← Product Code [PQP](/productcode/PQP) · P170019S043 # FoundationOne CDx (F1CDx) (P170019S043) _Foundation Medicine, Inc. · PQP · Oct 6, 2023 · Pathology · APPR_ **Canonical URL:** https://fda-staging.innolitics.com/device/P170019S043 ## Device Facts - **Applicant:** Foundation Medicine, Inc. - **Product Code:** [PQP](/productcode/PQP.md) - **Decision Date:** Oct 6, 2023 - **Decision:** APPR - **Device Class:** Class 3 - **Review Panel:** Pathology - **Attributes:** Real-World Evidence ## Real-World Evidence | Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags | | --- | --- | --- | --- | --- | --- | | P170019S043 · Oct 6, 2023 | FoundationOne CDx (F1CDx) | Foundation Medicine, Inc. | FMI clinical archives (banked clinical samples); LIBRETTO-001 clinical trial (retrospective analysis of archived specimens) | Retrospective analysis of archived FFPE tumor specimens from the LIBRETTO-001 clinical trial and FMI clinical archives was used to establish clinical validity and concordance of the F1CDx assay for detecting RET fusions. | Retrospective analysis; Clinical bridging study; Clinical archives; Companion diagnostic validation | ### Clinical Evidence | Study Design | Population | Comparator | Key Endpoints | | --- | --- | --- | --- | | FoundationOne CDx Retrospective Analysis of RET fusions in LIBRETTO-001; Retrospective clinical bridging study; Follow-up/Duration: Not applicable (retrospective testing); Study Period: LIBRETTO-001 initiated May 2, 2017 | Patients with advanced solid tumors (NSCLC, thyroid, pancreas, colorectal, etc.) enrolled in LIBRETTO-001; Sample Size: 175 patients from LIBRETTO-001; 311 samples from FMI clinical archives; Number of Sites: Multi-center (LIBRETTO-001) | Clinical Trial Assays (CTAs) used for enrollment | Objective Response Rate (ORR) using RECIST 1.1; Concordance (PPA, NPA) | ## 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 when using the DNAx extraction method. The test is intended for detection of substitutions and indels in 324 genes, CNAs in 15 genes and select gene rearrangements, as well as genomic signatures including MSI and TMB using DNA isolated from FFPE tumor tissue specimens when using the CoExtraction method for DNA isolation. 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. ## Device Story F1CDx is a NGS-based IVD test for somatic/germline variant detection in solid tumors. Input: DNA from FFPE tumor tissue (DNAx or CoExtraction methods). Process: Whole-genome shotgun library construction; hybridization-based capture of 324 cancer-related genes; sequencing on Illumina HiSeq 4000. Analysis: Proprietary pipeline detects substitutions, indels, CNAs, and rearrangements; calculates MSI, TMB, and HRD status. Output: Clinical report identifying genomic alterations and companion diagnostic (CDx) status for specific therapies. Used in clinical laboratories by trained personnel. Results assist oncologists in selecting targeted therapies (e.g., RET inhibitors, TKIs, immunotherapy) based on biomarker status. Benefits include personalized treatment selection for patients with advanced solid tumors. ## Clinical Evidence Retrospective clinical bridging study using 175 LIBRETTO-001 trial samples and 311 archival samples. Primary endpoint: ORR by BIRC. F1CDx-positive population ORR was 75.0% (95% CI [64.4%, 85.6%]). Concordance analysis showed PPA 90.1% and NPA 100% for pooled pan-tumor population. Sensitivity analysis using multiple imputation confirmed robustness of ORR estimates. ## Technological Characteristics NGS-based targeted hybridization capture. DNA isolated from FFPE via DNAx or CoExtraction. Sequencing on Illumina HiSeq 4000. Targets 324 genes (coding exons, select introns/promoters). Bioinformatics pipeline uses BWA, GATK, Picard, and proprietary algorithms for variant calling, MSI (fraction-based), TMB, and HRD (LOH/tBRCA). ## Regulatory Identification A next generation sequencing (NGS) oncology panel is a device used for the qualitative detection of germline or somatic variants in one or more cancer-related genes. The device is intended to be used on DNA or RNA isolated from human clinical specimens. ## Reference Devices - FoundationFocus CDxBRCA - BRACAnalysis CDx - Myriad myChoice® CDx - Oncomine Dx Target Test - cobas EGFR Mutation Test v2 - therascreen EGFR RGQ PCR Kit - therascreen KRAS RGQ PCR Kit - cobas KRAS Mutation Test - Praxis Extended RAS Panel - therascreen BRAF V600E RGQ PCR Kit - cobas 4800 BRAF V600 Mutation Test - THxID BRAF Kit - Vysis ALK Break Apart FISH Probe Kit - ALK (D5F3) CDx Assay - PathVysion HER-2 DNA Probe Kit - PATHWAY Anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody - InSite HER-2/neu Kit - SPOT-Light HER2 CISH Kit - Bond Oracle HER2 IHC System - HER2 CISH pharmDx Kit - INFORM HER2 Dual ISH DNA Probe Cocktail - HercepTest - HER2 FISH pharmDx Kit - therascreen PIK3CA RGQ PCR Kit ## Submission Summary (Full Text) > This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com. > > Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a PMA](https://innolitics.com/services/regulatory/), [a 510(k)](https://innolitics.com/services/510ks/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/). {0} # SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) ## I. GENERAL INFORMATION | Device Generic Name: | Next 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/S043 | | Date of FDA Notice of Approval: | October 6, 2023 | The original PMA (P170019) 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 *RET* fusions in patients with solid tumors who may benefit from treatment with RETEVMO® (selpercatinib). ## 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 when using the DNAx extraction method. The test is intended for detection of ---PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 1 of 54 {1} substitutions and indels in 324 genes, CNAs in 15 genes and select gene rearrangements, as well as genomic signatures including MSI and TMB using DNA isolated from FFPE tumor tissue specimens when using the CoExtraction method for DNA isolation. 