← Product Code [PQP](/productcode/PQP) · P170019S048 # FoundationOne CDx (F1CDx) (P170019S048) _Foundation Medicine, Inc. · PQP · Nov 16, 2023 · Pathology · APPR_ **Canonical URL:** https://fda-staging.innolitics.com/device/P170019S048 ## Device Facts - **Applicant:** Foundation Medicine, Inc. - **Product Code:** [PQP](/productcode/PQP.md) - **Decision Date:** Nov 16, 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 | | --- | --- | --- | --- | --- | --- | | P170019S048 · Nov 16, 2023 | FoundationOne CDx (F1CDx) | Foundation Medicine, Inc. | Retrospective testing of banked FFPE tumor tissue specimens from the CAPItello-291 clinical trial; Foundation Medicine clinical archives | Retrospective analysis of clinical trial samples and archival clinical samples was used to establish analytical accuracy/concordance and support the companion diagnostic claim for PIK3CA/AKT1/PTEN alterations. | Retrospective analysis; Archival tissue; Companion diagnostic validation; Clinical trial cohort | ### Clinical Evidence | Study Design | Population | Comparator | Key Endpoints | | --- | --- | --- | --- | | Analytical Accuracy/Concordance Study; Retrospective concordance study | Breast cancer patients enrolled in CAPItello-291 and samples from FMI clinical archives; Sample Size: 237 samples | Externally validated NGS assay (evNGS) | Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), Positive Predictive Value (PPV), Negative Predictive Value (NPV) | ## 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 16 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 that analyzes DNA from FFPE tumor tissue to detect genomic alterations. The device uses hybridization-based capture of 324 cancer-related genes; libraries are sequenced on the Illumina HiSeq 4000. Proprietary software processes sequence data to identify base substitutions, indels, CNAs, and rearrangements. The assay is performed as a laboratory service at FMI sites. Results are reviewed by bioinformatics personnel and pathologists. The output provides tumor mutation profiling and identifies specific biomarkers (e.g., PIK3CA/AKT1/PTEN alterations) to guide treatment decisions for FDA-approved therapies. This helps clinicians select targeted treatments, potentially improving patient outcomes by matching patients to therapies based on their tumor's molecular profile. ## Clinical Evidence Clinical evidence for the PIK3CA/AKT1/PTEN CDx claim was derived from the CAPItello-291 Phase III trial. The study randomized 708 patients with HR-positive/HER2-negative breast cancer to capivasertib+fulvestrant or placebo+fulvestrant. F1CDx retrospectively tested 594 patients. In the F1CDx-altered population (n=287), median PFS was 7.3 months for capivasertib+fulvestrant vs 3.1 months for placebo+fulvestrant (HR 0.49; 95% CI: 0.38-0.64; p<0.001). Analytical validation included concordance studies against an externally validated NGS assay (n=236), demonstrating PPA of 96.00% and NPA of 99.93%. ## Technological Characteristics Targeted NGS assay using hybridization-based capture of 324 genes. DNA extracted from FFPE tissue via DNAx or CoExtraction methods. Sequencing performed on Illumina HiSeq 4000. Detects SNVs, indels, CNAs, and rearrangements. Bioinformatics pipeline uses BWA, Picard, SAMtools, and GATK. Reports MSI and TMB. Validated for 50-1000 ng DNA input. ## 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. ## 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/S048 | | Date of FDA Notice of Approval: | November 16, 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) claim for the detection of *PIK3CA/AKT1/PTEN*-alterations in patients with breast cancer who may benefit from treatment with TRUQAP™ (capivasertib) in combination with FASLODEX® (fulvestrant). ## 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 substitutions and indels in 324 genes, CNAs in 16 genes and select gene rearrangements, ---PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 1 of 49 {1} 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 | BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) | | | | 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® | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 2 of 49 {2} | | | (cobimetinib) and ZELBORAF® (vemurafenib) | | --- | --- | --- | | Breast cancer | ERBB2 (HER2) amplification | HERCEPTIN® (trastuzumab), KADCYLA® (ado-trastuzumabemtansine), or PERJETA® (pertuzumab) | | | PIK3CA C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y alterations | PIQRAY® (alpelisib) | | | AKT1 E17K, PIK3CA R88Q, N345K, C420R, E542K, E545A, E545D, E545Q, E545K, E545G, Q546E, Q546K, Q546R, Q546P, M1043V, M1043I, H1047Y, H1047R, H1047L, and G1049R; and PTEN alterations | TRUQAP™ (capivasertib) in combination with FASLODEX® (fulvestrant) | | 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) | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 3 of 49 {3} | | TMB ≥ 10 mutations per megabase | KEYTRUDA® (pembrolizumab) | | --- | --- | --- | | | *NTRK1/2/3* fusions | ROZLYTREK® (entrectinib) or 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 F1CDx assay is performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found in the FoundationOneCDx assay labeling. ### V. DEVICE DESCRIPTION FoundationOneCDx (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. FoundationOneCDx (F1CDx) is performed exclusively as a laboratory service using DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples. The assay employs two extraction methods (either DNAx or CoExtraction, an automated DNA/RNA co-extraction methodology) for DNA extraction from routine FFPE biopsy or surgical resection specimens; 50-1000 ng of DNA will undergo whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, one promoter region, one non-coding (ncRNA), and select intronic regions from 34 commonly rearranged genes, 21 of which also include the coding exons (refer to Table 2 and Table 3 for the complete list of genes included in F1CDx). In total, the assay 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 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 selected