FoundationOne Liquid CDx (F1 Liquid CDx)

P190032 · Foundation Medicine, Inc. · PQP · Aug 26, 2020 · Pathology

Device Facts

• Record ID: P190032
• Device Name: FoundationOne Liquid CDx (F1 Liquid CDx)
• Applicant: Foundation Medicine, Inc.
• Product Code: PQP · Pathology
• Decision Date: Aug 26, 2020
• Decision: APPR
• Device Class: Class 3

Intended Use

FoundationOne® Liquid CDx is a qualitative next generation sequencing based in vitro diagnostic test that uses targeted high throughput hybridization-based capture technology to detect and report substitutions, insertions and deletions (indels) in 311 genes, including rearrangements and copy number losses only in BRCA1 and BRCA2. FoundationOne® Liquid CDx utilizes circulating cell-free DNA (cfDNA) isolated from plasma derived from anti-coagulated peripheral whole blood of cancer patients collected in FoundationOne® Liquid CDx cfDNA blood collection tubes included in the FoundationOne® Liquid CDx Blood Sample Collection Kit. The test is intended to be used 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, FoundationOne® Liquid CDx is intended to provide tumor mutation profiling for substitutions and indels to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms.

Device Story

FoundationOne Liquid CDx is a laboratory-service NGS assay for solid malignant neoplasms. It processes cfDNA isolated from plasma of anti-coagulated peripheral whole blood. The workflow involves cfDNA extraction, whole-genome shotgun library construction, and hybridization-based capture of 324 cancer-related genes. Libraries are sequenced on the Illumina NovaSeq 6000. Proprietary software analyzes sequence data to detect genomic alterations (substitutions, indels, BRCA1/2 rearrangements/copy number losses). Results are annotated and merged with patient data for clinical review. The test identifies patients eligible for specific targeted therapies (e.g., EGFR inhibitors for NSCLC, rucaparib for prostate cancer) and provides tumor mutation profiling. It is performed at a single-site laboratory (Foundation Medicine, Inc.). Healthcare professionals use the output to guide treatment decisions, potentially benefiting patients by matching them to appropriate targeted therapies.

Clinical Evidence

Clinical validation included a bridging study for BRCA1/BRCA2 in mCRPC (TRITON2 trial, N=209) and a non-inferiority study for EGFR alterations in NSCLC (N=280). BRCA1/2 concordance between F1 Liquid CDx and CTA showed PPA 82.4% and NPA 98.6% (≥30ng input). ORR in BRCA+ patients was 46.3% (F1 Liquid CDx) vs 43.5% (CTA). EGFR non-inferiority study demonstrated F1 Liquid CDx is non-inferior to the cobas EGFR Mutation Test v2. Analytical studies (LoD, LoB, precision, interference, stability) supported performance across 324 genes.

Technological Characteristics

NGS-based in vitro diagnostic; utilizes hybridization-based capture of 324 cancer-related genes. Sequencing performed on Illumina NovaSeq 6000. Materials include FoundationOne Liquid CDx cfDNA Blood Collection Tubes. Workflow: cfDNA extraction (KingFisher Flex), library construction (NEBNext, Bravo Benchbot), hybrid capture (biotinylated DNA oligonucleotides), and sequencing by synthesis. Software: proprietary analysis pipeline (BWA, Samtools, Picard, Biopython).

Indications for Use

Indicated for cancer patients with solid malignant neoplasms. Used as a companion diagnostic to identify patients eligible for targeted therapies (NSCLC patients for EGFR Exon 19 deletions/L858R; prostate cancer patients for BRCA1/BRCA2 alterations). Provides tumor mutation profiling for substitutions and indels.

Regulatory Classification

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

• Roche cobas® EGFR Mutation Test v2
• FoundationACT (FACT)
• FoundationOne® Liquid LDT

Submission Summary (Full Text)

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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® Liquid 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: P190032

Date of FDA Notice of Approval: 8/26/2020

Breakthrough Device: Granted breakthrough device status (formerly known as the Expedited Access Pathway, or EAP) on April 25, 2018 because (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® Liquid CDx is a qualitative next generation sequencing based *in vitro* diagnostic test that uses targeted high throughput hybridization-based capture technology to detect and report substitutions, insertions and deletions (indels) in 311 genes, including rearrangements and copy number losses only in BRCA1 and BRCA2. FoundationOne® Liquid CDx utilizes circulating cell-free DNA (cfDNA) isolated from plasma derived from anti-coagulated peripheral whole blood of cancer patients collected in FoundationOne® Liquid CDx cfDNA blood collection tubes included in the FoundationOne® Liquid CDx Blood Sample Collection Kit. The test is intended to be used 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, FoundationOne® Liquid CDx is intended to provide tumor mutation profiling for substitutions and indels to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms.

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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 alteration | IRESSA® (gefitinib)
TAGRISSO® (osimertinib)
TARCEVA® (erlotinib) |
| Prostate cancer | BRCA1, BRCA2 alterations | RUBRACA® (rucaparib) |

A negative result from a plasma specimen does not mean that the patient’s tumor is negative for genomic findings. Patients who are negative for the mutations listed in Table 1 should be reflexed to routine biopsy and their tumor mutation status confirmed using an FDA-approved tumor tissue test, if feasible.

Genomic findings other than those listed in Table 1 of the intended use statement are not prescriptive or conclusive for labeled use of any specific therapeutic product.

FoundationOne® Liquid CDx is a single-site assay performed at Foundation Medicine, Inc. in Cambridge, MA.

## III. CONTRAINDICATIONS

There are no known contraindications.

## IV. WARNINGS AND PRECAUTIONS

- Alterations reported may include somatic (not inherited) or germline (inherited) alterations; however, the test does not distinguish between germline and somatic alterations. If a reported alteration is suspected to be germline, confirmatory testing should be considered in the appropriate clinical context.
- The test is not intended to replace germline testing or to provide information about cancer predisposition.
- Patients for whom no companion diagnostic alterations are detected should be considered for confirmation with an FDA-approve tumor tissue test, if possible.

## V. DEVICE DESCRIPTION

The FoundationOne Liquid CDx assay is performed exclusively as a laboratory service using circulating cell-free DNA (cfDNA) isolated from plasma derived from anti-coagulated peripheral whole blood from patients with solid malignant neoplasms. The assay employs a single DNA extraction method to obtain cfDNA from plasma from whole blood. Extracted cfDNA undergoes whole-genome shotgun library construction and hybridization-based capture of 324 cancer-related genes. All coding exons of 309 genes are targeted; select intronic or non-coding regions are targeted in BRCA1 and BRCA2 (refer to Table 2 for the complete list of genes reported by FoundationOne Liquid CDx). Hybrid-capture selected libraries are sequenced with deep coverage using the NovaSeq® 6000 platform. Sequence data are processed using a custom analysis pipeline designed to detect genomic alterations, including base substitutions and indels in 311

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genes, and copy number variants and genomic rearrangements in BRCA1 and BRCA2. A subset of targeted regions in 75 genes is baited for increased sensitivity.

