← Product Code [PQP](/productcode/PQP) · P170019S029

# FoundationOne CDx (F1CDx) (P170019S029)

_Foundation Medicine, Inc. · PQP · Feb 18, 2022 · Pathology · APPR_

**Canonical URL:** https://fda-staging.innolitics.com/device/P170019S029

## Device Facts

- **Applicant:** Foundation Medicine, Inc.
- **Product Code:** [PQP](/productcode/PQP.md)
- **Decision Date:** Feb 18, 2022
- **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 |
| --- | --- | --- | --- | --- | --- |
| P170019S029 · Feb 18, 2022 | FoundationOne CDx (F1CDx) | Foundation Medicine, Inc. | Foundation Medicine clinical commercial database; Commercially procured tumor bank specimens | Retrospective chart review of clinical commercial database samples and analysis of tumor bank specimens were used to demonstrate analytical accuracy and concordance of the device's MSI calling against MMR IHC assays and to support the negative percent agreement (NPA) assessment. | Retrospective chart review; Clinical commercial database; Tumor bank; Concordance analysis |

### Clinical Evidence

| Study Design | Population | Comparator | Key Endpoints |
| --- | --- | --- | --- |
| Retrospective chart review | 134 colorectal cancer (CRC) and 52 uterus endometrial cancer patients from the FMI clinical commercial database; Sample Size: 178 samples (128 CRC and 50 endometrial) | Mismatch repair (MMR) immunohistochemistry (IHC) assays | Concordance between MSI classification (MSI-H/dMMR vs non-MSI-H/pMMR) |
| Retrospective analysis of tumor tissue FFPE specimens | Patients from KEYNOTE-158 and KEYNOTE-164 clinical trials and commercially procured tumor bank specimens; Sample Size: 1664 tumor samples tested | Local clinical trial assays (CTAs) (PCR and IHC) | Concordance between local CTAs and F1CDx; ORR in efficacy population |

## Indications for Use

FoundationOne®CDx (F1CDx) is a qualitative next generation sequencing based in vitro diagnostic test that uses targeted high throughput hybridization-based capture technology for detection of substitutions, insertion and deletion alterations (indels) and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. The test is intended as a companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Additionally, F1CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product.

## Device Story

F1CDx is a NGS-based IVD performed at centralized laboratories (Cambridge, MA; Morrisville, NC). It accepts FFPE tumor tissue; extracts DNA; performs whole-genome shotgun library construction; uses hybrid-capture to target 324 cancer-related genes. Sequencing occurs on Illumina HiSeq 4000. Proprietary software processes data to detect base substitutions, indels, CNAs, and rearrangements; calculates MSI, TMB, and HRD status. Results are reviewed by bioinformatics personnel and pathologists. Output is a clinical report identifying genomic alterations and companion diagnostic biomarker status. Healthcare providers use this to select targeted therapies (e.g., pembrolizumab for MSI-H/TMB-H) or for general tumor profiling. Benefits include identifying patients eligible for specific targeted treatments, potentially improving clinical outcomes.

## Clinical Evidence

Clinical evidence derived from a bridging study using retrospective samples from KEYNOTE-158 and KEYNOTE-164 trials. 165 patients had evaluable F1CDx results. ORR in F1CDx-positive/CTA-positive patients was 43.0% (95% CI: 33.5, 52.9) vs 12.1% (95% CI: 5.0, 23.3) in F1CDx-negative/CTA-positive patients. Concordance analysis (n=1174) showed PPA 69.8% and NPA 99.3% against local CTA results. Analytical validation included LoD (15.67% tumor purity), LoB (zero), and precision studies.

## Technological Characteristics

Targeted NGS using hybridization-based capture of 324 genes. DNA input 50-1000 ng from FFPE. Sequencing on Illumina HiSeq 4000. Analysis pipeline uses BWA, Picard, GATK, and proprietary algorithms for variant calling, MSI (fraction-based), TMB, and HRD. Software is proprietary, cloud-based/server-hosted. Instruments include Agilent Bravo, Hamilton STAR, KingFisher Flex, Covaris LE220-Plus.

## 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.

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- cobas EGFR Mutation Test v2
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- FoundationFocus CDxBRCA
- 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/).

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# 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/S029

Date of FDA Notice of Approval: February 18, 2022

The original PMA (P170019) was approved on November 30, 2017 and is indicated for the detection of genetic alterations in patients who may benefit from one of fifteen FDA-approved therapies for non-small cell lung cancer (NSCLC), melanoma, breast cancer, colorectal cancer (CRC), and ovarian cancer. The SSED to support the indication is available on the CDRH website and is incorporated by reference here. Subsequently, thirteen PMA supplements were approved for expanding the indications for use of F1CDx since its original approval. A PMA supplement (P170019/S005) for adding genomic loss of heterozygosity (LOH) was approved on April 10, 2019. A PMA supplement (P170019/S004) for adding an indication for LYNPARZA® (olaparib) in ovarian cancer patients with *BRCA1/2* alterations was approved on July 1, 2019. A PMA supplement (P170019/S008) for adding an indication for TAGRISSO® (osimertinib) in NSCLC patients with *EGFR* exon 19 deletions and *EGFR* exon 21 L858R alterations was approved on July 1, 2019. A PMA supplement (P170019/S006) for adding an indication for PIQRAY® (alpelisib) in breast cancer patients with *PIK3CA* alterations was approved on December 3, 2019. A PMA supplement (P170019/S010) for adding a second site in Morrisville, NC, where the F1CDx assay will be performed, was approved on December 16, 2019. A PMA supplement (P170019/S013) for adding an indication for PEMZYRE® (pemigatinib) in cholangiocarcinoma patients with *FGFR2* fusions was approved on April 17, 2020. A PMA supplement (P170019/S011) for adding an indication for TABRECTA® (capmatinib) in NSCLC patients with *MET* single nucleotide variants (SNVs) and indels that lead to *MET* exon 14 skipping was approved on May 6, 2020. A PMA supplement (P170019/S015) for adding an indication for LYNPARZA® (olaparib) in metastatic castration resistant prostate cancer (mCRPC) patients with mutations in homologous recombination repair (HRR) genes was approved on May 19, 2020. A PMA

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supplement (P170019/S016) for adding an indication for KEYTRUDA® (pembrolizumab) in patients with solid tumors high tumor mutational burden (TMB) at the cut-off of 10 mutations per megabase (mut/Mb) was approved on June 16, 2020. A PMA supplement (P170019/S017) for adding an indication for VITRAKVI® (larotrectinib) in patients with *NTRK1/2/3* fusions was approved on October 23, 2020. PMA supplement (P170019/S021) for adding an indication for TRUSELTIQ™ (infigratinib) in cholangiocarcinoma patients with FGFR2 fusions and select rearrangements was approved on May 28, 2021. A PMA supplement (P170019/S023) for adding an indication for ALUNBRIG® (brigatinib) in NSCLC patients with *ALK* rearrangements was approved on June 30, 2021. A PMA supplement (P170019/S026) was approved July 30, 2021 for a tumor profiling claim to replace the Principal Component Analysis (PCA) MSI caller used within F1CDx to the Fraction-Based (FB) MSI caller. A PMA supplement (P170019/S025) was approved on November 10, 2021, for the class labeling for BRAF Inhibitors approved by FDA. A PMA supplement (P170019/S030) was approved on January 19, 2022, for adding an indication TECENTRIQ® (atezolizumab) in combination with COTELLIC® (cobimetinib) and ZELBORAF® (vemurafenib) in melanoma patients with BRAF V600 mutations.

The current supplement was submitted to expand the indication for F1CDx to include a companion diagnostic (CDx) indication for the detection of microsatellite instability – High (MSI-H) status in patients with solid tumors who may benefit from treatment with KEYTRUDA® (pembrolizumab).

## II. INDICATIONS FOR USE

FoundationOne®CDx (F1CDx) is a qualitative next generation sequencing based *in vitro* diagnostic test that uses targeted high throughput hybridization-based capture technology for detection of substitutions, insertion and deletion alterations (indels) and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. The test is intended as a companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Additionally, F1CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product.