genomic rearrangements (e.g., gene fusions). Rearrangements in one of the targeted genes included in Table 2 may be reported along with their uniquely identified genomic partners, which can be any gene in PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 4 of 49 {4} 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) will be reported. Table 2. Genes with full coding exonic regions included in FoundationOneCDx 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 | | ARAF | CARD11 | CRKL | EZH2 | GATA6 | KDM5C | MLH1 | P2RY8 | PTEN* | SMAD2 | VEGFA | | ARFRP1 | CASP8 | CSF1R | FAM46C | GID4 (C17orf39) | KDM6A | MPL | PALB2* | PTPN11 | SMAD4 | VHL | | ARID1A | CBFB | CSF3R | FANCA | GNA11 | KDR | MRE11A | PARK2 | PTPRO | SMARC A4 | WHSC1 | | ASXL1 | CBL | CTCF | FANCC | GNA13 | KEAP1 | MSH2 | PARP1 | QKI | SMARC B1 | WHSC1L1 | | ATM* | CCND1 | CTNNA1 | FANCG | GNAQ | KEL | MSH3 | PARP2 | RAC1 | SMO | WT1 | | ATR | CCND2 | CTNNB1 | FANCL* | GNAS | KIT | MSH6 | PARP3 | RAD21 | SNCAIP | XPO1 | | ATRX | CCND3 | CUL3 | FAS | GRM3 | KLHL6 | MST1R | PAX5 | RAD51 | SOCS1 | XRCC2 | | AURKA | CCNE1 | CUL4A | FBXW7 | GSK3B | KMT2A (MLL) | MTAP | PBRM1 | RAD51B* | SOX2 | ZNF217 | | AURKB | CD22 | CXCR4 | FGF10 | H3F3A | KMT2D (MLL2) | MTOR | PDCD1 | RAD51C* | SOX9 | ZNF703 | | AXIN1 | CD274 | CYP17A1 | FGF12 | HDAC1 | KRAS | MUTYH | PDCD1L G2 | RAD51D* | SPEN | | | AXL | CD70 | DAXX | FGF14 | HGF | LTK | MYC | PDGFRA | RAD52 | SPOP | | | BAP1 | CD79A | DDR1 | FGF19 | HNF1A | LYN | MYCL | PDGFRB | RAD54L* | SRC | | | BARD1* | CD79B | DDR2 | FGF23 | HRAS | MAF | MYCN | PDK1 | RAF1 | STAG2 | | | BCL2 | CDC73 | DIS3 | FGF3 | HSD3B1 | MAP2K1 | MYD88 | PIK3C2B | RARA | STAT3 | | | BCL2L1 | CDH1 | DNMT3A | FGF4 | ID3 | MAP2K2 | NBN | PIK3C2G | RB1 | STK11 | | | BCL2L2 | CDK12* | DOT1L | FGF6 | IDH1 | MAP2K4 | NF1 | PIK3CA | RBM10 | SUFU | | | BCL6 | CDK4 | EED | FGFR1 | IDH2 | MAP3K1 | NF2 | PIK3CB | REL | SYK | | | BCOR | CDK6 | EGFR | FGFR2 | IGF1R | MAP3K13 | NFE2L2 | PIK3R1 | RET | TBX3 | | | BCORL1 | CDK8 | EP300 | FGFR3 | IKBKE | MAPK1 | NFKBIA | PIM1 | RICTOR | TEK | | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 5 of 49 {5} *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, and select non-coding regions that include a 3'UTR, a promoter region, and a 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 | | BRAF introns 7- 10 | EGFR introns 7, 15, 24-27 | EWSR1 introns 7- 13 | FGFR3 intron 17 | MYB intron 14 | NTRK2 Intron 12 | RARA intron 2 | SDC4 intron 2 | TMPRSS2 introns 1- 3 | *ETV6 is a common rearrangement partner for NTRK3 ### Test Output The output of the test includes: Category 1: CDx Claims noted in Table 1 of the Intended Use Category 2: Cancer Mutations with Evidence of Clinical Significance Category 3: Cancer Mutations with Potential Clinical Significance Genomic findings other than those listed in Table 1 of the intended use statement (i.e., Categories 2 and 3) are not prescriptive or conclusive for labeled use of any specific therapeutic product. ### Test Kit Contents The test includes a sample shipping kit, which is sent to ordering laboratories. The shipping kit contains the following components: - Specimen Preparation Instructions - Shipping Instructions - Return Shipping Label ### Instruments The F1CDx assay is intended to be performed with serial number-controlled instruments as indicated in Table 4, below. All instruments are qualified by Foundation Medicine, Inc. (FMI) under FMI's Quality System. PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 6 of 49 {6} 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 | ### 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 PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 7 of 49 {7} 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. 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, PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 8 of 49 {8} 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. 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). PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 9 of 49 {9} 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) 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 PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 10 of 49 {10} 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. For patients with solid tumors whose samples have FB-MSI scores >0.0041 and <0.0124, an MSI “Cannot be Determined” result is reported. Patients with this result should be re-tested with a validated orthogonal (alternative) method as these MSI scores represent a range of scores with low reliability. Patients with solid tumors may also receive an MSI status reported as MSI-Cannot Be Determined due to a quality control (QC) failure. Patients with this result should consider re-testing with FoundationOne CDx or an orthogonal (alternative) method, if clinically appropriate. Tumor mutational burden (TMB) is measured by counting all synonymous and non-synonymous substitution and indel variants present at 5% allele frequency or greater and filtering out potential germline variants according to published databases of known germline polymorphisms including Single Nucleotide Polymorphism database (dbSNP) and Exome Aggregation Consortium (ExAC). Additional germline alterations still present after database querying are assessed for potential germline status and filtered out using a somatic-germline/zygosity (SGZ) algorithm. Furthermore, known and likely driver mutations are filtered out to exclude bias of the data set. The resulting mutation number is then divided by the coding region corresponding to the number of total variants counted, or 793 kb. The resulting number is communicated as mutations per Mb unit (mut/Mb). After completion of the Analysis Pipeline, variant data are displayed in the FMI custom developed CATi software applications with sequence QC metrics. As part of data analysis QC for every sample, the F1CDx assay assesses cross-contamination through the use of a SNP profile algorithm, reducing the risk of false-positive calls that could occur as a result of an unexpected contamination event. Sequence data are reviewed by trained bioinformatics personnel. Samples failing any QC metrics are automatically held and not released. ### G. Report Generation Approved results are annotated by automated software with CDx relevant information and are merged with patient demographic information and any additional information provided by FMI as a professional service prior to approval and release by the laboratory director or designee. ### H. Internal Process Controls Related to the System #### Positive Control Each assay run includes a control sample run in duplicate. The control sample contains a pool of ten HapMap cell lines and is used as a positive mutation detection control. 