FoundationOne CDx (F1CDx)

{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/S017 | | Date of FDA Notice of Approval: | October 23, 2020 | The original PMA (P170019) for FoundationOne CDx was approved on November 30, 2017 for the detection of genetic alterations in patients who may benefit from one of fifteen FDA-approved therapies for non-small cell lung cancer (NSCLC), melanoma, breast cancer, colorectal cancer (CRC), and ovarian cancer. Subsequently, nine PMA supplements were approved for expanding the indications for use of F1CDx since its original approval. PMA supplement (P170019/S005) for adding genomic loss of heterozygosity (LOH) was approved on April 10, 2019. PMA supplement (P170019/S004) for adding an indication for LYNPARZA® (olaparib) in ovarian cancer patients with *BRCA1/2* alterations was approved on July 1, 2019. PMA supplement (P170019/S008) for adding an indication for TAGRISSO® (osimertinib) in NSCLC patients with *EGFR* exon 19 deletions and *EGFR* exon 21 L858R alterations was approved on July 1, 2019. PMA supplement (P170019/S006) for adding an indication for PIQRAY® (alpelisib) in breast cancer patients with *PIK3CA* alterations was approved on December 3, 2019. PMA supplement (P170019/S010) for adding a second site in Morrisville, NC, where the F1CDx assay will be performed, was approved on December 16, 2019. PMA supplement (P170019/S013) for adding an indication for PEMZYRE® (pemigatinib) in cholangiocarcinoma patients with *FGFR2* fusions was approved on April 17, 2020. PMA supplement (P170019/S011) for adding an indication for TABRECTA® (capmatinib) in NSCLC patients with *MET* single nucleotide variants (SNVs) and indels that lead to *MET* exon 14 skipping was approved on May 6, 2020. ---PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 1 of 59 {1} PMA supplement (P170019/S015) for adding an indication for LYNPARZA® (olaparib) in metastatic castration resistant prostate cancer (mCRPC) patients with mutations in homologous recombination repair (HRR) genes was approved on May 19, 2020. PMA supplement (P170019/S016) for adding an indication for KEYTRUDA® (pembrolizumab) in patients with solid tumors high tumor mutational burden (TMB) at the cut-off of 10 mutations per megabase (mut/Mb) was approved on June 16, 2020. The current supplement was submitted to expand the indication for the F1CDx to include a companion diagnostic indication for fusions of neurotrophic tyrosine receptor kinases genes NTRK1, NTRK2, or NTRK3 fusions in patients with solid tumors who may benefit from treatment with VITRAKVI® (larotrectinib). ## 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 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 | Indication | Biomarker | Therapy | | --- | --- | --- | | Non-small cell lung cancer (NSCLC) | EGFR exon 19 deletions and EGFR exon 21 L858R alterations | Gilotrif® (afatinib), Iressa® (gefitinib), Tagrisso® (osimertinib), or Tarceva® (erlotinib) | | | EGFR exon 20 T790M alterations | Tagrisso® (osimertinib) | | | ALK rearrangements | Alecensa® (alectinib), Xalkori® (crizotinib), or Zykadia® (ceritinib) | | | BRAF V600E | Tafinlar® (dabrafenib) in combination with Mekinist® (trametinib) | | | MET single nucleotide variants (SNVs) and indels that lead to MET exon 14 skipping | Tabrecta™ (capmatinib) | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 2 of 59 {2} | Indication | Biomarker | Therapy | | --- | --- | --- | | Melanoma | BRAF V600E | Tafinlar® (dabrafenib) or Zelboraf® (vemurafenib) | | | BRAF V600E and V600K | Mekinist® (trametinib) or Cotellic® (cobimetinib) in combination with Zelboraf® (vemurafenib) | | Breast cancer | ERBB2 (HER2) amplification | Herceptin® (trastuzumab), Kadcyla® (ado-trastuzumab-emtansine), or Perjeta® (pertuzumab) | | | PIK3CA C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y alterations | Piqray® (alpelisib) | | Colorectal cancer | KRAS wild-type (absence of mutations in codons 12 and 13) | Erbitux® (cetuximab) | | | KRAS wild-type (absence of mutations in exons 2, 3, and 4) and NRAS wild-type (absence of mutations in exons 2, 3, and 4) | Vectibix® (panitumumab) | | Ovarian cancer | BRCA1/2 alterations | Lynparza® (olaparib) or Rubraca® (rucaparib) | | Cholangiocarcinoma | FGFR2 fusions and select rearrangements | Pemazyre™ (pemigatinib) | | Prostate cancer | Homologous Recombination Repair (HRR) gene (BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D and RAD54L) alterations | Lynparza® (olaparib) | | Solid tumors | TMB ≥ 10 mutations per megabase | Keytruda® (pembrolizumab) | | | NTRK1/2/3 fusions | Vitrakvi® (larotrectinib) | The test is also used for detection of genomic loss of heterozygosity (LOH) from formalin-fixed, paraffin-embedded (FFPE) ovarian tumor tissue. Positive homologous recombination deficiency (HRD) status (F1CDx HRD defined as tBRCA-positive and/or LOH high) in ovarian cancer patients is associated with improved progression-free survival (PFS) from Rubraca (rucaparib) maintenance therapy in accordance with the Rubraca product label. The F1CDx assay will be performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 3 of 59 {3} ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found 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. The assay employs a single DNA extraction method from routine FFPE biopsy or surgical resection specimens, 50-1000 ng of which undergoes whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, 1 promoter region, 1 non-coding RNA (ncRNA), and select intronic regions from 34 commonly rearranged genes, 21 of which also include the coding exons (refer to Table 2 and Table 3, below, for the complete list of genes included in F1CDx). In total, the assay therefore detects alterations in 324 genes. Using the Illumina® HiSeq 4000 platform, hybrid-capture selected libraries are sequenced to high uniform depth (targeting > 500X median coverage with > 99% of exons at coverage > 100X). Sequence data are processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number alterations (amplifications and homozygous deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic signatures including microsatellite instability (MSI), tumor mutational burden (TMB), and positive homologous recombination deficiency (HRD) status (tBRCA-positive and/or LOH high) will be reported. Table 2. Genes with full coding exonic regions included in F1CDx for the detection of substitutions, 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 | | 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 | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 4 of 59 {4} | 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 | | 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 | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 5 of 59 {5} | *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 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | ### 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. **Table 4. Instruments for use with the F1CDx assay** | Instrument | | --- | | Illumina® HiSeq 4000 | | Illumina cBot® System | | Agilent Technologies Benchbot Workstation with Integrated Bravo Automated Liquid Handler | | Beckman Biomek NX^{P} Span-8 Liquid Handler | | Hamilton Microlab STAR/STARlet Liquid Handling Workstation | | Thermo Fisher Scientific KingFisher™ Flex with 96 Deep-well Head | | Covaris LE220-Plus Focused-ultrasonicator | ### 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). PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 6 of 59 {6} ### 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 Specimens passing pathology review are queued for DNA extraction which begins with lysis of cells from FFPE tissue by digestion with a proteinase K buffer followed by automated purification using the 96-well KingFisher™ Flex Magnetic Particle Processor. After completion of DNA extraction, double-stranded DNA (dsDNA) is quantified by the Quant-iT™ PicoGreen® fluorescence assay using the provided lambda DNA standards (Invitrogen) prior to Library Construction (LC). The sample must yield a minimum of 55 ng of genomic DNA to ensure sufficient DNA for quality control (QC) and to proceed with LC. ### C. Library Construction Library Construction (LC) begins with normalization of DNA to 50-1000 ng. Normalized DNA samples are randomly sheared (fragmented) to ~200 bp by adaptive focused acoustic sonication using the Covaris LE220-Plus 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 NEB), including mixes for end repair, dA addition and ligation, are performed in 96-well plates (Eppendorf) on the Bravo Benchbot (Agilent) or Microlab STAR (Hamilton) using the “with-bead” protocol¹ to maximize reproducibility and library yield. Indexed (6 bp barcodes) sequencing libraries are PCR amplified with HiFi™ (Kapa) for 10 cycles and subsequently 1.8X SPRI purified. Purification and dilution for QC are performed. Following LC, a QC procedure is performed by quantifying single-stranded DNA (ssDNA) from purified libraries using the Quant-iT™ OliGreen® ssDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Libraries yielding insufficient sequencing library are failed. ### D. Hybrid Capture Hybrid Capture (HC) begins with normalization of each library to 500-2000 ng. Normalized samples then undergo solution hybridization which is performed using a > 50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 7 of 59 {7} 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 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 off-board clustering on the Illumina cBot with patterned flow cell technology to generate monoclonal clusters from a single DNA template followed by sequencing using sequencing by synthesis (SBS) chemistry on the Illumina HiSeq 4000. Fluorescently labeled 3'-blocked dNTPs along with a polymerase are incorporated through the flow cell to create a growing nucleotide chain that is excited by a laser. A camera captures the emission color of the incorporated base and then is cleaved off. The terminator is then removed to allow the nucleotide to revert to its natural form and to allow the polymerase to add another base to the growing chain. A new pool of fluorescently labeled 3'-blocked dNTPs are added with each new sequencing cycle. The color changes for each new cycle as a new base is added to the growing chain. This method allows for millions of discrete clusters of clonal copies of DNA to be sequenced in parallel. ### F. Sequence Analysis Sequence data are 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. PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 8 of 59 {8} 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. - 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) PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 9 of 59 {9} and distribution of alignment positions. Rearrangements are annotated for predicted function (e.g., creation of fusion gene). To determine microsatellite instability (MSI) status, 95 intronic homopolymer repeat loci (10-20 bp long in the human reference genome) with adequate coverage on the F1CDx assay are analyzed for length variability and compiled into an overall MSI score via principal components analysis (PCA). Using the 95 loci, for each sample the repeat length is calculated in each read that spans the locus. The means and variances of repeat lengths are recorded. PCA is used to project the 190-dimension data onto a single dimension (the first principal component) that maximizes the data separation, producing an MSI score. Each sample is assigned a qualitative status of MSI-High (MSI-H) or MSI-Stable (MSS); ranges of the MSI score are assigned MSI-H or MSS by manual unsupervised clustering. Samples with low coverage (< 250X median) are assigned a status of MSI-unknown. 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 PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 10 of 59 {10} 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. 100 different germline SNPs present across the entire targeted region are required to be detected by the analysis pipeline. If SNPs are not detected as expected, this results in a QC failure, as it indicates a potential processing error. ## Sensitivity Control The HapMap control pool used as the positive control is prepared to contain variants at 5%-10% MAF which must be detected by the analysis pipeline to ensure 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 PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 11 of 59 {11} 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). 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, 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, BRCA1, and BRCA2 genes | ATM | BRCA1 | BRCA2 | | --- | --- | --- | | M1T | M1V | M1R | | R2032K | M1I | M1I | | R2227C | C61G | V159M | | R2547_S2549del | C64Y | V211L | | G2765S | R71G | V211I | | R2832C | R71K | R2336P | | S2855_V2856delinsRI (annotated as S2855_V2856>RI) | R1495M | R2336H | | R3008C | E1559K | | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 12 of 59 {12} | ATM | BRCA1 | BRCA2 | | --- | --- | --- | | R3008H | D1692N | | | 8418+5_8418+8delGTGA or 8418+1_8418+4delGTGA | D1692H | | | | R1699W | | | | A1708E | | | | G1788V | | # **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. Unidentified partners (encoded as N/A) or LINC non-coding partners are also considered CDx biomarker negative. ### VI. ALTERNATIVE PRACTICES AND PROCEDURES There are FDA-approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using FFPE tumor specimens, as listed in Table 1 of the F1CDx intended use statement. The approved CDx tests are listed in Table 7, below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle. Table 7. List of FDA approved CDx assays for genes targeted by F1CDx | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | HER-2 | PathVysion HER-2 DNA Probe Kit | Abbott Molecular, Inc. | FISH | HERCEPTIN (trastuzumab) | Breast cancer | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 13 of 59 {13} Table 7. List of FDA approved CDx assays for genes targeted by F1CDx | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | | PATHWAY Anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody | Ventana Medical Systems, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | InSite HER-2/neu Kit | Biogenex Laboratories, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | SPOT-Light HER2 CISH Kit | Life Technologies, Inc. | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | Bond Oracle HER2 IHC System | Leica Biosystems | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | HER2 