← Product Code PQP · P170019

# FoundationOne CDx (P170019)

_Foundation Medicine, Inc. · PQP · Nov 30, 2017 · Pathology · APPR_

**Canonical URL:** https://fda.innolitics.com/device/P170019

## Device Facts

- **Applicant:** Foundation Medicine, Inc.
- **Product Code:** PQP
- **Decision Date:** Nov 30, 2017
- **Decision:** APPR
- **Device Class:** Class 3
- **Review Panel:** Pathology
- **Attributes:** AI/ML, Expedited Review

## Intended Use

FoundationOne CDx™ (F1CDx) is a next generation sequencing based in vitro diagnostic device 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 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 cancer patients with solid malignant neoplasms. The F1CDx test is a single-site assay performed at Foundation Medicine, Inc.

## Device Story

F1CDx is a single-site NGS assay performed at Foundation Medicine, Inc. using DNA from FFPE tumor tissue. Input: 50-1000 ng DNA. Process: whole-genome shotgun library construction; hybrid-capture of 324 cancer-related genes; sequencing on Illumina HiSeq 4000. Output: report of genomic alterations (substitutions, indels, CNAs, rearrangements) and signatures (MSI, TMB). Used by oncologists to identify patients eligible for specific targeted therapies. Output affects clinical decision-making by matching patients to approved therapies or providing tumor mutation profiling for oncology guidelines. Benefits include identifying potential treatment options for patients with solid tumors.

## Clinical Evidence

Clinical concordance studies compared F1CDx to approved CDx tests (cobas EGFR v2, Ventana ALK, therascreen KRAS, Dako HER2 FISH, cobas BRAF, FoundationFocus CDxBRCA). Studies used retrospective samples (N=175-406 per study). Primary endpoints were PPA/NPA. Results demonstrated non-inferiority to comparators. Analytical studies (bench-only) established LoD, LoB, precision, and stability.

## Technological Characteristics

NGS-based assay using hybrid-capture of 324 genes. DNA extracted from FFPE tissue. Sequencing on Illumina HiSeq 4000. Employs biotinylated DNA oligonucleotide baits for target enrichment. Software pipeline performs variant calling, MSI scoring via PCA, and TMB calculation. Single-site assay performed at Foundation Medicine, Inc.

## Regulatory Identification

A next generation sequencing (NGS) oncology panel is a device used for the qualitative detection of germline or somatic variants in one or more cancer-related genes. The device is intended to be used on DNA or RNA isolated from human clinical specimens.

## Reference Devices

- PathVysion HER-2 DNA Probe Kit
- Pathway Anti-HER-2/neu (4B5)
- Insite HER-2/neu Kit
- Spot-Light HER2 CISH Kit
- Bond Oracle Her2 IHC System
- HER2 CISH pharmDx Kit
- INFORM HER2 DUAL ISH DNA Probe Cocktail
- HercepTest
- HER2 FISH pharmDx Kit
- THxID BRAF Kit
- cobas BRAF V600 Mutation Test
- Oncomine Dx Target Test
- Praxis Extended Ras Panel
- cobas KRAS Mutation Test
- therascreen KRAS RGQ PCR Kit
- Vysis ALK Break Apart FISH Probe Kit
- ALK (D5F3) CDx Assay
- cobas EGFR Mutation Test v2
- therascreen EGFR RGQ PCR Kit
- FoundationFocus CDxBRCA

## Submission Summary (Full Text)

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>
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PMA P170019: FDA Summary of Safety and Effectiveness Data

# 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™

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

Date of FDA Notice of Approval: November 30, 2017

Breakthrough Device: Granted breakthrough device status [previously Expedited Access Pathway (EAP)] on June 15, 2016 because the device (1) is intended to diagnose a life threatening or irreversibly debilitating disease or condition (2) represents a breakthrough technology that provides a clinically meaningful advantage over existing legally marketed technology, and (3) the availability of the device is in the best interest of patients.

## II. INDICATIONS FOR USE

FoundationOne CDx™ (F1CDx) is a next generation sequencing based in vitro diagnostic device 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 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 cancer patients with solid malignant neoplasms. The F1CDx test is a single-site assay performed at Foundation Medicine, Inc.

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Table 1. Companion diagnostic indications

|  Indication | Biomarker | Therapy  |
| --- | --- | --- |
|  Non-small cell lung cancer (NSCLC) | EGFR exon 19 deletions and EGFR exon 21 L858R alterations | Gilotrif® (afatinib), Iressa® (gefitinib), or Tarceva® (erlotinib)  |
|   |  EGFR exon 20 T790M alterations | Tagrisso® (osimertinib)  |
|   |  ALK rearrangements | Alecensa® (alectinib), Xalkori® (crizotinib), or Zykadia® (ceritinib)  |
|   |  BRAF V600E | Tafinlar® (dabrafenib) in combination with Mekinist® (trametinib)  |
|  Melanoma | BRAF V600E | Tafinlar® (dabrafenib) or Zelboraf® (vemurafenib)  |
|   |  BRAF V600E and V600K | Mekinist® (trametinib) or Cotellic® (cobimetinib) in combination with Zelboraf® (vemurafenib)  |
|  Breast cancer | ERBB2 (HER2) amplification | Herceptin® (trastuzumab), Kadcyla® (ado-trastuzumab-emtansine), or Perjeta® (pertuzumab)  |
|  Colorectal cancer | KRAS wild-type (absence of mutations in codons 12 and 13) | Erbitux® (cetuximab)  |
|   |  KRAS (exons 2, 3, and 4) and NRAS (exons 2, 3, and 4) | Vectibix® (panitumumab)  |
|  Ovarian cancer | BRCA1/2 alterations | Rubraca® (rucaparib)  |

III. CONTRAINDICATIONS

There are no known contraindications.

IV. WARNINGS/PRECAUTIONS AND LIMITATIONS

The warnings/precautions and limitations are included in the FoundationOne CDx assay labeling.

V. DEVICE DESCRIPTION

FoundationOne CDx (F1CDx) is a single-site assay performed at Foundation Medicine, Inc. 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

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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 complete list of genes included in F1CDx). In total, the assay therefore detects alterations in a total of 324 genes. Using the Illumina® HiSeq 4000 platform, hybrid-capture-selected libraries will be sequenced to high uniform depth (targeting  $>500\mathrm{X}$  median coverage with  $>99\%$  of exons at coverage  $>100\mathrm{X}$ ). Sequence data is 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) and tumor mutational burden (TMB) will be reported.

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

|  ABL1 | BRAF | CDKN1A | EPHA3 | FGFR4 | IKZF1 | MCL1 | NKX2-1 | PMS2 | RNF43 | TET2  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  ACVR1B | BRCA1 | CDKN1B | EPHB1 | FH | INPP4B | MDM2 | NOTCH1 | POLD1 | ROS1 | TGFBR2  |
|  AKT1 | BRCA2 | CDKN2A | EPHB4 | FLCN | IRF2 | MDM4 | NOTCH2 | POLE | RPTOR | TIPARP  |
|  AKT2 | BRD4 | CDKN2B | ERBB2 | FLT1 | IRF4 | MED12 | NOTCH3 | PPARG | SDHA | TNFAIP3  |
|  AKT3 | BRIP1 | CDKN2C | ERBB3 | FLT3 | IRS2 | MEF2B | NPM1 | PPP2R1A | SDHB | TNFRSF14  |
|  ALK | BTG1 | CEBPA | ERBB4 | FOXL2 | JAK1 | MEN1 | NRAS | PPP2R2A | SDHC | TP53  |
|  ALOX12B | BTG2 | CHEK1 | ERCC4 | FUBP1 | JAK2 | MERTK | NT5C2 | PRDM1 | SDHD | TSC1  |
|  AMER1 | BTK | CHEK2 | ERG | GABRA6 | JAK3 | MET | NTRK1 | PRKAR1A | SETD2 | TSC2  |
|  APC | C11orf30 | CIC | ERRF11 | 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 | SMARCA4 | WHSC1  |
|  ASXL1 | CBL | CTCF | FANCC | GNA13 | KEAP1 | MSH2 | PARP1 | QKI | SMARCB1 | 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 | PDCD1LG2 | RAD51D | SPEN |   |
|  AXL | CD70 | DAXX | FGF14 | HGF | LTK | MYC | PDGFRA | RAD52 | SPOP |   |
|  BAP1 | CD79A | DDR1 | FGF19 | HNF1A | LYN | MYCL | PDGFRB | RAD54L | SRC |   |

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|  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 a ncRNA gene.