100 different germline SNPs present across the entire targeted region are PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 11 of 49 {11} 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 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 PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 12 of 49 {12} 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. 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 | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 13 of 49 {13} | 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 | | 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 PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 14 of 49 {14} 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). #### **Biomarker Rules for Rearrangements that Lead to *ROS1* Fusions:** Rearrangements in *ROS1* shall be considered CDx biomarker positive, i.e., to lead to *ROS1* RNA fusion, if the following condition is met: - In-strand rearrangement events that may lead to a *ROS1* RNA fusion with another protein coding gene in which the *ROS1* kinase domain is not disrupted. *ROS1* must be on the 3' end of the detected fusion. In this regard, out-of-strand events are considered as non-fusion rearrangements and are classified as CDx biomarker negative. Intragenic fusions in which genomic rearrangement events are wholly internal to the *ROS1* (i.e., *ROS1-ROS1* events) are also considered biomarker negative. Unidentified partners (encoded as N/A) or LINC non-coding partners are also considered CDx biomarker negative. ROS1 fusions with novel partners are required to be in frame. #### **Biomarker Rules for *RET* Fusions:** Fusions in *RET* shall be considered CDx biomarker positive if the following criteria are met: - Any fusion event involving *RET* and another protein-coding gene - *RET* and the partner gene must be in the same 5'-3' orientation - *RET* must be on the 3' end of the detected rearrangement - The *RET* breakpoint must occur before the start of the kinase domain (amino acids 724-1016) PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 15 of 49 {15} # **Biomarker Rules for *AKT1*, *PIK3CA*, and *PTEN* Alterations:** Alterations in *AKT1*, *PIK3CA*, and/or *PTEN* are considered Companion biomarker positive if the following criteria are met: Table 9. Biomarker definition for AKT1, PIK3CA, and PTEN | Gene (Transcript) | Alteration Type | Protein/Transcript Effect | | --- | --- | --- | | *AKT1* (NM_001014431) | SNV | E17K | | *PIK3CA* (NM_006218) | SNV | R88Q, N345K, C420R, E542K, E545A, E545D, E545Q, E545K, E545G, Q546E, Q546K, Q546R, Q546P, M1043V, M1043I, H1047Y, H1047R, H1047L, and G1049R | | *PTEN* (NM_000314) | SNV and indels | C124R, G129E, R130Q, C136R, S170R, R173C, C124S, G129V, R130G C136Y, G129R, R130L, R130P | | | | Any nonsense, frameshift, or splice site alteration • Missense mutations in the start codon and short variant deletions spanning from upstream of the start codon (annotated as M1?) are included in this category. | | | Copy Number Alteration | Homozygous deletion (HD) represents a deletion of one or more exons regardless of transcript in both alleles | | | Rearrangement (RE) | Any rearrangement that disrupts protein function, regardless of transcript • Intragenic events including duplications of only part of the gene, deletions, or inversions. • Translocations, deletions, or inversions where one breakpoint is in *PTEN* and the other breakpoint is in another gene or intergenic region. | ### 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 10, below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 16 of 49 {16} 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 10. List of FDA approved CDx assays for genes targeted by F1CDx | | Device | Company | Technology | Therapy | Indication | | --- | --- | --- | --- | --- | --- | | HER2-Amplification | PathVysion HER-2 DNA Probe Kit | Abbott Molecular, Inc. | FISH | HERCEPTIN (trastuzumab) | Breast cancer | | | PATHWAY Anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody | Ventana Medical Systems, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | InSite HER-2/neu Kit | Biogenex Laboratories, Inc. | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | SPOT-Light HER2 CISH Kit | Life Technologies, Inc. | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | Bond Oracle HER2 IHC System | Leica Biosystems | IHC | HERCEPTIN (trastuzumab) | Breast cancer | | | HER2 CISH pharmDx Kit | Dako Denmark A/S | CISH | HERCEPTIN (trastuzumab) | Breast cancer | | | INFORM HER2 Dual ISH DNA Probe Cocktail | Ventana Medical Systems, Inc. | Dual ISH | HERCEPTIN (trastuzumab) | Breast cancer | | | 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 | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 17 of 49 {17} | 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 | BRACAnalysis CDx | Myriad Genetic Laboratories, Inc. | Sanger and PCR | \ LYNPARZA (olaparib) - treatment/maintenance | Ovarian cancer | | PIK3CA | therascreen PIK3CA RGQ PCR Kit | QIAGEN | PCR | PIQRAY (alpelisib) | Breast cancer | | RET | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | RETEVMO (selpercatinib) | NSCLC and Thyroid Cancer | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 18 of 49 {18} **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 approved PMA supplements that affected the Intended Use are listed in Table 11. **Table 11. 