CISH pharmDx Kit | Dako Denmark A/S | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | INFORM HER2 Dual ISH DNA Probe Cocktail | Ventana Medical Systems, Inc. | Dual ISH | HERCEPTIN (trastuzumab) | Breast cancer | | | HercepTest | Dako Denmark A/S | IHC | HERCEPTIN (trastuzumab) PERJETA (pertuzumab) KADCYLA (ado-trastuzumab emtansine) | Breast cancer Gastric or Gastroesophageal junction adenocarcinoma | | | HER2 FISH pharmDx Kit | Dako Denmark A/S | FISH | HERCEPTIN (trastuzumab) PERJETA (pertuzumab) KADCYLA (ado-trastuzumab emtansine) | Breast cancer Gastric or Gastroesophageal junction adenocarcinoma | | BRAF-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 | | BRAF-V600E | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | TAFINLAR (dabrafenib) MEKINIST (trametinib) | NSCLC | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 14 of 59 {14} Table 7. List of FDA approved CDx assays for genes targeted by F1CDx | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | | 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 | | ALK - fusion | Vysis ALK Break Apart FISH Probe Kit | Abbott Molecular, Inc. | FISH | XALKORI (crizotinib) | NSCLC | | | ALK (D5F3) CDx Assay | Ventana Medical Systems, Inc. | IHC | XALKORI (crizotinib) | NSCLC | | EGFR - Exon 19 deletions & L858R | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TARCEVA (erlotinib) TAGRISSO (osimertinib) IRESSA (gefitinib) | NSCLC | | | therascreen EGFR RGQ PCR Kit | QIAGEN | PCR | GILOTRIF (afatinib) IRESSA (gefitinib) | NSCLC | | | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | IRESSA (gefitinib) | NSCLC | | EGFR T790M | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TAGRISSO (osimertinib) | NSCLC | | BRCA1/2 | FoundationFocus CDx_{BRCA} | Foundation Medicine, Inc. | NGS | RUBRACA (rucaparib) | Advanced ovarian cancer | | PIK3CA | therascreen PIK3CA RGQ PCR Kit | QIAGEN | PCR | PIQRAY (alpelisib) | Breast cancer | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 15 of 59 {15} **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. initially designed and developed the FoundationOne® laboratory developed test (F1 LDT), and the first commercial sample was tested in 2012. The F1 LDT has been used to detect the presence of genomic alterations in FFPE tumor tissue specimens. The F1 LDT is not FDA-cleared or -approved. The F1CDx Premarket Approval (PMA) was originally approved on November 30, 2017 by FDA (P170019) and is commercially available in the U.S. since March 30, 2018. The following PMA supplements affecting the Intended Use were approved by FDA. - • P170019/S005 was approved on April 10, 2019. - • P170019/S004 and P170019/S008 were approved on July 1, 2019. - • P170019/S009 was approved on August 21, 2019. - • P170019/S006 was approved on December 3, 2019. - • P170019/S010 was approved on December 16, 2019. - • P170019/S013 was approved on April 17, 2020. - • P170019/S011 was approved on May 6, 2020. - • P170019/S015 was approved on May 19, 2020. - • P170019/S016 was approved on June 16, 2020. ## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect test results and, subsequently, inappropriate patient management decisions. Patients with false positive results may undergo treatment with one of the therapies listed in the above intended use statement without clinical benefit and may experience adverse reactions associated with the therapy. Patients with false negative results may not be considered for treatment with the indicated therapy. There is also a risk of delayed results, which may lead to delay of treatment with the indicated therapy. For the specific adverse events related to the approved therapeutics, please see the approved drug product labels. ## IX. SUMMARY OF NONCLINICAL STUDIES ### A. Laboratory Studies The primary evidence for supporting the performance of F1CDx in detecting *NTRK1*, *NTRK2*, and *NTRK3* fusions, hereafter referred to as *NTRK1/2/3* fusions was from the data presented using intended use specimens across all validation studies. In addition to the existing platform-level validation results (P170019), analytical accuracy, intermediate precision, and limit of detection (LoD) studies as well as *in silico* analyses ---PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 16 of 59 {16} of real-world data from the Foundation Medicine clinical database were conducted to support the indication for NTRK1/2/3 fusions. Table 8 shows the distribution of tumor types in analytical studies. The counts corresponding to NTRK-positive or NTRK-negative status are with respect to the CDx rules for NTRK1/2/3 (restricting to fusions only). Table 9 shows the distribution of F1CDx NTRK1/2/3 fusions per NTRK gene evaluated in the analytical validation studies. For F1CDx platform-level validation (P170019), performance characteristics were established using DNA derived from a wide range of FFPE tissue types; tissue types associated with CDx indications were included in each study. Each study included CDx variants as well as a broad range of representative alteration types (substitution, insertion and deletion, copy number alterations, rearrangements) in various genomic contexts across several genes. Analyses of genomic signatures including MSI and TMB were also conducted. Precision data from a Conditions of Approval (CoA) study for P170019 included two NTRK samples with additional precision data to support the NTRK1/2/3 fusion CDx indication. Table 8. Distribution of Tumor Types in Analytical Studies. The counts corresponding to NTRK-positive or NTRK- negative status are with respect to the CDx rules for NTRK1/2/3 (restricting to fusions only) | Tumor Type | Accuracy | | Precision (N=7) | LoD (N=7) | | --- | --- | --- | --- | --- | | | NTRK positive (N=83) | NTRK negative (N=543) | | | | Appendix | 0 | 0 | 0 | 0 | | Breast | 6 | 17 | 0 | 1 | | Bone Sarcoma | 0 | 2 | 0 | 0 | | Cholangiocarcinoma^{1} | 2 | 2 | 1 | 0 | | CRC^{2} | 19 | 51 | 3 | 1 | | GIST | 2 | 6 | 0 | 1 | | Head and Neck | 11 | 10 | 0 | 0 | | IFS | 2 | 0 | 0 | 0 | | Lung | 7 | 246 | 0 | 1 | | Skin (includes Melanoma) | 1 | 1 | 0 | 0 | | Neuroendocrine^{3} | 1 | 2 | 0 | 0 | | Oral | 0 | 1 | 0 | 0 | | Other^{4} | 0 | 0 | 0 | 1 | | Pancreas | 0 | 2 | 0 | 0 | | Primary CNS | 0 | 1 | 0 | 0 | | Reproductive | 5 | 171 | 1 | 0 | | Salivary Gland | 11 | 11 | 1 | 1 | | Soft Tissue Sarcoma | 4 | 8 | 0 | 0 | | Thyroid | 6 | 2 | 1 | 1 | | Unknown | 5 | 1 | 0 | 0 | | Urinary^{5} | 1 | 9 | 0 | 0 | $^{1}$ Cholangiocarcinoma includes hepatic cancer PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 17 of 59 {17} ² CRC includes colon and anal cancers ³ Neuroendocrine includes thymus cancers ⁴ Other includes malignant neoplasm with neuroendocrine features and unspecified primary serous carcinoma ⁵ Urinary includes congenital mesoblastic nephroma Table 9. Distribution of CDx NTRK1/2/3 Fusions (as determined by F1CDx per F1CDx's NTRK1/2/3 Fusion Rules) | Study | NTRK1 | NTRK2 | NTRK3 | | --- | --- | --- | --- | | Accuracy | 46 | 6 | 31 | | Precision | 2 | 1 | 4 | | LoD | 2 | 2 | 3 | ### 1. Analytical Accuracy/Concordance #### a. Comparison to an Orthogonal Method for NTRK1/2/3 rearrangements Analytical accuracy was evaluated by demonstrating concordance between F1CDx and an externally validated NGS