, 20.72%] | | | Sampling Site | | | | 0.72 | | Metastatic | 12 (38.71%) | 2 (25%) | 13.71% [-23.51%, 38.68%] | | | Primary | 14 (45.16%) | 2 (25%) | 20.16% [-17.48%, 44.86%] | | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 25 of 34 {25} | Covariate | F1LCDx Evaluable/CTA+ | F1LCDx Unevaluable/CTA+ | Difference in mean/proportion (95% CI) | p-value comparing the two subsets* | | --- | --- | --- | --- | --- | | Plasma | 5 (16.13%) | 2 (25%) | -8.87% [-44.12%, 15.44%] | | | N/A | 0 (0%) | 2 (25%) | -25% [-59.07%, -4.02%] | | *p-value was from nonparametric Mann-Whitney Test for continuous measures, and Fisher-Freeman-Halton Test for categorical measures between the CDx-evaluable and CDx-unevaluable sets Q1: first quartile, Q3: third quartile, SD: standard deviation, ECOG: Eastern Cooperative Oncology Group® Height BL: Height baseline, Weight BL: Weight baseline, TBSTAT: Tobacco use status ### D. Safety and Effectiveness Results #### 1. Safety Results The safety with respect to treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib) was addressed during the review of the supplemental New Drug Application (sNDA) and is not addressed in detail in this SSED. The evaluation of safety was based on the analysis of adverse events (AEs), clinical laboratory evaluations, physical examinations, and vital signs. No adverse events were reported in the conduct of the diagnostic studies used to support this sPMA as these involved retrospective testing of banked plasma specimens only. Please refer to Drugs@FDA for complete safety information on BRAFTOVI (encorafenib) and MEKTOVI® (binimetinib). #### 2. Effectiveness Results a. Concordance Between F1LCDx and enrolling CTAs Concordance between F1LCDx and enrolling CTA testing using matched plasma and tissue samples from PHAROS clinical study, is shown in Table 17 below. While all samples from the PHAROS clinical study population were positive for BRAF V600E as a requirement for enrollment in the clinical study, biomarker negative subject samples were identified and commercially procured or from FMI archives to represent the BRAF V600E-negative population, and their plasma samples were tested by F1LCDx. The PPA was 59.26% (48/81) with two-sided 95% confidence interval (48.38%, 69.3%) and NPA was 100% (99/99) with two-sided 95% confidence interval (96.26%, 100%) after excluding F1LCDx-unevaluable results when considering both patients that were treatment naïve. Since patients were enrolled and initially tested by local CTAs, the positive and negative predictive values (PPV and NPV) were calculated using the PPA and NPA, after adjusting for the prevalence of BRAF V600E among the intention-to-treat (ITT) population. The prevalence estimates used in the adjusted agreement were 0.5%, 1%, 2%, and 8%. In this analysis with 1% prevalence, F1LCDx demonstrated an adjusted PPV of 100% with two-sided 95% confidence interval (92.59%, 100%) and NPV of 99.59% with two-sided 95% confidence interval (99.48%, 99.69%). PPAs of 62% (31/50) and 55% (17/31) were observed for patients that were from treatment naïve and previously treated patient Cohorts, respectively, indicating PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 26 of 34 {26} comparable detection of the *BRAF* V600E in plasma by F1LCDx among the two patient populations. **Table 17. Concordance Between F1LCDx and CTA Testing** | | CTA Testing | | | | --- | --- | --- | --- | | | *BRAF* V600E + | *BRAF* V600E - | Total | | **F1LCDx** | | | | | *BRAF* V600E + | 48 | 0 | 48 | | *BRAF* V600E - | 33 | 99 | 132 | | Unevaluable | 17 | 21 | 38 | | Total | 98 | 120 | 218 | | PPA (95% CI*) | 59.26% (48.38% - 69.3%) | | | | NPA (95% CI*) | 100.0% (96.26% - 100.0%) | | | \*Two-sided 95% confidence intervals for PPA/NPA were calculated with Wilson score method. The discordance between the CTA and F1LCDx among *BRAF* V600E positive patients in the primary analysis were evaluated. Of the 33 CTA-positive/F1LCDx-negative samples, 27 had no *BRAF* V600E detected by the F1LCDx pipeline. Six (6) samples had *BRAF* V600E detected by F1LCDx but were filtered out for failing the pipeline's quality threshold. All the 33 CTA-positive/F1LCDx-negative samples were from patients in the NDA population of the PHAROS trial and had clinical