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/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/S023 | June 30, 2021 | *ALK* Rearrangements | Non-Small Cell Lung Cancer | ALUNBRIG® (brigatinib) | | P170019/S025 | November 10, 2021 | *BRAF* V600E | Melanoma | BRAF Inhibitor Monotherapy Group Claim | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 19 of 49 {19} | Submission No. | Date of Approval | Biomarker/Update | Patient Population | Drug | | --- | --- | --- | --- | --- | | | | *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) | | P170019/S043 | October 6, 2023 | *RET* fusions | Solid Tumors | RETEVMO® (selpercatinib) | | P170019/S039 | October 11, 2023 | *BRAF V600E* | NSCLC | BRAFTOVI® (encorafenib) in combination with MEKTOVI® (binimetinib) | ## 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 *AKT1*, *PIK3CA*, and *PTEN* alterations in breast cancer patients was from the data presented PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 20 of 49 {20} using intended use specimens across all 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 accuracy/concordance, limit of blank (LoB), limit of detection (LoD), intermediate precision, and site-to-site precision studies were conducted to support the indication for AKT1, PIK3CA, and PTEN alterations. Table 12 shows the distribution of genes and alteration types that were represented in all the analytical validation studies to support performance of the assay for detection of all the variant types in the AKT1, PIK3CA, and PTEN genes. The analytical studies were performed using the DNAx extraction method. The F1CDx device was updated to include addition of an automated DNA/RNA CoExtraction methodology (CoEx method) to enable isolation of DNA and RNA from the same FFPE tumor specimens. The F1CDx was authorized for DNA only when using the CoEx method. Use of the CoEx method for the new intended use in this supplement 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 PIK3CA and PTEN positive samples. The comparability study between the DNAx and CoEx DNA extraction methods assessed a total of 11 clinical specimens from patients with breast cancer harboring 10 PIK3CA SNVs and one PTEN HD. The observed PPA was 100% when using the DNAx method as reference, while the NPA ranged from 90% to 100% since one PIK3CA SNV was detected by the CoEx method but not the DNAx method for one of the specimens. To assess precision, 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 study evaluated 6 specimens from breast cancer patients with PIK3CA SNVs with variant allele frequencies ranging from 3.1% to 45.3 %. The precision study also included one specimen with a PTEN homozygous deletion with tumor purity of 60%. One replicate from one specimen with a PIK3CA SNV failed to yield a valid result. The agreement for reproducibility and repeatability of the PIK3CA and PTEN positive specimens from patients with breast cancer that yielded valid results was 100%. 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 PIK3CA/AKT1/PTEN alterations in breast cancer by F1CDx were performed using previous versions of the analytical pipeline that has undergone further iteration with modification in the final device design. To demonstrate the F1CDx analytical pipeline changes continue to support the performance for which the test is approved, regression testing using the most current analytical pipeline version will be performed post-market to confirm the robust performance for F1CDx for the detection of PIK3CA/AKT1/PTEN alterations (see section XIII). PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 21 of 49 {21} Table 12: Alterations evaluated in the Analytical Validation Studies | Analytical Validation Study | Gene | Alteration Type | Number of Alterations | | --- | --- | --- | --- | | LoD | PTEN | HD | 1 | | | PTEN | RE | 3 | | Intermediate Precision | AKT1 | SNV | 2 | | | PIK3CA | SNV* | 12 | | | PTEN | HD | 3 | | | | Indel* | 1 | | | | SNV | 4 | | Site-to-Site Reproducibility | AKT1 | SNV | 2 | | | PIK3CA | SNV | 2 | | | PTEN | RE | 3 | | Accuracy** | AKT1 | SNV | 12 | | | PIK3CA | SNV | 87 | | | PTEN | SNV | 22 | | | | Indel | 17 | | | | HD | 16 | | | | RE | 5 | *There are 4 non-target alterations in the intermediate precision study, PIK3CA C420R, PIK3CA E545Q, and PTEN C124S and PTEN_77_78insTG AC. Non-target variants are defined as variants that meet the biomarker definition but were not specified in the initial analytical validation protocol as being a target of the analysis. ** The number of alterations evaluated in the accuracy study correspond to alterations detected as positive by F1CDx, or externally validated orthogonal method, or both. ### 1. Analytical Accuracy/Concordance #### a. Comparison to an Orthogonal Method for PIK3CA/AKT1/PTEN alterations To demonstrate analytical accuracy, comparisons between F1CDx and an externally validated NGS assay (evNGS) to detect AKT1, PIK3CA, and PTEN alterations was performed. The concordance study was conducted with available residual DNA previously extracted from 188 FFPE tissue from breast cancer patients enrolled in the CAPItello-290 (NCT03997123) and CAPItello-291 (NCT04305496) clinical studies that supported TRUQAP (capivasertib) approval (refer to Section X Summary of Primary Clinical Study for study details). The study was supplemented with 49 samples selected from the FMI clinical archives. In total the available samples for the analytical accuracy PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 22 of 49 {22} study included 127 *PIK3CA/AKT1/PTEN* -altered (biomarker positive) and 110 non-altered (biomarker negative) samples. In total, 237 samples were processed in the concordance study with representation from all genes (*PIK3CA*, *AKT1* and *PTEN*) and variant types (SNVs, indels, RE, and HD) included in the capivasertib CDx biomarker definition. Of the 49 samples selected from FMI's archives, all were processed successfully from LC to sequencing by F1CDx, including two that were processed conditionally as they did not meet the required minimum DNA input for library