assay (evNGS) for the detection of NTRK1/2/3 rearrangements. The analytical accuracy study was performed with available residual DNA previously extracted from FFPE clinical specimens (38 samples with sufficient remaining material) from patients with solid tumors enrolled in the clinical studies LOXO-TRK - 14001 (NCT02122913), -15002 (NAVIGATE, NCT02576431), and -15003 (SCOUT, NCT02637687) that supported VITRAKVI (larotrectinib) approval, refer to Section X for study details. The 38 samples from the clinical studies LOXO-TRK - 14001, - 15002, and -15003 included the following cancer types: bone sarcoma (1), breast (1), cholangiocarcinoma (2), colorectal cancer (7), infantile fibrosarcoma (2), lung (4), melanoma (1), oral cancer (1), primary CNS (1), salivary gland (3), soft tissue sarcoma (10), neuroendocrine (2), thyroid (2), and urinary (1) cancer. Due to the limited number of available clinical trial samples, the study also included NTRK1/2/3 rearrangement positive (N=94) samples from the FMI clinical archives which were previously evaluated at FMI. These 94 samples included samples from breast (14), cholangiocarcinoma (1), colorectal cancer (15), gastrointestinal stromal tumor GIST (2), head and neck (14), lung (10), neuroendocrine (1), reproductive (11), salivary gland (9), thyroid (6), and urinary (6) cancer. For five (5) samples, the cancer type was not known. The study also included NTRK1/2/3 rearrangement negative samples previously processed and tested by the evNGS in prior concordance validation studies. The previously executed concordance studies were newly evaluated for the presence or absence of NTRK1/2/3 rearrangements. This evaluation included 474 samples that were tested in previous concordance analyses. PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 18 of 59 {18} NTRK1/2/3 rearrangement positive and negative sample sets included disease ontologies that were representative of the intended use population. However, an additional ten (10) pediatric samples and ten (10) salivary gland samples were also included as part of the NTRK1/2/3 rearrangement negative sample set to better align with the intended use population. The ten (10) pediatric samples included the following cancer types: bone sarcoma (1), GIST (1), head and neck (1), pancreas (1), soft tissue sarcoma (1), and urinary cancer (1). For one sample the cancer type was unknown. In total, 626 clinical FFPE samples (543 NTRK fusion negative and 83 NTRK positive fusion samples) from patients with solid tumors were evaluated in this analytical accuracy study. Table 10 shows a breakdown of the sample selection based on NTRK rearrangements (includes fusion and non-fusion rearrangements). Table 10. Samples Evaluated for NTRK1/2/3 Rearrangement Concordance. | Sample Subset | Disease Cohort Represented | Number of Samples | | --- | --- | --- | | NTRK1/2/3 Rearrangement Positive^{a} | Pan-tumor | 94 | | NTRK1/2/3 Rearrangement Negative^{a} | Pediatric | 10 | | | Salivary Gland | 10 | | NTRK1/2/3 Rearrangement Positive and Rearrangement Negative (from LOXO 14001, 15002, and 15003)^{a} | Pan-tumor | 38 | | Total Samples Processed by F1CDx and evNGS | | | | NTRK1/2/3 Rearrangement Negative for evNGS Re-analysis^{b} | Reproductive | 152 | | | Reproductive | 123 (121^{c}) | | | Lung, Neuroendocrine | 34 | | | Pan-tumor | 34 | | | Breast | 74 | | | Lung | 15 | | | Lung | 187 | | Total Samples for evNGS Re-analysis Only | | 474 | | Total Samples Evaluated for Concordance | | 626 | $^{a}$ Samples processed by F1CDx and evNGS assays. $^{b}$ Samples previously processed as part of prior concordance validations with evNGS; only reanalysis by evNGS performed for this validation. $^{c}$ Two (2) samples were considered positive per the primary analysis approach. These two samples would be F1CDx biomarker negative per the F1CDx biomarker rules for NTRK 1/2/3 fusions PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 19 of 59 {19} There were no invalid results from either F1CDx or evNGS. Two analyses were conducted to assess concordance. ## Primary Analysis The primary analysis focused on the concordance of NTRK1/2/3 rearrangement detection by F1CDx and evNGS assay by considering a sample to be positive if any NTRK1/2/3 rearrangements was present, otherwise it was considered as negative. This analysis was conducted to determine the analytical accuracy of NTRK1/2/3 rearrangement detection as fusions are a subset of rearrangements, and the methodology to detect NTRK1/2/3 rearrangement (fusions and non-fusion rearrangements) is the same. The samples were categorized into two subsets based on the origin of the sample: 588 samples from the FMI clinical archives whose status was initially determined and ascertained by an F1CDx device, and 38 clinical samples from the larotrectinib clinical trial patients whose NTRK fusion status was initially determined and ascertained by the local clinical trial assays (LCTAs) prior to enrollment in the VITRAKVI clinical trials. As a result of the two distinct sample selection methods by assay, each subset of samples was evaluated separately for the calculation of percent positive agreement (PPA) and negative percent agreement (NPA) and adjusted according to a prevalence (0.32%) of NTRK1, NTRK2, and NTRK3 in the intended use population⁹. Table 11 and Table 12 summarize the results for the primary analysis for Subset 1, while Table 13 and Table 14 summarize the results for the primary analysis for Subset 2. Subset 1 refers to the 588 samples from the FMI database and Subset 2 refers to the 38 VITRAKVI clinical trial samples. Table 11. Contingency Table Comparing F1CDx and evNGS Results for the Detection of NTRK1/2/3 Rearrangements Based on the Primary Analysis - Sample Subset 1 | NTRK1/2/3 | evNGS | | | | | --- | --- | --- | --- | --- | | | | NTRK + | NTRK - | Total | | F1CDx | NTRK + | 78 | 18 | 96 | | | NTRK - | 0 | 492 | 492 | | | Total | 78 | 510 | 588 | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 20 of 59 {20} **Table 12. Summary of Agreement Statistics Based on the Primary Analysis -Sample Subset 1** | Agreement Statistic | Estimate (95% CI)^{a} | | --- | --- | | PPV | 81.25% (72.30%, 87.80%) | | NPV | 100.00% (99.23%, 100.00%) | | Adjusted PPA | 100.00% (95.31%, 100.00%)^{b} | | Adjusted NPA | 99.94% (99.91%, 99.96%) | | Unadjusted PPA | 100.00% (95.31%, 100.00%) | | Unadjusted NPA | 96.47% (94.49%, 97.76%) | $^{a}$ For unadjusted value, 95% 2-sided score CI was computed; for adjusted value, 95% 2-sided CI was calculated by selecting 2.5% and 97.5% percentile of bootstrapped (10000 times) samples. $^{b}$ 95% 2-sided score CI of unadjusted PPA was computed given adjusted PPA was 100%. **Table 13. Contingency Table Comparing F1CDx and evNGS Results for the Detection of NTRK1/2/3 Rearrangements Based on the Primary Analysis - Sample Subset 2** | NTRK1/2/3 | UW-OncoPlex | | | | | --- | --- | --- | --- | --- | | | | NTRK + | NTRK - | Total | | F1CDx | NTRK + | 16 | 0 | 16 | | | NTRK - | 2 | 20 | 22 | | | Total | 18 | 20 | 38 | **Table 14. Summary of Agreement Statistics Based on the Primary Analysis - Sample Subset 2** | Agreement Statistic | Estimate (95% CI)^{a} | | --- | --- | | PPA | 88.89% (67.20%, 96.90%) | | NPA | 100.00% (83.89%, 100.00%) | | Adjusted PPV | 