outcome data (refer to Section X.D.2.b, below). Based on the low PPA between F1LCDx and the enrolling CTAs, which were predominately tissue-based tests, as shown in Table 16 above, F1LCDx may miss a large proportion of patients with NSCLC with *BRAF* V600E who may derive benefit from BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib). Therefore, reflex testing using tissue specimens to an FDA approved tissue test will be required, if feasible, if the plasma test is negative. # b. ORR in Patients Positive by F1LCDx for *BRAF* V600E The efficacy of encorafenib in combination with binimetinib was evaluated in the PHAROS clinical study in those subjects positive for *BRAF* V600E by F1LCDx for the treatment naïve and previously treated cohorts (Table 18 and Table 19). For the treatment naïve cohort, the efficacy results for the F1LCDx+/CTA+ population (74.19%) is comparable to that for the CTA+ patients (74.58%), indicating the clinical utility of F1LCDx in identifying *BRAF* V600E positive NSCLC patients. For the previously treated cohort, the efficacy results for the F1LCDx+/CTA+ population (35.29%) is lower than the CTA+ efficacy (46.15%), which may be attributable to the small sample size (n=17, F1LCDx+/CTA+). The median duration of response (DoR) for F1LCDx PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 27 of 34 {27} clinical efficacy population for treatment naïve cohort was 23.1 months and for previously treated cohort was 16.72 months. **Table 18. Primary Efficacy in the Bridging Study Subpopulations (Treatment Naïve Cohort)** | | CTA+ (n=59) | CTA+/F1LCDx+ (n=31) | CTA+/F1LCDx- (n=19) | CTA+/F1LCDx unevaluable (n=9) | | --- | --- | --- | --- | --- | | **ORR (95% CI)^{#}** | 74.6% (61.6, 85.0) | 74.2% (55.4, 88.1) | 68.4% (43.4, 87.4) | 88.9% (51.8, 99.7) | | Complete response (CR) | 9 (15%) | 5 (16%) | 3 (16%) | 1 (11%) | | Partial response (PR) | 35 (59%) | 18 (58%) | 10 (53%) | 7 (78%) | | **Duration of Response (DoR)** | | | | | | Median DoR, months (95% CI) | NE* (23.1, NE^{1}) | 23.1 (12.0, NE^{1}) | NE* (NE^{1}, NE^{1}) | 16.13 (NE^{2}) | | % with DoR ≥6 months | 75 | 65.2 | 92.3 | 75 | | % with DoR ≥12 months | 59.1 | 47.8 | 84.6 | 50 | NE*Median DOR is not estimable since the response rate did not fall below 50% in the Kaplan-Meier estimate. NE$^{1}$ Not estimable in the upper 95% CI of median DOR due to lower (upper) 95% CI of the response rate did not fall below 50%. NE$^{2}$ CI was not calculated since the sample size is less than 10. $^{#}$CI calculated based on exact method to align with the drug label. **Table 19. Primary Efficacy in the Bridging Study Subpopulations (Previously treated Cohort)** | | CTA+ (n=39) | CTA+/F1LCDx+ (n=17) | CTA+/F1LCDx- (n=14) | CTA+/F1LCDx unevaluable (n=8) | | --- | --- | --- | --- | --- | | **ORR (95% CI)^{#}** | 46.2% (30.1, 62.8) | 35.3% (14.2, 61.7) | 42.9% (17.7, 71.1) | 75% (34.9, 96.8) | | Complete response (CR) | 4 (10%) | 3 (18%) | 0 (0%) | 1 (12%) | | Partial response (PR) | 14 (36%) | 3 (18%) | 6 (43%) | 5 (62%) | | **Duration of Response (DOR)** | | | | | PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 28 of 34 {28} | | CTA+ (n=39) | CTA+/F1LCDx+ (n=17) | CTA+/F1LCDx- (n=14) | CTA+/F1LCDx unevaluable (n=8) | | --- | --- | --- | --- | --- | | Median DoR, months (95% CI) | 16.72 (11.93, NE^{1}) | 16.72 (NE^{2}) | 11.93 (NE^{2}) | NE* (NE^{2}) | | % with DoR ≥6 months | 66.7 | 83.3 | 66.7 | 50.0 | | % with DoR ≥12 months | 33.3 | 33.3 | 33.3 | 33.3 | NE*Median DOR is not estimable since the response rate did not fall below 50% in the Kaplan-Meier estimate. NE$^{1}$ Not estimable in the upper 95% CI of median DOR due to lower (upper) 95% CI of the response rate did not fall below 50%. NE$^{2}$ CI was not calculated since the sample size is less than 10. $^{1}$CI calculated based on exact method to align with the drug label. Samples that were positive by F1LCDx for BRAF V600E alteration in the clinical bridging study had VAFs as low as 0.0099%. Since F1LCDx does not have a pre-specified VAF cut-off for positivity, but rather uses an algorithm that is based on select metrics for each variant position, it is possible that patients with VAFs below the limit of detection (approximately 0.4 to 0.9 %) and below that which