|  ALK
introns 18, 19 | BRCA1
introns 2, 7, 8, 12, 16, 19, 20 | ETV4
introns 5, 6 | EZR
introns 9-11 | KIT
intron 16 | MYC
intron 1 | NUTM1
intron 1 | RET
introns 7-11 | SLC34A2
intron 4  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  BCL2
3’UTR | BRCA2
intron 2 | ETV5
introns 6, 7 | FGFR1
intron 1, 5, 17 | KMT2A
(MLL)
introns 6-11 | NOTCH2
intron 26 | PDGFRA
introns 7, 9, 11 | ROS1
introns 31-35 | TERC
ncRNA  |
|  BCR
introns 8, 13, 14 | CD74
introns 6-8 | ETV6
introns 5, 6 | FGFR2
intron 1, 17 | MSH2
intron 5 | NTRK1
introns 8-10 | RAF1
introns 4-8 | RSPO2
intron 1 | TERT
Promoter  |
|  BRAF
introns 7-10 | EGFR
introns 7, 15, 24-27 | EWSR1
introns 7-13 | FGFR3
intron 17 | MYB
intron 14 | NTRK2
Intron 12 | RARA
intron 2 | SDC4
intron 2 | TMPRSS2
introns 1-3  |

## 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

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# 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  |
|  Beckman Biomek NXP Span-8 Liquid Handler  |
|  Thermo Scientific Kingfisher Flex DW 96  |

# 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 the normalization of DNA to 50-1000 ng. The normalized DNA samples are randomly sheared (fragmented) to ~200 bp by adaptive focused acoustic sonication using a Covaris LE220 before purification using 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,

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are performed in 96-well plates (Eppendorf) on a Bravo Benchbot (Agilent) using the "with-bead" protocol¹ to maximize reproducibility and library yield. Indexed (6 bp barcodes) sequencing libraries are PCR amplified with HiFi™ (Kapa) for 10 cycles, and subsequently 1.8X SPRI purified. Purification and dilution for QC are performed.

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

## D. Hybrid Capture

Hybrid Capture (HC) begins with normalization of each library to 500-2000 ng. Normalized samples then undergo solution hybridization which is performed using a > 50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 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.

Quality Control for Hybrid Capture is performed by measuring dsDNA yield using a 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 dNTP's 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

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new base is added to the growing chain. This method allows for millions of discrete clusters of clonal copies of DNA to be sequenced in parallel.

## F. Sequence Analysis

Sequence data is analyzed using proprietary software developed by FMI. Sequence data is mapped to the human genome (hg19) using Burrows-Wheeler Aligner (BWA) v0.5.9.⁴ PCR duplicate read removal and sequence metric collection is performed using Picard 1.47 (http://picard.sourceforge.net) and SAMtools 0.1.12a.⁵ Local alignment optimization is performed using Genome Analysis Toolkit (GATK) 1.0.4705.⁶ Variant calling is performed only in genomic regions targeted by the test.

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

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

- Collecting all read-pairs for which at least one read maps to the target region.
- Decomposing each read into constituent k-mers and constructing an enumerable graph representation (de-Bruijn) of all candidate non-reference haplotypes present.
- Evaluating the support of each alternate haplotype with respect to the raw read data to generate mutational candidates. All reads are compared to each of the candidate haplotypes via ungapped alignment, and a read 'vote' for each read is 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

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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 (5) chimeric pairs [three (3) 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, 95 intronic homopolymer repeat loci (10-20 bp long in the human reference genome) with adequate coverage on F1CDx Assay are analyzed for length variability and compiled into an overall MSI score via principal components analysis. Using the 95 loci, for each sample the repeat length is calculated in each read that spans the locus. The means and variances of repeat lengths is recorded. Principal components analysis (PCA) is used to project the 190-dimension data onto a single dimension (the first principal component) that maximizes the data separation, producing an MSI score. Each sample is assigned a qualitative status of MSI-High (MSI-H) or MSI-Stable (MSS); ranges of the MSI score are assigned MSI-H or MSS by manual unsupervised clustering. Samples with low coverage (< 250X median) are assigned a status of MSI-unknown.

Tumor mutational burden (TMB) is measured by counting all synonymous and non-synonymous 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 is displayed in the FMI custom-developed CATi software applications with sequence quality control 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 is reviewed by trained bioinformatics personnel. Samples failing any QC metrics are automatically held and not released.

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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 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%.

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 Cx tests are listed in Table 5 below; for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm301431.htm?source=govdelivery.

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Table 5. 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) | Ventana Medical Systems, Inc. | IHC | Herceptin (trastuzumab) | Breast cancer  |
|   |  Rabbit Monoclonal Primary Antibody | Biogenex Laboratories, Inc. | IHC | Herceptin (trastuzumab) | Breast cancer  |
|   |  Insite HER-2/neu Kit  |   |   |   |   |
|   |  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) | 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-V600 | THxID BRAF Kit | bioMerieux | PCR | Mekinist (tramatenib) | Melanoma  |
|   |  cobas BRAF V600 Mutation Test | Roche Molecular Systems, Inc. | PCR | Zelboraf (vemurafenib) | Melanoma  |
|  BRAF-600E | THxID BRAF Kit | bioMerieux | PCR | Tafinlar (dabrafemib) | Melanoma  |
|   |  Oncomine Dx Target Test | Life Technologies, Inc. | NGS | Tafinlar (dabrafemib) Mekinist (trametinib) | Non-small cell lung cancer  |
|  NRAS | Praxis Extended Ras Panel | Illumina | NGS | Vectibix (panitumumab) | Colorectal cancer  |

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|   | Device | Company | Technology | Therapy | Indication  |
| --- | --- | --- | --- | --- | --- |
|  KRAS | cobas KRAS Mutation Test | Roche Molecular Systems, Inc. | PCR | Erbitux (cetuximab) Vectibix (panitumumab) | Colorectal cancer  |
|   |  therascreen KRAS RGQ PCR Kit | QIAGEN | PCR | Erbitux (cetuximab) Vectibix (panitumumab) | Colorectal cancer  |
|   |  Praxis Extended Ras Panel | Illumina | NGS | Vectibix (panitumumab) | Colorectal cancer  |
|  ALK - fusion | Vysis ALK Break Apart FISH Probe Kit | Abbott Molecular, Inc. | FISH | Xalkori (crizotinib) | Non-small cell lung cancer  |
|   |  ALK (D5F3) CDx Assay | Ventana Medical Systems, Inc. | IHC | Xalkori (crizotinib) | Non-small cell lung cancer  |
|  EGFR - Exon 19 deletions & L858R | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | Tarceva (erlotinib) | Non-small cell lung cancer  |
|   |  therascreen EGFR RGQ PCR Kit | QIAGEN | PCR | Gilotrif (afatinib) Iressa (gefitinib) | Non-small cell lung cancer  |
|   |  Oncomine Dx Target Test | Life Technologies, Inc. | NGS | Iressa (gefitinib) | Non-small cell lung cancer  |
|  EGFR T790M | cobas EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | PCR | Tagrisso (osimertinib) | Non-small cell lung cancer  |
|  BRCA1/2 | FoundationFocus CDxBRCA | Foundation Medicine, Inc. | NGS | Rubraca (rucaparib) | Advanced Ovarian  |

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 assay has not been marketed in the United States or any foreign country.

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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 indicated therapy. For the specific adverse events related to the approved therapeutics, please see approved drug product labels.

# IX. SUMMARY OF NON-CLINICAL STUDIES

## A. Laboratory Studies

Performance characteristics were established using DNA derived from a wide range of FFPE tissue types; tissue types associated with CDx indications were included in each study. Each study included CDx variants as well as a broad range of representative alteration types (substitution, insertion-deletion, copy number alterations, rearrangements) in various genomic contexts across a number of genes. Analyses of genomic signatures including MSI and TMB were also conducted.

## 1. Analytical Accuracy/Concordance

### a. Comparison to an Orthogonal Method

The detection of alterations by F1CDx was compared to results of an externally validated NGS assay (evNGS). The comparison between short alterations, including base substitutions and short indels, detected by F1CDx and the orthogonal method included 188 samples from 46 different tumor tissue types including ovarian serous carcinoma, breast carcinoma, brain glioblastoma, vulva squamous cell carcinoma, cervix adenocarcinoma, lung, melanoma and colon adenocarcinoma.

A summary of Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) is provided in Table 6, below. Overall there were 157 overlapping genes between the F1CDx assay and the orthogonal method. None of the 188 samples were determined to be invalid on either platform.

Table 6. Concordance Summary for short variants (inclusive of both substitutions and indels), base substitutions, and indels with VUS included

|  Variant Type | F1CDx+/evNGS+ | F1CDx-/evNGS+ | F1CDx+/evNGS- | F1CDx-/evNGS- | PPA*(95% CI) | NPA*(95% CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  All short variants | 1282 | 73 | 375 | 284218 | 94.6% (93.3%-95.8%) | 99.9% (99.9%-99.9%)  |
|  Substitutions | 1111 | 39 | 334 | 242540 | 96.6% (95.4%-97.6%) | 99.9% (99.8%-99.9%)  |
|  Indels | 171 | 34 | 41 | 41678 | 83.4% (77.6%-88.2%) | 99.9% (99.9%-99.9%)  |

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Since the PPA and NPA were calculated without adjusting for the distribution of samples enrolled using F1 LDT, positive predictive value (PPV) and negative predictive value (NPV) were also estimated conditional on F1CDx. The total number of alterations detected across all 188 samples by either F1CDx and/or evNGS was used to determine NPV. The observed PPV for all short variants was  $77.4\%$  [95% confidence interval (CI):  $75.3\% - 79.4\%$ ] and for indels PPV was  $80.7\%$  [95% CI:  $74.7\% - 85.7\%$ ]. The NPV for all short variants was  $>99.9\%$ . Differences in variants of unknown significance (VUS) alteration calls between the platforms were noted, and is expected based on differences in filtering employed by F1CDx and the NGS comparator. Discordant alterations not related to VUS filtering were primarily caused by deletions with low allelic fraction in homopolymer regions. The F1CDx variant calling pipeline imposes a filter based on MAF of  $\geq 0.10\%$  for indels in homopolymer regions to reduce the likelihood of calling false positives resulting from artifacts introduced by the technology. As such, the difference observed was due to varying filter thresholds between the two platforms. For additional concordance for the CDx-associated variants, refer to the Clinical Concordance Studies described in Section X.