100.00% (80.64%, 100.00%)^{b} | | Adjusted NPV | 99.96% (99.91%, 100.00%) | | Unadjusted PPV | 100.00% (80.64%, 100.00%) | | Unadjusted NPV | 90.91% (72.19%, 97.47%) | $^{a}$ For unadjusted value, 95% 2-sided score CI was computed; for adjusted value, 95% 2-sided CI was calculated by selecting 2.5% and 97.5% percentile of bootstrapped (10000 times) samples. $^{b}$ 95% 2-sided score CI of unadjusted PPV was computed given adjusted PPV was 100%. The entire data set (n=626 samples) was evaluated by combining both sample subsets together to calculate the weighted PPA, NPA, positive predictive value (PPV), and negative predictive value (NPV), and bootstrapping the 626 samples 10000 times. Table 15 presents the weighted PPA, PPV, NPA, and NPV based on the bootstrapping the combined data sets 10000 times. The weighted PPA was 90.00% (75.00%, 100.00%) and the weighted NPA was 99.94% (99.92%, 99.97%). PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 21 of 59 {21} Table 15. Weighted PPA, PPV, NPA, and NPV for the Detection of NTRK1/2/3 Rearrangements Combining Sample Subsets 1 and 2 Based on the Primary Analysis. | Weighted Agreement Statistic | Estimate (95%CI)^{a} | | --- | --- | | Weighted PPA | 90.00% (75.00%, 100.00%) | | Weighted PPV | 98.13% (97.53%, 99.08%) | | Weighted NPA | 99.94% (99.92%, 99.97%) | | Weighted NPV | 100.00% (100.00%, 100.00%) | In total there were 20 samples that were discordant based on the primary analysis. In 18 of the discordant samples, the F1 CDx assay detected NTRK1/2/3 rearrangements, while the evNGS assay did not detect the rearrangements. In the remaining 2 discordant samples, NTRK1/2/3 rearrangements were detected by the evNGS assay but not the F1CDx assay. Of the 18 discordant calls that are F1CDx positive (present) and evNGS negative (absent), based on the primary analysis: - Nine (9) samples had breakpoints residing outside of evNGS baited regions - Five (5) samples had the event observed but were not reported due to being below the evNGS assay limit of detection - One (1) sample had an intragenic NTRK3-NTRK3 rearrangement that was intentionally not reported by evNGS due to being intragenic - Two (2) samples were not reported by evNGS likely due to low sample quality as reported by the evNGS assay - One sample (1) (NTRK3-KDM2A) was not detected by evNGS ## Secondary Analysis The secondary analysis focused on the concordance of NTRK1/2/3 rearrangement detection that results in an NTRK1/2/3 fusion event per the F1CDx biomarker rule for the NTRK1/2/3 fusion CDx biomarker. In the secondary analysis, a sample was considered F1CDx positive only if it met the NTRK1/2/3 biomarker rule, otherwise it was considered as F1CDx negative. Table 16 and Table 17 summarize the results for the secondary analysis for Subset 1, while Table 18 and Table 19 summarize the results for the secondary analysis for Subset 2. Subset 1 refers to the 588 samples from the FMI database and Subset 2 refers to the 38 VITRAKVI clinical trial samples. PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 22 of 59 {22} **Table 16. Contingency Table Comparing F1CDx and evNGS Results for the Detection of NTRK1/2/3 Rearrangements Based on the Secondary Analysis - Sample Subset 1** | *NTRK1/2/3* | EvNGS | | | | | --- | --- | --- | --- | --- | | | | *NTRK +* | *NTRK -* | *Total* | | F1CDx | *NTRK +* | 64 | 4 | 68 | | | *NTRK -* | 10 | 510 | 520 | | | *Total* | 74 | 514 | 588 | **Table 17. Summary of Agreement Statistics Based on the Secondary Analysis - Sample Subset 1** | Agreement Statistic | Estimate (95% CI)^{a} | | --- | --- | | PPV | 94.12% (85.83%, 97.69%) | | NPV | 98.08% (96.50%, 98.95%) | | Adjusted PPA | 13.58% (8.66%, 25.25%) | | Adjusted NPA | 99.98% (99.96%, 100.00%) | | Unadjusted PPA | 86.49 % (76.88%, 92.49%) | | Unadjusted NPA | 99.22% (98.02%, 99.70%) | $^{a}$ For unadjusted value, 95% 2-sided score CI was computed; for adjusted value, 95% 2-sided CI was calculated by selecting 2.5% and 97.5% percentile of bootstrapped (10000 times) samples. **Table 18. Contingency Table Comparing F1CDx and evNGS Results for the Detection of NTRK1/2/3 Rearrangements based on the Secondary Analysis - Sample Subset 2** | *NTRK1/2/3* | EvNGS | | | | | --- | --- | --- | --- | --- | | | | *NTRK +* | *NTRK -* | *Total* | | F1CDx | *NTRK +* | 15 | 0 | 15 | | | *NTRK -* | 3 | 20 | 23 | | | *Total* | 18 | 20 | 38 | **Table 19. Summary of Agreement Statistics Based on the Secondary Analysis - Sample Subset 2** | Agreement Statistic | Estimate (95% CI)^{a} | | --- | --- | | PPA | 83.33% (60.78%, 94.16%) | | NPA | 100.00% (83.89%, 100.00%) | | Adjusted PPV | 100.00% (79.61%, 100.00%)^{b} | | Adjusted NPV | 99.95% (99.88%, 100.00%) | | Unadjusted PPV | 100.00% (79.61%, 100.00%) | | Unadjusted NPV | 86.96% (67.87%, 95.46%) | $^{a}$ For unadjusted value, 95% 2-sided score CI was computed; for adjusted value, 95% 2-sided CI was calculated by selecting 2.5% and 97.5% percentile of bootstrapped (10000 times) samples. $^{b}$ 95% 2-sided score CI of unadjusted PPV was computed given adjusted PPV was 100%. The entire data set (n=626 samples) was evaluated by combining both sample subsets together to calculate the weighted PPA, NPA, PPV, and NPV, and PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 23 of 59 {23} bootstrapping the 626 samples 10000 times. Table 20 presents the weighted PPA, PPV, NPA, and NPV based on the bootstrapping the combined data sets 10000 times. The weighted PPA was 54.08% (37.94%, 71.37%) and the weighted NPA was 99.98% (99.96%, 100.00%). Table 20. Weighted PPA, PPV, NPA, and NPV for the Detection of NTRK1/2/3 Rearrangements Combining Sample Subsets 1 and 2 Based on the Secondary Analysis | Weighted Agreement Statistic | Estimate (95% CI) | | --- | --- | | Weighted PPA | 54.08% (37.94%, 71.37%) | | Weighted PPV | 97.53% (94.57%, 99.54%) | | Weighted NPA | 99.98% (99.96%, 100.00%) | | Weighted NPV | 98.14% (96.90%, 99.24%) | In total there were 17 samples that were discordant based on the secondary analysis. Of 17 discordant samples, 13 were F1CDx biomarker negative but evNGS biomarker positive. Of the 13 samples, 10 were non-fusion rearrangements and thus considered as F1CDx biomarker negative per the F1CDx biomarker rules. However, these 10 were considered evNGS biomarker positive as the evNGS assay cannot differentiate between fusions and rearrangements. Thus, these 10 were not considered as discordant per the primary analysis. The three clinical trial samples that were positive by the evNGS assay but negative by F1CDx were from patients enrolled in the clinical trials that did not respond to VITRAKVI, (two patients had stable disease and one had progressive disease). ## 2. Analytical Sensitivity ### a. Limit of Blank (LoB) The LoB was confirmed using DNA from nine (9) samples from patients with solid tumors that were biomarker negative. These comprised samples from the following diseases and specimen types in parenthesis: breast invasive ductal carcinoma (breast), colon adenocarcinoma (colon), stomach adenocarcinoma (stomach), pediatric brain medulloblastoma (brain), pleura mesothelioma (pleura), small intestine neuroendocrine carcinoma (small intestine), pancreas carcinoma (whipple resection), salivary gland adenoid cystic carcinoma (lung), and thyroid