was enrolled in the trial (e.g., 0.0099%) are reported as positive. The clinical effectiveness of F1LCDx has not been demonstrated for patients with NSCLC who have BRAF V600E with VAF below 0.0099%. A limitation addressing the uncertainty of the clinical effectiveness of the device for BRAF V600E with VAFs below those evaluated in the clinical study is included as a limitation to the device (also refer to Section XII. C. below). ### c. Sensitivity Analysis Sensitivity analyses with regard to missing values were conducted to evaluate the robustness of the ORR estimates considering F1LCDx unevaluable patients enrolled in the PHAROS trial. Samples were considered missing if the samples were not tested, if they were tested but returned an invalid result, or if they did not satisfy the cfDNA minimum input requirement (i.e., ≥ 20ng). Amongst all CTA-positive patients, 17.3% did not have a F1LCDx result (17/98). To evaluate the impact of the F1LCDx unevaluable population, the distribution of patients for baseline covariates and disease characteristics was compared among the CTA-positive population, the F1LCDx-evaluable/CTA-positive subpopulation, and F1LCDx-unevaluable/CTA-positive subpopulation. A PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 29 of 34 {29} multiple imputation method was utilized to account for patients with missing or non-evaluable F1LCDx results (n=17). The imputed ORR by IRC was estimated to be 75.73% (95% CI: 61.73%, 89.74%) for treatment naïve cohort, which is comparable to the ORR for the CTA-positive population for treatment naïve cohort [74.58% (62.20, 83.94)]. Similarly, the imputed ORR for previously treated cohort [43.28% (22.31%, 64.26%)] was comparable to the ORR for the CTA-positive population for previously treated cohort [46.15% (31.57, 61.42)]. Thus, the sensitivity analysis demonstrated the robustness of the clinical efficacy estimate to the missing F1LCDx results. 3. Pediatric Extrapolation In this premarket application, existing clinical data was not leveraged to support approval of a pediatric patient population. E. Financial Disclosure The Financial Disclosure by Clinical Investigators regulation (21 CFR 54) requires applicants who submit a marketing application to include certain information concerning the compensation to, and financial interests and arrangement of, any clinical investigator conducting clinical studies covered by the regulation. The pivotal clinical study included one (1) investigator which was a full-time employee of the sponsor and had disclosable financial interests/arrangements as defined in 21 CFR 54.2(a), (b), (c) and (f) and described below: - Compensation to the investigator for conducting the study where the value could be influenced by the outcome of the study: [0] - Significant payment of other sorts: [0] - Proprietary interest in the product tested held by the investigator: [1] - Significant equity interest held by investigator in sponsor of covered study: [0] The applicant has adequately disclosed the financial interest/arrangements with clinical investigators. Statistical analyses were conducted by FDA to determine whether the financial interests/arrangements had any impact on the clinical study outcome. The information provided does not raise any questions about the reliability of the data. XI. PANEL MEETING RECOMMENDATION AND FDA'S POST-PANEL ACTION In accordance with the provisions of section 515(c)(3) of the act as amended by the Safe Medical Devices Act of 1990, this PMA was not referred to the Molecular and Clinical Genetics Panel, an FDA advisory committee, for review and recommendation because the information in the PMA substantially duplicates information previously reviewed by this panel. PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 30 of 34 {30} # XII. CONCLUSIONS DRAWN FROM PRECLINICAL AND CLINICAL STUDIES ## A. Effectiveness Conclusions The effectiveness of the F1LCDx assay to identify a *BRAF* V600E in NSCLC patients to be treated with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib), was demonstrated through a clinical bridging study using specimens from patients enrolled into the PHAROS trial. The data from the analytical validation and clinical bridging studies support the reasonable assurance of safety and effectiveness of the F1LCDx assay when used in accordance with the indications for use. Data from the PHAROS trial show that patients who had a qualifying *BRAF* V600E received benefit from treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib) and support the addition of the CDx indication to F1LCDx. ## B. Safety Conclusions The risks of the device are based on data collected in the validation studies conducted to support PMA approval, as described above. The F1LCDx assay is an *in vitro* diagnostic test, which involves testing of cfDNA extracted from blood or plasma. 