# b. Comparison to FoundationOne LDT (F1 LDT)

To support the use of retrospective data generated using the F1 LDT as supplemental and supporting data for F1CDx, a concordance study was conducted with the F1CDx assay. This study evaluated a set of 165 specimens with variants in genes interrogated by both assays. The study included DNA derived from 35 different tumor types extracted from tissues with tumor content ranging from  $20\%$  to  $90\%$ . PPA and NPA between F1CDx and F1 LDT, using the F1 LDT assay as the reference method, was calculated for all alterations, as well as for alterations binned by type: short variants, CNAs and rearrangements. A total of 2325 variants, including 2026 short variants, 266 copy number alterations and 33 rearrangements met the variant inclusion criteria. The study results are in Table 7, below.

Table 7. Summary of concordance between F1CDx and F1 LDT

|  Variant Type | F1CDx+/ F1 LDT+ | F1CDx- /F1 LDT+ | F1CDx+/ F1 LDT- | F1CDx- /F1 LDT- | PPA(95%CI) | NPA(95%CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  All variants | 2246 | 33 | 46 | 322890 | 98.6% (98.0%-99.0%) | 99.99% (99.98%-99.99%)  |
|  All short variants | 1984 | 19 | 23 | 299099 | 99.1% (98.5%-99.4%) | 99.99% (99.9%-99.995%)  |
|  Substitutions | 1692 | 10 | 19 | 254854 | 99.4% (98.9%-99.7%) | 99.99% (99.99%-99.996%)  |
|  Indels | 292 | 9 | 4 | 44245 | 97.0% (94.4%-98.6%) | 99.99% (99.98%-99.998%)  |
|  All CNAs | 230 | 14 | 22 | 19204 | 94.3% (90.6%-96.8%) | 99.9% (99.8%-99.96%)  |
|  Amplifications | 157 | 10 | 12 | 14671 | 94.0% (89.3%-97.1%) | 99.9% (99.9%-99.96%)  |
|  Losses | 73 | 4 | 10 | 4533 | 94.8% (87.2%-98.6%) | 99.8% (99.6%-99.9%)  |

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|  Variant Type | F1CDx+/F1 LDT+ | F1CDx-/F1 LDT+ | F1CDx+/F1 LDT- | F1CDx-/F1 LDT- | PPA(95%CI) | NPA(95%CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  Rearrangements | 32 | 0 | 1 | 4587 | 100.0% (89.1%-100%) | 99.98% (99.99 -99.999%)  |

The qualitative output for MSI (MSI-H vs MSS) in F1 LDT and F1CDx were evaluated. PPA, NPA and Overall Percent Agreement (OPA) of MSI status between the two assays was calculated for all 165 samples. Of the 165 samples, 5 were MSI-H by F1 LDT and 160 were MSS by F1 LDT, there was one discordant sample observed. The discordant sample was called MSS by F1 LDT and MSI-H by F1CDx. After manual review, the discordant case had an MSI score close to the threshold used to classify MSI status, thus leading to discordant MSI status in the two assays. PPA was  $100\%$  with a  $95\%$  CI of  $47.8 - 100\%$ , NPA was  $99.5\%$  with a  $95\%$  CI of  $96.6\% - 99.98\%$  and the OPA was  $99.4\%$  with a  $95\%$  CI of  $96.7\% - 99.98\%$ .

Furthermore, additional MSI concordance data was reviewed including the following: 1) concordance between the F1 LDT MSI results and a validated mismatch repair immunohistochemistry (MMR IHC) panel and 2) concordance between the F1 LDT and a validated MSI-PCR test. Comparison to MMR IHC was performed on 30 colorectal cancer specimens, and comparison to PCR was performed on a total of 39 FFPE tissue samples (18 colorectal, 20 uterine and one cecum cancer specimen). In this sample set, there were two specimens noted as MSI-ambiguous which were not included in the concordance calculations. Combining IHC and PCR data, the overall concordance was  $97\%$  (65/67); see concordance Table 8, below. Additional concordance data will be provided post-market comparing MSI test results generated using F1CDx, to both a validated MMR IHC panel and a validated MSI-PCR test.

Table 8. Concordance Table with FoundationOne LDT (F1 LDT) and a validated MMR IHC and MSI-PCR assay

|   | IHC |   |   | PCR  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  F1 LDT | MSI-H | MSS | Total | MSI-H | MSS | Total  |
|  MSI-H | 1 | 0 | 1 | 17 | 1 | 18  |
|  MSS | 0 | 29 | 29 | 1 | 18 | 19  |
|  Total | 1 | 29 | 30 | 18 | 19 | 37  |

TMB concordance was evaluated by comparing the TMB output in terms of mutations per Mb. Analyses were conducted using all 153 non-zero TMB scores. From linear regression analysis using F1 LDT TMB as the predictor and F1CDx TMB as the outcome, the intercept is -0.27782 (95%CI: -0.662-0.106), and the slope is 0.94064 (95% CI: 0.919-0.963).

# 2. Analytical Sensitivity

# a. Limit of Detection (LoD)

The LoD of alterations assessed by F1CDx was evaluated. The LoDs of five (5) CDx biomarkers representing  $EGFR$  exon 21 L858R,  $EGFR$  Exon 20 T790M,  $EGFR$  exon 19 deletion,  $KRAS$  codons 12/13 substitution and  $BRAF$  V600E were determined and is summarized in Table 9, below.

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Table 9. Summary of LoD for alterations associated with CDx claims

|  Alteration | LoD*
Allele Fraction (%)  |
| --- | --- |
|  EGFR L858R | 2.4%  |
|  EGFR Exon 19 deletion | 5.1%  |
|  EGFR T790M | 2.5%  |
|  KRAS G12/G13 | 2.3%  |
|  BRAF V600E | 2.0%  |
|  BRCA1/2**
Alteration in non-repetitive regions or homopolymer repeats < 4 bp
Alterations in a homopolymer region > 4bp | 6%
15.3%  |

*LoD calculations for the CDx variants were based on the hit rate approach, as there were less than three levels with hit rate between 10% and 90% for all CDx variants (not including BRCA1/2 variants). LoD from the hit rate approach is defined as the lowest level with 100% hit rate (worst scenario).
**See Summary of Safety and Effectiveness Data for P160018. LoD calculated using probit approach.

Additional categories of alteration types were evaluated for the F1CDx assay platform validation. A single FFPE tumor sample was selected for each of the variant categories. For each sample, six levels of MAF, with 13 replicates per level, were evaluated (total of 78 aliquots per sample). To meet the number of required replicates proposed in CLSI EP17-A2, multiple different pooled DNA extractions from a single FFPE samples and banked DNA samples were used to meet the required variant characteristics. For platform-wide LoD assessment, the indels were grouped together (other than homopolymer repeat context) as they are similar in LoD characteristics. The indels ranged from 1 bp up to 42 bp insertions and deletions up to 276 bp. Indels at homopolymer repeat context had higher LoD, with a dependency on the length of the repeat context. When estimating the LoD of platform substitutions, the analyses excluded certain variants that were fully detected at all dilution levels (N = 13 substitutions, MAF Range 2.2%-15.2%) as they did not reach full dilution and were determined not to be representative of the remaining 166 alterations. Representative LoD for the range of variants detected by the F1CDx platform is summarized in Table 10.

Table 10. Summary representative LoD for platform alterations

|  Variant Category | Subcategory* | N | Range LoD**
Allele Fraction (%)  |
| --- | --- | --- | --- |
|  Base Substitutions | known | 21*** | 1.8-7.9  |
|   |  other | 166*** | 5.9-11.8  |
|  Indels at non-homopolymer context, including insertions up to 42bp and deletions up to 276bp | known | 3 | 4.5-6.5  |
|   |  other | 17 | 6.0-10.2  |

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|  Variant Category | Subcategory* | N | Range LoD**
Allele Fraction (%)  |
| --- | --- | --- | --- |
|  Indels at homopolymer context | 5bp repeat | 8 | 10.0-12.2  |
|   |  6bp repeat | 2 | 13.6-13.7  |
|   |  7bp repeat | 4 | 16.3-20.4  |
|   |  8bp repeat | 3 | 17.0-20.0  |

*Alterations classified as "known" are defined as those that are listed in COSMIC. Alterations classified as "other" include truncating events in tumor suppressor genes (splice, frameshift and nonsense) as well as variants that appear in hotspot locations but do not have a specific COSMIC association, or are considered variants of unknown significance (VUS) due to lack of reported evidence and conclusive change in function.