Table 2: Genomic Regions in which Variants are Reported by FoundationOne Liquid CDx¹

| ABL1
[Exons 4-9] | CALR | CYP17A1 | FGFR4 | KDM6A | MYCL
(MYCL1) | POLD1 | SMAD4 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| ACVR1B | CARD11 | DAXX | FH | KDR | MYCN | POLE | SMARCA4 |
| AKT1
[Exon 3] | CASP8 | DDR1 | FLCN | KEAP1 | MYD88
[Exon 4] | PPARG | SMARCB1 |
| AKT2 | CBFB | DDR2
[Exons 5, 17, 18] | FLT1 | KEL | NBN | PPP2R1A | SMO |
| AKT3 | CBL | DIS3 | FLT3
[Exons 14, 15, 20] | KIT
[Exons 8, 9, 11, 12, 13, 17] | NF1 | PPP2R2A | SNCAIP |
| ALK
[Exons 20-29] | CCND1 | DNMT3A | FOXL2 | KLHL6 | NF2 | PRDM1 | SOCS1 |
| ALOX12B | CCND2 | DOT1L | FUBP1 | KMT2A
(MLL) | NFE2L2 | PRKAR1A | SOX2 |
| AMER1
(FAM123B) | CCND3 | EED | GABRA6 | KMT2D
(MLL2) | NFKBIA | PRKCI | SOX9 |
| APC | CCNE1 | EGFR | GATA3 | KRAS | NKX2-1 | PTCH1 | SPEN |
| AR | CD22 | EP300 | GATA4 | LTK | NOTCH1 | PTEN | SPOP |
| ARAF
[Exons 4, 5, 7, 11, 13, 15, 16] | CD274
(PD-L1) | EPHA3 | GATA6 | LYN | NOTCH2 | PTPN11 | SRC |
| ARFRP1 | CD70 | EPHB1 | GNA11
[Exons 4, 5] | MAF | NOTCH3 | PTPRO | STAG2 |
| ARID1A | CD79A | EPHB4 | GNA13 | MAP2K1
(MEK1)
[Exons 2, 3] | NPM1
[Exons 4-6, 8, 10] | QKI | STAT3 |
| ASXL1 | CD79B | ERBB2 | GNAQ
[Exons 4, 5] | MAP2K2
(MEK2)
[Exons 2-4, 6, 7] | NRAS
[Exons 2, 3] | RAC1 | STK11 |
| ATM | CDC73 | ERBB3
[Exons 3, 6, 7, 8, 10, 12, 20, 21, 23, 24, 25] | GNAS
[Exons 1, 8] | MAP2K4 | NSD3
(WHSC1L1) | RAD21 | SUFU |
| ATR | CDH1 | ERBB4 | GRM3 | MAP3K1 | NT5C2 | RAD51 | SYK |
| ATRX | CDK12 | ERCC4 | GSK3B | MAP3K13 | NTRK1
[Exons 14, 15] | RAD51B | TBX3 |
| AURKA | CDK4 | ERG | H3F3A | MAPK1 | NTRK2 | RAD51C | TEK |
| AURKB | CDK6 | ERRF11 | HDAC1 | MCL1 | NTRK3
[Exons 16, 17] | RAD51D | TERC*
{ncRNA} |
| AXIN1 | CDK8 | ESR1
[Exons 4-8] | HGF | MDM2 | P2RY8 | RAD52 | TERT*
{Promoter} |
| AXL | CDKN1A | EZH2
[Exons 4, 16, 17, 18] | HNF1A | MDM4 | PALB2 | RAD54L | TET2 |
| BAP1 | CDKN1B | FAM46C | HRAS
[Exons 2, 3] | MED12 | PARK2 | RAF1 | TGFBR2 |

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| | | | | | | [Exons 3, 4, 6, 7, 10, 14, 15, 17] | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| BARD1 | CDKN2A | FANCA | HSD3B1 | MEF2B | PARP1 | RARA | TIPARP |
| BCL2 | CDKN2B | FANCC | ID3 | MEN1 | PARP2 | RB1 | TNFAIP3 |
| BCL2L1 | CDKN2C | FANCG | IDH1
[Exon 4] | MERTK | PARP3 | RBM10 | TNFRSF14 |
| BCL2L2 | CEBPA | FANCL | IDH2
[Exon 4] | MET | PAX5 | REL | TP53 |
| BCL6 | CHEK1 | FAS | IGF1R | MITF | PBRM1 | RET
[Exons 11, 13-16] | TSC1 |
| BCOR | CHEK2 | FBXW7 | IKBKE | MKNK1 | PDCD1
(PD-1) | RICTOR | TSC2 |
| BCORL1 | CIC | FGF10 | IKZF1 | MLH1 | PDCD1LG2
(PD-L2) | RNF43 | TYRO3 |
| BRAF
[Exons 11-18] | CREBBP | FGF12 | INPP4B | MPL
[Exon 10] | PDGFRA
[Exons 12, 18] | ROS1
[Exons 31, 36-38, 40] | U2AF1 |
| BRCA1 {Introns 2, 7, 8, 12, 16, 19, 20} | CRKL | FGF14 | IRF2 | MRE11A | PDGFRB
[Exons 12-21, 23] | RPTOR | VEGFA |
| BRCA2 {Intron 2} | CSF1R | FGF19 | IRF4 | MSH2 | PDK1 | SDHA | VHL |
| BRD4 | CSF3R | FGF23 | IRS2 | MSH3 | PIK3C2B | SDHB | WHSC1 |
| BRIP1 | CTCF | FGF3 | JAK1 | MSH6 | PIK3C2G | SDHC | WT1 |
| BTG1 | CTNNA1 | FGF4 | JAK2
[Exons 14] | MST1R | PIK3CA
[Exons 2, 3, 5-8, 10, 14, 19, 21] (Coding Exons 1, 2, 4-7, 9, 13, 18, 20) | SDHD | XPO1 |
| BTG2 | CTNNB1
[Exon 3] | FGF6 | JAK3
[Exons 5, 11, 12, 13, 15, 16] | MTAP | PIK3CB | SETD2 | XRCC2 |
| BTK
[Exons 2, 15] | CUL3 | FGFR1 | JUN | MTOR
[Exons 19, 30, 39, 40, 43-45, 47, 48, 53, 56] | PIK3R1 | SF3B1 | ZNF217 |
| C11orf30 (EMSY) | CUL4A | FGFR2 | KDM5A | MUTYH | PIM1 | SGK1 | ZNF703 |
| C17orf39 (GID4) | CXCR4 | FGFR3
[Exons 7, 9 (alternative designation exon 10), 14, 18] | KDM5C | MYC | PMS2 | SMAD2 | |

¹ As part of its FDA-approved intended use, the FoundationOne Liquid CDx assay interrogates 311 genes, including 309 genes with complete exonic (coding) coverage and 2 genes with only select non-coding coverage (indicated with an *). Select genes and select exons (indicated in bold) are captured with increased sensitivity.

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The output of the test includes:

Category 1: Companion Diagnostic (CDx) claims noted in Table 1 of the Intended Use

Category 2: ctDNA Biomarkers with Strong Evidence of Clinical Significance in ctDNA

Category 3: Biomarkers with Evidence of Clinical Significance in tissue supported by:

3A: strong analytical validation using ctDNA
3B: analytical validation using ctDNA

Category 4: Other Biomarkers with Potential Clinical Significance

## FoundationOne Liquid cfDNA CDx Blood Specimen Collection Kit Contents

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

- Specimen preparation and shipping instructions
- Two FoundationOne Liquid CDx cfDNA Blood Collection Tubes (8.5 mL nominal fill volume per tube)
- Return shipping label

## Instruments

The FoundationOne Liquid CDx assay is intended to be performed with the serial number-controlled instruments indicated in Table 3, below. All instruments are qualified by Foundation Medicine, Inc. (Foundation Medicine) under Foundation Medicine's Quality System.

Table 3: Instruments for use with the FoundationOne Liquid CDx assay

| Instrument |
| --- |
| Illumina NovaSeq 6000 |
| Beckman Biomek NXP Span-8 Liquid Handler |
| Thermo Scientific Kingfisher Flex DW 96 |
| Bravo Benchbot |
| Hamilton STARlet STAR Liquid Handling Workstation |

## Test Process

All assay reagents included in the FoundationOne Liquid CDx assay process are qualified by Foundation Medicine and are compliant with the medical device Quality System Regulation (QSR).

## A. Specimen Collection and Preparation

Whole blood specimens are collected in FoundationOne Liquid CDx cfDNA Blood Collection Tubes (BCT) provided as a component of the FoundationOne Liquid CDx specimen collection kit. Prior to cfDNA isolation, the plasma is separated from whole blood by centrifugation, which separates the plasma from the buffy coat (white blood cells) and red blood cells. The plasma layer is removed from the buffy coat to avoid contamination of cellular DNA into the plasma sample. A residual volume of plasma

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remains in the tube to avoid disturbing the buffy coat. A second spin of the separated plasma at high speed further pellets cell debris and protein.

