FoundationOne 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 CDx (F1CDx) | | Device Procode: | PQP | | Applicant's Name and Address: | Foundation Medicine, Inc. 150 Second Street Cambridge, MA 02141 | | Date(s) of Panel Recommendation: | None | | Premarket Approval Application (PMA) Number: | P170019/S054 | | Date of FDA Notice of Approval: | January 16, 2025 | The original PMA (P170019) for FoundationOne CDx (F1CDx) was approved on November 30, 2017 for the detection of genetic alterations in patients who may benefit from one of eighteen FDA-approved therapies for non-small cell lung cancer (NSCLC), melanoma, breast cancer, colorectal cancer (CRC), and ovarian cancer. Subsequently, additional PMA supplements were approved for expanding the indications for use of F1CDx since the original approval. See Section VII for more details. The current supplement was submitted to expand the indication for F1CDx to include a companion diagnostic (CDx) indication for the detection of *BRAF* V600 mutations and *BRAF* fusions in patients with pediatric low-grade glioma who may benefit from treatment with OJEMDA (tovorafenib). ## II. INDICATIONS FOR USE FoundationOne CDx (F1CDx) is a qualitative next generation sequencing based *in vitro* diagnostic test that uses targeted high throughput hybridization-based capture technology for detection of substitutions, insertion and deletion alterations (indels) and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. The test is intended as a companion diagnostic to identify ---PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 1 of 45 {1} patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Additionally, F1CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product. Table 1. Companion diagnostic indications | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Breast cancer | AKT1 E17K; PIK3CA R88Q, N345K, C420R, E542K, E545A, E545D, E545Q, E545K, E545G, Q546E, Q546K, Q546R, Q546P, M1043V, M1043I, H1047Y, H1047R, H1047L, and G1049R; and PTEN alterations | TRUQAP™ (capivasertib) in combination with FASLODEX® (fulvestrant) | | | ERBB2 (HER2) amplification | HERCEPTIN® (trastuzumab) | | | | KADCYLA® (ado-trastuzumab emtansine) | | | | PERJETA® (pertuzumab) | | | PIK3CA C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y alterations | PIQRAY® (alpelisib) | | Cholangiocarcinoma | FGFR2 fusions and select rearrangements | PEMAZYRE® (pemigatinib) | | Colorectal cancer | KRAS wild-type (absence of mutations in codons 12 and 13) | ERBITUX® (cetuximab) | | | KRAS wild-type (absence of mutations in exons 2, 3, and 4) and NRAS wild-type (absence of mutations in exons 2, 3, and 4) | VECTIBIX® (panitumumab) | | Melanoma | BRAF V600 mutation-positive | TECENTRIQ® (atezolizumab) in combination with COTELLIC® (cobimetinib) and ZELBORAF® (vemurafenib) | | | BRAF V600E | BRAF Inhibitor Approved by FDA* | | | BRAF V600E and V600K | BRAF/MEK Inhibitor Combinations Approved by FDA* | | | | MEKINIST® (trametinib) | | Non-small cell lung cancer (NSCLC) | ALK rearrangements | ALECENSA® (alectinib) | | | | ALUNBRIG® (brigatinib) | | | | XALKORI® (crizotinib) | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 2 of 45 {2} | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | | BRAF V600E | ZYKADIA® (ceritinib) | | | | BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) | | | | TAFINLAR® (dabrafenib) in combination with MEKINIST® (trametinib) | | | EGFR exon 19 deletions and EGFR exon 21 L858R alterations | EGFR Tyrosine Kinase Inhibitors (TKI) Approved by FDA* | | | EGFR exon 20 T790M alterations | TAGRISSO® (osimertinib) | | | MET single nucleotide variants (SNVs) and indels that lead to MET exon 14 skipping | TABRECTA® (capmatinib) | | | ROS1 fusions | ROZLYTREK® (entrectinib) | | Ovarian cancer | BRCA1, BRCA2 alterations | LYNPARZA® (olaparib) | | Pediatric low-grade glioma | BRAF V600 mutation-positive and BRAF fusions | OJEMDA™ (tovorafenib) | | Prostate cancer | BRCA1, BRCA2 alterations | AKEEGA® (niraparib + abiraterone acetate) | | | | LYNPARZA® (olaparib) in combination with abiraterone | | | Homologous Recombination Repair (HRR) gene (BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D and RAD54L) alterations | LYNPARZA® (olaparib) | | Solid tumors | MSI-High | KEYTRUDA® (pembrolizumab) | | | NTRK1/2/3 fusions | ROZLYTREK® (entrectinib) | | | | VITRAKVI® (larotrectinib) | | | RET fusions | RETEVMO® (selpercatinib) | | | TMB ≥ 10 mutations per megabase | KEYTRUDA® (pembrolizumab) | *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-diagnosticdevices-in-vitro-and-imaging-tools The F1CDx assay is performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 3 of 45 {3} ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings/precautions and limitations are included in the FoundationOne®CDx assay labeling. ### V. DEVICE DESCRIPTION FoundationOne CDx (F1CDx) is performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. The assay includes reagents, software, instruments, and procedures for testing DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples. F1CDx uses DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples. The assay employs two extraction methods, (either DNAx or CoExtraction, an automated DNA/RNA co-extraction methodology) for DNA extraction from routine FFPE biopsy or surgical resection specimens; 50-1000 ng of DNA will undergo whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, one promoter region, one non-coding (ncRNA), and select intronic regions from 34 commonly rearranged genes, 21 of which also include the coding exons (refer to Table 2 and Table 3 for the complete list of genes included in F1CDx). In total, the assay detects alterations in a total of 324 genes. Using the Illumina® NovaSeq 6000, hybrid capture-selected libraries are sequenced to high uniform depth (targeting >500X median coverage with >99% of exons at coverage >100X). Sequence data is then processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number alterations (amplifications and homozygous gene deletions), and select genomic rearrangements (e.g., gene fusions). Rearrangements in one of the targeted genes included in Table 3 may be reported along with their uniquely identified genomic partners, which can be any gene in the genome even if not explicitly targeted by the assay. Additionally, genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) are reported. Table 2. Genes with full coding exonic regions included in F1CDx for the detection of substitutions, insertions and deletions (indels), and copy number alterations (CNAs) | ABL1 | BRAF | CDKN1A | EPHA3 | FGFR4 | IKZF1 | MCL1 | NKX2-1 | PMS2 | RNF43 | TET2 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ACVR1B | BRCA1 | CDKN1B | EPHB1 | FH | INPP4B | MDM2 | NOTCH1 | POLD1 | ROS1 | TGFBR2 | | AKT1 | BRCA2 | CDKN2A | EPHB4 | FLCN | IRF2 | MDM4 | NOTCH2 | POLE | RPTOR | TIPARP | | AKT2 | BRD4 | CDKN2B | ERBB2 | FLT1 | IRF4 | MED12 | NOTCH3 | PPARG | SDHA | TNFAIP3 | | AKT3 | BRIP1 | CDKN2C | ERBB3 | FLT3 | IRS2 | MEF2B | NPM1 | PPP2R1A | SDHB | TNFRSF14 | | ALK | BTG1 | CEBPA | ERBB4 | FOXL2 | JAK1 | MEN1 | NRAS | PPP2R2A | SDHC | TP53 | | ALOX12B | BTG2 | CHEK1 | ERCC4 | FUBP1 | JAK2 | MERTK | NT5C2 | PRDM1 | SDHD | TSC1 | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 4 of 45 {4} | AMER1 | BTK | CHEK2 | ERG | GABRA6 | JAK3 | MET | NTRK1 | PRKAR1A | SETD2 | TSC2 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | APC | C11orf30 | CIC | ERRFI1 | GATA3 | JUN | MITF | NTRK2 | PRKCI | SF3B1 | TYRO3 | | AR | CALR | CREBBP | ESR1 | GATA4 | KDM5A | MKNK1 | NTRK3 | PTCH1 | SGK1 | U2AF1 | | ARAF | CARD11 | CRKL | EZH2 | GATA6 | KDM5C | MLH1 | P2RY8 | PTEN | SMAD2 | VEGFA | | ARFRP1 | CASP8 | CSF1R | FAM46C | GID4 (C17orf39) | KDM6A | MPL | PALB2 | PTPN11 | SMAD4 | VHL | | ARID1A | CBFB | CSF3R | FANCA | GNA11 | KDR | MRE11A | PARK2 | PTPRO | SMARC A4 | WHSC1 | | ASXL1 | CBL | CTCF | FANCC | GNA13 | KEAP1 | MSH2 | PARP1 | QKI | SMARC B1 | WHSC1L1 | | ATM | CCND1 | CTNNA1 | FANCG | GNAQ | KEL | MSH3 | PARP2 | RAC1 | SMO | WT1 | | ATR | CCND2 | CTNNB1 | FANCL | GNAS | KIT | MSH6 | PARP3 | RAD21 | SNCAIP | XPO1 | | ATRX | CCND3 | CUL3 | FAS | GRM3 | KLHL6 | MST1R | PAX5 | RAD51 | SOCS1 | XRCC2 | | AURKA | CCNE1 | CUL4A | FBXW7 | GSK3B | KMT2A (MLL) | MTAP | PBRM1 | RAD51B | SOX2 | ZNF217 | | AURKB | CD22 | CXCR4 | FGF10 | H3F3A | KMT2D (MLL2) | MTOR | PDCD1 | RAD51C | SOX9 | ZNF703 | | AXIN1 | CD274 | CYP17A1 | FGF12 | HDAC1 | KRAS | MUTYH | PDCD1L G2 | RAD51D | SPEN | | | AXL | CD70 | DAXX | FGF14 | HGF | LTK | MYC | PDGFRA | RAD52 | SPOP | | | BAP1 | CD79A | DDR1 | FGF19 | HNF1A | LYN | MYCL | PDGFRB | RAD54L | SRC | | | BARD1 | CD79B | DDR2 | FGF23 | HRAS | MAF | MYCN | PDK1 | RAF1 | STAG2 | | | BCL2 | CDC73 | DIS3 | FGF3 | HSD3B1 | MAP2K1 | MYD88 | PIK3C2B | RARA | STAT3 | | | BCL2L1 | CDH1 | DNMT3A | FGF4 | ID3 | MAP2K2 | NBN | PIK3C2G | RB1 | STK11 | | | BCL2L2 | CDK12 | DOT1L | FGF6 | IDH1 | MAP2K4 | NF1 | PIK3CA | RBM10 | SUFU | | | BCL6 | CDK4 | EED | FGFR1 | IDH2 | MAP3K1 | NF2 | PIK3CB | REL | SYK | | | BCOR | CDK6 | EGFR | FGFR2 | IGF1R | MAP3K13 | NFE2L2 | PIK3R1 | RET | TBX3 | | | BCORL1 | CDK8 | EP300 | FGFR3 | IKBKE | MAPK1 | NFKBIA | PIM1 | RICTOR | TEK | | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 