**Table 1. Companion diagnostic indications**

|  Tumor Type | Biomarker(s) Detected | Therapy  |
| --- | --- | --- |
|  Non-small cell lung cancer (NSCLC) | *EGFR* exon 19 deletions and *EGFR* exon 21 L858R alterations | Gilotrif® (afatinib), Iressa® (gefitinib), Tagrisso® (osimertinib), or Tarceva® (erlotinib)  |
|   |  *EGFR* exon 20 T790M alterations | Tagrisso® (osimertinib)  |
|   |  *ALK* rearrangements | Alecensa® (alectinib),  |

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|   |  | 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)  |
|  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)  |
|  Colorectal cancer | KRAS wild-type (absence of mutations in codons 12 and 13) | Erbitux® (cetuximab)  |
|   |  KRAS wild-type (absence of mutations in exons 2, 3, and 4) and NRAS wild type (absence of mutations in exons 2, 3, and 4) | Vectibix® (panitumumab)  |
|  Ovarian cancer | BRCA1/2 alterations | Lynparza® (olaparib) or Rubraca® (rucaparib)  |
|  Cholangiocarcinoma | FGFR2 fusions and select rearrangements | Pemazyre® (pemigatinib) 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)  |
|  Solid tumors | TMB ≥ 10 mutations per megabase | Keytruda® (pembrolizumab)  |
|   |  NTRK1/2/3 fusions | Vitrakvi® (larotrectinib)  |
|   |  MSI-High | Keytruda® (pembrolizumab)  |

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*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 will be 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 FoundationOne®CDx labeling.

### V. DEVICE DESCRIPTION

FoundationOne®CDx (F1CDx) is performed at Foundation Medicine, Inc. sites located in Cambridge, MA and Morrisville, NC. The assay includes reagents, software, instruments and procedures for testing DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples.

The assay employs a single DNA extraction method from routine FFPE biopsy or surgical resection specimens, 50-1000 ng of which undergoes whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, 1 promoter region, 1 non-coding RNA (ncRNA), and select intronic regions from 34 commonly rearranged genes, 21 of which also include the coding exons (refer to Table 2 and Table 3, below, for the complete list of genes included in F1CDx). In total, the assay therefore detects alterations in 324 genes. Using the Illumina® HiSeq 4000 platform, hybrid-capture selected libraries are sequenced to high uniform depth (targeting > 500X median coverage with > 99% of exons at coverage > 100X). Sequence data are processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number alterations (amplifications and homozygous deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic signatures including microsatellite instability (MSI), tumor mutational burden (TMB), and positive homologous recombination deficiency (HRD) status (tBRCA-positive and/or LOH high) will be reported.

Table 2. Genes with full coding exonic regions included in F1CDx for the detection of substitutions, insertions and deletions (indels), and copy number alterations (CNAs)

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|  ABL1 | BRAF | CDKN1A | EPHA3 | FGFR4 | IKZF1 | MCL1 | NKX2-1 | PMS2 | RNF43 | TET2  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  ACVR1B | BRCA1 | CDKN1B | EPHB1 | FH | INPP4B | MDM2 | NOTCH1 | POLD1 | ROS1 | TGFBR2  |
|  AKT1 | BRCA2 | CDKN2A | EPHB4 | FLCN | IRF2 | MDM4 | NOTCH2 | POLE | RPTOR | TIPARP  |
|  AKT2 | BRD4 | CDKN2B | ERBB2 | FLT1 | IRF4 | MED12 | NOTCH3 | PPARG | SDHA | TNFAIP3  |
|  AKT3 | BRIP1 | CDKN2C | ERBB3 | FLT3 | IRS2 | MEF2B | NPM1 | PPP2R1A | SDHB | TNFRSF14  |
|  ALK | BTG1 | CEBPA | ERBB4 | FOXL2 | JAK1 | MEN1 | NRAS | PPP2R2A | SDHC | TP53  |
|  ALOX12B | BTG2 | CHEK1 | ERCC4 | FUBP1 | JAK2 | MERTK | NT5C2 | PRDM1 | SDHD | TSC1  |
|  AMER1 | BTK | CHEK2 | ERG | GABRA6 | JAK3 | MET | NTRK1 | PRKAR1A | SETD2 | TSC2  |
|  APC | C11orf30 | CIC | ERRFI1 | GATA3 | JUN | MITF | NTRK2 | PRKCI | SF3B1 | TYRO3  |
|  AR | CALR | CREBBP | ESR1 | GATA4 | KDM5A | MKNK1 | NTRK3 | PTCH1 | SGK1 | U2AF1  |
|  ARAF | CARD11 | CRKL | EZH2 | GATA6 | KDM5C | MLH1 | P2RY8 | PTEN | SMAD2 | VEGFA  |
|  ARFRP1 | CASP8 | CSF1R | FAM46C | GID4 (C17orf39) | KDM6A | MPL | PALB2 | PTPN11 | SMAD4 | VHL  |
|  ARID1A | CBFB | CSF3R | FANCA | GNA11 | KDR | MRE11A | PARK2 | PTPRO | SMARC A4 | WHSC1  |
|  ASXL1 | CBL | CTCF | FANCC | GNA13 | KEAP1 | MSH2 | PARP1 | QKI | SMARC B1 | WHSC1L1  |
|  ATM | CCND1 | CTNNA1 | FANCG | GNAQ | KEL | MSH3 | PARP2 | RAC1 | SMO | WT1  |
|  ATR | CCND2 | CTNNB1 | FANCL | GNAS | KIT | MSH6 | PARP3 | RAD21 | SNCAIP | XPO1  |
|  ATRX | CCND3 | CUL3 | FAS | GRM3 | KLHL6 | MST1R | PAX5 | RAD51 | SOCS1 | XRCC2  |
|  AURKA | CCNE1 | CUL4A | FBXW7 | GSK3B | KMT2A (MLL) | MTAP | PBRM1 | RAD51B | SOX2 | ZNF217  |
|  AURKB | CD22 | CXCR4 | FGF10 | H3F3A | KMT2D (MLL2) | MTOR | PDCD1 | RAD51C | SOX9 | ZNF703  |
|  AXIN1 | CD274 | CYP17A1 | FGF12 | HDAC1 | KRAS | MUTYH | PDCD1L G2 | RAD51D | SPEN |   |
|  AXL | CD70 | DAXX | FGF14 | HGF | LTK | MYC | PDGFRA | RAD52 | SPOP |   |
|  BAP1 | CD79A | DDR1 | FGF19 | HNF1A | LYN | MYCL | PDGFRB | RAD54L | SRC |   |
|  BARD1 | CD79B | DDR2 | FGF23 | HRAS | MAF | MYCN | PDK1 | RAF1 | STAG2 |   |
|  BCL2 | CDC73 | DIS3 | FGF3 | HSD3B1 | MAP2K1 | MYD88 | PIK3C2B | RARA | STAT3 |   |
|  BCL2L1 | CDH1 | DNMT3A | FGF4 | ID3 | MAP2K2 | NBN | PIK3C2G | RB1 | STK11 |   |
|  BCL2L2 | CDK12 | DOT1L | FGF6 | IDH1 | MAP2K4 | NF1 | PIK3CA | RBM10 | SUFU |   |
|  BCL6 | CDK4 | EED | FGFR1 | IDH2 | MAP3K1 | NF2 | PIK3CB | REL | SYK |   |
|  BCOR | CDK6 | EGFR | FGFR2 | IGF1R | MAP3K13 | NFE2L2 | PIK3R1 | RET | TBX3 |   |
|  BCORL1 | CDK8 | EP300 | FGFR3 | IKBKE | MAPK1 | NFKBIA | PIM1 | RICTOR | TEK |   |

Table 3. Genes with select intronic regions for the detection of gene rearrangements, a promoter region, and an ncRNA gene

|  ALK introns 18, 19 | BRCA1 introns 2, 7, 8, 12, 16, 19, 20 | ETV4 introns 5, 6 | EZR introns 9- 11 | KIT intron 16 | MYC intron 1 | NUTM1 intron 1 | RET introns 7- 11 | SLC34A2 intron 4  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

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|  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  |

## Test Output

The output of the test includes:

Category 1: CDx Claims noted in Table 1 of the Intended Use

Category 2: Cancer Mutations with Evidence of Clinical Significance

Category 3: Cancer Mutations with Potential Clinical Significance

Genomic findings other than those listed in Table 1 of the intended use statement (i.e., Categories 2 and 3) are not prescriptive or conclusive for labeled use of any specific therapeutic product.

## Test Kit Contents

The test includes a sample shipping kit, which is sent to ordering laboratories. The shipping kit contains the following components:

- Specimen Preparation Instructions
- Shipping Instructions
- Return Shipping Label

## Instruments

The F1CDx assay is intended to be performed with serial number-controlled instruments as indicated in Table 4, below. All instruments are qualified by Foundation Medicine, Inc. (FMI) under FMI's Quality System.