## B. DNA Extraction

Following the separation of plasma from whole blood, cfDNA is isolated from plasma using the KingFisher™ Flex Magnetic Particle Processor, which uses an efficient and automated method to purify cfDNA. The KingFisher™ Instrument uses magnetic rods to move nucleic acid through purification phases of binding, washing, and elution to yield high purity cfDNA. After isolating cfDNA, the Agilent 4200 TapeStation is used to quantify cfDNA.

## C. Library Construction

Library Construction (LC) begins with the normalization of cfDNA. The samples are purified, using AMPure® XP Beads (Agencourt®). Solid-phase reversible immobilization (SPRI) purification is used subsequent to library construction with the NEBNext® kits (NEB), including mixes for end repair with blunt-end and 5'-phosphorylate the cfDNA fragments using T4 Polynucleotide Kinase and T4 DNA Polymerase. This step prepares the 3'-end for dA-addition while also preparing the 5'-end of the DNA fragment for ligation. Second, dA-addition will incorporate a single dAMP to the 3'-end of the End-Repaired material. After dA-addition, a universal Y-adaptor is ligated onto each end of the DNA fragment using a DNA ligase. These steps are performed in 96-well plates (Eppendorf) on a Bravo Benchbot (Agilent) using the "with-bead" protocol to maximize reproducibility and library yield. Indexed (Foundation Medicine customized six base pair barcodes) sequencing libraries are PCR amplified with a high-fidelity DNA polymerase (HiFi™, Kapa) for ten cycles, SPRI purified and quantified by PicoGreen® fluorescence assay (Invitrogen). Process matched control (PMC) is prepared and added to the plate with other cfDNA samples at the beginning of LC.

## D. Hybrid Capture

Hybrid Capture begins with the normalization of each library to 500 ng to 2000 ng. Solution hybridization is performed using a >50-fold molar excess of a pool of individually synthesized 5'-biotinylated DNA 120 base pair oligonucleotides (Integrated DNA Technology) for baits. The baits target regions from 324 cancer-related genes including all coding exons of 309 genes and only select introns or non-coding regions in 15 genes. Baits were designed by appointing 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; single nucleotide polymorphism (SNP) targets were allocated one bait each. Intronic baits were filtered for repetitive elements as defined by the University of California at Santa Cruz (UCSC) Genome Repeat Masker track. Hybrid selection of targets demonstrating reproducibly low coverage was boosted by increasing the number of baits for these targets.

Upon completion of the pre-capture normalization, blocking DNA (adaptor block, Cot, Salmon Sperm DNA) is added to the sequencing library and the mixture is lyophilized in a 96-well plate. The library is then re-suspended in nuclease-free water,

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heat denatured at 95°C for 5 minutes, temperature ramps from 95°C to 68°C to anneal blocking DNA, and then the samples are incubated at 68°C for a minimum of 5 minutes before the addition of the baitset reagent. After a 20-24-hour incubation, the library-bait duplexes are captured on paramagnetic MyOne™ streptavidin beads (Invitrogen) and off-target library is removed by washing one time with Saline Sodium Citrate (SSC) at 25°C and four times with SSC at 55°C. The PCR master mix is added to directly amplify the captured library from the washed beads. After amplification, the samples are SPRI purified and quantified by PicoGreen.

## E. Sequencing

Sequencing on the Illumina NovaSeq 6000 platform employs on-board cluster generation (OBCG) using patterned FC technology to generate monoclonal clusters via ExAmp from a single DNA template. The clusters are then sequenced using sequencing by synthesis (SBS) chemistry. The NovaSeq system is capable of sequencing up to two flowcells at a time. During OBCG, a single DNA template is introduced into each of the primer substrate layered nanowells of the flowcell, where the template is immediately and rapidly amplified by ExAmp. This rapid amplification prevents other DNA templates from binding, ensuring a monoclonal cluster is formed in each nanowell. The procedure allows for fixed size and spacing of the clusters which results in improved and more accurate resolution.

A growing nucleotide chain is created on the flowcell by incorporating fluorescently labeled, 3'-blocked dNTPs. After excitation by a laser, the camera captures the emission color of the incorporated, fluorescently labeled nucleotide. The 3'-block is then removed, reverting the nucleotide to its natural form, which allows the polymerase to add another base to the growing double strand of DNA. With each successive SBS cycle, a new fluorescently labeled 3'-blocked dNTP is added. SBS allows for millions of discrete clusters of clonal copies of DNA to be sequenced in parallel.

## F. Sequence Analysis

Sequence data is analyzed using mainly proprietary software developed by Foundation Medicine. External tools used include: 1) BWA (Burrows-Wheeler Aligner) v0.7.17, for aligning sequence reads to the genomic reference, 2) Samtools v1.6 for utility operations, 3) Picard tools v1.56 for metrics calculations, and 4) Biopython for the pairwise2 sequence alignment module.

Reads from each Illumina flowcell are demultiplexed (sorted into sets of reads deriving from distinct samples), and their fragment barcodes (FBCs) are extracted and encoded into the read names. For each sample, read pairs with matching, valid FBCs are aligned and processed together to: 1) identify clusters of reads originating from the same original fragment, 2) merge overlapping read pairs into single reads, where possible, and 3) generate consensus reads representing all information in the set of reads for each cluster, encoding positions with mismatches (errors) with base quality 20. The consensus reads are then aligned to the reference genome to generate the 'consensus' BAM.

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For the detection of short variants (e.g., substitutions and small indels) in each target region of interest, a de novo assembly is performed. This is done using proprietary software to generate a de Bruijn graph including all k-mers in reads mapping to a particular locus. The graph is parsed to identify paths that originate and terminate in reference nodes from the locus. Increased k-mer sizes may be used to account for ambiguities, cycles, and other problematic regions within the graph. The result of the graph traversal is a set of candidate variants. For each variant, there is a set of k-mers supporting the variant and a set of k-mers that would support the reference or another variant at the location.

Each candidate variant is then scanned against reads in the locus to identify which reads support either the candidate variant or a different variant or reference at the location. The cluster membership of the supporting reads is then assessed to determine which clusters show unambiguous support for the variant and which have conflicting assignments, indicating that the variant may have arisen as an error in sequencing or library preparation. The final variant calls are made based on a model that takes into account the coverage at the location, the number of supporting read clusters and their redundancy level, and the number of error-containing clusters.

## 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 Foundation Medicine as a professional service prior to approval and release by the laboratory director or designee.

## H. Internal Process Controls

### Positive Control

Each assay run includes a control sample run in duplicate. The control sample contains a pool of eleven HapMap cell lines and is used as a positive mutation detection control. 100 different germline SNPs present across the entire targeted region are required to be detected by the analysis pipeline.

### Sensitivity Control

The HapMap control pool used as the positive control is prepared to contain variants at 0.1%, 10% mutant allele frequency (MAF) which must be detected by the analysis pipeline to ensure expected sensitivity for each run.

### Negative Control

Samples are barcoded molecularly at the library construction (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.

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# I. CDx Classification Criteria

1. BRCA1, BRCA2 alterations to identify patients eligible for rucaparib in prostate cancer:

The CDx classification criteria and the list of BRCA1/BRCA2 missense mutations for rucaparib, based on the trial prespecifications are described in Table 4 and Table 5; however, not all of the missense mutations listed below were observed in the TRITON2 clinical study.