**LoD calculations for the platform variants were based on the hit rate approach for variants with less than three levels with hit rate between 10% and 90% and probit approach for variants with at least three levels with hit rate between 10% and 90%. LoD from the hit rate approach is defined as the lowest level with 100% hit rate (worst scenario).

***Data includes an alteration in the TERT promoter 124C&gt;T (LoD of 7.9%) TERT is the only promoter region interrogated by F1CDx and is highly enriched for repetitive context of poly-Gs, not present in coding regions.

## b. Limit of Blank (LoB)

The limit of blank (LoB) of zero was confirmed using the mutation calls from mutation-negative FFPE samples (19 distinct samples with four replicates per sample), with a percentage of false-positive results less than 5% (type I error risk α=0.05). Seventy-five (75) samples (19 × 4 − 1, because one failed to meet the Library Construction QC process specification) were used for the assessment of LoB. It was confirmed that each replicate of LoB sample was negative for variants included in the LoD analysis.

## c. Analytical Sensitivity – Tumor Purity

The minimum tumor fraction required to support the robustness of the F1CDx for select CDx alterations and platform validation was evaluated. For each sample, six levels of tumor content, with 13 replicates per level, were evaluated, so a total of 78 aliquots per sample. The analytical sensitivity of two (2) CDx biomarkers representing ALK fusion and ERBB2 amplification were determined and is summarized in Table 11, below. The data show that for ALK fusion the assay is robust down to 2.6% tumor purity, therefore at the minimal required tumor purity required for the assay, 20%, the assay is robust at detecting this alteration. For ERBB2 amplification, the data show that the assay is robust at detecting this alteration down 25.3% tumor purity. Similarly, additional categories representing four alteration types were also evaluated for the F1CDx assay platform validation and results summarized in Table 12. In addition, analytical sensitivity based on tumor purity was also evaluated for MSI-H. Performance of TMB at the recommended tumor purity of ≥ 20%, was found to be robust based on the LoD of short variants.

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Table 11. Summary of analytical sensitivity for tumor purity for alterations associated with CDx claims

|  Alteration | Tumor Purity (%)*  |
| --- | --- |
|  ALK fusion | 2.6%**  |
|  ERBB2 amplification | 25.3%***  |

* Sensitivity calculations for the CDx variants were based on the hit rate approach, as there were less than three levels with hit rate between 10% and 90%. LoD from the hit rate approach is defined as the lowest level with 100% hit rate (worst scenario).
**The number of chimeric reads for the sample evaluated is 16 at the indicated tumor fraction.
***The number of copy number amplifications for the sample evaluated is 6.

Table 12. Summary analytical sensitivity for tumor purity for platform validation

|  Variant Category | N | Range Tumor Purity (%)*  |
| --- | --- | --- |
|  Copy Number Amplifications (CN > 10) | 8 | 9.6%-18.5%  |
|  Copy Number Amplifications (6 ≤ CN ≤ 10) | 7 | 19.5%-58.3%**  |
|  Copy Number: Homozygous Deletions | 3 | 33.4%-33.4%  |
|  Genomic Rearrangements | 3 | 9.2%-14.9%  |
|  MSI-High | 3 | 8.3%-15.8%  |

* Sensitivity calculations for the platform variants were based on the hit rate approach for variants with less than three levels with hit rate between 10% and 90% and probit approach for variants with at least three levels with hit rate between 10% and 90%. LoD from the hit rate approach is defined as the lowest level with 100% hit rate (worst scenario).
**Max represents VUS alteration at calling threshold.

# 3. Analytical Specificity

# a. Interfering Substances

To evaluate the potential impact of endogenous and exogenous interfering substances on the performance of the F1CDx assay, this study evaluated five FFPE specimens representing five tumor types (ovary, lung, colorectal, breast cancer and melanoma). The specimens included representative variant types (substitution, indel, amplification, homozygous deletion and rearrangement), and were tested in duplicate (Table 13). An additional 54 short alterations (base substitutions and indels) were assessed. The addition of interfering substances including melanin (endogenous), ethanol (exogenous), proteinase K (exogenous), and molecular index barcodes (MIB) (exogenous) was evaluated to determine if they were impactful to F1CDx, and the results were compared to the control (no interferents) condition.

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Table 13. Summary of tumor types and variant types included in study

|  Tumor Type | Gene (and variant as relevant) | Variant type  |
| --- | --- | --- |
|  Colorectal | FGFR1 | Rearrangement  |
|   |  BCL2L1 | Amplification  |
|   |  AXIN1 c.1058G>A (R353H) | Substitution  |
|   |  SOX9 c.768_769insGG (R257fs*23) | Indel  |
|  Breast | ERBB2 | Amplification  |
|   |  AKT1 | Amplification  |
|   |  CCND1 | Amplification  |
|  Lung | CDKN2A | Homozygous Deletion  |
|   |  CDKN2B | Homozygous Deletion  |
|   |  EGFR | Amplification  |
|  Ovary | BRCA1 c.5263_5264insC (Q1756fs*74) | Indel  |
|   |  ERCC4 c.2395C>T | Substitution  |
|   |  TP53 c.779_779delC (S261fs*84) | Indel  |
|  Melanoma | BRAF c.1799T>A (V600E) | Substitution  |
|   |  TP53 c. 856G>A (E286K) | Substitution  |
|   |  IGF1R | Amplification  |

Interfering substances included melanin, ethanol, proteinase K, and molecular index barcodes, as noted in Table 14. below. Each of the five FFPE specimens were tested in either two or four replicates each, resulting in a total of 170 data points across the five specimens (10 without interferent, 80 for evaluation of melanin, ethanol and proteinase K and 80 for molecular index barcodes) were assessed in this study.

Table 14. Interfering Substance Evaluated

|  Substances | Level | # Samples | # Replicates/Sample  |
| --- | --- | --- | --- |
|  No interferent | - | 5 | 2  |
|  Melanin | 0.025 μg/mL | 5 | 2  |
|  Melanin | 0.05 μg/mL | 5 | 2  |
|  Melanin | 0.1 μg/mL | 5 | 2  |
|  Melanin | 0.2 μg/mL | 5 | 2  |
|  Proteinase K | 0.04 mg/mL | 5 | 2  |
|  Proteinase K | 0.08 mg/mL | 5 | 2  |
|  Ethanol | 5% | 5 | 2  |
|  Ethanol | 2.5% | 5 | 2  |
|  MIB | 0 | 5 | 4  |
|  MIB | 5% | 5 | 4  |
|  MIB | 15% | 5 | 4  |
|  MIB | 30% | 5 | 4  |

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Substances were considered as non-interfering if, when compared to no interferent, the DNA yield was sufficient to meet the standard processing requirements of DNA isolation (≥ 55 ng), the quality was sufficient to create products per the specification of library construction (≥ 545 ng) and hybrid capture (≥ 140 ng), and the sample success rate (fraction of samples that met all process requirements and specification), across all replicates in aggregate, was ≥ 90%. Sequence analysis was assessed as percent agreement for each specimen and was calculated as the number of replicates with the correct alteration call reported per the total number of replicates processed. Percent agreement (fraction of correct calls) was computed across the replicates. The results were aggregated across all variants in all five FFPE specimens, and concordance was assessed within each treatment category across variants. The acceptance for concordance required a minimum of 90% of correct calls within each treatment category. All samples tested at all interfering substance levels met all process requirements and specifications; indicating that the sample quality was not impacted by the interfering substances. The concordance for each variant for the melanin, proteinase K and MIB evaluations was 100%, and 95.3% for the ethanol evaluation. Each met the acceptance criterion of ≥ 90% indicating that the test performance was not affected by the tested interferents. In addition to the variants selected to represent specific alteration types summarized in Table 13 above, samples included in the study harbored 54 additional short alterations (substitutions and indels) which were found to be 100% concordant across all replicates for each variant.

See Summary of Safety and Effectiveness Data for P160018 for additional interference studies wherein the interference of necrotic tissue, triglycerides, hemoglobin, and xylene, in addition to ethanol, proteinase K, and MIBs, was evaluated in ovarian tissue and assessed BRCA1/2 alterations.

## b. In silico Analysis – Hybrid Capture Bait Specificity

Bait specificity was addressed through an assessment of coverage at the base level for targeted regions included in F1CDx. Lack of bait specificity and/or insufficient bait inclusion would result in regions of diminished high quality mapped reads due to the capture of off-target content. This analysis showed that all regions that may harbor alterations associated with companion diagnostic claims consistently have high quality (MQS ≥ 30), deep coverage ≥ 250X. When assessing the entire gene set, 91.45% of individual bases within targeted introns platform-wide had ≥ 100X coverage.