Table 4. Instruments for use with the F1CDx assay

|  Instrument  |
| --- |
|  Illumina® HiSeq 4000  |
|  Illumina cBot® System  |
|  Agilent Technologies Benchbot Workstation with Integrated Bravo Automated Liquid Handler  |
|  Beckman Biomek NX® Span-8 Liquid Handler  |
|  Hamilton Microlab STAR/STARlet Liquid Handling Workstation  |
|  Thermo Fisher Scientific KingFisher™ Flex with 96 Deep-well Head  |
|  Covaris LE220-Plus Focused-ultrasonicator  |

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# 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

Specimens passing pathology review are queued for DNA extraction which begins with lysis of cells from FFPE tissue by digestion with a proteinase K buffer followed by automated purification using the 96-well KingFisher™ Flex Magnetic Particle Processor.

After completion of DNA extraction, double-stranded DNA (dsDNA) is quantified by the Quant-iT™ PicoGreen® fluorescence assay using the provided lambda DNA standards (Invitrogen) prior to Library Construction (LC). The sample must yield a minimum of 55 ng of genomic DNA to ensure sufficient DNA for quality control (QC) and to proceed with LC.

# C. Library Construction

Library Construction (LC) begins with normalization of DNA to 50-1000 ng. Normalized DNA samples are randomly sheared (fragmented) to ~200 bp by adaptive focused acoustic sonication using the Covaris LE220-Plus before purification with a 1.8X volume of AMPure® XP Beads (Agencourt®). Solid-phase reversible immobilization (SPRI) purification and subsequent library construction with the NEBNext® reagents (custom-filled kits by NEB), including mixes for end repair, dA addition and ligation, are performed in 96-well plates (Eppendorf) on the Bravo Benchbot (Agilent) or Microlab STAR (Hamilton) using the “with-bead” protocol¹ to maximize reproducibility and library yield. Indexed (6 bp barcodes) sequencing libraries are PCR amplified with HiFi™ (Kapa) for 10 cycles and subsequently 1.8X SPRI purified. Purification and dilution for QC are performed.

Following LC, a QC procedure is performed by quantifying single-stranded DNA (ssDNA) from purified libraries using the Quant-iT™ OliGreen® ssDNA Assay Kit (Life Technologies) read on a Molecular Devices Multimode SpectraMax M2 plate Reader. Libraries yielding insufficient sequencing library are failed.

# D. Hybrid Capture

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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.

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 SAM tools 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.

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Base substitution detection is performed using a Bayesian methodology, which allows for the detection of novel somatic alterations at low mutant allele frequency (MAF) and increased sensitivity for alterations at hotspot sites through the incorporation of tissue-specific prior expectations.7 Reads with low mapping (mapping quality < 25) or base calling quality (base calls with quality ≤ 2) are discarded. Final calls are made at MAF ≥ 5% (MAF ≥ 1% at hotspots).

To detect indels, de novo local assembly in each targeted exon is performed using the de-Bruijn approach.8 Key steps are:

- Collecting all read-pairs for which at least one read maps to the target region.
- Decomposing each read into constituent k-mers and constructing an enumerable graph representation (de-Bruijn) of all candidate non-reference haplotypes present.
- Evaluating the support of each alternate haplotype with respect to the raw read data to generate mutational candidates. All reads are compared to each of the candidate haplotypes via ungapped alignment, and a read 'vote' for each read is assigned to the candidate with best match. Ties between candidates are resolved by splitting the read vote, weighted by the number of reads already supporting each haplotype. This process is iterated until a 'winning' haplotype is selected.
- Aligning candidates against the reference genome to report alteration calls.

Filtering of indel candidates is carried out similarly to base substitutions, with an empirically increased allele frequency threshold at repeats and adjacent sequence quality metrics as implemented in GATK: % of neighboring bases mismatches < 25%, average neighboring base quality > 25, average number of supporting read mismatches ≤ 2. Final calls are made at MAF ≥ 5% (MAF ≥ 3% at hotspots).

Copy number alterations (CNAs) are detected using a comparative genomic hybridization (CGH)-like method. First, a log-ratio profile of the sample is acquired by normalizing the sequence coverage obtained at all exons and genome-wide SNPs (~3,500) against a process-matched normal control. This profile is segmented and interpreted using allele frequencies of sequenced SNPs to estimate tumor purity and copy number at each segment. Amplifications are called at segments with ≥ 6 copies (or ≥ 7 for triploid/≥ 8 for tetraploid tumors) and homozygous deletions at 0 copies, in samples with tumor purity ≥ 20%. Amplifications in ERBB2 are called positive at segments with ≥ 5 copies for diploid tumors.

Genomic rearrangements are identified by analyzing chimeric read pairs. Chimeric read pairs are defined as read pairs for which reads map to separate chromosomes, or at a distance of over 10 megabase (Mb). Pairs are clustered by genomic coordinate of the pairs, and clusters containing at least five chimeric pairs (three for known fusions) are identified as rearrangement candidates. Filtering of candidates is performed by mapping quality (average read mapping quality in the cluster must be 30 or above) and distribution of alignment positions. Rearrangements are annotated for predicted function (e.g., creation of fusion gene).

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To determine a patient's 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. 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 FoundationOneCDx 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). Per the F1CDx assay, a patient whose tumor has a TMB ≥ 10 mut/Mb is reported as eligible for treatment with KEYTRUDA:

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

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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 reviewed by trained bioinformatics personnel. Samples failing any QC metrics are automatically held and not released.

### G. Report Generation

Approved results are annotated by automated software with CDx relevant information and are merged with patient demographic information and any additional information provided by FMI as a professional service prior to approval and release by the laboratory director or designee.

### H. Internal Process Controls Related to the System

#### Positive Control

Each assay run includes a control sample run in duplicate. The control sample contains a pool of ten HapMap cell lines and is used as a positive mutation detection control. One hundred (100) different germline SNPs present across the entire targeted region are required to be detected by the analysis pipeline. If SNPs are not detected as expected, this results in a QC failure, as it indicates a potential processing error.

#### 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:

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1. Deletions greater than or equal to 5 bp that affect positions -3 to -30 in the intronic region immediately adjacent to the splice acceptor site at the 5' boundary of MET exon 14.
2. Indels affecting positions -1 or -2 at the splice acceptor site of the 5' boundary of MET exon 14.
3. Base substitutions and indels affecting positions 0, +1, +2, or +3 at the splice donor site of the 3' boundary of MET exon 14.

### Homologous Recombination Repair (HRR) Genes

A clinical report is provided to the ordering physician for each F1CDx test performed at Foundation Medicine, Inc. Each report is generated and reviewed by an internal team consisting of clinical bioinformatics analysts, scientists, curators, and pathologists for mutations positive for the therapies identified. Each sample is assessed for mutations in the 14 HRR genes, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, and RAD54L (Table 5). For these genes, both deleterious and suspected deleterious mutations in short variant, copy number alteration, and rearrangement variant classes are determined by an in-house software pipeline. Alterations listed in the COSMIC database and homozygous deletions are considered deleterious. Suspected deleterious mutations include truncating events (i.e., splice, frameshift, and nonsense alterations), as well as large rearrangements that disrupt the coding sequence. The COSMIC check is a second layer of check for HRR positive suspected deleterious alterations. All splice, nonsense, and frameshift alterations in HRR genes are considered biomarker positive and would be considered as suspected deleterious mutations (or “likely” status in FMI reporting rules). If these mutations are additionally reported in COSMIC, they would be listed as deleterious mutations (or “known” status in FMI reporting).

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  |

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*For BRCA2, truncating mutations must occur upstream of bases encoding amino acid 3326. Additionally, the frameshift mutation T367fs*13 in FANCL is ineligible. All short variants must occur in the canonical transcript.

The specific deleterious mutation (DM) and suspected deleterious mutation (SDM) missense mutations or non-frameshift mutations for BRCA1, BRCA2, and ATM are shown in Table 6, below. However, any missense or non-frameshift mutations in the other 12 genes would not be considered HRR positive.