Table 4: Classification Criteria for Deleterious Tumor BRCA Variants

| Qualification Criteria | Sequence Classification | Methodology |
| --- | --- | --- |
| A BRCA1 or BRCA2 alteration that includes any of the sequence classifications | Protein truncating mutations | Sequence analysis identifies premature stop codons anywhere in the gene coding region, except: 3’ of and including BRCA2 K3326* |
| | Splice site mutations | Sequence analysis identifies variant splice sequences at intron/exon junctions -/+ 2bp of exon starts/ends |
| | Homozygous deletions | Sequence analysis identifies deletions in both gene alleles of ≥1 exon in size |
| | Large protein truncating rearrangements | Sequence analysis identifies protein truncating rearrangements |
| | Deleterious missense mutations | Curated list (Table 5) |

Table 5: Deleterious BRCA Missense Alterations

| BRCA1 Alterations (Protein Change) | | | | | BRCA2 Alterations (Protein Change) | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| M1V | C44Y | R71T | R1699W | G1770V | M1V | R2336P | T2722R |
| M1T | C44F | R71M | R1699Q | M1775K | M1T | R2336L | D2723H |
| M1R | C47S | S770L | G1706R | M1775R | M1R | R2336H | D2723G |
| M1I | C47Y | R1495T | G1706E | C1787S | M1I | T2412I | G2724W |
| M18T | C47F | R1495M | A1708E | G1788V | D23N | R2602T | G2748D |
| L22S | C61S | R1495K | S1715R | P1812A | D23Y | W2626C | A2911E |
| I26N | C61G | E1559K | S1722F | A1823T | S142N | I2627F | E3002K |
| T37K | C61Y | E1559Q | V1736A | V1833M | S142I | R2659T | R3052W |
| C39R | C64R | T1685A | G1738R | W1837R | V159M | R2659K | D3095G |
| C39G | C64G | T1685I | G1738E | V1838E | V211I | E2663V | D3095E |
| C39Y | C64Y | D1692N | K1759N | | V211L | S2670L | N3124I |
| C39W | C64W | M1689R | L1764P | | Y600C | I2675V | N3187K |
| H41R | R71G | D1692H | I1766N | | K1530N | T2722K | |
| C44S | R71K | D1692Y | I1766S | | | | |

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VI. ALTERNATIVE PRACTICES AND PROCEDURES

The Roche cobas® EGFR Mutation Test v2 from Roche Molecular Systems, Inc. is an FDA-approved companion diagnostic intended for the detection of EGFR Exon 19 deletions (Exon 19del) and L858R substitution in plasma obtained from patients with advanced and metastatic NSCLC for treatment with TARCEVA® (erlotinib), TAGRISSO® (osimertinib), and IRESSA® (gefitinib).

There are no FDA-approved CDx alternatives for the detection of genomic alterations of BRCA1 or BRCA2 for the identification of prostate cancer patients eligible for treatment with RUBRACA® (rucaparib).

VII. MARKETING HISTORY

Foundation Medicine designed and developed FoundationOne® Liquid CDx based on previous versions of the assay, including the FoundationACT (FACT) and FoundationOne® Liquid laboratory developed test (LDT), a revised version of FACT. The first commercial sample was tested in 2016. The FACT and FoundationOne Liquid LDTs have been used to detect the presence of genomic alterations in blood and plasma specimens. Neither the FACT nor FoundationOne Liquid LDTs were FDA-cleared or - approved.

The FoundationOne Liquid CDx assay has not been marketed in the United States or any foreign country.

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 FoundationOne Liquid CDx test results, and subsequently, inappropriate patient management decisions. Patients with false positive CDx biomarker results may undergo treatment with one of the therapies listed in the 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 targeted 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 approved drug product labels.

For the specific adverse events that occurred in the clinical study, please see the RUBRACA® (rucaparib) FDA approved package insert which is available at Drugs@FDA.

IX. SUMMARY OF NONCLINICAL STUDIES

A. Laboratory Studies

Performance characteristics were established using circulating cfDNA derived from blood specimens extracted from a wide range of tumor types. Table 6 below provides

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a summary of the number of tumor types and variants included in each study. As summarized in the table below, each study included a broad range of representative alteration types (substitutions, insertion-deletions, copy number alterations, rearrangements) in various genomic contexts across a number of genes. The validation studies included $>7,000$ sample replicates, $>31,000$ unique variants, $>30$ tumor types, representing all 324 genes targeted by the assay. Table 7 provides a summary of the representation of unique variants in BRCA1 and BRCA2, and EGFR, genes associated with CDx indications for FoundationOne Liquid CDx.

Table 6. Representation of tumor types and variants* across validation studies

| Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # Unique | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Targeted Genes | Subs | Indels | Rearrang. | Copy Number Losses |
| Contrived Sample Functional Characterization (CSFC) Study | Breast cancer Colorectal Cancer (CRC) Lung cancer Contrived samples | 13 | 1843 | 228 | 563 | 81 | 11 | 1 |
| F1 Liquid CDx to Validated Tumor Tissue Test Concordance: BRCA1 and BRCA2 Variants | Prostate cancer Ovarian cancer | 279 | N/A | 2 | 100 | 87 | 9 | 2 |
| Orthogonal Concordance | 23 cancer types Contrived samples | 278 | N/A | 64 | 541 | 12 | 11 | 0 |
| LoD Estimation | Prostate Contrived samples | 10 | 877 | 286 | 1490 | 247 | 32 | 3 |
| LoB | Healthy Donors | 28 | 79 | 322 | 26134 | 4482 | 911 | 42 |
| Potentially Interfering Substances | Contrived samples | 9 | 336 | 18 | 16 | 11 | 11 | 2 |
| Hybrid Capture Bait Specificity | 25 cancer types Contrived samples | 3546 | N/A | 324 | N/A | N/A | N/A | N/A |
| Reagent Stability | Contrived samples | 8 | 142 | 279 | 1090 | 215 | 32 | 2 |
| Reagent Interchangeability | Contrived samples | 8 | 192 | 20 | 15 | 11 | 11 | 1 |
| Precision study 1 | Breast cancer CRC Lung cancer Ovarian cancer Prostate cancer Skin cancer Contrived samples | 47 | 1121 | 280 | 900 | 229 | 63 | 5 |

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| Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # Unique | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Targeted Genes | Subs | Indels | Rearrang. | Copy Number Losses |
| Precision study 2 | Lung cancer Prostate cancer Stomach cancer CRC Bile duct cancer Breast cancer | 10 | 230 | 6 | 6 | 4 | 0 | 0 |
| DNA Extraction | CRC Prostate cancer Breast cancer Lung cancer Skin cancer | 6 | 72 | 161 | 265 | 53 | 2 | 0 |
| Whole Blood Sample Stability | Lung cancer CRC Prostate cancer Breast cancer | 11 | 22 | 66 | 75 | 15 | 1 | 0 |
| Inverted Tube Whole Blood Sample Stability | Lung cancer CRC Breast cancer Ovarian cancer Prostate cancer | 130 | 260 | 237 | 594 | 91 | 5 | 0 |
| Cross Contamination | Contrived samples | 5 | 376 | 39 | 9 | 5 | 4 | 1 |
| Guard Banding | Contrived samples | 10 | 375 | 20 | 17 | 12 | 12 | 1 |
| Clinical validation for detection of EGFR Exon 19 deletions and L858R alterations: non-inferiority study | Lung cancer | 177 | N/A | 1 | 5 | 7 | N/A | N/A |
| Clinical validation study for detection of deleterious alterations in BRCA1 and BRCA2 in prostate cancer | Prostate cancer | 199 | N/A | 2 | 44 | 55 | 8 | 1 |
| Blood Collection Tube Equivalence | Ovarian cancer Breast cancer CRC Prostate cancer Lung cancer Skin cancer Stomach cancer | 60 | 192 | 116 | 135 | 39 | 13 | 0 |

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| Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # Unique | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Targeted Genes | Subs | Indels | Rearrang. | Copy Number Losses |
| Automation Line Equivalence | Contrived samples | 8 | 187 | 303 | 1926 | 337 | 63 | 4 |
| Variant Report Curation | Breast cancer CRC Lung cancer Prostate cancer Skin cancer | 19 | 57 | 183 | 300 | 104 | 15 | 2 |
| Pan-tumor performance (includes historical analysis) | 20 cancer types | 19868 | N/A | N/A | N/A | N/A | N/A | N/A |
| Molecular Index Barcode Performance | 25 cancer types Contrived samples | 7637 | N/A | 324 | N/A | N/A | N/A | N/A |
| F1 Liquid LDT to F1 Liquid CDx Concordance | 25 cancer types | 927 | N/A | 73 | 1815 | 376 | 109 | N/A |

*Variant result totals may include variants classified as VUS or benign.