5 of 45 {5} **Table 3. Genes with select intronic regions for the detection of gene rearrangements, a promoter region, and an ncRNA gene** | *ALK* introns 18, 19 | *BRCA1* introns 2, 7, 8, 12, 16, 19, 20 | *ETV4* introns 5, 6 | *EZR* introns 9-11 | *KIT* intron 16 | *MYC* intron 1 | *NUTM1* intron 1 | *RET* introns 7-11 | *SLC34A2* intron 4 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | *BCL2* 3'UTR | *BRCA2* intron 2 | *ETV5* introns 6, 7 | *FGFR1* intron 1, 5, 17 | *KMT2A* (MLL) introns 6-11 | *NOTCH2* intron 26 | *PDGFRA* introns 7, 9, 11 | *ROS1* introns 31-35 | *TERC* ncRNA | | *BCR* introns 8, 13, 14 | *CD74* introns 6-8 | *ETV6** introns 5, 6 | *FGFR2* intron 1, 17 | *MSH2* intron 5 | *NTRK1* introns 8-10 | *RAF1* introns 4-8 | *RSPO2* intron 1 | *TERT* Promoter | | *BRAF* introns 7-10 | *EGFR* introns 7, 15, 24-27 | *EWSR1* introns 7-13 | *FGFR3* intron 17 | *MYB* intron 14 | *NTRK2* Intron 12 | *RARA* intron 2 | *SDC4* intron 2 | *TMPRSS2* introns 1-3 | *ETV6 is a common rearrangement partner for NTRK3 ### Test Output The output of the test includes: Category 1: CDx Claims noted in Table 1 of the Intended Use Category 2: Cancer Mutations with Evidence of Clinical Significance Category 3: Cancer Mutations with Potential Clinical Significance Genomic findings other than those listed in Table 1 of the intended use statement (i.e., Categories 2 and 3) are not prescriptive or conclusive for labeled use of any specific therapeutic product. ### Test Kit Contents The test includes a sample shipping kit, which is sent to ordering laboratories. The shipping kit contains the following components: - Specimen Preparation Instructions - Shipping Instructions - Return Shipping Label ### Instruments The F1CDx assay is intended to be performed with serial number-controlled instruments as indicated in Table 4, below. All instruments are qualified by Foundation Medicine, Inc. (FMI) under FMI's Quality System. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 6 of 45 {6} Table 4. Instruments for use with the F1CDx assay | Instrument | | --- | | Hamilton STAR/STARlet Liquid Handling Workstation | | Covaris LE220-plus Focused ultrasonicator | | Thermo Fisher Scientific KingFisher™ Flex with 96 Deep-well Head | | Illumina® NovaSeq 6000 System | ### Test Process All assay reagents included in the F1CDx assay process are qualified by FMI and are compliant with the medical device Quality System Regulation (QSR). ### A. Specimen Collection and Preparation Formalin-fixed, paraffin-embedded (FFPE) tumor specimens are collected and prepared following standard pathology practices. FFPE specimens may be received either as unstained slides or as an FFPE block. Prior to starting the assay, a Hematoxylin and Eosin (H&E) stained slide is prepared, and then reviewed by a board-certified pathologist to confirm disease ontology and to ensure that adequate tissue (0.6 mm³), tumor content (≥ 20% tumor) and sufficient nucleated cells are present to proceed with the assay. ### B. DNA Extraction #### DNAx Extraction Method Specimens passing pathology review are queued for DNA extraction which begins with lysis of cells from FFPE tissue by digestion with a proteinase K buffer followed by automated purification using the 96-well KingFisher™ FLEX Magnetic Particle Processor. After completion of DNA extraction, double-stranded DNA (dsDNA) is quantified by the Quant-iT™ PicoGreen® fluorescence assay using the provided lambda DNA standards (Invitrogen) prior to Library Construction (LC). The sample must yield a minimum of 55 ng of genomic DNA to ensure sufficient DNA for quality control (QC) and to proceed with LC. #### CoEx Extraction Method Specimens passing pathology review are queued for nucleic acid extraction which begins with placement of the FFPE samples into an AutoLys tube, where using a pre-programmed automated method, the AutoLys STAR adds RNA digestion and proteinase K solutions. The RNA containing lysate is removed for downstream RNA extraction using the KingFisher RNA extraction process. The AutoLys Tubes containing partially digested tissue then receive DNA Lysis solution on the AutoLys STAR and are placed into a Vortemp for digestion. The sample is then centrifuged to separate sample-associated paraffin from the lysate, and returned to the AutoLys STAR where the lysate is transferred to a KingFisher dKF PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 7 of 45 {7} plate. The dKF plate is loaded onto the Hamilton STAR for automated addition of DNA binding buffer and magnetic beads, and DNA isolation is performed using the KingFisher Flex. The DNA samples are then transferred to matrix tubes on DNAE plates using Hamilton STAR before proceeding to DNA quantification. After completion of DNA extraction, double-stranded DNA (dsDNA) is quantified by the Quant-iT™ PicoGreen® fluorescence assay using the provided lambda DNA standards (Invitrogen) prior to Library Construction (LC). The sample must yield a minimum of 55 ng of genomic DNA to ensure sufficient DNA for quality control (QC) and to proceed with LC. ### C. Library Construction Library Construction (LC) begins with the normalization of DNA to 50-1000 ng. The normalized DNA samples are randomly sheared (fragmented) to ~200 bp by adaptive focused acoustic sonication using the Covaris LE220 before purification with a 1.8X volume of AMPure® XP Beads (Agencourt®). Solid-phase reversible immobilization (SPRI) purification and subsequent library construction with the NEBNext® reagents (custom-filled kits by New England Biolabs), including mixes for end repair, dA addition and ligation, are performed in 96-well plates (Eppendorf) on the Bravo Benchbot (Agilent) or Hamilton Microlab STAR/STARlet Liquid Handling Workshop using the “with-bead” protocol to maximize reproducibility and library yield. Indexed (6 bp barcodes) sequencing libraries are polymerase chain reaction (PCR) amplified with HiFi™ (Kapa) for 10 cycles, and subsequently 1.8X SPRI purified. Purification and dilution for QC are performed. Following LC, a QC procedure is performed by quantifying single-stranded DNA (ssDNA) from purified libraries using the Quant-iT™ PicoGreen® ssDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Libraries yielding insufficient sequencing library are failed. ### D. Hybrid Capture Hybrid Capture (HC) begins with normalization of each library to 500-2000 ng. Normalized samples then undergo solution hybridization which is performed using a > 50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 bp oligonucleotides. The baits target ~1.8 Mb of the human genome including all coding exons of 309 cancer-related genes, introns or non-coding regions of 35 genes, plus > 3,500 single nucleotide polymorphisms (SNPs) located throughout the genome. Baits are designed by tiling overlapping 120 bp DNA sequence intervals covering target exons (60 bp overlap) and introns (20 bp overlap), with a minimum of three baits per target; SNP targets are allocated one bait each. Intronic baits are filtered for repetitive elements² as defined by the UCSC Genome RepeatMasker track. After hybridization, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen), and off-target material is removed by washing one time with 1X SSC at 25°C and four times with 0.25X SSC at 55°C. The PCR master mix is added to directly amplify (12 cycles) the captured library from the washed PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 8 of 45 {8} beads.³ After 12 cycles of amplification, the samples are 1.8X SPRI purified. Purification and dilution for QC are performed. QC for HC is performed by measuring dsDNA yield using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Captured libraries yielding less than 140 ng of sequencing library are failed. # E. Sequencing Sequencing is performed using on-board cluster generation using patterned flow cell technology to generate monoclonal clusters from a single DNA template followed by sequencing using sequencing by synthesis (SBS) chemistry on the Illumina NovaSeq 6000. Fluorescently labeled 3'-blocked deoxynucleotide triphosphates (dNTPs) together with a polymerase are incorporated through the flow cell to create a growing nucleotide chain that is excited by a laser. A camera captures the emission color of the incorporated base and then is cleaved off. The terminator is then removed to allow the nucleotide to revert to its natural form and to allow the polymerase to add another base to the growing chain. A new pool of fluorescently labeled 3'-blocked dNTPs are added with each new sequencing cycle. The color changes for each new cycle as a new base is added to the growing chain. This method allows for millions of discrete clusters of clonal copies of DNA to be sequenced in parallel. # F. Sequence Analysis Sequence data is analyzed using proprietary software developed by FMI. Sequence data are mapped to the human genome (hg19) using Burrows-Wheeler Aligner (BWA) v0.5.9.⁴ PCR duplicate read removal and sequence metric collection are performed using Picard 1.47 (http://picard.sourceforge.net) and SAMtools 0.1.12a.⁵ Local alignment optimization is performed using Genome Analysis Toolkit (GATK) 1.0.4705.