construction, i.e., DNA levels less than 50ng. These were included in the concordance analysis because they harbored rare alterations. All but 1 of the 237 samples completed lab processing and passed the sample QC metrics for the evNGS assay; however, 8 samples were identified as having low tumor content (ranging from 13.2-20%). One sample failed library construction for multiple repeats due to low sample quality. A total of 236 samples were included in the concordance analysis. The F1CDx and externally validated NGS (evNGS) results for the detection of *AKT1*, *PIK3CA*, and *PTEN* alterations and stratified by alteration type, using evNGS as the comparator assay, are provided in the contingency table in Table 13. Variant-level concordance was assessed and the CDx biomarker definition was applied to the F1CDx and evNGS test results. The positive predictive value (PPV) and negative predictive value (NPV) were calculated along with positive percent agreement (PPA) and negative percent agreement (NPA) unadjusted for prevalence for all three genes and stratified by gene. **Table 13. Contingency Table Comparing the Detection of *AKT1*, *PIK3CA*, and *PTEN* Alterations by the F1CDx and evNGS** | *AKT1/PIK3CA/PTEN* alterations | | | | | | | --- | --- | --- | --- | --- | --- | | | evNGS + | evNGS - | Invalid | Total | | | **F1CDx +** | 144 | 9 | 1 | 154 | PPV:94.12% [95%CI*: 89.20%, 96.87%] | | **F1CDx -** | 6 | 13,529 | 57 | 13,592 | NPV:99.96% [95%CI*: 99.90%, 99.98%] | | **Invalid** | 0 | 0 | 0 | 0 | | | **Total** | | | 58 | 13,746 | | | | PPA: 96.00% [95%CI*: 91.55%, 98.15%] | NPA: 99.93% [95%CI*: 99.87%, 99.97%] | | | | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 23 of 49 {23} | **AKT1 E17K** | | | | | | | --- | --- | --- | --- | --- | --- | | | **evNGS +** | **evNGS -** | **Invalid** | **Total** | | | **F1CDx +** | 12 | 0 | 0 | 12 | PPV:100.00% [95%CI*: 75.75%, 100.00%] | | **F1CDx -** | 0 | 224 | 1 | 225 | NPV:100.00% [95%CI*: 98.31%, 100.00%] | | **Invalid** | 0 | 0 | 0 | 0 | | | **Total** | 0 | 0 | 1 | 237 | | | | PPA: 100.00% [95%CI*: 75.75%, 100.00%] | NPA: 100.00% [95%CI*: 98.31%, 100.00%] | | | | | **PIK3CA Alterations** | | | | | | | | **evNGS +** | **evNGS -** | **Invalid** | **Total** | | | **F1CDx +** | 84 | 2 | 1 | 87 | PPV: 97.67% [95%CI*: 91.91%, 99.36%] | | **F1CDx -** | 0 | 4398 | 18 | 4416 | NPV:100.00% [95%CI*: 99.91%, 100.00%] | | **Invalid** | 0 | 0 | 0 | 0 | | | **Total** | 84 | 4400 | 19 | 4503 | | | | PPA: 100.00% [95%CI*: 95.63%, 100.00%] | NPA: 99.95% [95%CI*: 99.83%, 99.99%] | | | | | **PTEN Alterations** | | | | | | | | **evNGS +** | **evNGS -** | **Invalid** | **Total** | | | **F1CDx +** | 48 | 7 | 0 | 55 | PPV:96.67% [95%CI*: 91.74%, 98.70%] | | **F1CDx -** | 6 | 8907 | 38 | 8951 | NPV:100.00% [95%CI*: 99.96%, 100.00%] | | **Invalid** | 0 | 0 | 0 | 0 | | | **Total** | 0 | 8914 | 38 | 9606 | | | | PPA: 88.89% [95%CI*: 77.81%, 94.81%] | NPA: 99.92% [95%CI*: 99.84%, 99.96%] | | | | | **PTEN SNVs** | | | | | | | | **evNGS +** | **evNGS -** | **Invalid** | **Total** | | | **F1CDx +** | 116 | 4 | 1 | 121 | PPV:96.67% [95%CI*: 91.74%, 98.70%] | | **F1CDx -** | 0 | 9084 | 38 | 9122 | NPV: 100.00% [95%CI*: 99.96%, 100.00%] | | **Invalid** | 0 | 0 | 0 | 0 | | | **Total** | 116 | 9088 | 39 | 9243 | | | | PPA: 100.00% [95%CI*: 96.79%, 100%] | NPA: 99.96% [95%CI*: 99.89 %, 99.98%] | | | | | **PTEN indels** | | | | | | | | **evNGS +** | **evNGS -** | **Invalid** | **Total** | | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 24 of 49 {24} | F1CDx + | 11 | 1 | 0 | 0 | PPV: 91.67% [95%CI*: %, %] | | --- | --- | --- | --- | --- | --- | | F1CDx - | 5 | 3759 | 16 | 16 | NPV: 99.87% [95%CI*: 99.69%, 99.94%] | | Invalid | 0 | 0 | 0 | 0 | | | Total | 16 | 3760 | 16 | 3792 | | | | PPA: 68.75% [95%CI*: 44.40%, 85.84%] | NPA: 99.97% [95%CI*: 99.85%, 100%] | | | | | PTEN RE | | | | | | | | evNGS + | evNGS - | Invalid | Total | | | F1CDx + | 2 | 3 | 0 | 5 | PPV: 40% [95%CI*: 11.76%, 76.93 %] | | F1CDx - | 0 | 467 | 2 | 469 | NPV 100%: % [95%CI*: 99.18%, 100.00%] | | Invalid | 0 | 0 | 0 | 0 | | | Total | 2 | 470 | 2 | 474 | | | | PPA: 100.00% [95%CI*: 34.24%, 100%] | NPA: 99.36% [95%CI*: 98.14%, 99.78%] | | | | | PTEN HD | | | | | | | | evNGS + | evNGS - | Invalid | Total | | | F1CDx + | 15 | 1 | 0 | 16 | PPV: 93.75% [95%CI*: 71.67%, 98.89%] | | F1CDx - | 1 | 219 | 1 | 221 | NPV: 99.55% [95%CI*: 97.47 %, 99.20%] | | Invalid | 0 | 0 | 0 | 0 | | | Total | 16 | 220 | 1 | 237 | | | | PPA: 93.75% [95%CI*: 71.67%, 98.89%] | NPA: 99.55% [95%CI*: 97.47%, 99.92%] | | | | *95% 2-sided confidence intervals (CI) were calculated using the Wilson score method. There were 13 samples with a total of 15 discordant calls between the F1CDx and evNGS test results (Two samples contained more than one alteration). The following was observed for the 15 discordant calls in the 13 samples: - The 4 discordant SNVs, 2 PIK3CA SNVs and 2 PTEN SNVs (F1CDx-positive/evNGS-negative) had low VAFs ranging from 0.93% to 2.34% by F1CDx. - One sample had a PTEN indel alteration detected at different splice sites by each assay - Four samples with PTEN indel alterations were not detected by F1CDx; three of those samples were filtered out by the analysis pipeline because the alterations were below the threshold. - One sample had a PTEN RE detected by F1CDx with low chimeric reads of 15. The evNGS assay called PTEN copy number alteration PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 25 of 49 {25} but could not determine if mono-allelic or bi-allelic, i.e., the call did not meet biomarker criteria. - Two additional discordant PTEN REs (F1CDx-positive/evNGS-negative) were called by F1CDx with 15 or fewer supporting chimeric read pairs. - The only PTEN HD F1CDx-positive/evNGS negative was in a sample that the evNGS assay labelled as low tumor content (15%); F1CDx test results also revealed that this sample had a low computational tumor purity (TP) of 15%. ## 2. Analytical Sensitivity ### a. Limit of Blank (LoB) The LoB of zero was confirmed by testing 3 biomarker negative FFPE tissue samples (breast carcinoma or invasive ductal