## 4. Carryover/Cross-Contamination

No carryover or cross-contamination was observed when alternating positive and negative samples for BRCA1 and BRCA2 variants, assessed in a checker-board pattern (see Summary of Safety and Effectiveness Data for P160018). In addition, data from plates evaluated in multiple analytical validation studies, containing high-level confirmed ERBB2 amplifications, EGFR T790M alterations or ALK fusions were examined for cross-contamination in adjacent wells containing confirmed negative samples. No contamination was detected.

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# 5. Precision and Reproducibility

Precision was evaluated for alterations associated with CDx claims, as well as representative alterations to support platform-level performance including, MSI, TMB and MAF of short variants. Repeatability including intra-run performance (run on the same plate under the same conditions) and reproducibility including inter-run performance (run on different plates under different conditions) were assessed and compared across three different sequencers and three different reagent lots, across multiple days (typical assay workflow spans 10 days) of performance by multiple operators. A full factorial design for this study was carried out with four (4) replicates per reagent lot/sequencer combination for samples with 36 replicates.

A set of 47 unique FFPE samples containing a variety of variants were evaluated. In total, 717 alterations were assessed, including variants associated with CDx claims (Table 15), and additional variants intended to demonstrate platform level validation (Table 16). The platform-level study included a total of 443 substitutions, 188 indels, 55 copy number amplifications, 13 copy number loss and 18 rearrangements in the variant set across the samples.

Table 15. Sample set with CDx variants

|  Gene | # of Unique Samples | Alteration | Tumor Type  |
| --- | --- | --- | --- |
|  EGFR | 3 | Exon 19 Deletion | NSCLC  |
|   |  2 | Exon 21 L858R  |   |
|   |  2 | Exon 20 T790M  |   |
|  KRAS | 3 | Codons 12/13 substitution | CRC  |
|  ALK | 3 | Fusion | NSCLC  |
|  BRAF | 3 | V600E/V600K | Melanoma  |
|  ERBB2 (HER2) | 3 | Amplification | Breast  |

Table 16. Sample set for platform validation

|  Alterations Type | # of Unique Samples | Alteration Size | Genomic Context  |
| --- | --- | --- | --- |
|  Substitution | 3 | - | -  |
|  Short Insertion | 2 | 1-2bp | Homopolymer Repeats  |
|  Short Insertion | 2 | 1-2bp | Dinucleotide Repeats  |
|  Short Insertion | 2 | 3-5bp | -  |
|  Short Insertion | 2 | >5bp | -  |
|  Short Deletion | 2 | 1-2bp | Homopolymer Repeats  |
|  Short Deletion | 2 | 1-2bp | Dinucleotide  |

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|  Alterations Type | # of Unique Samples | Alteration Size | Genomic Context  |
| --- | --- | --- | --- |
|   |  |  | Repeats  |
|  Short Deletion | 2 | 3-5bp | -  |
|  Short Deletion | 2 | >5bp | -  |
|  Amplification | 3 | - | -  |
|  Homozygous Deletion | 3 | - | -  |
|  Rearrangement | 3 | - | -  |

A contamination level of  $1.9\%$  was observed, wherein  $&lt; 1\%$  is required. As such, one sample and its associated aliquots were excluded from repeatability and reproducibility concordance and MSI analyses. In total, 18 samples to support CDx validation and 28 samples for platform validation were included in the final analysis. The data for process failures and no call rate are summarized in Table 17, below. There were no replicates that generated a no call result for base substitution, indel, CNA or rearrangement calls.

Table 17. Summary data including process failures and no call rates for samples included in the precision study

|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | Replicates with Pre-sequencing Process Failure | % Replicates with MSI No Call | % Replicates with TMB No Call | Mean TMB  |
| --- | --- | --- | --- | --- | --- | --- |
|  Sample 1 | SUB | EGFR T790M | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 2.1  |
|  Sample 2 | SUB | EGFR T790M | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 8.37  |
|  Sample 3 | RE | ALK Fusion | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 1.51  |
|  Sample 4 | RE | ALK Fusion | 0 | 0.0% (0 of 36) | 19.4% (7 of 36) | 3.36  |
|  Sample 5 | RE | ALK Fusion | 0 | 0.0% (0 of 36) | 11.1% (4 of 36) | 3.12  |
|  Sample 6 | Indel | EGFR Exon 19 Deletion | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 5.04  |
|  Sample 7 | Indel | EGFR Exon 19 Deletion | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 4.21  |
|  Sample 8 | Indel | EGFR Exon 19 Deletion | 0 | 0.0% (0 of 36) | 25.0% (9 of 36) | 2.8  |
|  Sample 9 | SUB | KRAS Codons 12/13 SUB | 0 | 0.0% (0 of 36) | 100.0% (36 of 36) | 3.33  |
|  Sample 10 | SUB | KRAS Codons 12/13 SUB | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 4.17  |
|  Sample 11 | SUB | BRAF V600E/V600K | 1 (HC) | 2.9% (1 of 35) | 0.0% (0 of 35) | 14.91  |
|  Sample 12 | SUB | BRAF V600E/V600K | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 23.54  |
|  Sample 13 | SUB | EGFR Exon 21 L858R | 2 (HC) | 0.0% (0 of 34) | 8.8% (3 of 34) | 3  |

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|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | Replicates with Pre-sequencing Process Failure | % Replicates with MSI No Call | % Replicates with TMB No Call | Mean TMB  |
| --- | --- | --- | --- | --- | --- | --- |
|  Sample 14 | Amplification | ERBB2 (HER2) Amplification | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 4  |
|  Sample 15 | Amplification | ERBB2 (HER2) Amplification | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 4.82  |
|  Sample 16 | Amplification | ERBB2 (HER2) Amplification | 2 (HC) | 0.0% (0 of 34) | 0.0% (0 of 34) | 7.9  |
|  Sample 17 | SUB | KRAS Codons 12/13 SUB | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 6.37  |
|  Sample 18 | SUB | EGFR Exon 21 L858R | 1 (HC) | 0.0% (0 of 35) | 2.9% (1 of 35) | 11.21  |
|  Sample 19 | CNA/RE/ SUB | Amplification | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 6.59  |
|  Sample 20 | CNA/SUB/ Indel | Homozygous Deletion | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 10.95  |
|  Sample 21 | SUB/Indel | Short Deletion (1-2bp with dinucleotide repeats) | 1 (LC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 42.91  |
|  Sample 22 | CNA/SUB/ Indel | Short Deletion (3-5bp) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 2.7  |
|  Sample 23 | SUB/Indel | Short Insertion (1-2bp with homopolymer repeats) | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 21.47  |
|  Sample 24 | SUB/Indel | Short Deletion (1-2bp with dinucleotide repeats) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 67.31  |
|  Sample 25 | CNA/SUB/ Indel | Short Deletion (1-2bp with homopolymer repeats) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 63.71  |
|  Sample 26 | SUB/Indel | Short Deletion (1-2bp with homopolymer repeats) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 51.83  |
|  Sample 27 | CNA/SUB/ Indel | SUB | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 12.44  |

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|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | Replicates with Pre-sequencing Process Failure | % Replicates with MSI No Call | % Replicates with TMB No Call | Mean TMB  |
| --- | --- | --- | --- | --- | --- | --- |
|  Sample 28 | RE/SUB/ Indel | Short Insertion (1-2bp with homopolymer repeats) | 1 (HC) | 0.0% (0 of 35) | 8.6% (3 of 35) | 5.87  |
|  Sample 29 | CNA/ SUB | SUB | 2 (1 LC, 1 HC) | 0.0% (0 of 34) | 0.0% (0 of 34) | 1.15  |
|  Sample 30 | CNA/RE/ SUB/Indel | RE | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 3.67  |
|  Sample 31 | RE/SUB/Indel | Short Insertion (1-2bp with dinucleotide repeats) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 1.26  |
|  Sample 32 | CNA/SUB/ Indel | Short Deletion (3-5bp) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 6.83  |
|  Sample 33 | SUB/Indel | Short Insertion (3-5bp) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 48.23  |
|  Sample 34 | CNA/RE/ SUB/Indel | Short Insertion (3-5bp) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 5.18  |
|  Sample 35 | RE/SUB/Indel | SUB | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 4.21  |
|  Sample 36 | RE/SUB/Indel | Short Insertion (>5bp) | 0 | 5.6% (2 of 36) | 94.4% (34 of 36) | 1.82  |
|  Sample 37 | CNA/RE/ SUB/Indel | Short Deletion (>5bp) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 8.09  |
|  Sample 38 | CNA/RE/ SUB/Indel | Short Insertion (>5bp) | 1 (LC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 2.2  |
|  Sample 39 | CNA /SUB | Amplification | 1 (HC) | 0.0% (0 of 35) | 0.00% (0 of 35) | 3.67  |
|  Sample 40 | CNA/RE/ SUB/Indel | RE | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 12.65  |
|  Sample 41 | CNA/ SUB/Indel | Homozygous Deletion | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 8.22  |
|  Sample 42 | CNA/RE/ SUB/Indel | Short Insertion (1-2bp with dinucleotide repeats) | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 10.16  |
|  Sample 43 | RE/SUB | Short Deletion (>5bp) | 0 | 0.0% (0 of 36) | 13.9% (5 of 36) | 2.24  |
|  Sample 44 | CNA /SUB | Homozygous Deletion | 1 (HC) | 0.0% (0 of 35) | 0.0% (0 of 35) | 7.28  |
|  Sample 45 | CNA/RE/ SUB/Indel | Amplification | 0 | 0.0% (0 of 36) | 0.0% (0 of 36) | 6.23  |
|  Sample 46 | CNA/RE/ | RE | 1 (HC) | 0.0% (0 of 35) | 5.7% (2 of 35) | 4.18  |

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|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | Replicates with Pre-sequencing Process Failure | % Replicates with MSI No Call | % Replicates with TMB No Call | Mean TMB  |
| --- | --- | --- | --- | --- | --- | --- |
|   | SUB/Indel |  |  |  |  |   |

*Samples 1-18 were selected to represent alterations associated with CDx claims and Samples 19-46 were selected for platform-wide analysis.
** While all possible alterations harbored in the sample were included in the analysis, each sample was selected based on a specific alteration in accordance with the study protocol.