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 | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Non-small cell lung cancer (NSCLC) | 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) | | | ALK rearrangements | Alecensa® (alectinib), Alunbrig® (brigatinib), 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) | | | ROS1 fusions | Rozlytrek® (entrectinib) | | Melanoma | BRAF V600E | BRAF Inhibitors approved by FDA* | | | BRAF V600E and V600K | Mekinist® (trametinib) or BRAF/MEK Inhibitor Combinations approved by FDA* | | | BRAF V600 mutation-positive | Tecentriq® (atezolizumab) in combination with Cotellic® (cobimetinib) and 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) | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 2 of 54 {2} | 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) | | Cholangiocarcinoma | FGFR2 fusions and select rearrangements | Pemazyre® (pemigatinib) or Truseltiq™ (infigratinib) | | 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) | | | BRCA1, BRCA2 alterations | Akeega® (niraparib + abiraterone acetate) | | Solid Tumors | MSI-High | Keytruda® (pembrolizumab) | | | TMB ≥ 10 mutations per megabase | Keytruda® (pembrolizumab) | | | NTRK1/2/3 fusions | Rozlytrek® (entrectinib) Vitrakvi® (larotrectinib) | | | RET fusions | Retevmo® (selpercatinib) | *For the most current information about the therapeutic products in this group, go to: https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools 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 is performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. ### III. CONTRAINDICATIONS There are no known contraindications. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 3 of 54 {3} # 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, qualified by FMI and procedures for testing 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, 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 detects alterations in a total of 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 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 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), tumor mutational burden (TMB), and positive homologous recombination deficiency (HRD) status (tBRCA-positive and/or LOH high) 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 | | AMER1 | BTK | CHEK2* | ERG | GABRA6 | JAK3 | MET | NTRK1 | PRKARA | 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/S043: FDA Summary of Safety and Effectiveness Data 4 of 54 {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 | | *Genes with copy number alteration reporting are limited to CDx variants when using the CoExtraction method 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 8 | 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/S043: FDA Summary of Safety and Effectiveness Data 5 of 54 {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 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | *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. Table 4. Instruments for use with the F1CDx assay | Instrument | | --- | | Agilent Technologies Benchbot Workstation with Integrated Bravo Automated Liquid Handler or Hamilton Microlab STAR/STARlet Liquid Handling Workstation | | Beckman Biomek NX^{P} Span-8 Liquid Handler or Hamilton Microlab STAR/STARlet Liquid Handling Workstation | | Hamilton AutoLys Liquid Handling Workstation | | Covaris LE220-plus Focused ultrasonicator | | Thermo Fisher Scientific KingFisher™ Flex with 96 Deep-well Head | | Illumina® cBot System | | Illumina® HiSeq 4000 System | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 6 of 54 {6} # 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 and are placed into a Vortemp for digestion. The sample is then centrifuged to separate sample-associated paraffin from the lysate, and the lysate is transferred to a KingFisher dKF 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. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 7 of 54 {7} 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. The 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 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 Workstation (Hamilton) using the “withbead” protocol¹ to maximize reproducibility and library yield. Indexed (6 bp barcodes) sequencing libraries are PCR amplified with HiFi™ (Kapa) for 10 cycles, and subsequently 1.8X SPRI purified. Purification and dilution for QC are performed. Following LC, a QC procedure is performed by quantifying single-stranded DNA (ssDNA) from purified libraries using the Quant-iT™ OliGreen® ssDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Libraries yielding insufficient sequencing library are failed. ### D. Hybrid Capture Hybrid Capture (HC) begins with normalization of each library to 500-2000 ng. Normalized samples then undergo solution hybridization which is performed using a > 50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 bp oligonucleotides. The baits target ~1.8 Mb of the human genome including all coding exons of 309 cancer-related genes, introns or non-coding regions of 35 genes, plus > 3,500 single nucleotide polymorphisms (SNPs) located throughout the genome. Baits are designed by tiling overlapping 120 bp DNA sequence intervals covering target exons (60 bp overlap) and introns (20 bp overlap), with a minimum of three baits per target; SNP targets are allocated one bait each. Intronic baits are filtered for repetitive elements² as defined by the UCSC Genome RepeatMasker track. After hybridization, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen), and off-target material is removed by 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. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 8 of 54 {8} 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. 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 PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 9 of 54 {9} 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 has an MSI-H score ≥ 0.0124 is reported as eligible for treatment with KEYTRUDA. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 10 of 54 {10} 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). To compute the percentage of genomic LOH for each tumor, LOH segments are inferred across the 22 autosomal chromosomes using the genome-wide aneuploidy/copy number profile and minor allele frequencies of the more than 3500 SNPs sequenced in the Foundation Medicine’s next-generation sequencing (NGS)-based platform. A comparative genomic hybridization (i.e., log-ratio profile of the sample) is obtained from the NGS sequencing data by normalizing the sequence coverage obtained at all exons and genome-wide SNPs against a process-matched normal control. This profile is segmented and interpreted using allele frequencies of sequenced SNPs to estimate copy number (Ci) and minor allele count (Mi) at each segment (i). A segment is determined to have LOH if Ci ≠ 0 and Mi = 0. Two types of LOH segments are excluded from the calculation of percent genomic LOH: (1) LOH segments spanning ≥ 90% of a whole chromosome or chromosome arm, as these LOH events usually arise through non-homologous recombination deficiency (HRD) mechanisms (e.g., mitotic nondisjunction), and (2) regions in which LOH inference is ambiguous (e.g., some small genomic regions that do not have sufficient heterozygous SNPs to support LOH calling). 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 PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 11 of 54 {11} 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. 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. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 12 of 54 {12} ## 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). 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 Tables 6-8, below. However, any missense or non-frameshift mutations in the other 12 genes would not be considered HRR positive. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 13 of 54 {13} **Table 6. Eligible deleterious mutations in the *ATM* gene (for olaparib CDx claim only)** | M1I | Y2470D | R2832C | | --- | --- | --- | | 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 | | **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 302-3C>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 | L2688P | 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 | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 14 of 54 {14} | 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. 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). PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 15 of 54 {15} # **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) ### 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 9, 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 9. List of FDA approved CDx assays for genes targeted by F1CDx | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | | 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 | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 16 of 54 {16} | | 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 | | | 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/S043: FDA Summary of Safety and Effectiveness Data 17 of 54 {17} | ALK-fusion | Vysis ALK Break Apart FISH Probe Kit | Abbott Molecular, Inc. | FISH | XALKORI (crizotinib) | NSCLC | | --- | --- | --- | --- | --- | --- | | ALK (D5F3) CDx Assay | Ventana Medical Systems, Inc. | IHC | XALKORI (crizotinib) | NSCLC | | EGFR – Exon 19 deletions & L858R | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TARCEVA (erlotinib) TAGRISSO (osimertinib) IRESSA (gefitinib) | NSCLC | | therascreen EGFR RGQ PCR Kit | QIAGEN | PCR | GILOTRIF (afatinib) IRESSA (gefitinib) | NSCLC | | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | IRESSA (gefitinib) | NSCLC | | EGFR T790M | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TAGRISSO (osimertinib) | NSCLC | | BRCA1/2 | FoundationFocus CDxBRCA | Foundation Medicine, Inc. | NGS | RUBRACA (rucaparib) | Advanced ovarian cancer | | 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 | | PIK3CA | 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 | Abbreviations: FISH – fluorescence in situ hybridization; IHC – immunohistochemistry; CISH chromogenic in situ hybridization; ISH – in situ hybridization; PCR – polymerase chain reaction; NGS – next-generation sequencing. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 18 of 54 {18} ## 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 approved PMA supplements that affected the Intended Use are listed in Table 10. **Table 10. 