carcinoma). Of the 60 replicates, 100% produced valid results and no AKT1, PIK3CA, or PTEN alterations were detected across all sample replicates; therefore, the false positive rate for calling alterations was determined to be 0.00%. The study demonstrated that the F1CDx assay does not produce false-positive results in detecting AKT1, PIK3CA, and PTEN alterations in biomarker-negative FFPE tissue samples from patients with breast cancer. ### b. Limit of Detection (LoD) The F1CDx LoD for the detection of PIK3CA, AKT1 and PTEN alterations was established through platform assessments of LoD and within CDx-specific LoD studies. The LoD of AKT1 E17K was established through a previously conducted study for the PMA approval (P170019) as 6.13% VAF. The LoD of PIK3CA SNVs was established previously as 4.91% VAF (refer to SSED for P170019/S006). The LoD of PTEN SNVs and indels was taken as approximately 5% based on aggregated data analysis of LoD for SNVs from the original PMA. The LoD was confirmed through a precision study that used samples at or near 5% VAF. The lowest observed average VAF that achieved 100% reproducibility for PTEN SNVs via the intermediate precision study is the claimed LoD for PTEN SNVs. This value is 5.72% VAF, see intermediate precision study results below. Similarly, for PTEN indels, the average VAF that achieved 100% reproducibility for a PTEN insertion variant is taken as the confirmed LoD. The LoDs of PTEN RE and HD were determined through a new study. DNA from three breast cancer samples harboring PTEN rearrangements were processed along with one breast cancer sample that had a PTEN HD. Each sample with a PTEN RE was assessed at five targeted chimeric read levels (30, 24, 18, 12, and 4). Twenty (20) replicates were assessed for each dilution level, except the 30 level, which examined 14 replicates. Ninety-four (94) replicates were tested per sample. Each sample with a PTEN homozygous PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 26 of 49 {26} deletion was assessed at five targeted tumor purity dilution levels at 50, 40, 35, 30, 20% of tumor purity with 14, 20, 20, 19, and 18 replicates, respectively. Ninety-one (91) replicates were tested per sample. Each specimen was evaluated close to the minimum input requirements of the assay (50 ng), thus representing the most challenging evaluation of PTEN alteration detection. The LoD for each sample was determined based on chimeric reads using hit rate method for PTEN RE and SNP-adjusted tumor purity (TP) using the probit method for PTEN homozygous deletions. The LoD for PTEN rearrangements was 20.45 chimeric reads and for PTEN HD was 42.28% tumor purity. The LoD for PTEN homozygous deletions was confirmed through an intermediate precision study where breast cancer samples targeted at or near platform LoD of 33.40% tumor purity were processed. The lowest observed tumor purity at 35.25% with >95% reproducibility was claimed as the LoD for PTEN homozygous deletions. The summary of LoD values established for AKT1 SNVs, PIK3CA SNVs, PTEN SNVs, PTEN rearrangements and PTEN homozygous deletions are captured in Table 14. Table 14. Summary of LoD for AKT1/PIK3CA/PTEN alterations | Gene | Variant Type | LoD | | --- | --- | --- | | AKT1 | SNVs | 6.13% VAF | | PIK3CA | SNVs | 4.91% VAF | | PTEN | SNVs | 5.72% VAF | | PTEN | Indel | 5.67% VAF | | PTEN | RE | 20.45 chimeric reads | | PTEN | HD | 35.25% tumor purity | ### 3. Precision #### a. Within-Laboratory (Intermediate) Precision To evaluate the precision of PIK3CA/AKT1/PTEN alteration detection, a within-laboratory (intermediate) precision study using 14 pre-extracted DNA samples from FFPE tissue from patients with breast cancer with 18 target alterations selected to be at 1-1.5x LoD was conducted at a challenging DNA input (close to 50 ng); refer to Table 15 for the samples evaluated in the study. In total, there were 321 sample replicates, of which, 219 were valid (i.e., passed processing QC criteria) and used in the analysis. PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 27 of 49 {27} Table 15. Samples evaluated in the Within-Laboratory (Intermediate) Precision Study. | Sample | Target Gene | Alteration | Source sample VAF (SNV)/TP (HD) | Alteration Type | Disease Ontology | | --- | --- | --- | --- | --- | --- | | 1 | PTEN | PTEN loss of exons 5 of 9 | 41.0% | HD | Breast invasive ductal carcinoma (IDC) | | 2 | PTEN | PTEN loss of exons 9 of 9 | 35.5% | HD | Breast invasive ductal carcinoma (IDC) | | 3 | PTEN | PTEN loss of exons 9 of 9 | 40.0% | HD | Breast carcinoma (NOS) | | 4 | AKT1 | E17K | 6.7% | SNV | Breast invasive ductal carcinoma (IDC) | | 5 | AKT1 | E17K | 2.1% | SNV | Breast invasive ductal carcinoma (IDC) | | 6 | PIK3CA | E542K | 3.6% | SNV | Breast invasive ductal carcinoma (IDC) | | 7 | PIK3CA | M1043I | 5.8% | SNV | Breast carcinoma (NOS) | | 8 | PIK3CA | E542K | 5.5% | SNV | Breast carcinoma (NOS) | | 9 | PIK3CA | M1043V | 5.6% | SNV | Breast carcinoma (NOS) | | 10 | PIK3CA | M1043V | 15.3% | SNV | Breast invasive lobular carcinoma (ILC) | | 11 | PIK3CA | N345K | 5.8% | SNV | Breast carcinoma (NOS) | | 12 | PIK3CA | E545K | 4.6% | SNV | Breast carcinoma (NOS) | | 13 | PIK3CA | Q546K | 1.6% | SNV | Breast invasive ductal carcinoma (IDC) | | 14 | PIK3CA | E542K | 2.34% | SNV | Breast invasive ductal carcinoma (IDC) | | 15 | PTEN | C124R | 12.4% | SNV | Breast invasive ductal carcinoma (IDC) | | 16 | PTEN | C124S | 37.3% | SNV | Breast invasive ductal carcinoma (IDC) | | 17 | PTEN | R130Q | 11.6% | SNV | Breast invasive ductal carcinoma (IDC) | | 18 | PTEN | R130X | 6.0% | SNV | Breast carcinoma (NOS) | For the assessment of reproducibility, sample replicates from the same source sample were processed under different conditions by varying one factor at a time. The conditions were applied on a plate-level basis and included the same operator, same day, same reagent lot, and same sequencer. The reproducibility performance was evaluated by comparing to the reference biomarker status (positive for targeted PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 28 of 49 {28} variants) across all valid replicates. Table 16 summarizes the reproducibility statistics across samples evaluated for the 14 samples selected to have 18 targeted variants at 1-1.5x LoD. Of the 18 targeted alterations 10 target alterations that had observed levels across all valid replicates for each source sample at ≥1x LoD (samples 1-10), while 8 target alterations had observed levels at <1x LoD (samples 11-18). All 10 target alterations with observed levels ≥ 1x LoD demonstrated 100% reproducibility, while the remaining 8 alterations with observed levels <1x LoD demonstrated 50% to 100% reproducibility. Table 16. Reproducibility for AKT1, PIK3CA, and PTEN alterations | Sample | Observed Average VAF (SNV) and TP (HD) | Fold LoD | Alteration | Variant Type | Positive Replicates | Valid Replicates | Reproducibility (95% 2-Sided Score CI) * | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 7.75% | 1.27x | AKT1 E17K | SNV | 23 | 23 | 100.00% [85.69%, 100.00%] | | 2 | 6.40% | 1.39x | PTEN R130X | SNV | 22 | 22 | 100.00% [85.13%, 100.00%] | | 3 | 6.20% | 1.35x | PTEN C124R | SNV | 20 | 20 | 100.00% [83.89%, 100.00%] | | 4 | 5.72% | 1.24x | PTEN R130Q | SNV | 23 | 23 | 100.00% [85.69%, 100.00%] | | 5 | 4.96% | 1.01x | PIK3CA M1043V | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | | 6 | 6.18% | 1.35x | PTEN C124S | SNV | 23 | 23 | 100.00% [85.69%, 100.00%] | | 7 | 5.13% | 1.05x | PIK3CA E545K | SNV | 22 | 22 | 100.00% [85.13%, 100.00%] | | 8 | 35.25% | 1.06x | PTEN loss exons 9 of 9 | HD | 24 | 24 | 100.00% [86.2%, 100.00%] | | 9 | 40.94% | 1.23x | PTEN loss exons 5 of 9 | HD | 24 | 24 | 100.0% [86.2%, 100.00%] | | 10 | 39.65% | 1.19x | PTEN loss exons 9 of 9 | HD | 23 | 23 | 100.00% [85.69 %, 100.00%] | | 11 | 1.35 % | 0.22x | AKT1 E17K | SNV | 11 | 22 | 50.00%, [30.72%, 69.28%] | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 29 of 49 {29} | 12 | 2.09 % | 0.43x | PIK3CA E542K | SNV | 19 | 22 | 86.36%, [66.67%,95.25% ] | | --- | --- | --- | --- | --- | --- | --- | --- | | 13 | 1.93 % | 0.39x | PIK3CA E542K | SNV | 21 | 22 | 95.45%, [78.20%,99.19% .] | | 14 | 0.68 % | 0.14x | PIK3CA Q546K | SNV | 14 | 22 | 63.64%, [42.95%,80.27% .] | | 15 | 4.31 % | 0.88x | PIK3CA M1043I | SNV | 24 | 24 | 100.00%, [86.20%, 100.00%.] | | 16 | 4.58 % | 0.93x | PIK3CA E542K | SNV | 24 | 24 | 100%, [86.20%, 100.00%.] | | 17 | 4.88 % | 1.00x | PIK3CA N345K | SNV | 22 | 22 | 100.00%, [85.13%, 100.00%.] | | 18 | 4.68 % | 0.96x | PIK3CA M1043V | SNV | 23 | 23 | 100.00%, [85.69%, 100.00%.] | *95% 2-sided CI were calculated using the Wilson score method. Another 3 SNVs in PIK3CA and 1 indel in PTEN with observed average VAF levels were found in the samples tested to meet the biomarker definition although they were not initially targeted for the evaluation of precision. All three variants achieved reproducibility of 100% as shown in Table 17. Table 17. Reproducibility of additional non-targeted alterations at LoD values ≥1x | Sample | Observed Average MAF | Fold LoD | Alteration | Variant Type | Positive Replicates | Valid Replicates | Reproducibility [95% 2-Sided Score CI] * | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 15.8% | 3.08x | PIK3CA E545K | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | | 2 | 5.67% | 1.23x | PTEN 77 78insTGA C | Indel | 23 | 23 | 100.00% [85.69%, 100.00%] | | 3 | 16.34% | 3.33x | PIK3CA C420R | SNV | 22 | 22 | 100.00% [85.13%, 100.00%] | | 4 | 33.60% | 6.86x | PIK3CA E545Q | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | *95% 2-sided CI were calculated using the Wilson score method. For the assessment of repeatability, sample replicates from the same source sample were processed under the same condition. The result was considered concordant PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 30 of 49 {30} when the alteration statuses are the same between the two replicates run under identical measurement conditions. Table 18 presents the repeatability statistics for the 10 target alterations at levels ≥1x LoD (samples 1-10), followed by samples at levels <1 LoD (samples 11-18). Table 18. Repeatability for AKT1, PIK3CA, and PTEN alterations | Sample | Observed Average VAF (SNV) and Tumor Purity (HD) | Fold LoD | Alteration | Variant Type | Positive Replicates | #Total Valid Replicates | Repeatability (95% 2-Sided Score CI)* | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 7.75% | 1.27x | AKT1 E17K | SNV | 11 | 11 | 100.00% [74.12%, 100.00%] | | 2 | 6.40% | 1.39x | PTENR130X | SNV | 11 | 11 | 100.00% [74.12%, 100.00%] | | 3 | 6.20% | 1.35x | PTEN C124R | SNV | 9 | 9 | 100.00% [N/A*] | | 4 | 5.72% | 1.24x | PTEN R130Q | SNV | 11 | 11 | 100.00% [74.12%, 100.00%] | | 5 | 4.96% | 1.01x | PIK3CA M1043V | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 6 | 6.18% | 1.35x | PTEN C124S | SNV | 11 | 11 | 100.00% [74.12%, 100.00%] | | 7 | 5.13% | 1.05x | PIK3CA E545K | SNV | 11 | 11 | 100.00% [74.12%, 100%] | | 8 | 35.25% | 1.06x | PTEN loss exons 9 of 9 | HD | 12 | 12 | 100.0% [75.75%, 100.00%] | | 9 | 40.94% | 1.23x | PTEN loss exons 5 of 9 | HD | 12 | 12 | 100.00% [75.75%, 100.00%] | | 10 | 39.65% | 1.19x | PTEN loss exons 9 of 9 | HD | 11 | 11 | 100.00% [74.12%, 100.00%] | PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 31 of 49 {31} | 11 | 1.35 | 0.22x | *AKT1* E17K | SNV | 2 | 11 | 18.18% [5.14%, 47.70%] | | --- | --- | --- | --- | --- | --- | --- | --- | | 12 | 2.09 | 0.43x | *PIK3CA* E542K | SNV | 8 | 11 | 72.73% [43.44%, 90.25%] | | 13 | 1.93 | 0.39x | *PIK3CA* E542K | SNV | 10 | 10 | 100.00% [N/A**] | | 14 | 0.68 | 0.14x | *PIK3CA* Q546K | SNV | 5 | 10 | 50.0% [N/A**] | | 15 | 4.31 | 0.88x | *PIK3CA* E542K | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 16 | 4.58 | 0.93x | *PIK3CA* M1043I | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 17 | 4.88 | 1.00x | *PIK3CA* N345K | SNV | 11 | 11 | 100.00% [74.12%, 100.00%] | | 18 | 4.68 | 0.96x | *PIK3CA* M1043V | SNV | 11 | 11 | 100.00% [74.12%, 100.00%] | *95% 2-sided CI were calculated using the Wilson score method. **CI is not estimated for sample sizes ≤10. Table 19 presents the repeatability statistics