⁶ Variant calling is performed only in genomic regions targeted by the test. Base substitution detection is performed using a Bayesian methodology, which allows for the detection of novel somatic alterations at low mutant allele frequency (MAF) and increased sensitivity for alterations at hotspot sites through the incorporation of tissue-specific prior expectations.⁷ Reads with low mapping (mapping quality < 25) or base calling quality (base calls with quality ≤ 2) are discarded. Final calls are made at MAF ≥ 5% (MAF ≥ 1% at hotspots). To detect indels, de novo local assembly in each targeted exon is performed using the de-Bruijn approach.⁸ Key steps are: - Collecting all read pairs for which at least one read maps to the target region. - Decomposing each read into constituent k-mers and constructing an enumerable graph representation (de-Bruijn) of all candidate non-reference haplotypes present. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 9 of 45 {9} - Evaluating the support of each alternate haplotype with respect to the raw read data to generate mutational candidates. All reads are compared to each of the candidate haplotypes via ungapped alignment, and a read 'vote' for each read is assigned to the candidate with best match. Ties between candidates are resolved by splitting the read vote, weighted by the number of reads already supporting each haplotype. This process is iterated until a 'winning' haplotype is selected. - Aligning candidates against the reference genome to report alteration calls. Filtering of indel candidates is carried out similarly to base substitutions, with an empirically increased allele frequency threshold at repeats and adjacent sequence quality metrics as implemented in GATK: % of neighboring bases mismatches < 25%, average neighboring base quality > 25, average number of supporting read mismatches ≤ 2. Final calls are made at MAF ≥ 5% (MAF ≥ 3% at hotspots). Copy number alterations (CNAs) are detected using a comparative genomic hybridization (CGH)-like method. First, a log-ratio profile of the sample is acquired by normalizing the sequence coverage obtained at all exons and genome-wide SNPs (~3,500) against a process-matched normal control. This profile is segmented and interpreted using allele frequencies of sequenced SNPs to estimate tumor purity and copy number at each segment. Amplifications are called at segments with ≥ 6 copies or ≥ 7 for triploid/≥ 8 for tetraploid tumors) and homozygous deletions at 0 copies, in samples with tumor purity ≥ 20%. Amplifications in ERBB2 are called positive at segments with ≥ 5 copies for diploid tumors. Genomic rearrangements are identified by analyzing chimeric read pairs. Chimeric read pairs are defined as read pairs for which reads map to separate chromosomes, or at a distance of over 10 megabase (Mb). Pairs are clustered by genomic coordinate of the pairs, and clusters containing at least five chimeric pairs (three for known fusions) are identified as rearrangement candidates. Filtering of candidates is performed by mapping quality (average read mapping quality in the cluster must be 30 or above) and distribution of alignment positions. Rearrangements are annotated for predicted function (e.g., creation of fusion gene). To determine microsatellite instability (MSI) status, F1CDx employs a fraction based (FB) MSI algorithm to categorize a tumor specimen as MSI-High (MSI-H) or microsatellite stable (MSS). The FB-MSI algorithm calculates the fraction of microsatellite loci determined to be altered or unstable (i.e., the fraction unstable loci score) based on a genome-wide analysis across >2000 microsatellite loci. For a given microsatellite locus, non-somatic alleles are discarded, and the microsatellite is categorized as unstable if remaining alleles differ from the reference genome. The final fraction unstable loci score is calculated as the number of unstable microsatellite loci divided by the number of evaluable microsatellite loci. Two FB-MSI score thresholds are applied to classify a tumor specimen as having MSI-H or MSS status. MSI-H status is reported for patients with solid tumors whose samples have FB-MSI scores ≥ 0.0124 while MSS status is reported for patients with solid tumors whose samples have FB-MSI scores ≤ 0.0041. Per the F1CDx assay, a patient whose tumor PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 10 of 45 {10} has an MSI-H score ≥ 0.0124 is reported as eligible for treatment with KEYTRUDA. For patients with solid tumors whose samples have FB-MSI scores >0.0041 and <0.0124, an MSI “Cannot be Determined” result is reported. Patients with this result should be re-tested with a validated orthogonal (alternative) method as these MSI scores represent a range of scores with low reliability. Patients with solid tumors may also receive an MSI status reported as MSI-Cannot Be Determined due to a quality control (QC) failure. Patients with this result should consider re-testing with FoundationOne CDx or an orthogonal (alternative) method, if clinically appropriate. Tumor mutational burden (TMB) is measured by counting all synonymous and non-synonymous substitution and indel variants present at 5% allele frequency or greater and filtering out potential germline variants according to published databases of known germline polymorphisms including Single Nucleotide Polymorphism database (dbSNP) and Exome Aggregation Consortium (ExAC). Additional germline alterations still present after database querying are assessed for potential germline status and filtered out using a somatic-germline/zygosity (SGZ) algorithm. Furthermore, known and likely driver mutations are filtered out to exclude bias of the data set. The resulting mutation number is then divided by the coding region corresponding to the number of total variants counted, or 793 kb. The resulting number is communicated as mutations per Mb unit (mut/Mb). After completion of the Analysis Pipeline, variant data are displayed in the FMI custom developed CATi software applications with sequence QC metrics. As part of data analysis QC for every sample, the F1CDx assay assesses cross-contamination through the use of a SNP profile algorithm, reducing the risk of false-positive calls that could occur as a result of an unexpected contamination event. Sequence data are reviewed by trained bioinformatics personnel. Samples failing any QC metrics are automatically held and not released. ### G. Report Generation Approved results are annotated by automated software with CDx relevant information and are merged with patient demographic information and any additional information provided by FMI as a professional service prior to approval and release by the laboratory director or designee. ### H. Internal Process Controls Related to the System #### Positive Control Each assay run includes a control sample run in duplicate. The control sample contains a pool of ten HapMap cell lines and is used as a positive mutation detection control. One hundred (100) different germline SNPs present across the entire targeted region are required to be detected by the analysis pipeline. If SNPs are not detected as expected, this results in a QC failure, as it indicates a potential processing error. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 11 of 45 {11} # Sensitivity Control The HapMap control pool used as the positive control is prepared to contain variants at 5%-10% MAF which must be detected by the analysis pipeline to ensure the expected sensitivity for each run. # Negative Control Samples are barcoded molecularly at the LC stage. Only reads with a perfect molecular barcode sequence are incorporated into the analysis. The Analysis Pipeline includes an algorithm that analyzes the SNP profile of each specimen to identify potential contamination that may have occurred prior to molecular barcoding and can detect contamination lower than 1%. # I. Variant Classification # Biomarker Rules for SNVs and indels that lead to MET exon 14 skipping An 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. # Homologous Recombination Repair (HRR) Genes A clinical report is provided to the ordering physician for each F1CDx test performed at Foundation Medicine, Inc. Each report is generated and reviewed by an internal team consisting of clinical bioinformatics analysts, scientists, curators, and pathologists for mutations positive for the therapies identified. Each sample is assessed for mutations in the 14 HRR genes, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, and RAD54L (Table 5). For these genes, both deleterious and suspected deleterious mutations in short variant, copy number alteration, and rearrangement variant classes are determined by an in-house software pipeline. Alterations listed in the COSMIC database and homozygous deletions are considered deleterious. Suspected deleterious mutations include truncating events (i.e., splice, frameshift, and nonsense alterations), as well as large rearrangements that disrupt the coding sequence. The COSMIC check is a second layer of check for HRR positive suspected deleterious alterations. All splice, nonsense, and frameshift alterations in HRR genes are considered biomarker positive and would be considered as suspected deleterious mutations (or "likely" status in FMI reporting rules). If these