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) | | 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) | | | | 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) | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 19 of 54 {19} | P170019/S025 | November 10, 2021 | *BRAF* V600E | 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 | Atezolizumab (Tecentriq) 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) | ## **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 the detection of *RET* fusions in solid tumor patients was from data using intended use specimens across the validation studies. In addition to the existing platform-level validation results (P170019), refer to Section IX.A. in P170019 Summary of Safety and Effectiveness Data, analytical concordance, limit of blank (LoB), limit of detection (LoD), and site-to-site precision PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 20 of 54 {20} studies were conducted to support the indication for RET fusions. Table 11 shows the distribution of tumor types evaluated in the studies. The F1CDx device was modified after the analytical validation studies were completed. The update included addition of an automated DNA/RNA CoExtraction methodology (CoExtraction method) to enable isolation of DNA and RNA from the same FFPE tumor specimens. Addition of the new CoEx method for DNA isolation which can be used in lieu of the DNA extraction method for F1CDx approved under the original PMA, was supported by validation studies demonstrating comparability between the two nucleic acid extraction methods, and precision studies at and above the LoD levels of the RET-positive fusion samples. The comparability study between the DNAx and CoEx DNA extraction methods assessed a total of two (2) clinical samples harboring RET fusions from patients with thyroid papillary carcinoma and observed a PPA of 100%. The precision study assessed two specimens positive for RET fusions with chimeric read levels at 17x and 21x LoD (151.5 and 185.9 chimeric reads respectively) from two patients with thyroid cancer. Six (6) curls were cut from each source block and processed with the CoExtraction method using a combination of two unique CoExtraction reagent lots and two unique CoExtraction instrument lines. The extracted DNA from each curl was subdivided into four (4) DNA sub-aliquots, for a total of 24 total replicates per source block entering precision testing (6 curl extractions x 4 extracted DNA subaliquots each). The agreement for reproducibility and repeatability of two RET fusion positive samples was 100%. An additional precision study with two RET fusion positive specimens from patients with thyroid cancer was conducted with chimeric read levels closer to LoD (3.2x and 2.8xLoD). For this study, previously extracted DNA derived from FFPE specimens using the CoExtraction method was used. Selected DNA samples were diluted to varying levels using biomarker-negative DNA (diluent DNA) to achieve levels closer to LoD. A total of 24 replicates (2 reagent lots x 3 sequencers x 2 plates x 2 replicates per sample) were assessed and a 100% agreement for reproducibility and repeatability was achieved for these two samples. Further, 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 RET fusions in solid tumors by F1CDx were performed using previous versions of the analytical pipeline that has undergone further iteration with modification in the final device design. Additionally, during review, it was identified that a NSCLC RET fusion-positive sample evaluated in the clinical concordance study failed the tumor purity QC metric, but was reported as passing the QC metric and reported as RET fusion-negative. This was a curation error and the sample should have been considered invalid per the F1CDx Genomic Analysis and Review protocol. To address the source of the error, the analytical pipeline was updated to include an automatic QC flag for low tumor purity, a process that was previously performed manually, and implemented as a mitigation. 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 PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 21 of 54 {21} post-market to confirm the robust performance for F1CDx for the detection of RET fusions (see section XIII). Table 11. Distribution of disease ontologies (DO) in the analytical validation studies. | Cancer Type | Disease Ontology | LoB | LoD | Precision | Concordance | | --- | --- | --- | --- | --- | --- | | Biliary | Ampullary adenocarcinoma | 0 | 0 | 0 | 1 | | Breast | Breast carcinoma (NOS) | 0 | 0 | 0 | 17 | | | Breast invasive ductal carcinoma (IDC) | 2 | 1 | 0 | 3 | | Cervix | Cervix squamous cell carcinoma (SCC) | 0 | 0 | 0 | 1 | | Colorectal | Colon adenocarcinoma (CRC) | 1 | 1 | 6 | 50 | | | Colon neuroendocrine carcinoma | 0 | 0 | 0 | 1 | | | Rectum adenocarcinoma (CRC) | 0 | 0 | 0 | 1 | | Neuroendocrine | Adrenal gland neuroblastoma | 0 | 0 | 0 | 1 | | Endometrial | Uterus endometrial adenocarcinoma | 1 | 0 | 0 | 0 | | Glioma | Brain glioblastoma (GBM) | 0 | 0 | 0 | 1 | | | Brain gliosarcoma | 0 | 0 | 0 | 1 | | Kidney | Kidney clear cell carcinoma | 1 | 0 | 0 | 0 | | Lung | Lung adenocarcinoma | 6 | 2 | 3 | 121 | | | Lung non-small cell lung carcinoma (NOS) | 3 | 0 | 0 | 37 | | | Lung squamous cell carcinoma | 5 | 0 | 0 | 2 | | Melanoma | Eye intraocular melanoma | 0 | 0 | 0 | 1 | | | Skin melanoma | 0 | 0 | 0 | 1 | | Ovary | Ovary carcinoma mixed histology | 0 | 0 | 0 | 1 | | | Ovary clear cell carcinoma | 0 | 0 | 0 | 2 | | | Ovary epithelial carcinoma (NOS) | 0 | 0 | 0 | 5 | | | Ovary high grade serous carcinoma | 0 | 0 | 0 | 7 | | | Ovary mucinous carcinoma | 0 | 0 | 0 | 1 | | | Ovary serous carcinoma | 1 | 0 | 1 | 8 | | Pancreas | Pancreas carcinoma (NOS) | 0 | 0 | 0 | 3 | | | Pancreas ductal adenocarcinoma | 0 | 0 | 0 | 9 | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 22 of 54 {22} | | Pancreatobiliary carcinoma (NOS) | 0 | 0 | 0 | 1 | | --- | --- | --- | --- | --- | --- | | | Pancreas neuroendocrine carcinoma | 0 | 0 | 1 | 1 | | Parathyroid | Parathyroid carcinoma | 0 | 0 | 0 | 1 | | Prostate | Prostate acinar adenocarcinoma | 0 | 0 | 0 | 8 | | Salivary Gland | Salivary gland adenocarcinoma | 0 | 0 | 0 | 2 | | | Salivary gland carcinoma (NOS) | 0 | 0 | 0 | 4 | | | Salivary gland mammary analogue secretory carcinoma (MASC) | 0 | 0 | 0 | 2 | | Skin | Skin basal cell carcinoma | 0 | 0 | 0 | 2 | | Skin sarcoma | Skin sarcoma | 1 | 0 | 0 | 0 | | Small intestine | Small intestine adenocarcinoma | 0 | 0 | 0 | 3 | | Soft tissue sarcoma | Soft tissue sarcoma (NOS) | 0 | 0 | 0 | 1 | | | Unknown primary sarcoma (NOS) | 0 | 0 | 0 | 1 | | Thyroid | Medullary Thyroid