Table 6. Eligible deleterious mutations in the ATM, BRCA1, and BRCA2 genes

|  ATM | BRCA1 | BRCA2  |
| --- | --- | --- |
|  M1T | M1V | M1R  |
|  R2032K | M1I | M1I  |
|  R2227C | C61G | V159M  |
|  R2547_S2549del | C64Y | V211L  |
|  G2765S | R71G | V211I  |
|  R2832C | R71K | R2336P  |
|  S2855_V2856delinsRI (annotated as S2855_V2856>RI) | R1495M | R2336H  |
|  R3008C | E1559K |   |
|  R3008H | D1692N |   |
|  8418+5_8418+8delGTGA or 8418+1_8418+4delGTGA | D1692H |   |
|   | R1699W |   |
|   | A1708E |   |
|   | G1788V |   |

### Biomarker Rules for Rearrangements that Lead to NTRK1, NTRK2, or NTRK3 Fusions:

Rearrangements in NTRK1, NTRK2, or NTRK3 shall be considered CDx biomarker positive, that is, to lead to a NTRK1, NTRK2, or NTRK3 RNA fusion, if the following criterion is met:

- In-strand rearrangement events that may lead to an NTRK1, NTRK2 or NTRK3 RNA fusion with a previously reported or novel partner gene in which the kinase domain is not disrupted. This also includes rearrangement events that result in reciprocal fusions (NTRK-3' and 5'-NTRK events).

In this regard out-of-strand events are considered as non-fusion rearrangements and are classified as CDx biomarker negative. Intragenic fusions in which genomic rearrangement events are wholly internal to the NTRK1, NTRK2, or NTRK3 genes (i.e., NTRK1-NTRK1, NTRK2-NTRK2, NTRK3-NTRK3 events) are also considered biomarker negative. Unidentified partners (encoded as N/A) or LINC non-coding partners are also considered CDx biomarker negative.

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## VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are FDA-approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using FFPE tumor specimens, as listed in Table 1 of the F1CDx intended use statement. The approved CDx tests are listed in Table 7, below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.

**Table 7. List of FDA approved CDx assays for genes targeted by F1CDx**

|   | Device | Company | Technology | Therapy | Indication  |
| --- | --- | --- | --- | --- | --- |
|  *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* | THxID BRAF Kit | bioMerieux | PCR | MEKINIST (tramatenib) | Melanoma  |

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Table 7. List of FDA approved CDx assays for genes targeted by F1CDx

|   | Device | Company | Technology | Therapy | Indication  |
| --- | --- | --- | --- | --- | --- |
|  *BRAF-V600E* | cobas 4800 BRAF V600 Mutation Test | Roche Molecular Systems, Inc. | PCR | COTELLIC (cobimetinib) ZELBORAF (vemurafenib) | Melanoma  |
|   |  cobas 4800 BRAF V600 Mutation Test | Roche Molecular Systems, Inc. | PCR | ZELBORAF (vemurafenib) | Melanoma  |
|   |  THxID BRAF Kit | bioMerieux | PCR | TAFINLAR (dabrafenib) | Melanoma  |
|  *BRAF-V600E* | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | TAFINLAR (dabrafenib) MEKINIST (trametinib) | NSCLC  |
|   |  *therascreen* BRAF V600E RGQ PCR Kit | QIAGEN | PCR | BRAFTOVI (encorafenib) Erbitux (cetuximab) | CRC  |
|  *NRAS* | Praxis Extended RAS Panel | Illumina, Inc. | NGS | VECTIBIX (panitumumab) | CRC  |
|  *KRAS* | **cobas** KRAS Mutation Test | Roche Molecular Systems, Inc. | PCR | ERBITUX (cetuximab) VECTIBIX (panitumumab) | CRC  |
|   |  *therascreen* KRAS RGQ PCR Kit | QIAGEN | PCR | ERBITUX (cetuximab) VECTIBIX (panitumumab) | CRC  |
|   |  Praxis Extended RAS Panel | Illumina, Inc. | NGS | VECTIBIX (panitumumab) | CRC  |
|  *ALK – fusion* | Vysis ALK Break Apart FISH Probe Kit | Abbott Molecular, Inc. | FISH | XALKORI (crizotinib) ALUNBRIG (brigatinib) | 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  |

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**Table 7. List of FDA approved CDx assays for genes targeted by F1CDx**

|   | Device | Company | Technology | Therapy | Indication  |
| --- | --- | --- | --- | --- | --- |
|   | Oncomine Dx Target Test | Life Technologies, Inc. | NGS | IRESSA (gefitinib) | NSCLC  |
|  *EGFR T790M* | **cobas** EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | TAGRISSO (osimertinib) | NSCLC  |
|  *BRCA1/2* | FoundationFocus CDx_{BRCA} | Foundation Medicine, Inc. | NGS | RUBRACA (rucaparib) | Advanced ovarian cancer  |
|  *PIK3CA* | *therascreen* PIK3CA RGQ PCR Kit | QIAGEN | PCR | PIQRAY (alpelisib) | Breast cancer  |

**Abbreviations:** FISH – fluorescence *in situ* hybridization; IHC – immunohistochemistry; CISH – chromogenic *in situ* hybridization; ISH – *in situ* hybridization; PCR – polymerase chain reaction; NGS – next generation sequencing.

## VII. MARKETING HISTORY

Foundation Medicine, Inc. initially designed and developed the FoundationOne® laboratory developed test (F1 LDT), and the first commercial sample was tested in 2012. The F1 LDT has been used to detect the presence of genomic alterations in FFPE tumor tissue specimens. The F1 LDT is not FDA-cleared or -approved.

The F1CDx Premarket Approval (PMA) was originally approved on November 30, 2017 by FDA (P170019) and is commercially available in the U.S. since March 30, 2018. The following PMA supplements affecting the Intended Use were approved by FDA.

- • P170019/S005 was approved on April 10, 2019.
- • P170019/S004 and P170019/S008 were approved on July 1, 2019.
- • P170019/S009 was approved on August 21, 2019.
- • P170019/S006 was approved on December 3, 2019.
- • P170019/S010 was approved on December 16, 2019.
- • P170019/S013 was approved on April 17, 2020.
- • P170019/S011 was approved on May 6, 2020.
- • P170019/S015 was approved on May 19, 2020.
- • P170019/S016 was approved on June 16, 2020.
- • P170019/S017 was approved on October 23, 2020.
- • P170019/S021 was approved on May 28, 2021.
- • P170019/S023 was approved on June 30, 2021.
- • P170019/S026 was approved on July 30, 2021.
- • P170019/S025 was approved on November 10, 2021
- • P170019/S030 was approved on January 19, 2022

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## **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**

Analytical validation for F1CDx platform-level validation (P170019), performance characteristics were established using DNA derived from a wide range of FFPE tumor tissue types in support of companion diagnostic (CDx) indications and have been described previously (P170019). The validation studies performed to gain approval for the fraction-based MSI (FB-MSI) caller on the F1CDx platform included samples across multiple tumor types representing a pan-solid tumor intended use population, and across the range of MSI scores, including samples with MSI-H status. Analytical validation studies conducted to gain platform and CDx approval for the FB-MSI caller included limit of detection (LoD), limit of blank (LoB), precision, and analytical accuracy.

This section summarizes the analytical studies conducted to support the identification of MSI-H status in patients with solid tumors using the FB-MSI caller.

#### **1. Analytical Accuracy/Concordance**

To demonstrate the analytical accuracy of MSI calling three independent studies were conducted. Two studies evaluated the concordance of F1CDx MSI calling to a PCR based comparator. A third study was conducted to evaluate concordance between F1CDx MSI calling and mismatch repair (MMR) immunohistochemistry (IHC) assays through a retrospective chart review of a randomly selected set of specimens from colorectal and endometrial cancer patients.

##### **1. Comparison to an PCR-based Orthogonal Method for Detecting MSI – Study I**

To demonstrate the analytical accuracy of the MSI calling, an analysis to assess concordance of F1CDx MSI calling was performed using a PCR-based comparator assay. The study included 186 FFPE tumor specimens representing various disease ontologies (see Table 8), of which 86 were

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selected with the PCR-based comparator assay, while 100 were selected with F1CDx.