for the 3 SNVs in *PIK3CA* and 1 indel in *PTEN* with observed average VAF levels were found in the samples tested to meet the biomarker definition although they were not initially targeted for the evaluation of precision. All three variants achieved repeatability of 100% as shown in Table 19. **Table 19. Repeatability of additional samples at LoD values ≥1x LoD** | Sample | Observed Average MAF | Fold LoD | Alteration | Variant Type | Positive Replicates | Valid Replicates | Repeatability [95% 2-Sided Score CI]* | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 15.8% | 3.08x | *PIK3CA* E545K | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 2 | 5.67% | 1.23x | *PTEN* 77 78insTGAC | Indel | 11 | 11 | 100.00% [74.12%, 100.00%] | | 3 | 16.34% | 3.3x | *PIK3CA* C420R | SNV | 10 | 10 | 100.00% [N/A**] | | 4 | 33.60% | 6.86x | *PIK3CA* E545Q | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | *95% 2-sided CI were calculated using the Wilson score method. **CI is not estimated for sample sizes ≤10. PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 32 of 49 {32} ## b. Site-to-site Precision Site-to-site precision for PIK3CA, AKT1 and PTEN alteration detection was evaluated via two site-to-site precision studies. A total of 7 biomarker positive samples consisting of pre-extracted DNA from FFPE tissue from breast cancer patients targeting 1-1.5x LoD alteration levels were selected for evaluation (see Table 20). These samples were selected based on harboring AKT1 E17K mutation (2 samples), PIK3CA SNVs (2 samples), and PTEN REs (3 samples) that meet the CDx biomarker definition. Together the studies evaluated 7 biomarker-positive samples at a challenging DNA input (close to 50 ng), at two laboratory locations in Cambridge, MA (CAM) and Morrisville, NC (RTP). The first study evaluated 2 samples with AKT1 E17K mutation and 2 samples with PIK3CA SNVs. Each sample tested in duplicate at the CAM and RTP sites, with two (2) reagent lots, and three (3) unique start days (sequencer runs) per site per reagent lot. A total of 24 replicates were evaluated per sample. The second study evaluated the site-to-site precision of the F1CDx assay in calling PTEN RE in DNA derived from FFPE tumor tissue from patients with breast cancer. Samples with PTEN REs and at least 1,200 ng remaining mass of DNA were selected for testing. Samples were titrated with biomarker-negative diluent DNA extracted from FFPE samples from breast cancer patients to achieve the desired targeted chimeric reads (1x - 1.5x LoD) and DNA input close to 50 ng. Each sample was tested in duplicate at 2 sites on 3 library construction start days with 2 reagent lots for a total of 24 replicates. Table 20. Samples evaluated in the Site-to-Site Precision Studies | Sample | Target Gene | Alteration | Source sample VAF (SNV)/Chimeric reads (RE) | Alteration Type | Disease Ontology | | --- | --- | --- | --- | --- | --- | | 1 | AKT1 | E17K | 9.13% | SNV | Breast Cancer | | 2 | AKT1 | E17K | 9.78% | SNV | Breast Cancer | | 3 | PIK3CA | E545K | 7.34% | SNV | Breast Cancer | | 4 | PIK3CA | Q546E | 7.37% | SNV | Breast Cancer | | 5 | PTEN | PTEN_PTEN duplication | 153* | RE | Breast carcinoma (NOS) | | 6 | PTEN | PTEN_N/A truncation | 113* | RE | Breast invasive ductal carcinoma (IDC) | | 7 | PTEN | PTEN_N/A truncation | 92* | RE | Breast invasive ductal carcinoma (IDC) | *Chimeric read data were from initial testing of the clinical samples. The targeted number of chimeric reads for each sample was 1x – 1.5x LoD. PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 33 of 49 {33} The point estimates and 95% two-sided CIs for reproducibility of each sample are detailed in Table 21. Only five samples were ≥1x LoD and included in the reproducibility results below. Table 21. Reproducibility for AKT1, PIK3CA and PTEN alterations | Sample | Observed Average VAF (SNV) and Chimeric Reads (RE) | Fold LoD | Alteration | Variant Type | Positive Replicates | Valid Replicates | Reproducibility (95% 2-Sided CI) * | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 9.46% | 1.54x | AKT1 E17K | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | | 2 | 10.66% | 1.74x | AKT1 E17K | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | | 3 | 7.41% | 1.51x | PIK3CA E545K | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | | 4 | 9.23% | 1.88x | PIK3CA Q546E | SNV | 24 | 24 | 100.00% [86.20%, 100.00%] | | 5 | 23.00 | 1.12x | PTEN_PTEN duplication | RE | 24 | 24 | 100.00% [86.20%, 100.00%] | | 6 | 19.42 | 0.95x | PTEN N/A_truncation | RE | 24 | 24 | 100.00% [86.20%, 100.00%] | | 7 | 20.08 | 0.98x | PTEN N/A_truncation | RE | 23 | 24 | 95.83% [79.76%, 99.26%] | *95% 2-sided CI were calculated using the Wilson score method. Repeatability was evaluated in the breast cancer samples by processing samples with 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 alteration. The point estimates and 95% two-sided CIs for repeatability of each sample are detailed in Table 22. PMA P170019/S048: FDA Summary of Safety and Effectiveness Data 34 of 49 {34} Table 22. Repeatability for AKT1, PIK3CA and PTEN alterations | Sample | Observed Average VAF (SNV) and Chimeric Reads (RE) | Fold LoD | Alteration | Variant Type | Positive Replicates | Valid Replicates | Repeatability (95% 2-Sided CI)* | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | 9.46% | 1.54x | AKT1 E17K | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 2 | 10.66% | 1.74x | AKT1 E17K | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 3 | 7.41% | 1.51x | PIK3CA E545K | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 4 | 9.23% | 1.88x | PIK3CA Q546E | SNV | 12 | 12 | 100.00% [75.75%, 100.00%] | | 5 | 23.00 | 1.12x | PTEN_PTEN duplication | RE | 12 | 12 | 100.00% [75.75%, 100.00%] | | 6 | 19.42 | 0.95x | PTEN N/A truncation | RE | 12 | 12 | 100.00% [75.75%, 100.00%] | | 7 | 2… --- **Source:** [https://fda-staging.innolitics.com/device/P170019S048](https://fda-staging.innolitics.com/device/P170019S048) **Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. 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