Table 7. Representation unique variants* in BRCA1, BRCA2, and EGFR

| Study | Gene | # of Unique Substitutions | # of Unique Indels | # of Unique Rearrangements | # of Unique Copy Number Loss |
| --- | --- | --- | --- | --- | --- |
| CSFC Study | BRCA1 | 0 | 0 | 0 | 0 |
| | BRCA2 | 0 | 2 Other DO 2 Contrived | 0 | 0 |
| | EGFR | 1 Other DO 1 Contrived | 1 NSCLC 1 Contrived | 0 | 0 |
| Limit of Blank (LoB) | BRCA1 | 258 HD | 80 HD | 18 HD | 1 HD |
| | BRCA2 | 433 HD | 127 HD | 14 HD | 1 HD |
| | EGFR | 228 HD | 33 HD | 14 HD | 0 |
| Limit of Detection (LoD) | BRCA1 | 6 Other DO 10 Contrived | 4 Contrived | 1 Contrived | 0 |
| | BRCA2 | 16 Other DO 7 Contrived | 5 Contrived | 0 | 1 Prostate Cancer |
| | EGFR | 8 Contrived | 3 Contrived | 0 | 0 |
| Interfering Sub | BRCA1 | 1 Contrived | 2 Contrived | 0 | 0 |
| | BRCA2 | 0 | 5 Contrived | 2 Contrived | 1 Contrived |
| | EGFR | 2 Contrived | 1 Contrived | 0 | 0 |
| Reagent Stability | BRCA1 | 10 Contrived | 3 Contrived | 2 Contrived | 0 |
| | BRCA2 | 6 Contrived | 5 Contrived | 1 Contrived | 0 |
| | EGFR | 9 Contrived | 3 Contrived | 0 | 0 |
| Reagent interchangeability | BRCA1 | 1 Contrived | 2 Contrived | 0 | 0 |
| | BRCA2 | 0 Contrived | 5 Contrived | 0 | 0 |

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| Study | Gene | # of Unique Substitutions | # of Unique Indels | # of Unique Rearrangements | # of Unique Copy Number Loss |
| --- | --- | --- | --- | --- | --- |
| | EGFR | 2 Contrived | 1 Contrived | 0 | 0 |
| Precision study 1 | BRCA1 | 12 Prostate Cancer
12 Other DO
12 Contrived | 11 Prostate Cancer
12 Other DO
11 Contrived | 4 Prostate Cancer
4 Other DO
4 Contrived | 0 |
| | BRCA2 | 13 Prostate Cancer
14 Other DO
13 Contrived | 7 Prostate Cancer
7 Other DO
7 Contrived | 1 Prostate Cancer
1 Other DO
1 Contrived | 1 Prostate Cancer
1 Other DO
1 Contrived |
| | EGFR | 5 NSCLC
5 Other DO
5 Contrived | 3 NSCLC
3 Other DO
3 Contrived | 1 NSCLC
1 Other DO
1 Contrived | 0 |
| Precision study 2 | BRCA1 | 1 Prostate Cancer | 1 Prostate Cancer | 0 | 0 |
| | BRCA2 | 1 Other DO | 1 Other DO | 0 | 0 |
| | EGFR | 1 Other DO | 1 Other DO | 0 | 0 |
| DNA Extraction | BRCA1 | 2 Other DO | 0 | 0 | 0 |
| | BRCA2 | 2 Prostate Cancer
3 Other DO | 1 Prostate Cancer | 0 | 0 |
| | EGFR | 1 NSCLC | 1 Other DO | 0 | 0 |
| Whole Blood Sample Stability | BRCA1 | 1 Other DO | 1 Other DO | 0 | 0 |
| | BRCA2 | 2 Other DO | 0 | 0 | 0 |
| | EGFR | 1 NSCLC | 0 | 0 | 0 |
| Inverted Tube Whole Blood Sample Stability | BRCA1 | 3 Other DO | 1 Prostate Cancer
2 Other DO | 0 | 0 |
| | BRCA2 | 3 Prostate Cancer
4 Other DO | 1 Other DO | 0 | 0 |
| | EGFR | 1 Other DO | 0 | 0 | 0 |
| Cross Contamination | BRCA1 | N/A | 1 contrived | N/A | N/A |
| | BRCA2 | N/A | N/A | N/A | N/A |
| | EGFR | N/A | N/A | N/A | N/A |
| Guard Banding | BRCA1 | 1 Contrived | 2 Contrived | 0 | 0 |
| | BRCA2 | 0 | 4 Contrived | 1 Contrived | 0 |
| | EGFR | 2 Contrived | 2 Contrived | 0 | 0 |
| F1 Liquid CDx to Validated Tumor Tissue Test Concordance: BRCA1 and BRCA2 Variants | BRCA1 | 6 Prostate Cancer
43 Ovarian Cancer | 2 Prostate Cancer
37 Ovarian Cancer | 2 Prostate Cancer
3 Ovarian Cancer | 1 Prostate Cancer
1 Ovarian Cancer |
| | BRCA2 | 23 Prostate Cancer
31 Ovarian Cancer | 29 Prostate Cancer
25 Ovarian Cancer | 5 Prostate Cancer | 1 Prostate Cancer
1 Ovarian Cancer |
| | EGFR | N/A | N/A | N/A | N/A |
| Orthogonal Concordance | BRCA1 | 1 Prostate Cancer
1 Other DO
3 Contrived | N/A | N/A | N/A |
| | BRCA2 | 4 Other DO
2 Contrived | N/A | N/A | N/A |
| | EGFR | 7 NSCLC
4 Other DO
Contrived | 5 NSCLC
1 Other DO
2 Contrived | N/A | N/A |
| | BRCA1 | 16 Prostate Cancer
60 Other DO | 5 Prostate Cancer
3 Other DO | 3 Prostate Cancer | |

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| Study | Gene | # of Unique Substitutions | # of Unique Indels | # of Unique Rearrangements | # of Unique Copy Number Loss |
| --- | --- | --- | --- | --- | --- |
| F1 Liquid LDT to F1 Liquid CDx Concordance | BRCA2 | 38 Prostate Cancer
92 Other DO | 39 Prostate Cancer
10 Other DO | 6 Prostate Cancer
1 Other DO | N/A
(copy loss not evaluated) |
| | EGFR | 50 NSCLC
37 Other DO | 15 NSCLC
2 Other DO | 3 NSCLC
2 Other DO | |
| Clinical validation study for detection of deleterious alterations in BRCA1 and BRCA2 in prostate cancer | BRCA1 | 21 Prostate Cancer | 3 Prostate Cancer | 3 Prostate Cancer | 0 |
| | BRCA2 | 53 Prostate Cancer | 53 Prostate Cancer | 5 Prostate Cancer | 1 Prostate Cancer |
| | EGFR | 17 Prostate Cancer | 1 Prostate Cancer | 3 Prostate Cancer | 0 |
| Blood Collection Tube Equivalence | BRCA1 | 1 Contrived | 1 Other DO
2 Contrived | 0 | 0 |
| | BRCA2 | 4 Other DO | 1 Other DO
5 Contrived | 0 | 0 |
| | EGFR | 2 Other DO
2 Contrived | 1 Contrived | 0 | 0 |
| Automation Line Equivalence | BRCA1 | 11 Contrived | 2 Contrived | 1 Contrived | 0 |
| | BRCA2 | 13 Contrived | 6 Contrived | 1 Contrived | 1 Contrived |
| | EGFR | 7 Contrived | 3 Contrived | 0 | 0 |
| Variant Report Curation | BRCA1 | 2 Prostate Cancer
1 Other DO | 8 Prostate Cancer
1 Other DO | 1 Prostate Cancer | 0 |
| | BRCA2 | 2 Prostate Cancer
5 Other DO | 3 Other DO | 0 | 1 Prostate Cancer |
| | EGFR | 1 NSCLC
2 Other DO | 0 | 0 | 0 |