Table 8. Distribution of Organ System and Disease Ontologies in Analytical Accuracy Study I

|  Organ System | Disease Ontology | Number of Samples Tested  |   |   |
| --- | --- | --- | --- | --- |
|   |   |  PCR Comparator Enrolled | F1CDx Enrolled | Total (n)  |
|  Gastrointestinal | Colon adenocarcinoma (CRC) | 1 | 12 | 13  |
|   |  Rectum adenocarcinoma (CRC) | - | 8 | 8  |
|   |  Esophagus squamous cell carcinoma (SCC) | - | 1 | 1  |
|   |  Appendix adenocarcinoma | - | 3 | 3  |
|   |  Ampullary adenocarcinoma | - | 2 | 2  |
|   |  Small intestine adenocarcinoma | 4 | - | 4  |
|   |  Stomach adenocarcinoma (NOS) | 10 | - | 10  |
|   |  Esophagus adenocarcinoma | 2 | - | 2  |
|   |  Esophagus carcinoma (NOS) | 8 | - | 8  |
|  Reproductive | Breast invasive ductal carcinoma (IDC) | - | 1 | 1  |
|   |  Vagina adenocarcinoma | - | 2 | 2  |
|   |  Breast carcinoma (NOS) | - | 5 | 5  |
|   |  Prostate acinar adenocarcinoma | - | 7 | 7  |
|   |  Ovary endometrioid adenocarcinoma | - | 2 | 2  |
|   |  Testis germ cell tumor (non-seminoma) | - | 2 | 2  |
|   |  Cervix adenocarcinoma | - | 1 | 1  |
|   |  Cervix squamous cell carcinoma (SCC) | - | 1 | 1  |
|   |  Penis squamous cell carcinoma (SCC) | - | 2 | 2  |
|   |  Uterus endometrial adenocarcinoma (NOS) | 15 | - | 15  |

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|   | Ovary cancer (NOS) | 2 | - | 2  |
| --- | --- | --- | --- | --- |
|   |  Ovary epithelial carcinoma (NOS) | 4 | - | 4  |
|   |  Ovary serous carcinoma | 1 | - | 1  |
|   |  Vulva squamous cell carcinoma (SCC) | - | 1 | 1  |
|   |  Uterus endometrial adenocarcinoma endometrioid | - | 4 | 4  |
|   |  Peritoneum serous carcinoma | - | 1 | 1  |
|  Hepato-Pancreatobiliary | Gallbladder adenocarcinoma | 6 | 1 | 7  |
|   |  Liver cholangiocarcinoma | 1 | 1 | 2  |
|   |  Bile duct adenocarcinoma |  | 1 | 1  |
|   |  Gallbladder carcinoma | 7 | - | 7  |
|   |  Liver hepatocellular carcinoma (HCC) | - | 2 | 2  |
|   |  Pancreas ductal adenocarcinoma | - | 4 | 4  |
|  Endocrine | Pancreas neuroendocrine tumor (pNET) | - | 1 | 1  |
|   |  Adrenal gland cortical carcinoma | - | 2 | 2  |
|   |  Thyroid anaplastic carcinoma | - | 1 | 1  |
|   |  Thyroid papillary carcinoma | 2 | 1 | 3  |
|   |  Thyroid follicular carcinoma | 1 | - | 1  |
|   |  Thyroid medullary carcinoma | 1 | - | 1  |
|   |  Thyroid carcinoma (NOS) | 8 | - | 8  |
|   |  Lung large cell neuroendocrine carcinoma | - | 1 | 1  |
|  Urinary | Kidney chromophobe carcinoma | - | 1 | 1  |
|   |  Bladder urothelial (transitional cell) carcinoma | - | 6 | 6  |
|   |  Kidney clear cell carcinoma | - | 1 | 1  |
|   |  Kidney urothelial carcinoma | - | 2 | 2  |

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|  Thoracic | Lung adenocarcinoma | - | 5 | 5  |
| --- | --- | --- | --- | --- |
|   |  Lung small cell undifferentiated carcinoma | - | 2 | 2  |
|   |  Lung squamous cell carcinoma (SCC) | - | 2 | 2  |
|  Skin/Soft Tissue | Skin squamous cell carcinoma (SCC) | - | 3 | 3  |
|   |  Pleura mesothelioma | - | 2 | 2  |
|   |  Soft tissue sarcoma (NOS) | 3 | - | 3  |
|  Other | Brain glioblastoma (GBM) | - | 4 | 4  |
|   |  Brain meningioma | - | 1 | 1  |
|   |  Head and neck squamous cell carcinoma (HNSCC) | - | 1 | 1  |
|   |  Unknown | 1 | - | 1  |
|   |  Unknown primary adenocarcinoma | 1 | - | 1  |
|   |  Unknown primary carcinoma (NOS) | 5 | - | 5  |
|   |  Unknown primary squamous cell carcinoma (SCC) | 1 | - | 1  |
|   |  Unknown primary malignant neoplasm (NOS) | 2 | - | 2  |
|  Total |   | 86 | 100 | 186  |

Of the 186 FFPE tumor specimens, 159 were evaluable with valid MSI results from both F1CDx and the comparator assay. Of the 86 samples selected with the PCR comparator assay, 24 failed to provide evaluable or valid F1CDx results due to QC failures, i.e., a 28% failure rate was observed primarily due to contamination for the samples that were procured externally using the PCR-based comparator. Of the 100 samples selected with F1CDx, i.e., internally procured, three (3) failed to provide evaluable or valid F1CDx results, i.e., a 3% failure rate was observed.

The PCR-based assay-selected subset contained 62 samples and the F1CDx assay-selected subset contained 97 samples that were available for concordance analysis. For concordance analysis, samples with F1CDx MSI scores ≥ 0.0124 were treated as MSI-H/positive in the concordance analysis. Samples with F1CDx FB- MSI scores <0.0124

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were treated as non-MSI-H or negative results. The PCR assay includes MSI-High (MSI-H), MSI-Low (MSI-L), and microsatellite stable (MSS) results, which were further dichotomized into MSI-H or positive and non-MSI-H (MSI-L and MSS) or negative in the concordance analysis.

Concordance results between F1CDx and PCR for MSI-H calling within the combined datasets, i.e., samples selected with the PCR comparator assay and F1CDx assay are summarized in Table 10 below. Positive percent agreement (PPA) and negative percent agreement (NPA) are estimates of agreement between the assays, calculated by determining the proportion of comparator positive and negative samples that F1CDx was able to call concordantly. PPA and NPA were calculated using the observed, unadjusted data from the sample subset enrolled with the F1CDx assay, therefore, results may be biased due to how samples were selected.

The combined PPA and NPA using the $\geq 0.0124$ threshold for MSI-H calling was 98.46% (95% CI [91.79% - 99.73%]) and 96.81% (95% CI [91.03% - 98.91%]) respectively. A total of four (4) discordant cases were observed (Table 9). A F1CDx-/PCR+ sample was from a patient with kidney chromophobe carcinoma and had an FB-MSI score of 0.0105. Three (3) F1CDx+/PCR- results were from patients with uterus endometrial carcinoma, thyroid anaplastic carcinoma, or brain glioblastoma, and had the following FB-MSI scores 0.0135, 0.0163 and 0.0174 respectively. Therefore, the four discordant samples had FB-MSI scores near the 0.0124 threshold.

**Table 9: Concordance Results for FB-MSI Detection Using PCR-based Comparator Assay**

|   | F1CDx+/PCR+ | F1CDx-/PCR + | F1CDx+/PCR - | F1CDx-/PCR - | PPA (95% CI) | NPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  **Combined datasets (PCR and F1CDx enrolled)** | 64 | 1 | 3 | 91 | 98.46% (91.79% - 99.73%) | 96.81% (91.03% - 98.91%)  |

While the concordance was high for samples from colorectal cancer (CRC) patients for the combined data sets, both PPA and NPA were 100.00%, concordance was lower for other disease ontologies (non-CRC). For non-CRC patient samples, the observed combined PPA and NPA using the $\geq 0.0124$ threshold for MSI-H calling were 98.04% and 97.53% respectively. Although the number of samples from patients with uterus endometrial adenocarcinoma that had valid F1CDx results was low, 8 out of 19 were valid per F1CDx, the observed combined PPA and NPA using the $\geq 0.0124$ threshold for MSI-H calling were 100% and 66.67%, respectively.

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In this study there were 10 samples with MSI-C cannot be Determined status due to FB-MSI scores >0.0041 and <0.0124, one (1) was MSI-H, seven (7) were MSS, while the remaining two (2) samples had an MSI-L status per the PCR-based comparator. All samples were from non-CRC tumors. The MSI-H sample per the PCR comparator was from a patient with kidney chromophobe carcinoma. The two (2) MSI-L samples per the PCR comparator were from patients with uterus endometrial adenocarcinoma, or brain glioblastoma. The seven (7) samples that were MSS per the PCR comparator were from patients with adrenal gland cortical carcinoma (1), appendix adenocarcinoma (1), kidney urothelial carcinoma (1), adrenal gland cortical carcinoma (1), liver cholangiocarcinoma (1), ampullary adenocarcinoma (1), or thyroid follicular carcinoma (1).