mutations are additionally reported in COSMIC, they would be listed as deleterious mutations (or "known" status in FMI reporting). PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 12 of 45 {12} The F1CDx assay is intended as an aid in selecting prostate cancer patients with deleterious or suspected deleterious HRR variants, identified by the rules below, and who may be eligible for treatment with Lynparza® (olaparib). Table 5. Mutation types identified in the HRR genes | Variant Class | Alteration Type | Description* | | --- | --- | --- | | Short Variant | Nonsense, frameshift or splice site | Any deleterious nonsense, frameshift, or splicing event that spans or occurs within ±2 bases of the intron/exon junction | | | Missense or non-frameshift | Any of the mutations listed in Table 6 for ATM, BRCA1, and BRCA2 | | Copy Number Alteration | Homozygous copy number loss | Deleterious homozygous copy number loss of one or more exons | | Rearrangement | Rearrangement | Any rearrangement that disrupts protein function | *For BRCA2, truncating mutations must occur upstream of bases encoding amino acid 3326. Additionally, the frameshift mutation T367fs*13 in FANCL is ineligible. All short variants must occur in the canonical transcript. The specific deleterious mutation (DM) and suspected deleterious mutation (SDM) missense mutations or non-frameshift mutations for BRCA1, BRCA2, and ATM are shown in Table 6-8, below. However, any missense or non-frameshift mutations in the other 12 genes would not be considered HRR positive. Table 6. Eligible deleterious mutations in the ATM gene (for olaparib CDx claim only) | M1I | Y2470D | 2832C | | --- | --- | --- | | M1L | R2547_S2549del | S2855_V2856>RI | | M1T | A2622V | D2913Y | | P292L | D2625_A2626>EP | R3008C | | D2016G | D2708N | R3008H | | R2032K | V2716A | splice site 331 +5G>A | | A2067D | G2765S | splice site 8418+5_8418+8delGTGA | | R2227C | F2827C | | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 13 of 45 {13} **Table 7. List of short variants in *BRCA1*** | M1R | C44S | R1495T | D1692Y* | G1738E | Y1853C | | --- | --- | --- | --- | --- | --- | | M1I | C44Y | E1559K | C1697R | G1738R | C1787_G1788>SD | | M1V | C47F | E1559Q | R1699Q | L1764P | Splice site 212+3A>G | | M1T | C61G | A1623G | R1699W | I1766S | Splice site 213-11T>G | | M18T | C61Y* | S1655F | L1705P | G1770V | Splice site 213-12A>G | | L22S | C64G | T1685A | G1706E | M1775K | Splice site 302-2C>G | | C24R | C64R | T1685I | G1706R | M1775R | Splice site 4986+3G>C | | T37K | C64W* | H1686R | A1708E | L1780P | Splice site 4986+5G>A | | C39G* | C64Y | V1688del | S1715N | G1788V | Splice site 4986+6T>G | | C39R | R71K | M1689R | S1715R | P1812A | Splice site 4986+6T>C | | C39W* | R71G | T1691I | W1718C | V1833M | Splice site 5074+3A>G | | C39Y | R170Q* | T1691K | S1722F | W1837R | Splice site 5194-12G>A | | H41R | R1495K | D1692N | V1736A | W1837C | Splice site 5406+4A>G | | C44F | R1495M | D1692H | V1736G | V1838E | | \*variants are part of the biomarker definition for the niraparib CDx claim only **Table 8. List of short variants in *BRCA2*** | M1R | R2336L* | R2659G | L2686P | Y2726C | N3124I | | --- | --- | --- | --- | --- | --- | | M1I | R2336P | R2659K | L2686P | G2748D | Splice site 316+4delA | | S142I* | L2510P | R2659T | T2722R | G2793R | Splice site 316+5G>A | | V159M | H2623R | Y2660D | D2723A | E3002K | Splice site 8487+3A>G | | V211I | W2626C | E2663V | D2723G | R3052W | Splice site 8754+4A>G | | V211L | I2627F | S2670L | D2723H | G3076V | Splice site 8754+5G>A | | R2336H | L2653P | I2675V | D2723V | D3095E | Splice site 8754+3G>C | \*variants are part of the biomarker definition for the niraparib CDx claim only ### **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-3'* and *5'*-*NTRK* events). 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. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 14 of 45 {14} Unidentified partners (encoded as N/A) or LINC non-coding partners are also considered CDx biomarker negative. ### Biomarker Rules for ALK Rearrangements: Rearrangements in ALK shall be considered CDx biomarker positive if the following criterion is met: - Any oncogenic ALK rearrangement whose breakpoint occurs within ALK intron 19 or whose partner gene is EML4. ### Biomarker Rules for FGFR2 Fusions and Select Rearrangements: Rearrangements in FGFR2 shall be considered CDx biomarker positive if the following criteria are met: - The rearrangement event involved FGFR2 and a literature-derived known partner gene regardless of strand or frame, - The rearrangement event involved FGFR2 and a novel partner gene that is both in-frame and in-strand, - Any FGFR2 rearrangement with one breakpoint in the hotspot region (intron 17-18) and the other breakpoint in the intergenic region or within another gene. This rule excludes 3' duplications of only exon 18, - Intragenic duplication of kinase domain (exon 9-17). ### 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. ### Biomarker Rules for RET Fusions: Fusions in RET shall be considered CDx biomarker positive if the following criteria are met: - Any fusion event involving RET and another protein-coding gene - RET and the partner gene must be in the same 5'-3' orientation - RET must be on the 3' end of the detected rearrangement - The RET breakpoint must occur before the start of the kinase domain (amino acids 724-1016) PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 15 of 45 {15} # **Biomarker Rule for AKT1, PIK3CA, and PTEN Alterations:** Alterations in *AKT1*, *PIK3CA*, and/or *PTEN* are considered Companion biomarker positive if the following criteria are met. Table 9 Biomarker definition for AKT1, PIK3CA, and PTEN | Gene (Transcript) | Alteration Type | Protein/Transcript Effect | | --- | --- | --- | | *AKT1* (NM_001014431) | SNV | E17K | | *PIK3CA* (NM_006218) | SNV | R88Q, N345K, C420R, E542K, E545A, E545D, E545Q, E545K, E545G, Q546E, Q546K, Q546R, Q546P, M1043V, M1043I, H1047Y, H1047R, H1047L, and G1049R | | *PTEN* (NM_000314) | SNV and indels | C124R, G129E, R130Q, C136R, S170R, R173C, C124S, G129V, R130G C136Y, G129R, R130L, R130P | | | | Any nonsense, frameshift, or splice site alteration - Missense mutations in the start codon and short variant deletions spanning from upstream of the start codon (annotated as M1?) are included in this category | | | Copy Number Alteration | Homozygous deletion (HD) represents a deletion of one or more exons regardless of transcript in both alleles | | | Rearrangement (RE) | Any rearrangement that disrupts protein function, regardless of transcript - Intragenic events including duplications of only part of the gene, deletions, or inversions. - Translocations, deletions, or inversions where one breakpoint is in PTEN and the other breakpoint is in another gene or intergenic region. | # **Biomarker Rule for *BRAF* V600 mutations and *BRAF* fusions:** *BRAF* V600 and *BRAF* fusion alterations are considered companion diagnostic biomarker positive if the following criteria are met. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 16 of 45 {16} Table 10. Biomarker definition for BRAF V600X and BRAF fusion alterations | Gene (Transcript) | Variant Type | Biomarker rules | | --- | --- | --- | | BRAF (NM_004333) | Short Variant | Any missense alteration resulting in V600X^{1} | | | Rearrangement | Any fusion event that involves BRAF and another protein-coding gene and meets all the following criteria: - BRAF and the partner gene must be in the same 5'-3' orientation - BRAF must be on the 3' end of the detected fusion - The BRAF breakpoint must occur after the autoinhibitory domain and before the kinase domain | $^{1}$X refers to any single amino acid change resulting from a missense alteration. ### VI. ALTERNATIVE PRACTICES AND PROCEDURES There are FDA-approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using FFPE tumor specimens, as listed in Table 1 of the F1CDx intended use statement. The approved CDx tests are listed in Table 11 below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle. Table 11. List of FDA approved CDx assays for genes targeted by F1CDx | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | HER2-Amplification | PathVysion HER-2 DNA Probe Kit | Abbott Molecular, Inc. | FISH | HERCEPTIN (trastuzumab) | Breast cancer | | | PATHWAY Anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody | Ventana Medical Systems, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | InSite HER-2/neu Kit | Biogenex Laboratories, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | SPOT-Light HER2 CISH Kit | Life Technologies, Inc. | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | Bond Oracle HER2 IHC System | Leica Biosystems | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | HER2 CISH pharmDx Kit | Dako Denmark A/S | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | INFORM HER2 Dual ISH DNA Probe Cocktail | Ventana Medical Systems, Inc. | Dual ISH | HERCEPTIN (trastuzumab) | Breast cancer | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 17 of 45 {17} | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | | HercepTest | Dako Denmark A/S | IHC | HERCEPTIN (trastuzumab) PERJETA (pertuzumab) KADCYLA (ado-trastuzumab emtansine) | Breast cancer Gastric