papillary carcinoma (thyroid). Each biomarker-negative sample was assessed in replicates of seven (7), resulting in a total of 63 sample aliquots to assess LoB. The number of replicate samples with incorrect calls was counted and converted into a percentage with respect to the number of all replicate LoB samples and reported as the percentage of false-positive results. If the percentage of false-positive results did PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 24 of 59 {24} not exceed 5% (type I error risk a=0.05), then at least 95% of the result was zero and LoB = zero was confirmed. For sample aliquots evaluated to assess LoB, rearrangements in the NTRK1/2/3 genes were not reported in any of the 63 sample aliquots. The percent of samples with incorrect calls was zero and confirmed the LoB = zero. # b. Limit of Detection (LoD) The F1CDx LoD for the detection of NTRK1, NTRK2, and NTRK3 fusions was investigated by assessing seven (7) samples (see Table 21). Selection of specimens for assessment of NTRK fusions represented various tumor types as shown in Table 21. Table 21. Samples assessed in the LoD study for NTRK fusions | Sample | Target NTRK Gene | Partner Gene | Alteration Type | Fusion Partner or Alteration Description | Disease Indication (Specimen Site) | | --- | --- | --- | --- | --- | --- | | 1 | NTRK1 | TPR | Fusion (CDx - positive) | 5'-TPR(x1-21)-NTRK1(x10-17) | NSCLC (Lung) | | 2 | NTRK1 | LMNA | Fusion (CDx - positive) | 5'-LMNA(ex1-2 NM_005572)-NTRK1(ex10-17 NM_002529) | CRC (Colon) | | 3 | NTRK2 | BCR | Fusion (CDx - positive) | 5'-BCR(ex1-14 NM_004327)-NTRK2(ex12-21 NM_006180) | Malignant neoplasm w/ neuroendocrine feature (Brain) | | 4 | NTRK2 | GARNL3 | Fusion (CDx - positive) | 5'-GARNL3 (NM_032293 exons 1-2) – NTRK2 (NM_006180 exons 16-21) | Breast carcinoma (NOS) (Adrenal Gland) | | 5 | NTRK3 | ETV6 | Fusion (CDx - positive) | 5'-ETV6 (x1-5) – NTRK3 (x14-19 NM_002530) | Small intestine GIST Sarcoma (Small Intestine) | | 6 | NTRK3 | ETV6 | Fusion (CDx - positive) | 5'-ETV6(ex1-5 NM_001987)-NTRK3(ex14-19 NM_002530) | Thyroid papillary carcinoma (Lymph Node) | | 7 | NTRK3 | ETV6 | Fusion (CDx - positive) | 5'-ETV6(ex1-5 NM_001987)-NTRK3(ex15-19 NM_002530) | Salivary gland carcinoma (Parotid Gland) | To establish the LoD each sample was assessed at five (5) targeted computational tumor purity levels (2.5%, 5%, 10%, 15%, and 20%). Computational tumor purity is calculated by fitting the observed log-ratio and minor allele frequency data with statistical models that predict a genome-wide copy number profile, tumor ploidy, and tumor purity (i.e., computational tumor purity). The log-ratio profile is obtained by normalizing aligned tumor sequence reads by dividing read depth by that of a process-matched normal control, followed by a GC-content bias correction using Loess regression. The minor PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 25 of 59 {25} allele frequency profile is obtained from the heterozygous genome-wide SNPs. Twenty (20) replicates were assessed for each dilution level, except 20%, which examined fourteen (14) replicates. Ninety-four (94) replicates were tested per sample. Each specimen was evaluated close to the minimum input requirements of the assay (50 ng), representing the most challenging evaluation of rearrangement detection. LoDs were determined based on tumor purity and reads using the empirical hit rate method for NTRK1, NTRK2, and NTRK3: - Empirical hit rate approach was used when there were less than three hit rate levels. The LoD is defined as the lowest level (tumor purity or reads) with at least 95% detection. A summary of the LoD results based on reads and tumor purity using the empirical hit rate method for NTRK gene alteration in each sample evaluated are shown in Table 22. Table 22. LoD of NTRK1/2/3 Rearrangements Based on the Empirical Hit rate Method by Sample | Sample | Target NTRK Gene | Partner Gene | Alteration Type | NTRK1/2/3 LoD (mean % Tumor Purity) | NTRK1/2/3 LoD (# of chimeric reads) | | --- | --- | --- | --- | --- | --- | | 1 | NTRK1 | TPR | Fusion (CDx - positive) | 12.10%^{1} | 9.55 | | 2 | NTRK1 | LMNA | Fusion (CDx - positive) | 11.90% | 24.55^{1} | | 3 | NTRK2 | BCR | Fusion (CDx - positive) | 11.50%^{1} | 19.75 | | 4 | NTRK2 | GARNL3 | Fusion (CDx - positive) | 6.70% | 24.16^{1} | | 5 | NTRK3 | ETV6 | Fusion (CDx - positive) | 4.10% | 9.05 | | 6 | NTRK3 | ETV6 | Fusion (CDx - positive) | 5.70% | 14.65^{1} | | 7 | NTRK3 | ETV6 | Fusion (CDx - positive) | 6.10%^{1} | 8.65 | $^{1}$Final LoD for each NTRK gene fusion. The final LoD for each NTRK gene fusion presented was determined as the highest LoD observed per gene. The LoD for each gene are: - NTRK1 fusion: 12.10% tumor purity and 24.55 for chimeric reads - NTRK2 fusion: 11.50% tumor purity and 24.16 for chimeric reads - NTRK3 fusion: 6.10 % tumor purity and 14.65 for chimeric reads Given the limited number of samples with NTRK1 and NTRK2 fusions evaluated to determine the assay LoD, a post-market study is planned with additional NTRK1 and NTRK2 fusions samples (see section XIII). ### 3. Analytical Specificity Refer to the Summary of Safety and Effectiveness Data P710019 (Section IX.A.3) for F1CDx platform validation of analytical specificity, including interfering substances and in silico hybrid capture bait specificity. PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 26 of 59 {26} #### 4. Carryover/Cross-Contamination Refer to the Summary of Safety and Effectiveness Data P170019 (Section IX.A.4) for F1CDx platform validation of carryover/cross-contamination. #### 5. Precision and Reproducibility ##### a. Intermediate Precision To support the F1CDx performance characteristics for the detection of *NTRK1*, *NTRK2*, and *NTRK3* fusions, the precision of seven (7) samples from patients with solid tumors were evaluated at a challenging DNA input (close to 50 ng); refer to Table 23 for the samples evaluated in this study. These samples evaluated had computational tumor purity ranging from 17.2% to 40.1%. The cut-off for a passing sample based on computational tumor purity is 20% and samples evaluated in the precision study included samples near the computational tumor purity input specification of 20% tumor purity for the F1CDx assay (see Table 23 and Table 24). The mean chimeric read count for these samples ranged from 8.48 to 159.04. The fold LoD based on chimeric reads ranged from 0.58 xLoD to 10.86 xLoD **Table 23. Samples Evaluated in the Intermediate Precision Study.