*Variant result totals may include variants classified as VUS or benign.
HD = apparently healthy donor; DO = Disease Ontology
Note: The number of unique variants in each sample type may add up to more than the total of unique variants as the same variants may be represented in multiple sample types

Clinical oncology blood specimens can be constrained by factors such as limitations in blood draw volumes and cfDNA concentration. For studies where clinical samples carrying CDx biomarkers/alteration types were not evaluated due to limitations in sample availability, a postmarket study is planned to confirm the performance of the FoundationOne Liquid CDx test using intended use clinical specimens. In some studies when use of clinical specimens was not feasible due to volume limitations, contrived samples were used which consisted of enzymatically sheared cell line DNA spiked into human plasma from healthy donors, extracted according to the assay's standard procedure, and the isolated cfDNA was then diluted with cfDNA. To support such use, a contrived sample functional characterization (CSFC) study was conducted to demonstrate comparable performance of sheared cell line DNA samples as compared to cfDNA isolated from plasma.

Highly actionable alterations were identified in the 39 contrived samples representing 17 genes and included 17 substitutions, 6 indels, 6 copy number losses, and 9 rearrangements that were used across validation studies. The 39 contrived samples

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included 7 EGFR (positive for 3 EGFR Ex 19 deletions, 2 EGFR L858R substitutions), 2 BRCA1 (positive for 2 indels and 1 substitutions), and 3 BRCA2 samples (positive for 5 indels). These 12 samples were used to supplement the samples used to support the performance of the EGFR and BRCA1/BRCA2 CDx indications listed in Table 1 as well as the tumor mutation profiling claim.

1. **Contrived Sample Functional Characterization (CSFC) Study:**
Similar performance between clinical cfDNA samples and contrived samples was confirmed by demonstrating equivalent hit rates across comparable dilutions between the two sample types, including the LoD level. Similar performance between clinical and contrived samples was established by testing a dilution series of contrived and clinical specimens harboring substitutions, insertions, deletions, rearrangements, and copy number losses, totaling 924 clinical (cfDNA) sample replicates and 1069 enzymatically fragmented cell-line genomic DNA (gDNA) contrived sample replicates. All matching alterations present in both the contrived and clinical specimens at comparable levels of variant allele fraction (for substitutions, indels, rearrangements) or tumor fraction (for copy number losses) were used to determine the similar performance for a total of 654 unique variants.

While all matching alterations were used in the analysis, clinical specimens were selected to target some highly relevant alterations for each alteration type, including some CDx biomarkers. Comparable hit rates at targeted dilution levels between clinical and contrived samples for these targeted alterations demonstrate similar performance between contrived and clinical samples for processing with FoundationOne Liquid CDx.

An additional evaluation of BRCA2 indels (BRCA2 5351delA and BRCA2 9097_9098insA) between 6 clinical replicates of each variant and 27 and 14 contrived sample replicates, respectively, for each contrived variant was performed using a regression analysis. A probit model was used to evaluate the relationship between the positive call rate and variant allele frequency. A regression analysis and formal hypothesis test confirmed no significant difference in test performance between contrived and clinical specimens as determined by a p-value > 0.05.

A post-market study will be conducted to confirm the functional comparability between contrived and clinical samples positive for other specific BRCA1 and BRCA2 alterations.

2. **Analytical Accuracy/Concordance with an Orthogonal Method:**
a. Concordance with Orthogonal cfDNA-based NGS Method #1:
The detection of short variants and rearrangements by the FoundationOne Liquid CDx assay was compared to that of an externally validated NGS assay in 74 genes common to both assays across 278 samples that represented an array of tumor types (>50 unique disease ontologies across 23 cancer types.

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The cancer types (# samples) included lung [NSCLC (75) and other (3)]; breast (54); prostate (32); colorectal [colon (27) and rectal (6)]; liver (11); ovarian (6); pancreas (9); gastrointestinal (7); bile duct (2); esophageal (5); skin (6); cervical (1); anal (1); bladder (1); gallbladder (1); salivary gland (2); thymus (1); thyroid (3); uterine (2); fallopian tube (1); head and neck (1); soft tissue (1); and unknown primary (19). The study included samples selected from clinical FoundationOne Liquid CDx testing (n=268) and contrived samples consisting of fragmented gDNA diluted in clinical cfDNA to represent rare alterations (n=10).

Using the externally validated NGS assay as the comparator, the analysis demonstrated a short variant Positive Percent Agreement (PPA) of 96.2% with a 95% two-sided CI of (94.8%, 97.4%). The short variant Negative Percent Agreement (NPA) was >99.9% with a 95% two-sided CI of (99.9%, 100.0%). The respective PPA of base substitutions and indels with a 95% two-sided CI was 96.1% (94.6%, 97.3%) and 100.0% (85.2%, 100.0%). The respective NPA and 95% two-sided CI of base substitutions and indels was >99.9% (99.9%, 100.0%) and 100.0% (99.9%, 100.0%) (Table 8).

Table 8: Concordance of short variants* called in FoundationOne Liquid CDx and the comparator assay

| Variant Type | CDx(+)/ Comp(+) | CDx(-)/ Comp (+) | CDx(+)/ Comp (-) | CDx(-)/ Comp (-) | PPA (95% CI) | NPA (95% CI) | OPA (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| All Short Variants | 868 | 34 | 8 | 152824 | 96.2% (94.8%, 97.4%) | >99.9% (99.9%, 100.0%) | >99.9% (99.9%, 100.0%) |
| Base Substitutions | 845 | 34 | 8 | 149511 | 96.1% (94.6%, 97.3%) | >99.9% (99.9%, 100.0%) | >99.9% (99.9%, 100.0%) |
| Indels | 23 | 0 | 0 | 3313 | 100.0% (85.2%, 100.0%) | 100.0% (99.9%, 100.0%) | 100.0% (99.9%, 100.0%) |

CDx = FoundationOne Liquid CDx; Comp = Comparator
N = 902 positive variants, N = 152,832 negative variants by the comparator assay)
*Variant result totals may include variants classified as VUS or benign.

For the rearrangement detection concordance between the FoundationOne Liquid CDx and the comparator assay, the observed rearrangement PPAComp was 100.0%, with a 95% two-sided CI of (59.04%, 100.0%). The NPAComp was 99.8%, with a 95% two-sided CI (99.5%, 100.0%).

Assessment of a subset of highly-actionable alterations were compared between the two assays. The analysis resulted in a PPA of 100% across all eligible highly-actionable alterations called in the comparator assay (Table 9) and a NPA of 99.8% (99.5%, 100.0%).

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Table 9. Concordance of highly actionable alterations called between FoundationOne Liquid CDx and the comparator assay

| Targeted Alteration | n | PPA (95% CI) | NPA (95% CI) |
| --- | --- | --- | --- |
| EGFR L858R | 10 | 100% (69.2%, 100.0%) | 100% (98.7%, 100.0%) |
| EGFR Exon 19 deletions | 11 | 100% (71.5%, 100.0%) | 100% (99.7%, 100.0%) |
| BRCA1 short variants | 1 | 100% (2.5%, 100.0%) | 100% (98.7%, 100.0%) |
| BRCA2 short variants | 2 | 100% (15.8%, 100.0%) | 100% (99.3%, 100.0%) |

These data demonstrate that the FoundationOne Liquid CDx assay and an externally validated NGS assay are highly concordant across the 74 genes common between the two panels.