or Gastroesophageal junction adenocarcinoma | | | HER2 FISH pharmDx Kit | Dako Denmark A/S | FISH | HERCEPTIN (trastuzumab) PERJETA (pertuzumab) KADCYLA (ado-trastuzumab emtansine) | Breast cancer Gastric or Gastroesophageal junction adenocarcinoma | | BRAF-V600E and V600K | THxID BRAF Kit | bioMerieux | PCR | MEKINIST (tramatenib) | Melanoma | | | cobas 4800 BRAF V600 Mutation Test | Roche Molecular Systems, Inc. | PCR | COTELLIC (cobimetinib) ZELBORAF (vemurafenib) | Melanoma | | BRAF-V600E | cobas 4800 BRAF V600 Mutation Test | Roche Molecular Systems, Inc. | PCR | ZELBORAF (vemurafenib) | Melanoma | | | THxID BRAF Kit | bioMerieux | PCR | TAFINLAR (dabrafenib) | Melanoma | | | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | TAFINLAR (dabrafenib) MEKINIST (trametinib) | NSCLC | | | therascreen BRAF V600E RGQ PCR Kit | QIAGEN | PCR | BRAFTOVI (encorafenib) Erbitux (cetuximab) | CRC | | NRAS | Praxis Extended RAS Panel | Illumina, Inc. | NGS | VECTIBIX (panitumumab) | CRC | | KRAS | cobas KRAS Mutation Test | Roche Molecular Systems, Inc. | PCR | ERBITUX (cetuximab) VECTIBIX (panitumumab) | CRC | | | therascreen KRAS RGQ PCR Kit | QIAGEN | PCR | ERBITUX (cetuximab) VECTIBIX (panitumumab) | CRC | | | Praxis Extended RAS Panel | Illumina, Inc. | NGS | VECTIBIX (panitumumab) | CRC | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 18 of 45 {18} | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | **ALK – fusion** | Vysis ALK Break Apart FISH Probe Kit | Abbott Molecular, Inc. | FISH | XALKORI (crizotinib) | NSCLC | | | ALK (D5F3) CDx Assay | Ventana Medical Systems, Inc. | IHC | XALKORI (crizotinib) | NSCLC | | **EGFR – Exon 19 deletions & L858R** | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TARCEVA (erlotinib) TAGRISSO (osimertinib) IRESSA (gefitinib) | NSCLC | | | *therascreen* EGFR RGQ PCR Kit | QIAGEN | PCR | GILOTRIF (afatinib) IRESSA (gefitinib) | NSCLC | | | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | IRESSA (gefitinib) | NSCLC | | **EGFR T790M** | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TAGRISSO (osimertinib) | NSCLC | | **BRCA1/2** | BRACAnalysis CDx | Myriad Genetic Laboratories, Inc. | NGS | LYNPARZA (olaparib) LYNPARZA (olaparib)-treatment/maintenance TALZENNA (talazoparib) | Breast, pancreatic, and prostate cancers Ovarian cancer Breast cancer | | | Myriad myChoice® CDx | Myriad Genetic Laboratories, Inc. | NGS | ZEJULA (niraparib) or Lynparza (olaparib) | Ovarian cancer | | | *therascreen* PIK3CA RGQ PCR Kit | QIAGEN | PCR | PIQRAY (alpelisib) | Breast cancer | | **ROS1** | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | XALKORI (crizotinib) | NSCLC | | **RET** | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | RETEVMO (selpercatinib) GAVRETO (pralsetinib) | NSCLC and Thyroid Cancer NSCLC | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 19 of 45 {19} **Abbreviations:** FISH – fluorescence in situ hybridization; IHC – immunohistochemistry; CISH chromogenic in situ hybridization; ISH – in situ hybridization; PCR – polymerase chain reaction; NGS – next generation sequencing. ## VII. MARKETING HISTORY Foundation Medicine, Inc. indicates the F1CDx assay was offered as a laboratory developed test (LDT) with the name “F1 LDT” since 2012. As described in the preamble to FDA’s LDT final rule, FDA uses the term 'IVDs offered as LDTs' for IVDs that are manufactured and offered as LDTs by laboratories that are certified under CLIA and that meet the regulatory requirements under CLIA to perform high complexity testing, and used within such laboratories, even if those IVDs do not fall within FDA’s traditional understanding of an LDT because they are not designed, manufactured, and used within a single laboratory. The F1CDx Premarket Approval (PMA) was originally approved on November 30, 2017 by FDA (P170019) and is commercially available in the U.S. since March 30, 2018. The approved PMA supplements that affected the Intended Use are listed in Table 12. **Table 12: Marketing History** | Submission No. | Date of Approval | Biomarker/Update | Patient Population | Drug | | --- | --- | --- | --- | --- | | P170019/S004 | July 1, 2019 | *BRCA1/2* alterations | Ovarian Cancer | LYNPARZA® (olaparib) | | P170019/S005 | April 10, 2019 | genomic loss of heterozygosity (LOH) | Ovarian Cancer | N/A | | P170019/S006 | December 3, 2019 | *PIK3CA* alterations | Breast Cancer | PIQRAY® (alpelisib) | | P170019/S008 | July 1, 2019 | *EGFR* exon 19 deletions and *EGFR* exon 21 L858R alterations | Non-Small Cell Lung Cancer | TAGRISSO® (osimertinib) | | P170019/S011 | May 6, 2020 | *MET* single nucleotide variants (SNVs) and indels that lead to *MET* exon 14 skipping | Non-Small Cell Lung Cancer | TABRECTA® (capmatinib) | | P170019/S013 | April 17, 2020 | *FGFR2* fusions | Cholangiocarcinoma | PEMZYRE® (pemigatinib) | | P170019/S015 | May 19, 2020 | mutations in homologous recombination repair (HRR) genes | metastatic castration resistant prostate cancer (mCRPC) | LYNPARZA® (olaparib) | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 20 of 45 {20} | Submission No. | Date of Approval | Biomarker/Update | Patient Population | Drug | | --- | --- | --- | --- | --- | | P170019/S016 | June 16, 2020 | high tumor mutational burden (TMB) at the cut-off of 10 mutations per megabase (mut/Mb) | Solid Tumors | KEYTRUDA® (pembrolizumab) | | P170019/S017 | October 23, 2020 | *NTRK1, NTRK2, or NTRK3* fusions | Solid Tumors | VITRAKVI® (larotrectinib) | | P170019/S021 | May 28, 2021 | *FGFR2* Fusion/Rearrangements | Cholangiocarcinoma | TRUSELTIQ® (infigratinib) | | P170019/S022 | July 21, 2021 | Additional variants to *BRCA1* and *BRCA2* | Ovarian Cancer | LYNPARZA® (olaparib) or RUBRACA® (rucaparib) | | | | Additional variants to *BRCA1, BRCA2* and *ATM* | Prostate Cancer | LYNPARZA® (olaparib) | | P170019/S023 | June 30, 2021 | *ALK* Rearrangements | Non-Small Cell Lung Cancer | ALUNBRIG® (brigatinib) | | P170019/S025 | November 10, 2021 | *BRAF* V600E Alterations | Melanoma | BRAF Inhibitor Monotherapy Group Claim | | | | *BRAF* V600E or V600K Alterations | Melanoma | BRAF/MEK Inhibitor Combination Group Claim | | P170019/S029 | February 18, 2022 | Microsatellite Instability High (MSI-H) Status | Solid Tumors | KEYTRUDA® (Pembrolizumab) | | P170019/S030 | January 19, 2022 | *BRAF* V600 Mutation-Positive | Unresectable Or Metastatic Melanoma | TECENTRIQ® (atezolizumab) In Combination with Cobimetinib and Vemurafenib | | P170019/S033 | March 16, 2022 | *EGFR* Exon 19 Deletions or *EGFR* Exon 21 L858R Mutations | Non-Small Cell Lung Cancer | Any One of The FDA-Approved EGFR Tyrosine Kinase Inhibitors (TKI) | | P170019/S014 | June 7, 2022 | *NTRK1, NTRK2, or NTRK3* fusions | Solid Tumors | ROZLYTREK® (entrectinib) | | | | *ROS1* fusions | NSCLC | | | P170019/S042 | August 11, 2023 | *BRCA1, BRCA2* alterations | Prostate Cancer | AKEEGA® (niraparib + abiraterone acetate) | | P170019/S043 | October 6, 2023 | *RET* fusions | Solid Tumors | RETEVMO® (selpercatinib) | | P170019/S039 | October 11, 2023 | *BRAF* V600E | NSCLC | BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) | | P170019/S048 | November 16, 2023 | *AKT1, PIK3CA, PTEN* alterations | Breast Cancer | TRUQAP™ (capivasertib) in combination with | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 21 of 45 {21} | Submission No. | Date of Approval | Biomarker/Update | Patient Population | Drug | | --- | --- | --- | --- | --- | | | | | | FASLODEX® (fulvestrant) | | P170019/S052 | August 30, 2024 | BRCA1, BRCA2 alterations | Prostate Cancer | LYNPARZA® (olaparib) in combination with abiraterone | ## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect test results, and subsequently, inappropriate patient management decisions. Patients with false positive results may undergo treatment with one of the therapies listed in the above intended use statement without clinical benefit and may experience adverse reactions associated with the therapy. Patients with false negative results may not be considered for treatment with the indicated therapy. There is also a risk of delayed results, which may lead to delay of treatment with the indicated therapy. For the specific adverse events related to the approved therapeutics, please see the approved drug product labels. ## IX. SUMMARY OF NON-CLINICAL STUDIES ### A. Laboratory Studies The primary evidence for supporting the performance of F1CDx in the detection of *BRAF* V600 mutations and *BRAF* fusions in pediatric low-grade glioma (pLGG) patients was from data generated using glioma specimens within the validation studies described herein. Given the low prevalence of pLGG, for analytical studies, while prioritizing the inclusion of low-grade gliomas and ≤25-year-old patient samples, *BRAF* mutation and fusion samples from both low- and high-grade gliomas and from all patient ages were used. In addition to the existing platform-level validation results (P170019), refer to Section IX.A. in P170019 Summary of Safety and Effectiveness Data, wherein, analytical concordance, site-to-site precision, limit of blank (LoB), and limit of detection (LoD) studies were conducted to support the indication for *BRAF* V600 mutations and *BRAF* fusions. Glioma samples evaluated in the analytical