## 2. Comparison to an PCR-based Orthogonal Method for Detecting MSI – Study II

An additional study to assess concordance of MSI-H calling to the PCR-based comparator assay was conducted to demonstrate the analytical validity of F1CDx. This study included the evaluation of a test set of 56 screen failure samples from Merck’s clinical trial study KEYNOTE-158, which supported the clinical validity of the F1CDx, see Section X below. An additional set of 161 commercially procured samples were included to support the analytical accuracy F1CDx. In total 217 specimens across multiple tumor types were evaluated in the study. Table 10 shows the distribution of tumor types in the second analytical accuracy study.

Of the 161 commercially procured samples, 154 were selected by an external vendor who coordinated mismatch repair (MMR) immunohistochemistry (IHC) testing to enrich the population of potential MSI-H samples for procurement prior to testing with the PCR comparator method and F1CDx. To support the pan-tumor assessment, seven (7) additional commercially procured samples with MSI status previously determined by F1CDx were selected by FMI and subsequently tested with the PCR comparator assay.

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Table 10: Distribution of Disease Ontologies in Analytical Accuracy Study II

|  Organ System | Disease Ontology | Number of Samples Tested  |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   |   |  PCR Comparator-Enrolled, Commercially Procured | PCR Comparator Enrolled, Clinical Trial Screen Failure | F1CDx-Enrolled, Commercially Procured | Total (n)  |
|  Endocrine | Thyroid carcinoma (NOS) | 1 | 7 | - | 8  |
|   |  Unknown primary neuroendocrine tumor (NET) | - | 5 | - | 5  |
|   |  Unknown primary undifferentiated neuroendocrine carcinoma | 2 | - | - | 2  |
|  Gastrointestinal | Anus squamous cell carcinoma | - | 3 | - | 3  |
|   |  Bile duct adenocarcinoma | - | 5 | - | 5  |
|   |  Colon adenocarcinoma (CRC) | 53 | - | - | 53  |
|   |  Esophagus carcinoma (NOS) | 1 | - | - | 1  |
|   |  Stomach adenocarcinoma (NOS) | 9 | - | - | 9  |
|   |  Stomach carcinoma (NOS) | 18 | - | - | 18  |
|  Hepato-Pancreatobiliary | Unknown primary adenocarcinoma | - | - | 1 | 1  |
|  Reproductive | Breast invasive ductal carcinoma (IDC) | 8 | - | - | 8  |
|   |  Cervix squamous cell carcinoma (SCC) | 2 | 6 | - | 8  |
|   |  Ovary epithelial carcinoma (NOS) | 2 | - | - | 2  |
|   |  Prostate acinar adenocarcinoma | 2 | - | - | 2  |
|   |  Uterus endometrial adenocarcinoma (NOS) | 50 | 8 | - | 58  |
|   |  Vulva squamous cell carcinoma (SCC) | - | 14 | - | 14  |
|  Soft Tissue/Skin | Skin melanoma | - | - | 1 | 1  |
|  Thoracic | Lung adenocarcinoma | - | - | 1 | 1  |

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|   | Lung small cell undifferentiated carcinoma | 2 | 7 | - | 9  |
| --- | --- | --- | --- | --- | --- |
|   |  Lung squamous cell carcinoma (SCC) | - | - | 4 | 4  |
|   |  Unknown primary mesothelioma | - | 1 | - | 1  |
|  Urinary | Kidney renal cell carcinoma (NOS) | 4 | - | - | 4  |
|  Total |   | 154 | 56 | 7 | 217  |

After sample processing and screening externally at the vendor and internally at FMI, a total of 111 samples failed to yield an evaluable or valid F1CDx result, while one (1) sample that underwent F1CDx sample processing was not evaluable by the PCR assay due to poor amplification. Of the 111 samples that failed to yield F1CDx evaluable results, three (3) samples failed for having less than 20% tumor nuclei, two (2) samples failed for having an insufficient quantity of slides. In addition, two (2) samples failed DNA extraction specifications due to low yield and four (4) samples failed HC QC specifications due to insufficient DNA mass. One hundred (100) samples failed F1CDx Post-Sequencing QC. Among the 100 sample failures identified, 91 samples displayed low-level contamination (≥1.01%, ≤3.57%) which was above the F1CDx specification of 1% and were removed from the primary analysis. Of note, 89 of the 91 contaminated samples were commercially procured. Of these 91 samples, 3 samples in addition to failing to meet the contamination QC also failed other F1CDx post-sequencing QC. An exploratory analysis including these samples was performed and is discussed below.

105 samples that met all sample selection and testing QC criteria and were included in the primary analysis. MSI assessment was dichotomized to MSI-H or non-MSI-H for both F1CDx, and the PCR assay results. Samples with F1CDx MSI scores ≥ 0.0124 were treated as MSI-H/positive in the concordance analysis. Samples with F1CDx FB-MSI scores <0.0124 were treated as non-MSI-H or negative results. As for the accuracy Study I, the PCR assay includes MSI-H, MSI-L, and MSS, which was further dichotomized into MSI-H or positive and non-MSI-H (MSI-L and MSS) or negative in the concordance analysis.

Among the 105 samples, 98 were PCR-enrolled and seven (7) were F1CDx-enrolled. Because concordance in the F1CDx-enrolled set was 100%, a prevalence adjustment does not change estimates of PPA or NPA for the dataset. As such, the analysis was simplified, and the two (2) datasets (PCR-enrolled and F1CDx-enrolled) were combined

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directly. Point estimates for PPA and NPA were calculated directly, along with 95% two-sided Wilson Score confidence intervals (CIs).

The combined PPA was 100.00% with 2-sided 95% CI of [87.54% - 100.00%], see Table 11. The combined NPA was 97.37% with 2-sided 95% CI of [91.12% - 99.29%], see Table 11. Two (2) samples, both from endometrial cancer patients, exhibited discordant MSI status results between the assays. Both discordant samples were PCR-enrolled and exhibited F1CDx MSI-H results (FB-MSI score of 0.0216 and 0.0192) and PCR non-MSI-H results (MSS).

**Table 11. Concordance Results MSI Detection Using PCR-based Comparator Assay**

|   | F1CDx+/ PCR+ | F1CDx- /PCR + | F1CDx+ /PCR - | F1CDx- /PCR - | PPA (95% CI) | NPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  **Combined data sets (PCR and F1CDx enrolled)** | 27 | 0 | 2 | 76 | 100.00% (87.54% - 100.00%) | 97.44% (91.12% - 99.29%)  |

For CRC patients in the combined data sets, for samples that yielded evaluable results (21 out of 53, 39.6%), PPA was 100% (10/10) and NPA was 100.00% (11/11) using the $\geq 0.0124$ threshold for MSI-H calling. For non-CRC patient samples with evaluable results, the observed PPA and NPA in the combined data sets using the $\geq 0.0124$ threshold for MSI-H calling was 100.00% (17/17) and 97.01% (63/65).

For the samples from patients with uterus endometrial adenocarcinoma that had evaluable results (25 out of 58, 43.10%) the observed PPA and NPA in the combined data sets using the $\geq 0.0124$ threshold for MSI-H calling was 100% (9/9) and 87.50% (14/16) respectively.

There were two (2) samples, both from patients with thyroid carcinoma, with MSI-Cannot be Determined status due to FB-MSI scores $>0.0041$ and $<0.0124$; both were MSS per the PCR based comparator.

An exploratory analysis was conducted where the MSI QC rule for contamination was not imposed, enabling 88 samples that were removed from the primary analysis to be included for analysis. This exploratory analysis increases the number of evaluable samples from 105 to 193. All sample failures, other than exclusively for contamination, remained excluded from the analysis. Among the 193 samples, 191 samples had concordant MSI status results between the F1CDx and PCR assays. All 88 additional samples included in the exploratory analysis produced

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100% concordant MSI results between F1CDx and PCR assay. The same two (2) samples, that were discordant in the primary analysis remained discordant in the exploratory analysis. The combined PPA was 100.00% with 95% two-sided CI of (94.58% - 100.00%). The combined NPA was 98.41% with 95% two-sided Wilson Score CI of (94.40% - 99.56%). In the exploratory analysis, there were five (5) samples with MSI-Cannot be Determined status due to FB-MSI scores >0.0041 and <0.0124, the same two samples in the primary analysis and three additional ones, from patients with uterus endometrial adenocarcinoma (1), bile duct adenocarcinoma (1) or cervix squamous cell carcinoma (1). All five patient samples had MSS status per the PCR comparator. Although the study showed a high failure rate by F1CDx due to the strict contamination QC metric, exclusion of the samples that failed the contamination QC metric does not impact concordance results.