Abbreviations: HC – Hybrid Capture, LC – Library Construction SUB – substitution, Indel – Insertion or Deletion, CNA – Copy Number Alteration, RE – Rearrangement.

Within the assessment of repeatability and reproducibility for CDx variants, all variants from all 18 samples had  $100.0\%$  positive call (PC) rates with two-sided  $95\%$  CIs ranging from  $(89.7\%, 100.0\%)$  to  $(90.3\%, 100.0\%)$ . Percent of negative calls at each CDx variant location for wild-type samples was  $100.0\%$  with two-sided  $95\%$  CIs ranging from  $(99.0\%, 100.0\%)$  to  $(99.1\%, 100.0\%)$ .

The platform-level repeatability and reproducibility, across the 717 variants, showed high overall negative call rates ranging from  $99.7\%$  to  $100.0\%$  for all 28 samples, and the overall positive call rates were also high for 27 samples ranging from  $95.3\%$  to  $100.0\%$  (except for sample TRF147811, with an overall positive call rate  $85.4\%$ $(82.3\%, 88.2\%)$ . The overall positive call rates and negative call rates are summarized in Table 18, below. The platform-level study included a total of 443 substitutions, 188 indels, 55 copy number amplifications, 13 copy number loss, and 18 rearrangements that were detected at least once by F1CDx in any replicate tested in the precision study.

Table 18. Positive and negative call rates for platform variants (N=717)

|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | PC Rate (exact 95% CI) | PC Rate (exact 95% CI)  |
| --- | --- | --- | --- | --- |
|  Sample 19 | CNA/RE/SUB | Amplification | 100.00% (99.40%-100.00%) | 99.98% (99.95%-99.99%)  |
|  Sample 20 | CNA/SUB/Indel | Homozygous Deletion | 99.37% (98.38%-99.83%) | 99.96% (99.92%-99.98%)  |
|  Sample 21 | SUB/Indel | Short Deletion (1-2bp with dinucleotide repeats) | 100.00% (99.10%-100.00%) | 99.97% (99.95%-99.99%)  |
|  Sample 22 | CNA/SUB/Indel | Short Deletion (3-5bp) | 97.84% (96.89%-98.56%) | 99.84% (99.78%-99.89%)  |
|  Sample 23 | SUB/Indel | Short Insertion (1-2bp with homopolymer repeats) | 99.81% (98.94%-100.00%) | 99.98% (99.95%-99.99%)  |
|  Sample 24 | SUB/Indel | Short Deletion (1-2bp with dinucleotide repeats) | 99.60% (97.81%-99.99%) | 99.94% (99.90%-99.97%)  |
|  Sample 25 | CNA/SUB/Indel | Short Deletion (1-2bp with homopolymer repeats) | 98.33% (97.11%-99.14%) | 99.98% (99.96%-100.00%)  |

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|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | PC Rate (exact 95% CI) | PC Rate (exact 95% CI)  |
| --- | --- | --- | --- | --- |
|  Sample 26 | SUB/Indel | Short Deletion (1-2bp with homopolymer repeats) | 100.00% (99.83%-100.00%) | 99.97% (99.94%-99.99%)  |
|  Sample 27 | CNA/SUB/Indel | SUB | 100.00% (99.32%-100.00%) | 99.98% (99.96%-100.00%)  |
|  Sample 28 | RE/ SUB/Indel | Short Insertion (1-2bp with homopolymer repeats) | 96.46% (94.14%-98.05%) | 99.96% (99.92%-99.98%)  |
|  Sample 29 | CNA/ SUB | SUB | 98.67% (97.27%-99.46%) | 99.98% (99.96%-100.00%)  |
|  Sample 30 | CNA/RE/SUB/ Indel | RE | 96.27% (95.39%-97.02%) | 99.87% (99.82%-99.91%)  |
|  Sample 31 | RE/SUB/Indel | Short Insertion (1-2bp with dinucleotide repeats) | 98.23% (97.48%-98.80%) | 99.66% (99.58%-99.73%)  |
|  Sample 32 | CNA/ SUB/Indel | Short Deletion (3-5bp) | 98.32% (97.57%-98.89%) | 99.92% (99.88%-99.95%)  |
|  Sample 33 | SUB/Indel | Short Insertion (3-5bp) | 99.30% (98.90%-99.58%) | 99.90% (99.86%-99.94%)  |
|  Sample 34 | CNA/RE/SUB/ Indel | Short Insertion (3-5bp) | 85.42% (82.27%-88.20%) | 99.89% (99.84%-99.93%)  |
|  Sample 35 | RE/SUB/Indel | SUB | 97.75% (96.42%-98.68%) | 99.98% (99.95%-99.99%)  |
|  Sample 36 | RE/SUB/Indel | Short Insertion (>5bp) | 95.30% (92.97%-97.03%) | 99.96% (99.93%-99.98%)  |
|  Sample 37 | CNA/RE/SUB/ Indel | Short Deletion (>5bp) | 100.00% (98.31%-100.00%) | 99.89% (99.84%-99.93%)  |
|  Sample 38 | CNA/RE/SUB/ Indel | Short Insertion (>5bp) | 100.00% (99.25%-100.00%) | 99.96% (99.93%-99.98%)  |
|  Sample 39 | CNA /SUB | Amplification | 96.83% (94.90%-98.17%) | 99.94% (99.90%-99.97%)  |
|  Sample 40 | CNA/RE/SUB/ Indel | RE | 95.97% (94.06%-97.40%) | 99.98% (99.96%-100.00%)  |
|  Sample 41 | CNA/ SUB/Indel | Homozygous Deletion | 100.00% (99.42%-100.00%) | 99.93% (99.89%-99.96%)  |
|  Sample 42 | CNA/RE/SUB/ Indel | Short Insertion (1-2bp with dinucleotide repeats) | 100.00% (99.30%-100.00%) | 99.95% (99.91%-99.97%)  |
|  Sample 43 | RE/SUB | Short Deletion (>5bp) | 100.00% (99.05%-100.00%) | 100.00% (99.98%-100.00%)  |
|  Sample 44 | CNA /SUB | Homozygous Deletion | 96.99% (95.39%-98.15%) | 99.84% (99.79%-99.89%)  |
|  Sample 45 | CNA/RE/SUB/ Indel | Amplification | 100.00% (98.95%-100.00%) | 99.93% (99.89%-99.96%)  |
|  Sample 46 | CNA/RE/SUB/ Indel | RE | 99.80% (99.29%-99.98%) | 99.98% (99.96%-100.00%)  |

*Samples 1-18 were selected to represent alterations associated with CDx claims and Samples 19-46 were selected for platform-wide analysis.
** While all possible alterations harbored in the sample were included in the analysis, each sample was selected based on a specific alteration in accordance with the study protocol.

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Abbreviations: PC - Positive Call, NC - Negative Call, SUB - substitution, Indel - Insertion or Deletion, CNA - Copy Number Alteration, RE - Rearrangement.

For the assessment of MSI,  $100.0\%$  positive call rates and negative call rates for all samples were observed, with the two sided  $95\%$  CIs ranging from  $(88.2\%, 100.0\%)$  to  $(88.5\%, 100.0\%)$  for positive call rates and  $(87.9\%, 100.0\%)$  to  $(88.55\%, 100.0\%)$  for negative call rates. For TMB determination, 46 samples included non-zero TMB scores and were included in the assessment of repeatability and reproducibility. Summary of the performance, including the standard deviation (SD) and coefficient of variation (CV) is shown Table 19, below. Twenty-three samples met the  $\leq 20\%$  CV requirements for this study.