Due to the rarity of some variant types in the $BRCA1$ and $BRCA2$ prostate cancer patient population, $BRCA1$ and $BRCA2$ samples from other cancers as well as other clinical samples that were shown to perform similarly were used to leverage for those samples not represented in the current study. While those samples were shown to perform similarly, an additional post-market study is planned to support the above described analytical accuracy study intended to provide additional data to confirm the performance of those $BRCA1$ and $BRCA2$ variants in prostate cancer specimens which were not represented in the above study either due to limitations in sample availability or due to absence of a well-validated comparator assay for robust detection of all $BRCA1$ and $BRCA2$ variant types. This post-market study will also include additional samples to represent other genes and variants tested for by the FoundationOne Liquid CDx test.

b. Concordance data for CDx-associated alterations:

i. Comparison with Validated NGS Tumor Tissue Assay: Samples from a total of 279 BRCA1 and BRCA2 positive prostate and ovarian cancer patients were tested and the concordance evaluated between matched FoundationOne Liquid CDx and a validated tumor tissue test for the detection of deleterious alterations in BRCA1 or BRCA2. The sample set was comprised of 100 unique substitutions, 87 unique indels, 9 unique rearrangements, and 2 unique copy number loss. As summarized in Table 10 below, a PPA of $88.0\%$ and an NPA of $95.7\%$ were observed.

Table 10. Concordance of FoundationOne Liquid CDx and Validated NGS Tumor Tissue Test #1 in prostate and ovarian cancer patients for the detection of alterations in BRCA1 or BRCA2

| | Validated Tumor Tissue Method #1 | | | |
| --- | --- | --- | --- | --- |
| | | (+) | (-) | |
| F1 Liquid CDx | (+) | 103 | 7 | PPA: 88.0% (80.9%, 92.7%) |
| | (-) | 14 | 155 | NPA: 95.7% (91.4%, 97.9%) |

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As summarized in Table 11 an overall PPA of 87.28% and an NPA of 99.83% were observed.

Table 11. Concordance of FoundationOne Liquid CDx and Validated NGS Tumor Tissue Test #1 in prostate and ovarian cancer patients for the detection of alterations* in BRCA1 or BRCA2

| | F1 Liquid CDx(+)/Tissue(+) | F1 Liquid CDx(-)/Tissue(+) | F1 Liquid CDx(+)/Tissue(-) | F1 Liquid CDx(-)/Tissue(-) | PPA (%) CI^{1} | NPA (%) CI^{1} |
| --- | --- | --- | --- | --- | --- | --- |
| Substitutions | 77 | 6 | 29 | 20255 | 92.77 (85.1%, 96.6%) | 99.86 (99.8%, 99.9%) |
| Indels | 65 | 3 | 31 | 16362 | 95.59 (87.8%, 98.5%) | 99.81 (99.7%, 99.9%) |
| Rearrangements | 4 | 3 | 7 | 1939 | 57.14 (25.1%, 84.2%) | 99.64 (99.3%, 99.8%) |
| Copy number loss | 5 | 10 | 1 | 263 | 33.33 (15.2%, 58.3%) | 99.62 (97.9%, 99.9%) |
| Total | 151 | 22 | 68 | 38819 | 87.28 (81.5%, 91.5%) | 99.83 (99.8%, 99.9%) |

*Variant result totals may include variants classified as VUS or benign.

An analysis was also performed for the subset of patients with prostate cancer as summarized in Table 12. This sample set was comprised of 24 substitutions, 27 indels, 6 rearrangements, and 1 copy number loss. An overall PPA of 80.00% and an NPA of 99.69% were observed.

Table 12. Concordance of FoundationOne Liquid CDx and Validated NGS Tumor Tissue Test #1 in the subset of patients with prostate cancer for the detection of alterations* in BRCA1/BRCA2

| | F1 Liquid CDx(+)/Tissue(+) | F1 Liquid CDx(-)/Tissue(+) | F1 Liquid CDx(+)/Tissue(-) | F1 Liquid CDx(-)/Tissue(-) | PPA (%) CI^{1} | NPA (%) CI^{1} |
| --- | --- | --- | --- | --- | --- | --- |
| Substitutions | 23 | 1 | 5 | 1459 | 95.83 (79.8%, 99.3%) | 99.66 (99.2%, 99.9%) |
| Indels | 25 | 3 | 4 | 1642 | 89.29 (72.8%, 96.3%) | 99.76 (99.4%, 99.9%) |
| Rearrangements | 3 | 3 | 1 | 365 | 50.00 (18.8%, 81.2%) | 99.73 (98.5%, 99.95%) |
| Copy number loss | 5 | 7 | 1 | 49 | 41.67 (19.3%, 68.1%) | 98.00 (89.5%, 99.7%) |
| Total | 56 | 14 | 11 | 3515 | 80.00 (69.2%, 87.7%) | 99.69 (99.4%, 99.8%) |

*Variant result totals may include variants classified as VUS or benign.

Some discordance is expected based on biological differences and sampling times between tumor tissue and plasma samples. Considering the impact of biological differences between analytes, these data demonstrate

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a good concordance for base substitutions but poor performance with rearrangements and copy number losses between the FoundationOne Liquid CDx and the validated tumor tissue test for the detection of deleterious alterations in BRCA1 or BRCA2.

ii. Comparison with Second Orthogonal Tumor Tissue NGS Assay: A separate exploratory analysis was also performed to demonstrate the concordance between matched plasma and FFPE samples using the FoundationOne Liquid CDx to an externally validated tissue-based orthogonal NGS method. In this study, 3 prostate specimens obtained from FMI's archives, which showed 1 BRCA1 rearrangement, 1 BRCA1 deletion, and 1 BRCA2 deletion in the FFPE specimens. The results of this study showed discordant results between the plasma and tissue results for the two BRCA deletions. For one of the discordant samples, based on the FoundationOne Liquid CDx results there appeared to be little evidence of cfDNA, suggesting that the discordance was most likely due to biological differences between the sample analytes (i.e., low ctDNA shedding) and for the other discordant sample, the orthogonal tissue assay detected a monoallelic loss of exons 5 - 7 in the BRCA1 gene. Hemizygous focal loss events are not expected to be called by the FoundationOne Liquid CDx assay.

# 3. Analytical Sensitivity:

a. Limit of Blank (LoB):

The LoB was established by sequencing plasma samples from 30 apparently healthy donors with no diagnosis of cancer using 4 replicates per sample. All donors were over the age of 60 with a median age of 68, and included 15 smokers and 15 non-smokers.

Across 30,622 short variants, 58 variants had a detection rate of greater than $5\%$ . Of those, five variants of unknown significance (VUS) (TSC1 965T>C, IRF4 1ins87, MSH3 186_187insGCCGCAGCGCCCGCAGCG, IGF1R 568C>T, WHSC1 1582C>A) had a detection rate significantly exceeding $5\%$ , up to $35\%$ for one variant, on an individual variant basis. Across 264 copy number alterations and 894 rearrangements, zero variants were detected.

Table 13: Detection Rate in LoB Study

| Category | Per Variant* Detection Rate |
| --- | --- |
| Level 1 | 0% (0 of 292) |
| Level 2 | 0% (0 of 10) |
| Level 3 | 0% (0 of 18) |
| Level 4 | 0.82% (47 of 5760) |
| VUS | 0.83% (203 of 24542) |
| All categories | 0.82% (250 of 30622) |

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*Variant result totals may include variants classified as VUS or benign. Note: Per variant detection rate was calculated as the number of unique variants detected at least once across all replicates divided by the total number of unique variants included in the analysis.

All other variants were assigned an LoB of 0, as the detection rate not significantly exceeding 5%. Each cancer-related alteration detected in this study was detected in replicates from a single donor, indicating that these are likely true variants present in the sample.