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 in cancer treatment. Patients with false positive results may undergo treatment with one of the therapies listed in Table 1 of 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 therapy. There is also a risk of delayed results, which may lead to delay of treatment with indicated therapy. ## C. Benefit-Risk Determination The probable benefit of the F1LCDx assay in identifying patients with NSCLC with *BRAF* V600E for treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib) was demonstrated through clinical bridging studies using specimens from patients enrolled into the PHAROS trial. For patients with NSCLC with *BRAF* V600E positive status, for the treatment naïve cohort, the ORR for the F1LCDx+/CTA+ population (74.58%) was comparable to ORR for the CTA+ patients (74.19%), indicating a meaningful clinical benefit of F1LCDx in identifying *BRAF* V600E positive NSCLC patients, for treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib). For the previously treated cohort, the ORR for the F1LCDx+/CTA+ populations (35.29%) was lower than the CTA+ ORR (46.15%), which could be attributed to the small sample size; but nevertheless, provides evidence of a meaningful clinical benefit of this device, for the indicated use. Of note, in the concordance analysis, the NPA and PPV were 100%. The observed ORRs for the F1LCDx *BRAF* V600E-positive patients in the treatment naïve and previously treated cohorts, supports probable benefit of F1LCDx in selecting *BRAF* V600E positive patients with NSCLC for treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib), in these patient populations. ---PMA P190032.S011: FDA Summary of Safety and Effectiveness Data 31 of 34 {31} There is potential risk associated with the use of this device, mainly due to 1) false positive, false negatives, or failure to provide a result, and 2) incorrect interpretation of test results by the user. The risks of the F1LCDx assay are associated with the potential mismanagement of patients resulting from false results of the test. Patients who are falsely determined to be positive by the test may be exposed to a drug that is not beneficial which may lead to adverse events or may have delayed access to treatments that could be more beneficial. A false negative result may prevent a patient from accessing a potentially beneficial drug. The risks of false results are partially mitigated by the analytical and clinical performance of the device, as summarized above, including the analytical accuracy, and clinical concordance and bridging efficacy studies. Of note, in the analytical accuracy study performed (n=304), the PPA was 98.91% and the NPA was 100%. In addition, the risks of false negative results are partially mitigated by a recommendation that those patients whose plasma generates a negative result for those alterations included in Table 1, including BRAF V600E, should have their tumor mutation status verified by using an FDA-approved tumor tissue test, if feasible. Additional factors to consider in determining probable risks and benefits for F1LCDx included: the availability of alternative tests. Of note, there are no FDA-approved CDx alternatives using cfDNA isolated from plasma for the detection of BRAF V600E to identify patients with NSCLC for treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib), and this device may meet an unmet clinical need; however, there is an FDA-approved tissue test available for the identification of patients with NSCLC with BRAF V600E for treatment with BRAFTOVI (encorafenib) in combination with MEKTOVI (binimetinib). The advantage of F1LCDx over the…