Cancer (MTC) | 1 | 0 | 0 | 0 | | | Thyroid anaplastic carcinoma | 0 | 0 | 0 | 12 | | | Thyroid carcinoma (NOS) | 0 | 0 | 2 | 33 | | | Thyroid follicular carcinoma | 4 | 0 | 0 | 2 | | | Thyroid follicular oncocytic carcinoma | 0 | 0 | 0 | 1 | | | Thyroid papillary carcinoma | 1 | 2 | 1 | 51 | | Unknown | Unknown primary malignant neoplasm (NOS) | 0 | 0 | 0 | 1 | | | Unknown primary adenocarcinoma | 0 | 0 | 0 | 5 | | | Unknown primary carcinoma (NOS) | 0 | 0 | 0 | 2 | | | Unknown primary urothelial carcinoma | 0 | 0 | 0 | 1 | | | Unknown primary neuroendocrine tumor (NET) | 0 | 0 | 0 | 1 | | | **Total** | **27** | **6** | **14** | **410** | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 23 of 54 {23} # 1. Analytical Accuracy/Concordance ## a. Comparison to Orthogonal Method for *RET* fusions An analytical accuracy study was performed to demonstrate the concordance between F1CDx and an externally validated NGS assay (evNGS) for the detection of *RET* fusions in solid tumors. The analytical accuracy study was performed with available residual DNA previously extracted from FFPE clinical specimens (31 samples with sufficient remaining material) from patients with solid tumors enrolled in the clinical study, LOXO-RET-17001 (LIBRETTO-001, NCT03157128) that supported the RETEVMO (selpercatinib) approval (refer to Section X for study details). Due to the limited number of available clinical trial samples, the study also included *RET* fusion-positive samples (N=125) and *RET* fusion-negative (N=154) from the FMI clinical archives that were previously evaluated at FMI. The study also included 100 *RET* fusion-negative samples from patients with NSCLC previously processed and tested by the evNGS in prior concordance validation studies; these previously executed concordance studies were newly evaluated for *RET* fusions. In total, 410 samples were processed in the analytical accuracy study: 160 NSCLC samples, 99 thyroid cancer (TC) samples, and 151 solid tumor samples. The solid tumor samples included samples from biliary tract (1), breast (20), cervix (1), colorectal (52), neuroendocrine (1), glioma (2), kidney (1), melanoma (2), ovary (24), pancreas (14), prostate (8), salivary gland (8), small intestine (3), soft tissue sarcoma (2) and unknown (10) cancer types. Table 12 presents a breakdown of the sample selection based on *RET* fusions. Table 12. Samples evaluated for *RET* fusion concordance | Source material | Biomarker Status | Disease Ontology | | | Total | | --- | --- | --- | --- | --- | --- | | | | NSCLC | TC | Solid tumors | | | FMI clinical archives | POSITIVE | 49 | 38 | 38 | 125 | | | NEGATIVE | 1 | 51 | 102 | 154 | | LIBRETTO-001 | POSITIVE | 10 | 10 | 11 | 31 | | Samples for evNGS re-analysis | NEGATIVE | 100 | - | - | 100 | | Total | | 160 | 99 | 151 | 410 | There were three invalid samples: one LIBRETTO-001 sample had insufficient remaining material after F1CDx testing and two solid tumor samples failed to meet QC metrics during F1CDx sequencing and post-sequencing steps. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 24 of 54 {24} The F1CDx and evNGS results for the detection of RET fusions, using evNGS as the reference method, are provided in the contingency table below (Table 13). Table 13. Contingency table comparing F1CDx and evNGS results for the detection of RET fusions | | evNGS | | | | | | --- | --- | --- | --- | --- | --- | | | | RET+ | RET- | Invalid | Total | | F1CDx | RET+ | 146 | 7 | 1 | 154 | | | RET- | 0 | 254 | 0 | 254 | | | Invalid | 0 | 2 | 0 | 2 | | | Total | 146 | 263 | 1 | 410 | The positive percent agreements (PPA), negative percent agreements (NPA), positive predictive value (PPV), and negative predictive value (NPV) with 95% confidence intervals (CI) are presented in Table 14 below. Table 14. Summary of agreement measures | Agreement Statistic | Estimate [95% CI] | | --- | --- | | PPA | 100% [97.44%, 100%] | | NPA | 97.32% [94.57%, 98.69%] | | PPV | 95.42% [90.86%, 97.77%] | | NPV | 100% [98.51%, 100%] | Since the PPA, NPA, PPV and NPV were calculated without adjusting for the distribution of samples enrolled, i.e., from the clinical trial or from FMI's clinical sample archives, the estimates of PPA, NPA, PPV, and NPV may be subject to potential bias. There were seven samples (all from FMI clinical archives) that were discordant between the F1CDx and evNGS test results. All discordant samples were determined to be RET fusion-positive by F1CDx and RET fusion-negative by evNGS. Of the seven discordant calls, there were four samples that were not reported by evNGS due to low tumor content or low sample quality, one sample that had a fusion detected in a biomarker-negative orientation by evNGS, and two samples that were not detected by evNGS. ## 2. Analytical Sensitivity ### a. Limit of Blank (LoB) The limit of blank was evaluated by testing matched normal samples from patients with solid tumors. The solid tumor samples comprised of the following diseases and specimen types: lung squamous cell carcinoma and lung adenocarcinoma (lung), ovary serous carcinoma (ovary), breast invasive ductal carcinoma (breast), kidney clear cell carcinoma (kidney), colon adenocarcinoma (colon), skin sarcoma (skin), and uterus endometrial adenocarcinoma (uterus). PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 25 of 54 {25} Each sample was assessed in replicates of four to 15, resulting in a total of 136 replicates. Of the 136 replicates, 135 were successfully sequenced and 126 passed the post-sequencing quality control (QC) criteria and were included in the analysis. Of the 126 valid sample replicates, there were no RET fusions detected, resulting in a false positive rate of 0%, and confirming the LoB to be zero. The limit of blank was also evaluated by testing FFPE tumor specimens from patients with NSCLC and