The presence of contamination in commercially procured samples often occurs at an elevated rate compared to clinical F1CDx samples and may be caused by FPPE block processing techniques used by outside FFPE tissue procurement vendors as well as the age of the specimen. This contamination level is low (~0-3%), but above the specified threshold of 1%. A retrospective analysis of the F1CDx commercial tests suggests that the high failure rate observed in the commercial lab (21.47%) is not as high as that seen in the accuracy study. A post market study will be conducted to confirm the failure rate of the F1CDx in the commercial setting, refer for Section XIII.

### 3. Retrospective Chart Review Concordance Results of FB-MSI Caller and IHC

To demonstrate the analytical accuracy of the MSI calling, an additional evaluation of the concordance between the MSI classification (MSI-High (MSI-H)/deficient mismatch repair (dMMR) versus non-MSI-H/proficient mismatch repair (pMMR)) determined by the FB-MSI caller and mismatch repair (MMR) immunohistochemistry (IHC) assays was performed through a retrospective chart review of a randomly selected set of 134 colorectal cancer (CRC) and 52 uterus endometrial cancer patients from the FMI clinical commercial database. Overall, a total of 178 samples (including 128 CRC and 50 uterus endometrial cases) passed the F1CDx QC assessment, i.e., provided evaluable or valid F1CDx results and were used as the analysis dataset. Samples with FB-MSI scores ≥ 0.0124 were treated as MSI-H/positive in the concordance analysis. Samples with F1CDx FB- MSI scores <0.0124 were treated as non-MSI-H or negative results. The IHC assays provide two outcomes, dMMR and pMMR. dMMR are considered positive results and pMMR are considered negative results for the analyses.

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The PPA and NPA for the overall sample set and CRC and uterus endometrial cancer datasets are shown in Table 12. Point estimates and 95% two-sided CI for PPA and NPA for the overall sample set were PPA 85.19% (95%CI [67.52, 94.08%]) and NPA 100.00% (95% CI [97.52%, 100.00%]).

**Table 12: Retrospective Chart Review Concordance Results Using IHC based Comparator Assays**

|  Dataset | F1CDx+/IHC+ | F1CDx-/IHC+ | F1CDx+/IHC- | F1CDx-/IHC- | PPA (95% CI) | NPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  Combined CRC and Endometrial | 23 | 4 | 0 | 151 | 85.19% (67.52, 94.08%) | 100.00% (97.52%, 100.00%)  |
|  CRC Only | 11 | 3 | 0 | 114 | 78.57% (52.41%, 92.43%) | 100.00% (96.74%, 100.00%)  |
|  Endometrial Only | 12 | 1 | 0 | 37 | 92.31% (66.69%, 98.63%) | 100.00% (90.59%, 100.00%)  |

Four (4) discordant samples were identified in this study, three (3) within the CRC subset and one (1) within the uterus endometrial cancer subset. All discordant samples were F1CDx non-MSI-H/dMMR.

MSI-Cannot be determined status for MSI cases with FB MSI >0.0041 and <0.0124 was observed in 11 samples, four CRC and seven uterus endometrial cancer samples. Of the 11 samples, 1 (9%) uterus endometrial cancer sample was dMMR while the remaining 10, (91%) had pMMR status.

## 2. Analytical Sensitivity

### 1. Limit of Blank (LoB)

To assess LoB for MSI, 111 test replicates from 10 individual tumor FFPE-derived DNA samples as well as a pool of biomarker-negative DNA derived from 30 unique FFPE tumor DNA samples were evaluated. The LoB was confirmed to be zero as 0 out of 111 test replicates yielded MSI-H results. The average, median, minimum, and maximum MSI scores observed were 0.0006, 0.0004, 0 and 0.0032 respectively.

### 2. Limit of Detection (LoD)

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The LoD for MSI-H calling using the FB-MSI caller in the F1CDx assay was assessed through evaluation of eight samples (see Table 14). The eight samples included five (5) disease ontologies that represent the organ systems where MSI-H cases are the most prevalent in the Intended Use (IU) population.

To establish LoD, five (5) to six (6) levels of varying computational tumor purity (TP) and 13 to 20 replicates per level were evaluated. To achieve the targeted tumor purity, biomarker-positive (MSI-H) FFPE-derived tumor DNA were mixed with unmatched biomarker-negative (microsatellite stable (MSS)) FFPE-derived tumor DNA from the same disease ontology. The hit rate for each dilution was computed as the number of replicates with MSI-H calls (MSI score was $\geq 0.0124$) per the total number of replicates tested at each level. The empirical hit rate approach was used since there were less than three hit rate levels with less than 95% detection rate. The LoD was defined as the lowest computational tumor purity with at least 95% detection.

Of the replicate measurements processed, 838 replicates across all eight (8) samples, 807 yielded F1CDx evaluable results. Twenty-five (25) replicates were unevaluable due to failure at the library construction (LC) quality control (QC), two (2) replicates, were unevaluable due to failure at the hybrid construction (HC) QC, two (2) replicates failed to meet MSI QC metrics, one (1) replicate failed to meet coverage requirements and one (1) replicate failed to meet both coverage requirements and MSI QC metrics.

Given that not all replicates were evaluable, LoD was established using 12 to 20 evaluable replicates per level. For each sample, the lowest computational tumor purity level measured at which MSI-H hit rates with at least 95% hit rate is shown in Table 13.

As 15.67% tumor purity is the maximal LoD value observed, the established MSI-H LoD is 15.67% tumor purity.

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Table 13: MSI-H Sample LoD Results

|  Sample | Organ System | Disease Ontology/Tissue Type | MSI-H LoD (Mean % Tumor Purity)  |
| --- | --- | --- | --- |
|  1 | Gastrointestinal | Colon adenocarcinoma (CRC) | 8.25%  |
|  2 | Gastrointestinal | Colon adenocarcinoma (CRC) | 8.33%  |
|  3 | Reproductive | Uterus endometrial adenocarcinoma | 8.85%  |
|  4 | Hepato-pancreatobiliary | Liver cholangiocarcinoma | 9.56%  |
|  5 | Thoracic | Lung squamous cell carcinoma | 9.96%  |
|  6 | Gastrointestinal | Colon adenocarcinoma (CRC) | 12.01%  |
|  7 | Urinary | Kidney urothelial carcinoma | 15.50%  |
|  8 | Gastrointestinal | Colon adenocarcinoma (CRC) | 15.67%  |

### 3. Analytical Specificity

#### 1. Interference

In an inference study for other biomarkers, eighteen (18) FFPE samples with MSS status were evaluated with four potentially interfering substances (hemoglobin, triglycerides, conjugated bilirubin, and unconjugated bilirubin) to evaluate whether these interfering substances impact the F1CDx MSI status calling. However, since MSI-H samples from various solid tumors were not included in this study, a post-market interfering substance study will be performed to evaluate the effects of endogenous interfering substances including necrotic tissue, melanin, and hemoglobin, and exogenous interference substances including molecular barcodes, proteinase K, and ethanol including MSI-H samples representing a range of solid tumors across the intended use population.

### 4. Precision (Within-Laboratory Precision) and Reproducibility (Site to Site Precision)

#### 1. Within-Laboratory Precision

To evaluate the performance of MSI detection using the FB-MSI caller, a prospectively designed retrospective analysis was performed using samples from various tumor types. FFPE-derived DNA samples were selected from banked Foundation Medicine DNA samples. From the 67 samples originally tested in the prospective study, 66 were considered in the retrospective analysis. One (1) unknown primary melanoma sample and associated replicates were excluded from the retrospective analysis study as the result of high laboratory failure rates observed in the original prospective study due to contamination present in the source sample material. Therefore, in total 66 samples were considered in the retrospective analysis. These samples consisted of a set of seven (7) MSI-H samples, six (6) from CRC patients and one (1) from a patient with uterus endometrial adenocarcinoma and 59 non-

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MSI-H samples from eight tumor types, ten (10) from patients with breast carcinoma, eight (8) from CRC patients, two (2) from patients with liver cholangiocarcinoma, eighteen (18) from patients with lung adenocarcinoma, four (4) from patients with lung non-small cell lung carcinoma, six (6) from patients with rectum adenocarcinoma, seven (7) patients with skin melanoma, three (3) with unknown primary melanoma, one (1) with thyroid carcinoma. Each sample was tested in two or three replicates in two (2) separate runs, with three (3) sequencers, and two (2) or three (3) reagent lots in a factorial design. A total of 36 replicates per sample were processed.