** | Sample | Target *NTRK Gene* | Partner Gene | Alteration Type | Fusion Partner or Alteration Description | Disease Ontology | | --- | --- | --- | --- | --- | --- | | 1 | *NTRK3-* | *ETV6* | Fusion (CDx-Positive) | 5'-NTRK3(ex1-13 NM_002530)-ETV6(ex6-8 NM_001987) | Thyroid carcinoma | | 2 | *NTRK3* | *ETV6* | Fusion (CDx-Positive) | 5'-ETV6(ex1-5 NM_001987)-NTRK3(ex15-19 NM_002530) | Salivary gland carcinoma | | 3 | *NTRK1* | *TPM3* | Fusion (CDx-Positive) | 5'-TPM3(NM_153649)-NTRK1(NM_002529) fusion (T7; N12) | CRC | | 4 | *NTRK3* | *ETV6* | Fusion (CDx-Positive) | 5'-ETV6(ex1-5 NM_001987)-NTRK3(ex15-19 NM_002530) | CRC | | 5 | *NTRK1* | *LMNA* | Fusion (CDx-Positive) | 5'-LMNA(NM_005572)-NTRK1(NM_002529) fusion (L4; N11) | CRC | | 6 | *NTRK2* | *DSTYK* | Fusion (CDx-Positive) | 5'-NTRK2(NM_006180)-DSTYK(NM_015375) fusion (N13*; D11) | Ovary Serous Carcinoma | | 7 | *NTRK3* | *ETV6* | Fusion (CDx-Positive) | 5'-NTRK3(NM_002530)-ETV6(NM_001987) fusion (N14; E6) | Liver cholangiocarcinoma | For the assessment of repeatability, each sample was divided into either 24 or 36 aliquots, with 12 aliquots processed in duplicate or triplicate under the same conditions. The conditions were applied on a plate-level and included the same operator, same day, same reagent lot and same sequencer. The result was PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 27 of 59 {27} considered concordant if all duplicates or triplicates matched the majority call for all aliquots of that sample. Table 24 summarizes the repeatability statistics across samples evaluated. **Table 24. Repeatability of variant calling.** | Sample | Mean TP | Mean Reads | Fold LoD based on Reads | Target NTRK Gene | Partner Gene | # Agree | Total # | Agreement (95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 40.1% | 39.25 | 2.68 | *NTRK3* | *ETV6* | 12 | 12 | 100.00% (73.50%, 100.00%) | | 2 | 20.0% | 35.39 | 2.42 | *NTRK3* | *ETV6* | 12 | 12 | 100.00% (73.50%, 100.00%) | | 3 | 17.2% | 22.71 | 0.93 | *NTRK1* | *TPM3* | 12 | 12 | 100.00% (73.50%, 100.00%) | | 4 | 34.4% | 159.04 | 10.86 | *NTRK3* | *ETV6* | 12 | 12 | 100.00% (73.50%, 100.00%) | | 5 | 18.2% | 41.52 | 1.69 | *NTRK1* | *LMNA* | 11 | 11 | 100.00% (71.50%, 100.00%) | | 6 | 35.2% | 35.04 | 1.45 | *NTRK2* | *DSTYK* | 11 | 11 | 100.00% (71.50%, 100.00%) | | 7 | 20.0% | 8.48 | 0.58 | *NTRK3* | *ETV6* | 11 | 12 | 91.70%, (61.50%, 99.80%) | Abbreviation: TP: tumor purity Reproducibility in the seven (7) samples was evaluated by processing aliquots originating from the same source DNA sample, under conditions where one factor was changed at a time (e.g., reagent lot and sequencers). The result was considered concordant when the aliquot matched the targeted and majority call of all 24 or 36 replicates. Table 25 summarizes the reproducibility statistics across the seven (7) samples evaluated. **Table 25. Reproducibility of variant calling** | Sample | Mean TP | Mean Reads | Fold LoD based on Reads | Target NTRK Gene | Partner Gene | # Agree | Total # | Agreement (95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 40.1% | 39.25 | 2.68 | *NTRK3* | *ETV6* | 36 | 36 | 100.00% (90.26%, 100.00%) | | 2 | 20.0% | 35.39 | 2.42 | *NTRK3* | *ETV6* | 36 | 36 | 100.00% (90.26%, 100.00%) | | 3 | 17.2% | 22.71 | 0.93 | *NTRK1* | *TPM3* | 24 | 24 | 100.0% (85.75%, 100.00%) | | 4 | 34.4% | 159.04 | 10.86 | *NTRK3* | *ETV6* | 23 | 23 | 100.0% (85.18%, 100.00%) | | 5 | 18.2% | 41.52 | 1.69 | *NTRK1* | *LMNA* | 23 | 23 | 100.0% (85.18%, 100.00%) | | 6 | 35.2% | 35.04 | 1.45 | *NTRK2* | *DSTYK* | 23 | 23 | 100.0% | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 28 of 59 {28} | | | | | | | | | (85.18%, 100.00%) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 7 | 20.0% | 8.48 | 0.58 | NTRK3 | ETV6 | 23 | 24 | 95.8% (78.90%, 99.90%) | Abbreviations: TP: tumor purity Given the limited number of samples with NTRK2 fusions evaluated in the precision study, a post-market study is planned to supplement the precision study with additional samples (see section XIII). # b. Site-to-Site reproducibility A reproducibility study to include the second site in Morrisville, North Carolina was not conducted to support the NTRK indication. Study results from a site-to-site reproducibility will be provided as a post-market study (see section XIII). # 6. Reagent Lot Interchangeability Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDxBRCA assay and F1CDx. For reagent lot interchangeability performance data, please see the Summary of Safety and Effectiveness Data for P160018. # 7. Stability Please refer to the Summary of Safety and Effectiveness Data P170019 [Section IX.A.7(a,b)] for F1CDx platform validation of reagent, DNA, and FFPE slide stability. # 8. General Laboratory Equipment and Reagent Evaluation # a. DNA Amplification Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDxBRCA assay and F1CDx. For DNA amplification performance data, see the Summary of Safety and Effectiveness Data for P160018. # b. DNA Extraction For F1CDx platform-level validation, the performance of DNA extraction from FFPE tumor specimens was evaluated. For details, refer to Section IX.A.8(b) of Summary of Safety and Effectiveness Data P170019. # c. Guard banding/Robustness Guard banding study results were leveraged from the F1CDx platform validation to evaluate the performance of the F1CDx assay and the impact of process variation with regard to uncertainty in the measurement of DNA concentration at various stages of the process. For details, refer to Section IX.A.9 in Summary of Safety and Effectiveness Data P170019. PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 29 of 59 {29} ## 9. Real-World Evidence Demonstrating Sequencing Quality Metrics for the Detection of *NTRK* Alterations A study analyzing F1CDx assay data was conducted to provide coverage analysis based on real-world data to support the pan-tumor indication for *NTRK1/2/3* rearrangements. Since F1CDx's approval, 76,597 samples have been processed by the DX1 baitset used by F1CDx for the most prevalent *NTRK* tumor specimen sites listed in Figure 1. An *in-silico* analysis was performed to present median exon coverage across these tumor specimen sites. Figure 1 and Table 26 show median exon coverage for all tumor specimen sites to be around 900x, which is above the quality control (QC) threshold of 250x.  **Figure 1. Median exon coverage for multiple tumor types in the F1CDx clinical database.** Table 26 summarizes critical post-DNA extraction QC metrics, namely, the mean of LC DNA yield, HC DNA yield, median exon coverage, % target 100X coverage, and the pass rate for the 15 different biopsy specimen sites. **Table 26. Critical Post-DNA Extraction QC Metrics** | Specimen Site | Mean LC Yield (ng) | Mean HC Yield (ng) | Mean Median Exon Coverage | % Target 100X Coverage | % Pass | | --- | --- | --- | --- | --- | --- | | Appendix | 7805.52 | 517.50 | 827 | 97.74 | 91.73 | PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 30 of 59 {30} | Specimen Site | Mean LC Yield (ng) | Mean HC Yield (ng) | Mean Median Exon Coverage | % Target 100X Coverage | % Pass | | --- | --- | --- | --- | --- | --- | | Brain | 8118.94 | 537.61 | 778 | 95.48 | 98.90 | | Breast | 7578.56 | 526.12 | 778 | 95.52 | 99.06 | | Colon | 7763.74 | 505.53 | 828 | 96.24 | 99.05 | | Esophagus | 7568.68 | 524.17 | 765.5 | 97.16 | 98.97 | | Gastro-esophageal junction | 7050.72 | 487.46 | 767 | 96.02 | 96.81 | | Liver | 7341.55 | 538.11 | 750 | 96.77 | 97.79 | | Lung | 7483.32 | 527.89 | 765 | 96.96 | 97.79 | | Pancreas | 7139.99 | 521.69 | 764 | 95.63 | 96.90 | | Pancreatobiliary | 7079.14 | 503.75 | 782 | 94.31 | 91.87 | | Prostate | 5652.69 | 434.15 | 753 | 91.93 | 92.82 | | Salivary Gland | 7753.89 | 518.89 | 822 | 95.22 | 99.20 | | Skin | 7526.18 | 514.82 | 785 | 96.02 | 99.04 | | Soft Tissue | 7724.26 | 533.95 | 768 | 95.69 | 98.32 | | Thyroid | 8173.36 | 506.99 | 827 | 96.67 | 97.00 | ### 10. In-silico Assessment of NTRK Bait set Coverage Analyses The DX1 baitset covers coding regions of NTRK1, NTRK2, and