Table 19. TMB precision analysis based on all samples with non-zero TMB scores

|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | Mean TMB Value | # Valid Results | Repeatability |   | Reproducibility |   | Average Median Exon Coverage  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |  SD | %CV | SD | %CV  |   |
|  Sample 1 | SUB | EGFR T790M | 2.1 | 36 | 0.9391 | 44.70% | 1.0447 | 49.80% | 648.7  |
|  Sample 2 | SUB | EGFR T790M | 8.37 | 36 | 2.0437 | 24.40% | 2.1012 | 25.10% | 697.9  |
|  Sample 3 | RE | ALK RE | 1.51 | 36 | 0.5815 | 38.60% | 0.6001 | 39.90% | 592.3  |
|  Sample 4 | RE | ALK RE | 3.36 | 36 | 0.594 | 17.70% | 0.6212 | 18.50% | 712.8  |
|  Sample 5 | RE | ALK RE | 3.12 | 36 | 0.7572 | 24.30% | 0.7608 | 24.40% | 722.3  |
|  Sample 6 | Indel | EGFR Exon 19 Deletion | 5.04 | 35 | 1.2615 | 25.00% | 1.2979 | 25.80% | 388.6  |
|  Sample 7 | Indel | EGFR Exon 19 Deletion | 4.21 | 35 | 0.9544 | 22.70% | 1.0088 | 24.00% | 622  |
|  Sample 8 | Indel | EGFR Exon 19 Deletion | 2.8 | 36 | 0.6641 | 23.70% | 0.6885 | 24.60% | 776.9  |
|  Sample 9 | SUB | KRAS Codons 12/13 SUB | 3.33 | 36 | 0.6062 | 18.20% | 0.7644 | 23.00% | 825.3  |
|  Sample 10 | SUB | KRAS Codons 12/13 SUB | 4.17 | 36 | 1.243 | 29.80% | 1.4987 | 36.00% | 707  |
|  Sample 11 | SUB | BRAF V600E/V600K | 14.91 | 35 | 3.1604 | 21.20% | 3.3605 | 22.50% | 319.2  |
|  Sample 12 | SUB | BRAF V600E/V600K | 23.54 | 36 | 1.8078 | 7.70% | 1.8277 | 7.80% | 706.3  |
|  Sample 13 | SUB | EGFR Exon 21 L858R | 3 | 34 | 1.0863 | 36.20% | 1.1141 | 37.10% | 743.2  |
|  Sample 14 | Amplification | ERBB2 (HER2) Amplification | 4 | 35 | 1.6585 | 41.50% | 1.8719 | 46.80% | 804.5  |
|  Sample 15 | Amplification | ERBB2 (HER2) Amplification | 4.82 | 35 | 0.5479 | 11.40% | 0.6082 | 12.60% | 535.1  |
|  Sample 16 | Amplification | ERBB2 (HER2) Amplification | 7.9 | 34 | 0.554 | 7.00% | 0.593 | 7.50% | 783.3  |
|  Sample 17 | SUB | KRAS Codons | 6.37 | 36 | 1.4682 | 23.00% | 1.5366 | 24.10% | 732.1  |

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|  Sample* | Alteration Type(s) Assessed | Alteration for Selection** | Mean TMB Value | # Valid Results | Repeatability |   | Reproducibility |   | Average Median Exon  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  | 12/13 SUB |  |  |  |  |  |  |   |
|  Sample 18 | SUB | EGFR Exon 21 L858R | 11.2 | 35 | 2.1352 | 19.10% | 2.1561 | 19.20% | 743.5  |
|  Sample 19 | CNA/RE/SUB | Amplification | 6.59 | 35 | 1.0807 | 16.40% | 1.3041 | 19.80% | 708.2  |
|  Sample 20 | CNA/SUB/Indel | Homozygous Deletion | 10.95 | 35 | 0.7975 | 7.30% | 0.936 | 8.50% | 429.2  |
|  Sample 21 | SUB/Indel | Short Deletion (1-2bp with dinucleotide repeats) | 42.91 | 35 | 0.6259 | 1.50% | 0.7054 | 1.60% | 765  |
|  Sample 22 | CNA/SUB/Indel | Short Deletion (3-5bp) | 2.7 | 36 | 0.4696 | 17.40% | 0.4696 | 17.40% | 741  |
|  Sample 23 | SUB/Indel | Short Insertion (1-2bp with homopolymer repeats) | 21.47 | 35 | 0.6681 | 3.10% | 0.7558 | 3.50% | 565.7  |
|  Sample 24 | SUB/Indel | Short Deletion (1-2bp with dinucleotide repeats) | 67.31 | 36 | 1.2633 | 1.90% | 1.7042 | 2.50% | 672.1  |
|  Sample 25 | CNA/SUB/Indel | Short Deletion (1-2bp with homopolymer repeats) | 63.71 | 36 | 1.7275 | 2.70% | 1.9587 | 3.10% | 730.6  |
|  Sample 26 | SUB/Indel | Short Deletion (1-2bp with homopolymer repeats) | 51.83 | 36 | 1.0879 | 2.10% | 1.2238 | 2.40% | 614.7  |
|  Sample 27 | CNA/SUB/Indel | SUB | 12.44 | 36 | 0.5556 | 4.50% | 0.6515 | 5.20% | 672.5  |
|  Sample 28 | RE/SUB/Indel | Short Insertion (1-2bp with homopolymer repeats) | 5.87 | 35 | 0.6259 | 10.70% | 0.6347 | 10.80% | 700.2  |
|  Sample 29 | CNA/SUB | SUB | 1.15 | 34 | 1.7122 | 149.00% | 1.8914 | 164.60% | 740.3  |
|  Sample 30 | CNA/RE/SUB/Indel | RE | 3.67 | 35 | 1.2768 | 34.80% | 1.5155 | 41.30% | 732.4  |
|  Sample 31 | RE/SUB/Indel | Short Insertion (1-2bp with dinucleotide repeats) | 1.26 | 36 | 0 | 0.00% | 0 | 0.00% | 732.3  |
|  Sample 32 | CNA/SUB/Indel | Short Deletion (3-5bp) | 6.83 | 36 | 2.4023 | 35.20% | 2.4413 | 35.80% | 689.7  |
|  Sample 33 | SUB/Indel | Short Insertion (3-5bp) | 48.23 | 36 | 1.1046 | 2.30% | 1.2945 | 2.70% | 824.1  |
|  Sample 34 | CNA/RE/SUB | Short Insertion | 5.18 | 36 | 0.5158 | 10.00% | 0.5668 | 10.90% | 574  |

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|  Sample* | Alteration Type(s) Assessed /Indel | Alteration for Selection** (3-5bp) | Mean TMB Value | # Valid Results | Repeatability |   | Reproducibility |   | Average Median Exon  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Sample 35 | RE/SUB/Indel | SUB | 4.21 | 35 | 0.6259 | 14.90% | 0.641 | 15.20% | 765.9  |
|  Sample 36 | RE/SUB/Indel | Short Insertion (>5bp) | 1.82 | 36 | 0.7857 | 43.20% | 0.8201 | 45.10% | 815.7  |
|  Sample 37 | CNA/RE/SUB /Indel | Short Deletion (>5bp) | 8.09 | 36 | 1.0665 | 13.20% | 1.2517 | 15.50% | 791.4  |
|  Sample 38 | CNA/RE/SUB /Indel | Short Insertion (>5bp) | 2.2 | 35 | 0.9301 | 42.40% | 0.9586 | 43.70% | 746.5  |
|  Sample 39 | CNA /SUB | Amplification | 3.67 | 35 | 0.9463 | 25.80% | 1.0407 | 28.30% | 704.5  |
|  Sample 40 | CNA/RE/SUB /Indel | RE | 12.65 | 35 | 0.4732 | 3.70% | 0.5019 | 4.00% | 645.9  |
|  Sample 41 | CNA/ SUB/Indel | Homozygous Deletion | 8.22 | 35 | 0.8349 | 10.20% | 0.8505 | 10.40% | 761.3  |
|  Sample 42 | CNA/RE/SUB /Indel | Short Insertion (1-2bp with dinucleotide repeats) | 10.16 | 36 | 0.2425 | 2.40% | 0.297 | 2.90% | 714.8  |
|  Sample 43 | RE/SUB | Short Deletion (>5bp) | 2.24 | 36 | 1.3058 | 58.30% | 1.4511 | 64.80% | 685.3  |
|  Sample 44 | CNA /SUB | Homozygous Deletion | 7.28 | 35 | 1.5508 | 21.30% | 1.6205 | 22.30% | 769.3  |
|  Sample 45 | CNA/RE/SUB /Indel | Amplification | 6.23 | 36 | 1.3755 | 22.10% | 1.3755 | 22.10% | 687.2  |
|  Sample 46 | CNA/RE/SUB /Indel | RE | 4.18 | 35 | 1.6427 | 39.30% | 1.7542 | 42.00% | 478.5  |

*Samples 1-18 were selected to represent alterations associated with CDx claims and Samples 19-46 were selected for platform-wide analysis.
** While all possible alterations harbored in the sample were included in the analysis, each sample was selected based on a specific alteration in accordance with the study protocol.