Of the 2,409 sample aliquots processed, 2,377 (98.7%) were successfully processed from LC to HC; 32 process failures were observed. Seven (7) failures were observed at LC QC step of the workflow, whereas additional 25 failures were observed at HC QC step. As noted above an unknown primary melanoma sample and associated replicates were excluded from retrospective analysis study due to high laboratory failure rates observed. Of the 2,344 replicates that underwent pipeline re-analysis to evaluate the precision of MSI calling, 2,273 (97.0%) passed post-sequencing MSI biomarker QC; 71 MSI biomarker QC failures were observed in replicates from five (5) source samples. Two (2) failures were observed due to low coverage; 68 were observed due to contamination, whereas an additional one was observed due to both low coverage and contamination. Out of all replicates from the 66 source samples (N=2373), a total of 100 replicates (4.2%) failed to meet F1CDx QC metrics at laboratory processing or post sequencing MSI biomarker QC. These replicates were removed from concordance data analysis. All 36 replicates of a skin melanoma sample failed to meet F1CDx QC metrics for MSI. Therefore, there were 65 evaluable samples for this study, 7 MSI-H samples and 58 non-MSI samples.

Data analysis was performed for each sample separately with replicates that passed QC, and yielded evaluable results. According to their MSI scores, samples were classified as MSI-H (≥ 0.0124) or non-MSI-H (<0.0124). An evaluation of within-laboratory (intermediate) precision for MSI status was performed by evaluation of within-laboratory precision and repeatability estimated as the percent agreement for each sample. The seven (7) MSI-H samples had MSI scores ranging from 0.0226 to 0.0682 and the 58 non-MSI-H samples had MSI scores ranging from 0.0043 to 0.0001. The agreement for within-laboratory precision and repeatability were 100% for the 65 evaluable samples. For 64 of 65 samples, the lower bound the two-sided 95% score CI ranged from 89.57% to 90.36% for within-laboratory precision and from 74.12% to 82.41% for repeatability. One sample with an average MSI score of 0.0009 had 7 evaluable replicates and agreement for within-laboratory precision (7/7) was 100%; the lower bound of the two-sided 95% score CI was 64.57%. For the same sample, the repeatability was 100%; the lower bound of the two-sided 95% score CI was 34.24% (see Table 14 for agreement results).

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Table 14: MSI-H vs non- MSI-H Within-Laboratory (Intermediate) Precision results

|  # | Disease Ontology | MSI-H status | Mean MSI Score | Mean Tumor Purity | Within-laboratory Precision |   |   | Repeatability  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |  Agree | Total | Agreement (95% 2-sided score CI) | Agree Pairs* | Total Pairs* | Agreement (95% 2-sided score CI)  |
|  1 | Colon adenocarcinoma (CRC) | MSI-H | 0.0682 | 30.19% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  2 | Colon adenocarcinoma (CRC) | MSI-H | 0.0588 | 46.63% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  3 | Colon adenocarcinoma (CRC) | MSI-H | 0.0579 | 30.97% | 30 | 30 | 100.00% (88.65%, 100.00%) | 12 | 12 | 100.00% (75.75%, 100.00%)  |
|  4 | Colon adenocarcinoma (CRC) | MSI-H | 0.0458 | 17.40% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  5 | Colon adenocarcinoma (CRC) | MSI-H | 0.0428 | 31.38% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  6 | Colon adenocarcinoma (CRC) | MSI-H | 0.0328 | 38.85% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  7 | Lung adenocarcinoma | non MSI-H | 0.0033 | 42.40% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  8 | Colon adenocarcinoma (CRC) | non MSI-H | 0.0024 | 44.34% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  9 | Breast invasive ductal carcinoma (IDC) | non MSI-H | 0.0022 | 48.28% | 34 | 34 | 100.00% (89.85%, 100.00%) | 16 | 16 | 100.00% (80.64%, 100.00%)  |
|  10 | Skin melanoma | non MSI-H | 0.0021 | 86.71% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  11 | Breast invasive ductal carcinoma (IDC) | non MSI-H | 0.0019 | 51.85% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  12 | Breast invasive ductal carcinoma (IDC) | non MSI-H | 0.0019 | 66.53% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  13 | Rectum adenocarcinoma (CRC) | non MSI-H | 0.0018 | 13.94% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  14 | Skin melanoma | non MSI-H | 0.0016 | 37.70% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  15 | Skin melanoma | non MSI-H | 0.0016 | 81.63% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |

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|  16 | Lung adenocarcinoma | non MSI-H | 0.0015 | 34.84% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  17 | Skin melanoma | non MSI-H | 0.0014 | 89.64% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  18 | Breast carcinoma (NOS) | non MSI-H | 0.0014 | 83.69% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  19 | Breast invasive ductal carcinoma (IDC) | non MSI-H | 0.0013 | 49.85% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  20 | Lung adenocarcinoma | non MSI-H | 0.0012 | 65.51% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  21 | Lung adenocarcinoma | non MSI-H | 0.0012 | 31.67% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  22 | Breast invasive ductal carcinoma (IDC) | non MSI-H | 0.0012 | 32.18% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  23 | Rectum adenocarcinoma (CRC) | non MSI-H | 0.0011 | 71.63% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  24 | Rectum adenocarcinoma (CRC) | non MSI-H | 0.0011 | 72.58% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  25 | Lung adenocarcinoma | non MSI-H | 0.0010 | 23.98% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  26 | Skin melanoma | non MSI-H | 0.0010 | 60.81% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  27 | Lung adenocarcinoma | non MSI-H | 0.0009 | 46.94% | 7 | 7 | 100.00% (64.57%, 100.00%) | 2 | 2 | 100.00% (34.24%, 100.00%)  |
|  28 | Rectum adenocarcinoma (CRC) | non MSI-H | 0.0009 | 33.06% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  29 | Lung adenocarcinoma | non MSI-H | 0.0008 | 33.21% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  30 | Rectum adenocarcinoma (CRC) | non MSI-H | 0.0008 | 58.89% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  31 | Lung adenocarcinoma | non MSI-H | 0.0008 | 27.87% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  32 | Unknown primary melanoma | non MSI-H | 0.0007 | 69.23% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  33 | Breast invasive ductal carcinoma (IDC) | non MSI-H | 0.0007 | 71.12% | 34 | 34 | 100.00% (89.85%, 100.00%) | 16 | 16 | 100.00% (80.64%, 100.00%)  |

PMA P170019-S029: FDA Summary of Safety and Effectiveness Data

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|  34 | Breast carcinoma (NOS) | non MSI-H | 0.0006 | 45.30% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  35 | Colon adenocarcinoma (CRC) | non MSI-H | 0.0006 | 40.82% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  36 | Lung squamous cell carcinoma (SCC) | non MSI-H | 0.0005 | 59.92% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  37 | Lung non-small cell lung carcinoma (NOS) | non MSI-H | 0.0004 | 30.74% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  38 | Breast invasive lobular carcinoma (ILC) | non MSI-H | 0.0003 | 48.87% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  39 | Colon adenocarcinoma (CRC) | non MSI-H | 0.0003 | 33.46% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  40 | Lung adenocarcinoma | non MSI-H | 0.0003 | 26.45% | 34 | 34 | 100.00% (90.36%, 100.00%) | 16 | 16 | 100.00% (80.64%, 100.00%)  |
|  41 | Colon adenocarcinoma (CRC) | non MSI-H | 0.0003 | 10.14% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  42 | Breast carcinoma (NOS) | non MSI-H | 0.0003 | 36.56% | 35 | 35 | 100.00% (90.11%, 100.00%) | 17 | 17 | 100.00% (81.57%, 100.00%)  |
|  43 | Skin melanoma | non MSI-H | 0.0002 | 23.83% | 36 | 36 | 100.00% (90.36%, 100.00%) | 18 | 18 | 100.00% (82.41%, 100.00%)  |
|  44 | Lung squamous cell ca…

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**Source:** [https://fda-staging.innolitics.com/device/P170019S029](https://fda-staging.innolitics.com/device/P170019S029)

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