TC. For the 6 NSCLC samples, a total of 60 replicates were assessed, and 58 replicates were valid. For the 6 TC samples, a total of 66 replicates were assessed; 64 replicates were valid. The false positive rate was 0%, and LoB of zero was confirmed in both studies. # b. Limit of Detection (LoD) The limit of detection (LoD) for the detection of RET fusions by F1CDx was determined by assessing six (6) samples. Selection of specimens for assessment of RET fusions represented various tumor types as shown in Table 15. Table 15. Samples assessed in LoD study for the detection of RET fusions | Sample | Target Gene | Partner Gene | Fusion Partner or Alteration Description | Disease Indication (Specimen Site) | | --- | --- | --- | --- | --- | | 1 | RET | TRIM24 | 5'-TRIM24(ex1-17 NM_003852)-RET(ex12-19 NM_020630) | Lung adenocarcinoma | | 2 | RET | ERC1 | 5'-ERC1(ex1-7 NM_178039)-RET(ex12-19 NM_020630) | Lung adenocarcinoma | | 3 | RET | NCOA4 | 5'-NCOA4(ex1-8 NM_005437)-RET(ex12-19 NM_020630) | Thyroid papillary carcinoma | | 4 | RET | CCDC6 | 5'-CCDC6(ex1-1 NM_005436)-RET(ex12-19 NM_020630) | Thyroid papillary carcinoma | | 5 | RET | KIF5B | 5'-KIF5B(ex1-15 NM_004521)-RET(ex12-19 NM_020630) | Breast invasive ductal carcinoma (IDC) | | 6 | RET | CCDC6 | 5'-CCDC6(ex1-1 NM_005436)-RET(ex11-19 NM_020630) | Colon adenocarcinoma (CRC) | Each sample was assessed at five targeted chimeric read levels (4, 12, 18, 24, and 30) with 20 replicates tested for each dilution level, except the dilution level of 30 chimeric reads, where 14 replicates were assessed. In total, 94 replicates were tested per sample, and each specimen was evaluated close to the minimum input requirements of the assay (50 ng). The LoD for each of the samples was determined based on chimeric reads using the hit rate method. A summary of the LoD results based on chimeric reads using the hit rate method are shown in Table 16. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 26 of 54 {26} Table 16. Summary of LoD analysis for RET fusions | Sample | Target Gene | Partner Gene | RET Fusion LoD (chimeric reads)^{1} | | --- | --- | --- | --- | | 1 | RET | CCDC6 | 4.90 | | 2 | RET | NCOA4 | N/A^{2} | | 3 | RET | TRIM24 | 8.50 | | 4 | RET | ERC1 | 10.85 | | 5 | RET | CCDC6 | 8.75 | | 6 | RET | KIF5B | 10.80 | $^{1}$LoD calculations were based on the hit rate approach; defined as the lowest level with ≥95% hit rate $^{2}$The LoD was not estimated because the criteria to determine LoD by hit rate were not met. The LoD of one sample was not estimated due to not meeting the hit rate method criteria, therefore, the LoD for RET fusions was determined using the median LoD from the five remaining samples. The LoD was determined to be 8.75 chimeric reads. ### 3. Precision and Reproducibility #### a. Site-to-site Precision and Reproducibility A site-to-site precision study was conducted to evaluate the inter-run reproducibility and intra-run repeatability of RET fusion detection. The study evaluated 14 different solid tumor samples at challenging DNA input (close to 50 ng) across different sites (Cambridge, MA and Morrisville, NC), reagent lots, and library construction start days. For each sample, 24 replicates were processed. Table 17 summarizes the disease ontologies (DO) and RET fusion status for the 14 samples included in the precision study. Each sample was mixed with RET fusion-negative, DO-matched DNA to dilute the samples to 1-3x LoD. Table 17. Samples selected for site-to-site precision study | Sample | Partner Gene | Target Gene | Fusion Partner or Alteration Description | Disease Ontology | | --- | --- | --- | --- | --- | | 1 | CCDC6 | RET | 5'-CCDC6(ex1-1 NM_005436)-RET(ex12-19 NM_020630) | Thyroid carcinoma | | 2 | CCDC6 | RET | 5'-CCDC6(ex1-1 NM_005436)-RET(ex12-19 NM_020630) | Thyroid papillary carcinoma | | 3 | CCDC6 | RET | 5'-CCDC6(ex1-1 NM_005436)-RET(ex12-19 NM_020630) | Thyroid carcinoma | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 27 of 54 {27} | 4 | *KIF5B* | *RET* | 5'-*KIF5B*(ex1-15 NM_004521)-*RET*(ex12-19 NM_020630) | Lung adenocarcinoma | | --- | --- | --- | --- | --- | | 5 | *KIF5B* | *RET* | 5'-*KIF5B*(ex1-15 NM_004521)-*RET*(ex12-19 NM_020630) | Lung adenocarcinoma | | 6 | *KIF5B* | *RET* | 5'-*KIF5B*(ex1-15 NM_004521)-*RET*(ex12-19 NM_020630) | Lung adenocarcinoma | | 7 | *NCOA4* | *RET* | 5'-*NCOA4*(ex1-8 NM_005437)-*RET*(ex12-19 NM_020630) | Colon adenocarcinoma | | 8 | *NCOA4* | *RET* | 5'-*NCOA4*(ex1-9 NM_005437)-*RET*(ex12-19 NM_020630) | Colon adenocarcinoma | | 9 | *NCOA4* | *RET* | 5'-*NCOA4*(ex1-9 NM_005437)-*RET*(ex12-19 NM_020630) | Colon adenocarcinoma | | 10 | *NCOA4* | *RET* | 5'-*NCOA4*(ex1-9 NM_005437)-*RET*(ex12-19 NM_020630) | Colon adenocarcinoma | | 11 | *TRIM24* | *RET* | 5'-*TRIM24*(ex1-9 NM_003852)-*RET*(ex12-19 NM_020630) | Colon adenocarcinoma | | 12 | *PRPF19* | *RET* | 5'-*PRPF19*(ex1-10 NM_014502)-*RET*(ex12-19 NM_020630) | Colon adenocarcinoma | | 13 | *ERC1* | *RET* | 5'-*ERC1*(ex1-7 NM_178039)-*RET*(ex12-19 NM_020630) | Pancreas neuroendocrine carcinoma | | 14 | *CCDC6* | *RET* | 5'-*CCDC6*(ex1-2 NM_005436)-*RET*(ex11-19 NM_020630) | Ovary serous carcinoma | Repeatability was evaluated in the 14 samples by processing two replicates from the same source sample and plate within each site, reagent lot, and start day. The result was considered in agreement if the duplicate replicates processed under identical conditions had the same detection status for the targeted *RET* fusion. The point estimates and 95% CIs for repeatability of each sample are detailed in Table 18. **Table 18. Repeatability for *RET* fusions** | Sample | Observed Average Chimeric Reads | Fold LoD | Partner Gene | Target Gene | # Positive Replicates | # Total Valid Replicates | Repeatability (95% CI*) | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 13.1 | 1.50x | *ERC1* | *RET* | 11 | 11 | 100% [74.1%, 100%] | | 2 | 12.0 | 1.37x | *CCDC6* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 3 | 18.3 | 2.09x | *CCDC6* | *RET* | 10 | 10 | 100% [N/A**] | | 4 | 13.4 | 1.53x | *NCOA4* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 5 | 15.4 | 1.76x | *KIF5B* | *RET* | 11 | 11 | 100% [74.1%, 100%] | | 6 | 18.7 | 2.14x | *CCDC6* | *RET* | 11 | 11 | 100% [74.1%, 100%] | PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 28 of 54 {28} | 7 | 19.0 | 2.18x | *KIF5B* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | --- | --- | --- | --- | --- | --- | --- | --- | | 8 | 21.1 | 2.41x | *NCOA4* | *RET* | 9 | 10 | 90.0% [N/A**] | | 9 | 16.6 | 1.90x | *CCDC6* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 10 | 13.0 | 1.48x | *KIF5B* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 11 | 18.4 | 2.10x | *TRIM24* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 12 | 21.8 | 2.49x | *NCOA4* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 13 | 21.4 | 2.45x | *NCOA4* | *RET* | 12 | 12 | 100% [75.8%, 100%] | | 14 | 12.4 | 1.41x | *PRPF19* | *RET* | 11 | 12 | 91.7% [64.6%, 98.5%] | *Two-sided 95% CI is calculated by the Wilson Score Method. **CI not provided for sample sizes ≤10. Reproducibility was evaluated by processing replicates from the same source sample under conditions where one factor was changed at a time (e.g., reagent lots, site, days). To be considered a positive call, the *RET* fusion had to be detected in each replicate of the source sample and meet the biomarker definition. The point estimates and 95% CIs for reproducibility of each sample are detailed in Table 19. **Table 19. Reproducibility for *RET* fusions** | Sample | Observed Average Chimeric Reads | Fold LoD | Partner Gene | Target Gene | # Positive Replicates | # Total Valid Replicates | Reproducibility (95% CI*) | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 13.1 | 1.50x | *ERC1* | *RET* | 23 | 23 | 100% [85.7%, 100%] | | 2 | 12.0 | 1.37x | *CCDC6* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 3 | 18.3 | 2.09x | *CCDC6* | *RET* | 22 | 22 | 100% [85.1%, 100%] | | 4 | 13.4 | 1.53x | *NCOA4* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 5 | 15.4 | 1.76x | *KIF5B* | *RET* | 23 | 23 | 100% [85.7%, 100%] | | 6 | 18.7 | 2.14x | *CCDC6* | *RET* | 23 | 23 | 100% [85.7%, 100%] | | 7 | 19.0 | 2.18x | *KIF5B* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 8 | 21.1 | 2.41x | *NCOA4* | *RET* | 21 | 22 | 95.5% [78.2%, 99.2%] | | 9 | 16.6 | 1.90x | *CCDC6* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 10 | 13.0 | 1.48x | *KIF5B* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 11 | 18.4 | 2.10x | *TRIM24* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 12 | 21.8 | 2.49x | *NCOA4* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 13 | 21.4 | 2.45x | *NCOA4* | *RET* | 24 | 24 | 100% [86.2%, 100%] | | 14 | 12.4 | 1.41x | *PRPF19* | *RET* | 23 | 24 | 95.8% [79.8%, 99.3%] | *Two-sided 95% CI is calculated by the Wilson Score Method PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 29 of 54 {29} # **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 FoundationOne CDx (F1CDx) for detecting RET fusions in patients with solid tumors was demonstrated in a retrospective analysis of specimens from patients enrolled in the LIBRETTO-001 clinical study of RETEVMO (selpercatinib). Data generated from the LIBRETTO-001 trial supported the clinical validation of the F1CDx assay for the identification of patients with RET fusion-positive solid tumors who may benefit from treatment with selpercatinib. A summary of the clinical study is presented below. # **A. FoundationOne CDx Retrospective Analysis of RET fusions in LIBRETTO-001** A reasonable assurance of safety and effectiveness for F1CDx for the detection of RET fusions in patients with solid tumors who may benefit from treatment with RETEVMO® (selpercatinib) was established through a clinical bridging study using tumor tissue FFPE specimens from patients enrolled in the LOXO-RET-17001 (LIBRETTO-001) clinical study with known RET fusion status, as well as RET fusion negative samples from the FMI archives. 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 finally determining the clinical outcome of the RET fusion positive population identified with F1CDx. # **1. LIBRETTO-001 Study Design** The LIBRETTO-001 clinical study is an open-label, multi-center Phase 1/2 study in patients with advanced solid tumors, including RET fusion-positive solid tumors (e.g., NSCLC, thyroid, pancreas, colorectal), RET-mutant MTC, and other tumors with RET activation (e.g., mutations in other tumor types or other evidence of RET activation). LIBRETTO-001 was initiated on May 2, 2017. This study included two parts: Phase 1 (dose escalation and dose expansion) and Phase 2 (dose expansion). The primary objective of the Phase 1 portion of the study was to determine the maximum tolerated dose (MTD)/recommended Phase 2 dose (RP2D) of RETEVMO. RP2D was determined to be 160 mg of RETEVMO orally twice daily (BID). Primary efficacy, the primary objective of Phase 2, was measured by the objective response rate (ORR) using Response Evaluation Criteria in Solid Tumors (RECIST 1.1) or Response Assessment in Neuro-Oncology (RANO), as appropriate for tumor type, as assessed by blinded independent review committee (BIRC). RETEVMO was approved by FDA for RET fusion positive non-small cell lung cancer (NSCLC), RET fusion positive thyroid PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 30 of 54 {30} cancer, and *RET* mutation positive medullary thyroid cancer in May 2020 based on safety and efficacy data from patients in LIBRETTO-001 with *RET* alterations. Further, RETEVMO was approved by FDA for *RET* fusion positive solid tumors in September 2022 based on