validation studies (site-to-site precision, LoB, and LoD) were extracted with DNAx extraction method using the HiSeq sequencer configuration. The F1CDx device was updated to include addition of an automated DNA/RNA CoExtraction methodology (CoEx method) to enable isolation of DNA and RNA from the same FFPE tumor specimens. The F1CDx was authorized for DNA only when using the CoEx method. The F1CDx device was also updated to replace the Illumina HiSeq 4000 sequencer with the Illumina NovaSeq 6000 sequencer. Analytical validation studies were conducted to demonstrate the comparable performance between the two nucleic acid extraction methods and two sequencer configurations, using samples from multiple disease ontologies to provide evidence of ---PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 22 of 45 {22} the robust detection of BRAF fusions and BRAF V600 mutations utilizing the NovaSeq configuration and the CoEx extraction method. The reproducibility and repeatability for BRAF mutations from the CoEx precision study included 5 BRAF V600 mutation samples. The aggregated reproducibility for BRAF mutations was 100.00% (104/104, 95% CI: [96.44%, 100.00%]) and the aggregated repeatability for BRAF mutations was 100.00% (50/50, 95% CI: [92.87%, 100.00%]). The CoExtraction concordance study included 19 samples with BRAF mutations, and 1 sample with BRAF fusion. The aggregated positive percent agreement (PPA) for BRAF mutation was 100.00% (19/19, 95% CI: [83.18%, 100.00%]) and the aggregated negative percent agreement (NPA) for BRAF mutation was 100.00% (20/20, 95% CI: [83.89%, 100.00%]). The aggregated PPA for BRAF mutation from the NovaSeq migration study using 19 samples with BRAF mutations, and 1 sample with BRAF fusion was 100.00% (19/19, 95% CI: [83.18%, 100.00%]) and the aggregated NPA for BRAF mutation was 100.00% (108/108, 95% CI: [96.57%, 100.00%]). The F1CDx analytical pipeline was modified after the analytical and clinical validation studies were completed. The analytical and clinical validation provided to support the detection of BRAF V600 mutations and BRAF fusions in low-grade glioma by F1CDx were performed using the analytical pipeline versions that have undergone further iteration with modification in the final device design. To demonstrate the F1CDx analytical pipeline changes continue to support the performance for which the test is approved, regression testing using the most current analytical pipeline version will be performed post-market to confirm the robust performance for F1CDx for the detection of BRAF V600 mutations and BRAF fusions (see section XIII). # i Analytical Accuracy / Concordance # Comparison to an Orthogonal Method To demonstrate analytical concordance, an analysis between F1CDx and an externally validated NGS assay (evNGS) to detect BRAF V600 mutations and BRAF fusions was performed. The concordance study was conducted using available residual DNA previously extracted by CoEx method from 15 FFPE tissue samples from patients with low-grade glioma (LGG) enrolled in the FIREFLY-1 (NCT04775485) clinical study that supported OJEMDA (tovorafenib) approval (Refer to Section X Summary of Primary Clinical Study for study details). The study was supplemented with DNA previously extracted using DNAx method from 189 FFPE glioma tissue samples from the FMI sample archives. In total, the available samples for the analytical concordance study included 103 BRAF V600 mutation and BRAF fusion (biomarker positive) and 101 non-altered (biomarker negative) samples. In total, 204 samples were processed in the concordance study with representation of both BRAF V600 mutations and BRAF fusions included in the tovorafenib CDx biomarker definition. Of the 204 samples, one sample was excluded due to improper storage conditions. In addition, 201 of the remaining 203 samples were processed successfully from library construction through PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 23 of 45 {23} sequencing by F1CDx. All 204 samples completed lab processing and passed the sample QC metrics for the evNGS assay. A total of 203 samples were included in the concordance analysis. The F1CDx and evNGS results for the detection of BRAF V600 mutations and BRAF fusions using evNGS as the comparator assay, are provided in the contingency table in Table 13. The statistical analysis using the evNGS assay results as the reference showed a positive predictive value (PPV) of 96.88% with a 95% confidence interval (CI) of (91.21%, 98.93%) and a negative predictive value (NPV) of 97.14% with 95% CI (91.93%, 99.02%) unadjusted for prevalence, and a positive percent agreement (PPA) of 96.88% (93/96) with 95% CI (91.21%, 98.93%), and a negative percent agreement (NPA) of 97.14% with 95% CI[(91.93%, 99.02%). Since the samples were selected from different sources based on different assays, the PPA/NPA and PPV/NPV may be subject to potential bias. Table 13. Contingency Table Comparing the Detection of BRAF V600 mutations and BRAF fusions by F1CDx and evNGS | | evNGS+ | evNGS- | Invalid | Total | | | --- | --- | --- | --- | --- | --- | | **F1CDx+** | 93 | 3 | 0 | 96 | PPV: 96.88% [95%CI*: 91.21%, 98.93%] | | **F1CDx-** | 3 | 102 | 0 | 105 | NPV:97.14% [95%CI*: 91.93%, 99.02%] | | **Invalid** | 1 | 1 | 0 | 2 | | | **Total** | 97 | 106 | 0 | 203 | | | | PPA: 96.88% [95%CI*: 91.21%, 98.93%] | NPA: 97.14% [95%CI*: 91.93%, 99.02%] | | | | *95% 2-sided confidence intervals (CI) were calculated using the Wilson score method. There were 6 discordant calls between F1CDx and evNGS test results. Of the 6 calls, the following was observed: - One sample was detected by F1CDx but was removed due to the contamination QC flag. - Two samples were detected by the evNGS, but not detected by F1CDx due to limited targeting in the region of the breakpoint. - Two samples were detected by F1CDx, but not detected by evNGS because BRAF intron 7 is not baited on the evNGS assay. - One sample was detected by F1CDx but not detected by evNGS because the biomarker definition requires the BRAF gene to be in the 3' orientation of the fusion product. For that sample, F1CDx detected both orientations, but the evNGS assay only detected the fusion in the biomarker-negative orientation. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 24 of 45 {24} # i. Analytical Sensitivity # a. Limit of Blank (LoB) The LoB of zero was confirmed by testing six biomarker negative FFPE glioma tissue samples. Ten replicates for each of the six DNA source samples were processed for a total of 60 sample replicates. Of the 60 replicates, 100% produced valid results and no BRAF V600 mutations or BRAF fusions were detected across all sample replicates; therefore, the false positive rate for calling alterations was determined to be 0.00%. The study demonstrated that the F1CDx assay does not produce false-positive results in detecting BRAF V600 mutations or BRAF fusions in biomarker-negative FFPE tissue samples from patients with glioma. # b. Limit of Detection (LoD) The F1CDx LoD for the detection of BRAF V600 mutations and BRAF fusions was established through confirmation of platform based LoD and within CDx-specific LoD studies. The LoD of BRAF single nucleotide variant (SNV) alterations was established through a previously conducted study for the PMA approval (P170019) as 3.2% VAF. The LoD was confirmed through a precision study that used samples at or near 1x LoD. The precision study processed at the laboratories in Cambridge, MA (CAM) and Research Triangle Park (RTP) in Morrisville, NC, determined the LoD for BRAF SNVs to be 2.49% VAF. The LoD of BRAF fusions was determined through a new LoD study. DNA from two unique glioma samples harboring BRAF fusions were processed. Each sample was assessed at five targeted chimeric read levels (30, 24, 18, 12, and 4). Twenty replicates were assessed for each dilution level, except the 30 reads level, which examined 14 replicates. Ninety-four replicates were tested per sample. A total of 188 sample replicates were processed, of which two failed F1CDx QC metrics. The remaining 186 sample replicates passed all QC criteria and were included in the final analysis. Each specimen was evaluated close to the minimum input requirements of the assay (50 ng), thus representing the most challenging evaluation of BRAF fusion detection. The LoD for each sample was determined based on chimeric reads using hit rate method for BRAF fusions. The LoD for the two individual BRAF fusion samples was 8.30 and 6.75 chimeric reads. The LoD for BRAF fusions was determined to be 7.53 chimeric reads, which was the median LoD from the two eligible BRAF fusion samples per the hit rate method. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 25 of 45 {25} ## ii. Precision To evaluate the performance of BRAF V600 mutation and BRAF fusion detection, a site-to-site precision study was conducted at the laboratories in Cambridge, MA (CAM) and Morrisville, NC (RTP) using 4 unique glioma samples harboring BRAF V600 mutations and BRAF fusions at challenging DNA input (close to 50 ng) conditions. Table 14 below lists the samples evaluated in this study. Samples were titrated with biomarker negative diluent DNA extracted from glioma FFPE DNA samples to achieve the targeted VAF and chimeric reads (BRAF SVs at ~1-1.5x LoD and BRAF fusions at ~1-3x LoD). In total, 96 sample replicates (24 replicates of each sample) were evaluated with two reagent lots at each of the two sites. Table 14. Samples Evaluated in the Precision Study | BRAF-positive samples | | | | BRAF-negative samples | | | --- | --- | --- | --- | --- | --- | | Sample ID | Disease Ontology | Biomarker-positive Alteration | LoD | Sample ID | Disease Ontology | | 1 | Brain pleomorphic xanthoastrocytoma | CDC42SE2-BRAF fusion | 7.53 reads | 5 | Brain pleomorphic xanthoastrocytoma | | 2 | Brain astrocytoma pilocytic | KIAA1549-BRAF fusion | 7.53 reads | 6 | Brain astrocytoma | | 3 | Brain astrocytoma pilocytic | BRAF 1799T>A/V600E | 3.2% VAF | 7 | Brain astrocytoma pilocytic | | 4 | Brain ganglioglioma | BRAF 1799T>A/V600E | 3.2% VAF | 8 | Brain ganglioglioma | The point estimates and 95% two-sided CIs for reproducibility of each sample at CAM and RTP are detailed in Table 15. Two targeted BRAF fusion samples had observed average chimeric reads >1x LoD and were included in the reproducibility results below. Table 15. Reproducibility for Targeted Variants Above LoD (CAM-RTP) | Source Sample | Targeted Variant | Variant Type | Observed Average Chimeric Reads | LoD (Reads) | # of Positive Replicates | # of Valid Replicates | Reproducibility (%) | 95% Two-Sided Score CI (%) | Fold LoD (x) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | CDC42SE2-BRAF | RE | 9.83 | 7.53 | 24 | 24 | 100.00 | [86.20, 100.00] | 1.31 | | 2 | KIAA1549-BRAF | RE | 9.25 | 7.53 | 24 | 24 | 100.00 | [86.20, 100.00] | 1.23 | Repeatability was evaluated in the two targeted BRAF fusions with observed chimeric reads ≥1x LoD. The point estimates and 95% two-sided CIs for repeatability of each sample are detailed in Table 16. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 26 of 45 {26} **Table 16. Repeatability for Targeted Variants above LoD (CAM-RTP)** | Source Sample | Targeted Variant | # of Agreed Pairs | # of Valid Pairs | Repeatability (%) | 95% Two-Sided Score CI (%) | | --- | --- | --- | --- | --- | --- | | 1 | *CDC42SE2-BRAF* | 12 | 12 | 100.00 | [75.75, 100.00] | | 2 | *KIAA1549-BRAF* | 12 | 12 | 100.00 | [75.75, 100.00] | An exploratory precision analysis was performed for two targeted *BRAF* single variant mutations (Sample ID 3 and 4), since they both had an observed average %VAF <1x LoD. Reproducibility and repeatability results were 100.00% and 100.00%, respectively, for both variants. #### **B. Animal Studies** No animal studies were conducted using the F1CDx assay. #### **C. Additional Studies** No additional studies were conducted using the F1CDx assay. ### **X. SUMMARY OF PRIMARY CLINICAL STUDY** The clinical performance of F1CDx for detecting *BRAF* V600 mutations and *BRAF* fusions in pediatric low-grade glioma (pLGG) patients was demonstrated in a retrospective analysis of specimens from patients enrolled in the FIREFLY-1 clinical study of OJEMDA (tovorafenib). Data generated from the trial supported the clinical validation of the F1CDx assay for identification of patients with *BRAF* V600 mutations and *BRAF* fusions who may benefit from treatment with tovorafenib. A summary of the clinical study is presented below. #### **FoundationOne CDx Clinical Bridging Study** A reasonable assurance of safety and effectiveness for F1CDx for detection of *BRAF* V600 mutations and *BRAF* fusions in pLGG patients who may benefit from treatment with OJEMDA (tovorafenib) was demonstrated through a clinical bridging study using tumor tissue FFPE specimens from patients enrolled in the FIREFLY-1 clinical study with known *BRAF* V600 mutation and *BRAF* fusion status, as well as *BRAF* V600 mutation and *BRAF* fusion negative samples that were commercially procured or available from the FMI DNA archives. The clinical bridging study utilized the NovaSeq instrument and CoEx extraction method. The clinical efficacy analysis was performed by analyzing concordance between F1CDx and the enrollment clinical trial assays (CTAs), followed by the imputation of the missing F1CDx results, and determining the clinical outcome of the *BRAF* V600 mutation and *BRAF* fusion positive population identified with F1CDx. ---PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 27 of 45 {27} # A. FIREFLY-1 Study Design The FIREFLY-1 clinical trial is a Phase 2 multi-center, non-randomized, open-label study of tovorafenib assessing the efficacy, safety, and pharmacokinetics in pediatric patients aged 6 months to ≤ 25 years with relapsed or refractory low-grade glioma (LGGs) or advanced solid tumors harboring a known activating RAF alteration, including BRAF V600 mutations and KIAA1549-BRAF fusions. FIREFLY-1 was initiated on April 22, 2021. This study included three arms: - Arm 1 (LGG): Patients with recurrent or progressive LGG harboring a known activating BRAF alteration, including BRAF V600 mutations and KIAA1549-BRAF fusions or rearrangements. - Arm 2 (LGG extension): Patients with recurrent or progressive LGG harboring a known or anticipated activating RAF alteration (e.g., BRAF or CRAF/RAF1 fusion or BRAF V600 mutation) or rearrangements. - Arm 3 (advanced solid tumor): Patients with advanced solid tumors harboring a known or anticipated activating RAF fusion (e.g., BRAF or CRAF/RAF1 fusion) or rearrangements. The primary analysis set for the New Drug Application (NDA) population and for the clinical validation studies includes patients enrolled in Arm 1 only. OJEMDA (tovorafenib) was granted accelerated approval by FDA for BRAF V600 mutation and BRAF fusion positive LGG patients in April 2024 based on data from patients using the Response Assessment in Pediatric Neuro-Oncology (RAPNO)-LGG criteria in the FIREFLY-1 trial. The clinical validation study results presented below follow the same efficacy analysis population used to support the approval of OJEMDA (tovorafenib). # BRAF V600 mutation and BRAF fusion Evaluation by F1CDx The clinical effectiveness of OJEMDA (tovorafenib) among patients with BRAF V600 mutations and BRAF fusions in pLGG patients as determined by F1CDx was demonstrated in a retrospective analysis of specimens from the FIREFLY-1 clinical trial. A bridging study was conducted to assess: (1) concordance between CTAs and F1CDx in identifying patients with BRAF V600 mutations and BRAF fusions, (2) the efficacy of OJEMDA (tovorafenib) in patients from the FIREFLY-1 clinical trial who have BRAF V600 mutations and BRAF fusions as determined by F1CDx and (3) the robustness of the concordance analysis and efficacy analysis with a sensitivity analyses that accounts for the uncertainty due to missing data for BRAF V600 mutation and BRAF fusion status as determined by F1CDx. # Clinical Bridging Study Design The clinical bridging study evaluated the clinical validity of F1CDx as a companion diagnostic (CDx) to identify BRAF V600 mutation and BRAF fusion positive patients from the FIREFLY-1 clinical trial. F1CDx testing was performed on FIREFLY-1 NDA patients who had samples with sufficient tissue material remaining and who PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 28 of 45 {28} tested positive for BRAF V600 mutations and BRAF fusions by the CTAs. BRAF V600 mutations and BRAF fusion negative samples that were commercially procured or from FMI's DNA archives were selected and identified as biomarker negative by an evNGS based assay or a FISH-based assay, and subsequently tested by F1CDx for the CTA negative (CTA-) results. # 1. Clinical Inclusion and Exclusion Criteria Samples meeting pre-defined criteria specified below were included in the clinical bridging study. # Inclusion Criteria for biomarker-positive samples: - Samples that are derived from LGG FFPE specimens. - Samples that meet minimum criteria for F1CDx testing requirements. - BRAF alteration-positive samples from the FIREFLY-1 clinical trial that have been obtained with proper informed consent. - Samples must be obtained in accordance with internal procedures and with Institutional Review Board (IRB) approval. # Inclusion Criteria for negative samples in the NGS cohort: - Samples must be derived from high confidence LGG FFPE specimens including the following disease ontologies (DOs): brain astrocytoma, brain astrocytoma pilocytic, brain astrocytoma pilomyxoid, brain dysembryonic neuroepithelial tumor, brain gangliocytoma, brain ganglioglioma, and brain oligodendroglioma. - Samples must be able to support both evNGS and F1CDx DNA input requirements. - Samples must have ≥ 20% tumor purity. - Samples must meet the minimum test requirements for the evNGS and F1CDx assays. - Samples must be obtained in accordance with internal procedures and IRB protocol. # Inclusion Criteria for negative samples in the FISH cohort: - Samples that are derived from LGG FFPE specimens. - Samples must meet the minimum test requirements for the FISH and F1CDx assays. - Samples must be obtained in accordance with internal procedures. # Exclusion Criteria for samples: - Samples failing to meet any of the inclusion criteria. - FIREFLY-1 clinical trial samples that lack clear identification or labeling. - Samples not obtained in accordance with internal procedures or not receiving IRB approval. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 29 of 45 {29} • Samples not obtained in accordance with IRB protocol. # 2. Follow-up Schedule The F1CDx clinical bridging study involved only retrospective testing of tissue tumor FFPE samples; as such, no additional patient follow-up was conducted. # 3. Clinical Bridging Study Endpoints The objectives of the F1CDx clinical bridging study were to: - Establish the clinical validity of F1CDx in identifying BRAF V600 mutations and BRAF fusions in glioma for treatment with OJEMDA (tovorafenib). - Evaluate the concordance of results for the BRAF V600 mutation and BRAF fusion status between the F1CDx assay and the CTAs used for enrollment into the FIREFLY-1 clinical trial. - Assess the robustness of the concordance and efficacy analyses to account for uncertainty due to missing data for the F1CDx BRAF V600 mutation and BRAF fusion status. A concordance analysis was performed for subjects identified with RAPNO-LGG criteria assessed by independent radiology review committee (IRC). Endpoints for the concordance analysis included positive percent agreement (PPA), negative percent agreement (NPA), prevalence adjusted positive predictive value (PPV) and prevalence-adjusted negative predictive value (NPV). # B. Accountability of PMA Cohort Arm 1 includes the patient population included in the NDA to support accelerated approval of tovorafenib. As of June 2023, 77 patients were enrolled in Arm 1 and were evaluated for the clinical bridging study (Note: 76 of the 77 patients were available by RAPNO-LGG criteria and is the basis for the approval of OJEMDA (tovorafenib)). An additional 120 BRAF V600 mutation and BRAF fusion negative samples were evaluated. Among the 197 samples evaluated, three CTA+ samples did not have available material for testing. A total of 194 samples were processed by F1CDx in this study. Out of 194 samples, 174 samples had valid test results and 20 samples failed during pre- and post- sequencing steps. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 30 of 45 {30} Table 17. Sample Accountability for the Clinical Bridging Study by Source Sample | CTA Status | Sample Source | # of Subjects (Samples) | # of F1CDx Unevaluable Samples | # of F1CDx Failed Samples | # of F1CDx Evaluable Samples (%) | | --- | --- | --- | --- | --- | --- | | Positive | FIREFLY-1 clinical trial | 77 | 3 | 19 | 55 (71.43%) | | Negative | Samples selected from FMI sample archive and tested negative by evNGS | 70 | 0 | 0 | 70 (100.00%) | | | Samples commercially procured tested by FISH based assay | 24 | 0 | 1 | 23 (95.83%) | | | Samples randomly selected from previous validation study that tested negative by evNGS based assay | 26 | 0 | 0 | 26 (100.00%) | | Total | | 197 | 3 | 20 | 174 (88.32%) | ### C. Study Population Demographics and Baseline Parameters In the clinical bridging study, baseline characteristics were compared between CDx-evaluable and CDx-unevaluable populations of the CTA+ population. The unevaluable population included patients who did not have a sample available to be tested by F1CDx or those whose sample failed F1CDx testing (invalid). The demographics and disease characteristics for the CDx-evaluable and CDx-unevaluable CTA+ patients are provided in Table 18. Differences in characteristics of the CDx-evaluable and CDx-unevaluable populations were identified by covariate analysis. The covariates that showed significant differences between the two groups at a significance level of α=0.2 were considered to be imbalanced between the F1CDx-evaluable and the F1CDx-unevaluable groups for patients enrolled based on RAPNO-LGG criteria. PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 31 of 45 {31} Table 18. Demographic and Clinical Characteristics for CTA+ Patients Identified based on RAPNO-LGG Criteria | Covariate | Group Name | CDx-unevaluable | CTA+ | CDx-evaluable | CTA+ | Difference | P-value | | --- | --- | --- | --- | --- | --- | --- | --- | | Age | Overall | Mean = 8.8, Min = 4, Q1 = 7, Median = 8, Q3 = 10, Max = 16, SD = 3.4 | Mean = 9.5, Min = 2, Q1 = 6, Median = 9, Q3 = 12, Max = 21, SD = 4.2 | 0.7 | 0.536 | | Height | Overall | Mean = 133.1, Min = 107.8, Q1 = 122.5, Median = 130.8, Q3 = 139.2, Max = 173, SD = 17 | Mean = 135.5, Min = 90.5, Q1 = 121, Median = 137.4, Q3 = 155, Max = 166.6, SD = 20.8 | 2.4 | 0.446 | | Weight | Overall | Mean = 33.3, Min = 14.2, Q1 = 20.7, Median = 29.2, Q3 = 41.8, Max = 78.9, SD = 15.5 | Mean = 38.7, Min = 14.6, Q1 = 26.3, Median = 33.8, Q3 = 47.2, Max = 93.4, SD = 17.3 | 5.5 | 0.151 | | BSA | Overall | Mean = 1.1, Min = 0.7, Q1 = 0.8, Median = 1, Q3 = 1.3, Max = 1.9, SD = 0.3 | Mean = 1.2, Min = 0.6, Q1 = 1, Median = 1.1, Q3 = 1.4, Max = 2.1, SD = 0.3 | 0.1 | 0.194 | | KPS | Overall | Mean = 95, Min = 90, Q1 = 92.5, Median = 95, Q3 = 97.5, Max = 100, SD = 7.1 | Mean = 77.5, Min = 70, Q1 = 70, Median = 75, Q3 = 82.5, Max = 90, SD = 9.6 | -17.5 | 0.153 | | LPS | Overall | Mean = 95.5, Min = 80, Q1 = 90, Median = 100, Q3 = 100, Max = 100, SD = 6 | Mean = 91, Min = 50, Q1 = 90, Median = 90, Q3 = 100, Max = 100, SD = 10.8 | -4.5 | 0.110 | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 32 of 45 {32} | Covariate | Group Name | CDx-unevaluable | CTA+ | CDx-evaluable | CTA+ | Difference | P-value | | --- | --- | --- | --- | --- | --- | --- | --- | | Time since specimen acquisition to clinical trial enrollment | Overall | Mean = 50.5, Min = 0.3, Q1 = 29, Median = 56.7, Q3 = 78.1, Max = 92.2, SD = 31.3 | Mean 44.6, Min = 0.1, Q1 = 8.3, Median = 34.8, Q3 = 68.8, Max = 157.6, SD = 41.1 | -6 | 0.370 | | Age Group | Overall | 22 | 53 | 31 | 0.311 | | 12 years to <16 years | 2(9.1%) | 14(26.4%) | 12(17.3%) | | | 16 years to <=25 years | 2(9.1%) | 4(7.5%) | 2(-1.5%) | | | 2 years to <6 years | 3(13.6%) | 10(18.9%) | 7(5.2%) | | | 6 years to <12 years | 15(68.2%) | 25(47.2%) | 10(-21.0%) | | | Race | Overall | 22 | 53 | 31 | 0.801 | | ASIAN | 2(9.1%) | 3(5.7%) | 1(-3.4%) | | | BLACK OR AFRICAN AMERICAN | 1(4.5%) | 1(1.9%) | 0(-2.7%) | | | MULTIPLE | 0(0.0%) | 3(5.7%) | 3(5.7%) | | | OTHER | 1(4.5%) | 4(7.5%) | 3(3.0%) | | | WHITE | 13(59.1%) | 27(50.9%) | 14(-8.1%) | | | N/A | 5(22.7%) | 15(28.3%) | 10(5.6%) | | | Race Group | Overall | 22 | 53 | 31 | 0.754 | | Others | 4(18.2%) | 11(20.8%) | 7(2.6%) | | | White | 13(59.1%) | 27(50.9%) | 14(-8.1%) | | | N/A | 5(22.7%) | 15(28.3%) | 10(5.6%) | | | Ethnicity | Overall | 22 | 53 | 31 | 1.000 | | HISPANIC OR LATINO | 0(0.0%) | 2(3.8%) | 2(3.8%) | | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 33 of 45 {33} | Covariate | Group Name | CDx-unevaluable | CTA+ | CDx-evaluable | CTA+ | Difference | P-value | | --- | --- | --- | --- | --- | --- | --- | --- | | | NOT HISPANIC OR LATINO | 17(77.3%) | 33(62.3%) | 16(-15.0%) | | | N/A | 5(22.7%) | 18(34.0%) | 13(11.2%) | | | Sex | Overall | 22 | 53 | 31 | 0.458 | | F | 9(40.9%) | 27(50.9%) | 18(10.0%) | | | M | 13(59.1%) | 26(49.1%) | 13(-10.0%) | | | Primary Tumor Site | Overall | 22 | 53 | 31 | 0.480 | | Brain Stem | 3(13.6%) | 3(5.7%) | 0(-8.0%) | | | Cerebellum | 0(0.0%) | 5(9.4%) | 5(9.4%) | | | Cerebral Hemisphere | 2(9.1%) | 3(5.7%) | 1(-3.4%) | | | Deep Midline Structures | 3(13.6%) | 6(11.3%) | 3(-2.3%) | | | Optical Pathway | 12(54.5%) | 26(49.1%) | 14(-5.5%) | | | Other | 2(9.1%) | 10(18.9%) | 8(9.8%) | | | Disease ontology subtype | Overall | 22 | 53 | 31 | 0.701 | | Astrocytic | 20(90.9%) | 50(94.3%) | 30(3.4%) | | | Mixed Glial-neuronal | 2(9.1%) | 2(3.8%) | 0(-5.3%) | | | Other | 0(0.0%) | 1(1.9%) | 1(1.9%) | | | Pre-operative staging | Overall | 22 | 53 | 31 | 1.000 | | Disseminated/Metastatic Disease | 2(9.1%) | 6(11.3%) | 4(2.2%) | | | Leptomeningeal Spread | 2(9.1%) | 6(11.3%) | 4(2.2%) | | | Localized Disease | 18(81.8%) | 41(77.4%) | 23(-4.5%) | | | | Overall | 22 | 53 | 31 | 0.726 | PMA P170019/S054: FDA Summary of Safety and Effectiveness Data 34 of 45 {34} | Covariate | Group Name | CDx-unevaluable | CTA+ | CDx-evaluable | CTA+ | Difference | P-value | | --- | --- | --- | --- | --- | --- | --- | --- | | Post-operative staging | Biopsy Only, Not Attempted | 13(59.1%) | 26(49.1%) | 13(-10.0%) | | | Gross Total Resection | 0(0.0%) | 1(1.9%) | 1(1.9%) | | | Sub-Total Resection | 9(40.9%) | 26(49.1%) | 17(8.1%) | | | Prior BRAF inhibitor therapy | Overall | 22 | 53 | 31 | 1.000 | | N | 20(90.9%) | 48(90.6%) | 28(-0.3%) | | | Y | 2(9.1%) | 5(9.4%) | 3(0.3%) | | | Prior MEK inhibitor therapy | Overall | 22 | 53 | 31 | 0.321 | | N | 12(54.5%) | 22(41.5%) | 10(-13.0%) | | | Y | 10(45.5%) | 31(58.5%) | 21(13.0%) | | | Number of prior lines of systemic therapies^{†} | Overall | 22 | 53 | 31 | 0.971 | | 1 | 5(22.7%) | 12(22.6%) | 7(-0.1%) | | | 2 | 6(27.3%) | 13(24.5%) | 7(-2.7%) | | | 3 | 5(22.7%) | 11(20.8%) | 6(-2.0%) | | | 4 | 3(13.6%) | 6(11.3%) | 3(-2.3%) | | | more than 5 | 3(13.6%) | 11(20.8%) | 8(7.1%) | | † More than 5 prior lines of therapies were aggregated into one group to increase number of data points within groups. ### D. Safety and Effectiveness Results #### 1. Safety Results The safety with respect to treatment with tovofarenib was addressed during the review of the tovofarenib NDA (No. 218033 and No. 21770) and is not addressed in detail in this Summary of Safety and Effectiveness Data. The evaluation of safety wa…