NTRK3, as well as select introns in NTRK1 (introns 8, 9, 10, and 11) and NTRK2 (intron 12). However NTRK1 intron 8, and NTRK2 intron 12 are not fully baited. Though NTRK3 introns are not baited, ETV6, the most common fusion partner for NTRK3, has introns that are baited allowing for detection of ETV6-NTRK3 fusions (ETV6 introns 5 and 6). However, ETV6 intron 5 is not fully baited. Further, a portion of fusion events between these two genes are likely undetected by F1CDx as a result of DX1 not having baits for intron 4 of ETV6. An in-silico analysis was performed with F1CDx test results of 12,464 clinical specimens across solid tumors to assess the baitset coverage levels of NTRK. Data from 12,464 clinical samples evaluated by F1CDx showed median exon coverage for the target regions in NTRK1, NTRK2, and NTRK3 >250X in >99% of specimens. A decrease in coverage, albeit to a small degree, was seen in NTRK1 intron 8 due that region being partially baited. ### 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 reasonable assurance of safety and effectiveness for F1CDx for detection of NTRK1, NTRK2 and NTRK3 fusions in patients with solid tumors who may benefit from treatment PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 31 of 59 {31} with VITRAKVI® (larotrectinib), was established through a clinical bridging study using tumor tissue FFPE specimens from patients enrolled in the LOXO-TRK-14001 (Bayer 20288, NCT02122913), -15002 (Bayer 20289, NAVIGATE, NCT02576431), and -15003 (Bayer 20290, SCOUT, NCT02637687), 14001, 15002, and 15003 clinical trials respectively, with known NTRK fusion status and an additional set of NTRK fusion negative samples from the FMI archives. A summary of the clinical study is presented below. ### A. Study Design The clinical effectiveness of F1CDx for detecting NTRK1, NTRK2, and NTRK3 fusions in patients with solid tumors who may benefit from treatment with VITRAKVI was demonstrated in a retrospective analysis of tumor tissue FFPE specimens from patients enrolled in studies 14001, 15002, and 15003 and an additional set of NTRK fusion negative tumor tissue FFPE specimens from the FMI archives. A bridging study was conducted to assess: 1) concordance of results for the NTRK gene fusion status between the F1CDx assay and the local clinical trial assays (LCTAs) used to determine patient eligibility for enrollment, and 2) the clinical utility of F1CDx in identifying patients with NTRK fusion positive status for treatment with VITRAKVI. Below is a summary of the clinical studies 140001, 15002 and 150003. ### Therapeutic Clinical Study TRK-14001 (Study 14001) Study 14001 is an ongoing multicenter open-label Phase 1 dose escalation study in adult patients with advanced solid tumors (all comers) unselected for the presence of NTRK gene fusions. Study objectives include characterization of safety, the identification of a maximum tolerated dose (MTD) and/or an appropriate dose for further study, and the characterization of pharmacokinetics. Planned dose levels ranged from 50 mg once daily (QD) to 200 mg twice daily (BID). Following the dose escalation portion of the study, a dose expansion was initiated for patients with solid PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 32 of 59 {32} tumors with documented NTRK fusions and for patients who the Investigator believed might benefit from a highly selective tropomyosin-related kinase (TRK) inhibitor. Diagnoses and main criteria for inclusion for Study LOXO-TRK-14001 included: - Adult patients with a locally advanced or metastatic solid tumor that has progressed or is nonresponsive to available therapies, are unfit for standard chemotherapy or for which no standard or available curative therapy exists. - Proof of a malignancy harboring an NTRK fusion as previously determined with prior testing from a Clinical Laboratory Improvement Amendments (CLIA)-certified or equivalent-certified laboratory. - At least 18 years of age. - Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, or 2 and life expectancy of at least 3 months. - Adequate hematologic, hepatic and renal status, as defined in the individual study protocols. ### Therapeutic Clinical Study TRK-15002 (Study 15002) Study 15002 is an ongoing multicenter open-label Phase 2 “basket” study in patients age 12 and older with recurrent advanced solid tumors with locally-advanced or metastatic malignancy with an NTRK1, NTRK2 or NTRK3 gene fusion as identified through molecular assays performed at CLIA or other similarly-certified laboratories. Study objectives include the characterization of antitumor activity, specifically best overall response, overall response rate (ORR), duration of response, progression-free survival (PFS), and overall survival (OS). Patients are enrolled into 1 of 8 tumor-defined cohorts consisting of non-small cell lung, thyroid, sarcoma, colorectal, salivary gland, biliary, primary central nervous system (CNS), and “all other”/ non-measurable cancers. All patients receive a starting dose of 100 mg BID, with dose modifications as needed for toxicity. Diagnoses and main criteria for inclusion for Study LOXO-TRK-15002 included: - Locally-advanced or metastatic malignancy with an NTRK1, NTRK2 or NTRK3 gene fusion, identified through molecular assays as routinely performed at CLIA or other similarly-certified laboratories. - Patients must have received prior standard therapy appropriate for their tumor type and stage of disease, or in the opinion of the investigator, would be unlikely to tolerate or derive clinically meaningful benefit from appropriate standard of care therapy. - Patients must have had at least 1 measurable lesion as defined by Response Evaluation Criteria in Solid Tumors, RECIST v1.1. Patients without RECIST v1.1 measurable disease (e.g., evaluable disease only) were eligible for enrollment to Cohort 8, regardless of tumor type. Patients in Cohort 7 (primary CNS tumors) should have met the following criteria: PMA P170019/S017: FDA Summary of Safety and Effectiveness Data 33 of 59 {33} ○ Had received prior treatment including radiation and/or chemotherapy, with radiation completed >12 weeks prior to Cycle 1 Day 1 (C1D1) of therapy, as recommended or appropriate for that CNS tumor type ○ Had ≥1 site of bi-dimensionally measurable disease (confirmed by magnetic resonance imaging [MRI] and evaluable by Response Assessment in Neuro-Oncology Criteria [RANO] criteria), with the size of at least 1 of the measurable lesions ≥1 cm in each dimension and noted on more than 1 imaging slice ○ Imaging study performed within 28 days before enrollment while on stable dose steroid medication for at least 5 days immediately before and during the imaging study. • At least 12 years of age. • ECOG score of ≤3. For those entered into Cohort 7, Karnofsky Performance Status of 50. • Adequate organ function as defined in the individual study protocols. ### Therapeutic Clinical Study TRK-15003 (Study 15003) Study 15003 is an ongoing multicenter open-label Phase 1/2 study in pediatric patients aged from birth to 21 years with advanced solid or primary CNS tumors. The study is divided into a Phase 1 dose escalation portion, a Phase 1 dose expansion portion, and a Phase 2 portion. In the Phase 2 portion, enrollment is rest…