safety and efficacy data from patients in LIBRETTO-001 with *RET* fusions. ## 2. *RET* fusion Evaluation by F1CDx The clinical effectiveness of F1CDx for detecting *RET* fusions in patients with solid tumors who may benefit from treatment with RETEVMO was demonstrated in a retrospective analysis of specimens from the LIBRETTO-001 clinical study. A bridging study was conducted to assess: (1) concordance between the clinical trial assays (CTAs) and F1CDx in identifying patients with *RET* fusions, (2) the efficacy of RETEVMO in patients from the LIBRETTO-001 clinical study who have *RET* fusions as determined by F1CDx, and (3) the robustness of the concordance analysis and efficacy analysis with a sensitivity analysis that accounts for the uncertainty due to missing data for *RET* 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 *RET* fusion-positive patients from the LIBRETTO-001 clinical study. F1CDx testing was performed on LIBRETTO-001 patients who had samples with sufficient tissue material remaining and who tested positive for *RET* fusions by the CTAs (CTA+). *RET* fusion negative samples from FMI's banked clinical samples archives were selected and tested with FoundationOne (F1) LDT, an NGS tissue assay, and subsequently tested by F1CDx for the CTA negative (CTA-) results. i. Clinical Inclusion and Exclusion Criteria Samples meeting the pre-defined criteria specified below were included in the clinical bridging study. Inclusion Criteria: - FFPE tissue samples (blocks or slides) - DNA derived from FFPE samples - Samples that meet F1CDx processing requirements - Samples that meet minimum criteria for F1CDx testing requirements - Samples from the LIBRETTO-001 clinical trial with proper informed consent Exclusion Criteria: - Samples failing to meet any of the inclusion criteria - LIBRETTO-001 clinical trial samples that lack clear identification or labeling PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 31 of 54 {31} • Samples not obtained in accordance with Institutional Review Board (IRB) approval # ii. 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. # iii. Clinical Endpoints The objectives of the F1CDx clinical bridging study were to: - Establish the clinical validity of F1CDx in identifying RET fusion-positive solid tumors for treatment with RETEVMO - Assess concordance of results for the RET fusion status between the F1CDx assay and the CTAs used for enrollment onto the LIBRETTO-001 clinical trial The efficacy analysis was performed using the primary efficacy outcome measure of the LIBRETTO-001 clinical study: Objective Response Rate (ORR) using RECIST 1.1, as appropriate for tumor type, as assessed by BIRC. Endpoints for the concordance analysis included PPA, NPA, and prevalence adjusted PPV and NPV. # B. Accountability of PMA Cohort There were 175 patients from the LIBRETTO-001 clinical trial with sufficient tissue samples available for testing with F1CDx. Of the 41 patients in the tissue agnostic (TA) supplemental new drug application (sNDA) population, 21 had samples available. Of the 144 patients in the non-small cell lung cancer (NSCLC) new drug application (NDA) population, 52 had samples available, and 74 additional samples were also available from the non-NDA efficacy patients for the concordance assessment. Additionally, of the 27 patients in the thyroid cancer (TC) NDA population, 21 had samples available, and 7 additional samples were also available from non-NDA efficacy patients for the concordance assessment. After sample processing, there were 15 TA sNDA samples, 87 NSCLC samples (35 NDA efficacy, 52 non-NDA efficacy), and 26 TC samples (21 NDA efficacy, 5 non-NDA efficacy) with valid F1CDx results. The pooled pan-tumor NDA efficacy population (PPT) consisted of the TA sNDA population, the NSCLC NDA population, and the TC NDA population, which yielded a total of 71 samples with valid F1CDx test results. In addition to the clinical study samples, 311 samples (100 pan-tumor, 107 NSCLC, and 104 TC) from FMI's clinical archives were processed by F1CDx for the RET fusion-negative population. There were six sample failures and 305 samples with valid F1CDx results; 138 samples (98 solid tumor, 20 NSCLC, and 20 TC) with valid F1CDx results were used in the PPT concordance analysis. PMA P170019/S043: FDA Summary of Safety and Effectiveness Data 32 of 54 {32} See Figure 1 for a schematic of the sample accountability in the F1CDx clinical bridging study. ![img-0.jpeg](img-0.jpeg) *Non-NDA efficacy patients are included for concordance analysis only. Figure 1: Clinical Bridging Study Sample Accountability ### 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 PPT population. The unevaluable population included patients who did not have a sample tested by F1CDx or those whose sample failed F1CDx testing (invalid). The demographics and disease characteristics for the CDx-evaluable and CDx-unevaluable PPT patients are provided in Table 20. 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 \( \alpha=0.2 \) were considered to be imbalanced. The number of patients without information collected or available are listed under NA (Missing). Table 20. Comparison of PPT demographics and clinical characteristics in the F1CDx evaluable-and F1CDx-unevaluable groups | Covariates | F1CDx-evaluable| CTA+ | F1CDx-unevaluable| CTA+ | Difference | P-value** | | --- | --- | --- | --- | --- | --- | --- | | Clinical Outcome (Response) | | | | 0.166 | | NO | 26.8% (19) | 36.9% (52) | -10.1% | | | YES | 73.2% (52) | 63.1% (89) | 10.1% | | | Age | | | | 0.607 | | Min | 20.0 | 21.0 | -1.0 |… --- **Source:** [https://fda-staging.innolitics.com/device/P170019S043](https://fda-staging.innolitics.com/device/P170019S043) **Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices. 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