Abbreviations: SD - Standard Deviation, CV - Coefficient of Variation, SUB - substitution, Indel - Insertion or Deletion, CNA - Copy Number Alteration, RE - Rearrangement.

# a. Reagent Lot-to-Lot Reproducibility

Three lots of critical reagents were assessed for four replicates in a full factorial design. Reagents were evaluated as internally prepared kits for each process step (LC, HC, sequencing). The variant level pairwise APA/ANAs among three reagent lots and the corresponding  $95\%$  confidence interval were calculated. The use of three different lots of reagents did not impact performance as 27 of 28 samples  $(96.4\%)$  had pairwise agreement estimates (APA and ANA) above  $95\%$ . One sample had APA estimates below  $90\%$ $(85.9\%$  to  $88.7\%)$ . The putative source of variability was determined to be non-focal copy number amplifications with low copy numbers close to the calling threshold; no specific reagent lot performed differently among three lots for this sample.

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b. Instrument-to-Instrument Reproducibility

Four replicates for each sample were sequenced on each of three Illumina HiSeq4000 sequencers, in a full factorial design. The variant level pairwise APA/ANAs among three sequencers and the corresponding 95% CI per sample were calculated. The use of three different sequencers did not impact performance as 27 of 28 samples (96.4%) had pairwise agreement estimates (APA and ANA) of at least 97%. One sample had APA estimates below 90% (86.6% to 89.2%). The putative source of variability was determined to be non-focal copy number amplifications with low copy numbers close to the calling threshold; no specific sequencer performed differently among three lots for this sample.

6. Reagent Lot Interchangeability

Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDx_BRCA assay and F1CDx. For reagent lot interchangeability performance data, see the Summary of Safety and Effectiveness for P160018.

7. Stability

a. Reagent Stability

Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus CDx_BRCA Assay and F1CDx. For reagent stability performance data, see the Summary of Safety and Effectiveness Data for P160018. The claimed reagent stability is 4 months for the LC and HC kits, and 3 months for the sequencing kits.

b. DNA Stability

Stability of DNA was evaluated through a retrospective review of data generated using the F1 LDT assay. Samples from 47 unique clinical specimens from 21 different tissues of origin were evaluated. The sample set covered 200 alterations inclusive of nucleotide changes, indels, CNAs (amplifications and losses) and rearrangements. Duration of DNA storage at time of testing ranged from 48 to 464 days, with a median of 184 days and a mean of 199 days. For this study, 199 of 200 alteration calls were concordant. A 242-day old sample with a single alteration call (FGF10 amplification) that met inclusion criteria was discordant; however, this sample was classified as not meeting all QC criteria due to other data quality issues. Sixteen other samples had concordant calls with DNA age &gt; 242 days. Based on this data, DNA stored in accordance with internal procedures can be considered stable for up to six months. An additional prospective DNA stability study is underway.

c. FFPE Slide Stability

The FFPE Slide Stability Study is an ongoing study with data summarized for T₀, T₁ (30 days) and T₂ (6 months) time points. This study evaluated the stability of FFPE tumor tissue prepared as slides prior to DNA extraction for use with F1CDx. The study evaluated five tumor samples including ovarian, lung, colorectal cancer, melanoma and breast cancer that contained a variety of DNA alterations, as described in Table 20, below. The five samples were selected to include specific alteration types

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that were reflective of the CDx-alterations (shown in Table 21); but were found to contain additional alterations as well (13 CNAs, one rearrangement, 53 base substitutions and five indels). To assess stability of pre-cut FFPE tissue for genomic alterations, the agreement between results from the defined time points for each sample were calculated by comparing the alteration call reported at each follow-up time point to the alteration call at baseline  $(\mathrm{T_0})$ . Alterations at the 30-day time point and the 6-month time point were in  $100\%$  agreement with the Day 0 baseline results  $(\mathrm{T_0})$ . Therefore, FFPE slides stored in accordance with internal procedures can be considered stable for at least 6 months. Further assessment at Months 12 and 15 will demonstrate stability of FFPE slides beyond 6 months.

Table 20. Stability Results at baseline, 30 days and 6 months

|  Tissue | Baseline Call (T0) |   | Percent Agreement to T0 | Percent Agreement to T0  |
| --- | --- | --- | --- | --- |
|   |  Gene | Variant Effect | 30 days (T1) | 6 months (T2)  |
|  Ovarian | BRCA1 | c.1340_1341insG, p.H448fs*8 | 100.0% (2/2) | 100.0% (2/2)  |
|  Lung | KRAS | c.34G>T, p.G12C | 100.0% (2/2) | 100.0% (2/2)  |
|  CRC | PIK3CA | c.3139C>T, p.H1047Y | 100.0% (2/2) | 100.0% (2/2)  |
|  Melanoma | CDKN2A | Homozygous Deletion | 100.0% (2/2) | 100.0% (2/2)  |
|  Melanoma | CDKN2B | Homozygous Deletion | 100.0% (2/2) | 100.0% (2/2)  |
|  Breast | ERBB2 | Amplification | 100.0% (1/1) | 100.0% (2/2)  |

Table 21. Percent agreement for each variant type

|  Variant type | # of variants | 30 days (T1) Percent Agreement (# agreement/total) | 95% 2-sided CI LB, UB | 6 months (T2) Percent Agreement (# agreement/total) | 95% 2-sided CI LB, UB  |
| --- | --- | --- | --- | --- | --- |
|  Copy Number | 13 | 100.0% (23/23) | 85.2%, 100.0% | 100.0% (26/26) | 86.8%, 100.0%  |
|  Rearrangement | 1 | 100.0% (2/2) | 15.8%, 100.0% | 100.0% (2/2) | 15.8%, 100.0%  |
|  Substitution | 53 | 100.0% (98/98) | 96.3%, 100.0% | 100.0% (106/106) | 96.6%, 100.0%  |
|  Ins/Dels | 5 | 100.0% (7/7) | 59.0%, 100.0% | 100.0% (10/10) | 69.2%, 100.0%  |

Abbreviations: LB - Lower Bound, CV - Upper Bound.

# 8. General Lab Equipment and Reagent Evaluation

# a. DNA Amplification

Identical reagents with the same specifications are used following the same protocols for both the FoundationFocus  $\mathrm{CDx}_{BRCA}$  assay and F1CDx. For DNA amplification performance data, see the Summary of Safety and Effectiveness Data for P160018.

# b. DNA Extraction

The performance of DNA extraction from FFPE tumor specimens was evaluated. The DNA extraction procedure for the F1CDx assay was assessed by testing FFPE specimens including two samples per tissue type for ten different tissue types including lung, breast, ovarian, melanoma, colorectal, brain, hepatic, pancreatic, thyroid, and bladder with different representative types of alterations. Samples were

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run in duplicate for a total of 240 extractions, employing two different KingFisher Flex Magnetic Particle Processors (120 extractions per processor) and comparing across three extraction reagent lots (80 extractions per reagent lot). Average DNA yield was calculated across 12 replicates for each sample. All average DNA yields were significantly above the minimum requirement of 55 ng, with the minimum being 758.3 ng. Only one sample aliquot out of 240 replicates failed the DNA yield specification, and the success rates based on the reagent lot and the equipment were 98.8% (79/80) and 99.2% (119/120), respectively, passing the acceptance criteria (≥ 90%). Concordance of all genomic alterations detected was also analyzed for all variant types across 12 replicates for each sample. Table 22 provides a summary of concordance across replicates. A study with and additional ten samples will be completed post-market.

Table 22. Summary of Concordance Across Replicates of DNA Extraction Study

|  Group | N_{concordance} | N_{total} | Concordance (95% CI)  |
| --- | --- | --- | --- |
|  Substitutions (All MAF) | 2700 | 2969 | 90.9% (89.9% 91.9%)  |
|  Substitutions (MAF > 10%) | 1631 | 1637 | 99.6% (99.2% 99.9%)  |
|  Substitutions (All MAF, excluding hypermutated sample)* | 1663 | 1685 | 98.7% (98% 99.1%)  |
|  Indel (All) | 465 | 476 | 97.7% (95.9% 98.8%)  |
|  Copy Number: Amplification | 307 | 314 | 97.8% (95.4% 99%)  |
|  Copy Number: Loss | 132 | 144 | 91.7% (85.9% 95.3%)  |
|  Rearrangement | 84 | 90 | 93.3% (85.9% 97.2%)  |

*One sample included in the study was hypermutated, harboring many alterations near LoD and exhibited evidence of external contamination. Concordance of substitutions was 80.8% for this sample.

## 9. Guard banding/Robustness

Guard banding studies were completed to evaluate the performance of the F1CDx assay and the impact of process variation with regard to uncertainty in the measurement of DNA concentration at various stages of the process. Guard bands were evaluated relative to observed and measured process variability for Library Construction (LC), Hybrid Capture (HC), and Sequencing. Each of the three guard banding experiments demon…

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**Source:** [https://fda.innolitics.com/device/P170019](https://fda.innolitics.com/device/P170019)

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