A post-market LoB study will be conducted to confirm the results in accordance with the FoundationOne Liquid CDx assay workflow.

b. Limit of Detection (LoD):

The LoD for each variant type was established by processing a total of 1,069 sample replicates across ten contrived (enzymatically fragmented cell-line gDNA) samples representing short variants, rearrangements, and copy number alterations (homozygous deletions). The LoD was determined using the conservative hit rate approach for the majority of variants. LoD by hit rate was defined as the mean VAF value (for short variants and rearrangements) or mean tumor fraction value (for copy number alterations) at the lowest dilution level tested with at least 95% detection across replicates. The hit rate was computed as the number of replicates with positive variant calls per the total number of replicates tested at each level. Short variants with hit rates of at least 95% at all dilution levels or hit rates below 95% for all dilution levels were excluded from analysis as LoD could not be reliably estimated.

The median estimated LoD for CDx alterations are presented in Table 14. The median LoD for targeted short variant, rearrangement, and copy number alterations were consistent with the platform LoD.

Table 14. LoD estimation for CDx alterations

| Gene | Alteration Subtype | # Samples Evaluated | Median LoD^{1} |
| --- | --- | --- | --- |
| BRCA1 | Indels | 1 | 0.38% VAF |
| | Substitutions | 8 | 0.34% VAF |
| | Rearrangement^{2} | 1 | 0.87% VAF |
| BRCA2 | Substitutions | 17 | 0.37% VAF |
| | Indels | 2 | 0.36% VAF |
| | BRCA2- EDA Truncation^{2} | 1 | 0.48% VAF |
| | Copy Number Loss^{1} | 1 | 48.1% TF |
| EGFR | Indels (Exon 19 deletions) | 2 | 0.27% VAF |
| | Substitutions (L858R substitutions) | 2 | 0.34% VAF |

The Estimated LoDs for BRCA1 and BRCA2 subs and indels were confirmed at values higher than the LoDs estimated for the non-CDx alterations. (see Precision: Reproducibility and Reproducibility section below, Tables 18 and 19 for confirmed LoD values).

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1 The accuracy of %VAF/%TF have not been analytically validated.
2 The LoD for these alterations was determined using clinical specimens.

The LoDs for other variants detected by the assay were determined to be similar to the median LoDs estimated for the CDx variants above. A total of 864 short variants were included in the platform LoD analysis. The enhanced sensitivity region of the bait set contains 269 of the short variants analyzed and the standard sensitivity region of the bait set contains 595 of the short variants analyzed. The median LoD for short variants was estimated at 0.40% for the enhanced sensitivity region and 0.82% of the standard sensitivity region. The median LoD is 30.4% tumor fraction for copy number losses. Rearrangement LoD was estimated as a median of 0.37% for the enhanced sensitivity region and 0.9% for the standard sensitivity region. The LoD for copy number losses was estimated at 30.4% based on tumor fraction.

Because a major component driving the detectability of a variant is genomic context (repetitiveness of the reference genomic region), an LoD analysis for short variants was also performed within categories based on genomic context.

4. Analytical Specificity:

a. Potentially Interfering Substances:

To evaluate the robustness of the FoundationOne Liquid CDx results in the presence of potentially interfering exogenous and endogenous substances, a total of 11 potential interferents were evaluated. These potential interferents included six endogenous substances (albumin, conjugated bilirubin, unconjugated bilirubin, cholesterol, hemoglobin and triglycerides) and five exogenous substances (DNA from another source [the microorganism Staphylococcus epidermidis], excess anticoagulant, proteinase K, ethanol and molecular index barcodes).

A total of 340 samples were tested to evaluate the potential interference of albumin, conjugated bilirubin, unconjugated bilirubin, cholesterol, hemoglobin, triglycerides, DNA from another source (the microorganism Staphylococcus epidermidis), excess anticoagulant, proteinase K, ethanol, and molecular index barcodes. An assessment of the cfDNA yield obtained during the DNA isolation, purification, and quantification steps, as well as at library construction QC (LCQC) and hybrid capture QC (HCQC) was performed. Substances were considered as non-interfering if ≥90% of samples across aggregated replicates per treatment level successfully met processing criteria. The process success rates for each step are listed in Table 15.

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Table 15: Process success rates with interfering substances

| Process | # Failed | # Pass | Total | Success Rate (%) | 95% CI LB (%) | 95% CI UB (%) |
| --- | --- | --- | --- | --- | --- | --- |
| DNA Extraction | 0 | 180 | 180 | 100.00 | 97.97 | 100.00 |
| LC | 1 | 339 | 340 | 99.71 | 98.37 | 99.99 |
| HC | 3 | 336 | 339 | 99.12 | 97.44 | 99.82 |
| Sequencing | 0 | 336 | 336 | 100.00 | 98.91 | 100.00 |

For each potential interferent, concordance of alteration calls was calculated relative to a control sample without interferent. The pre-defined variants included 27 short variants, 17 rearrangements, and 3 copy number variants. Of the 11 potential interferents tested across 16 conditions, concordance for all variant calls was 100% for 8 conditions and ≥97% for all conditions (Table 16). Potential interference from albumin (60 g/L), Bilirubin (unconjugated at 0.2 g/L), and cholesterol (at 150 mg/dL and 250 mg/dL) was observed for the single rearrangement evaluated for each of these four conditions.

Table 16: Concordance per substance for variants 1.5x LoD

| Substance | Concordance | 95% CI | N |
| --- | --- | --- | --- |
| Triglycerides, 37 mmol/L (or 33 g/L) | 100.00% | (91.00%, 100.00%) | 40 |
| Hemoglobin, 2.0 g/L | 100.00% | (91.00%, 100.00%) | 39 |
| Albumin, 60 g/L | 97.56% | (87.00%, 100.00%) | 41 |
| Bilirubin (conjugated), 0.2 g/L | 100.00% | (92.00%, 100.00%) | 42 |
| Bilirubin (unconjugated), 0.2 g/L | 97.44% | (87.00%, 100.00%) | 39 |
| Cholesterol Level 2, 3.88 mmol (150 mg/dL) | 97.56% | (87.00%, 100.00%) | 41 |
| Cholesterol Level 1, 6.47mmol (250 mg/dL) | 97.37% | (86.00%, 100.00%) | 38 |
| Staphylococcus epidermidis, 1 x 106 CFU/mL | 100.00% | (91.00%, 100.00%) | 39 |
| Anticoagulant, 5X nominal volume | 100.00% | (91.00%, 100.00%) | 41 |
| Proteinase K, +0.6 mg/mL | 98.00% | (89.00%, 100.00%) | 50 |
| Proteinase K, +0.3 mg/mL | 100.00% | (92.00%, 100.00%) | 46 |
| Ethanol, +2.5% | 97.96% | (89.00%, 100.00%) | 49 |
| Ethanol, +5.0% | 97.92% | (89.00%, 100.00%) | 48 |
| Molecular Index barcodes, +5% | 97.22% | (85.00%, 100.00%) | 36 |
| Molecular Index barcodes, +15% | 100.00% | (93.00%, 100.00%) | 48 |
| Molecular Index barcodes, +30% | 100.00% | (93.00%, 100.00%) | 49 |

Taken together, these data indicate that the FoundationOne Liquid CDx assay is robust to potential specimen-related endogenous substances and exogenous contaminants or interferents.

b. Hybrid Capture Bait Specificity:

Bait specificity was addressed through an assessment of coverage of targeted regions in FoundationOne Liquid CDx using 3,546 validation study samples. Results show that targeted genomic regions have consistently high, uniform coverage. For each genomic region associated with a predefined subset of

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highly-actionable alterations, between 94% to 100% of samples possessed the expected level of coverage. An in-depth, platform-wide examination of the FoundationOne Liquid CDx bait set through the analysis of HapMap process control samples revealed that, on average, 98.8% and 94.1% of platform-wide baited coding and non-coding regions, respectively, met their expected coverage levels. Samples assessed in this study consistently demonstrated high quality uniform and deep coverage across the entire genomic region targeted by the assay.

5. Carryover/Cross-Contamination:
The study demonstrated that the risk of cross contamination (intra-plate), and carry-over contamination (inter-plate) of samples during processing of the FoundationOne Liquid CDx assay is low. A total of 376 wells were examined for intra- and inter-plate contamination by processing and sequencing of contrived samples derived from cell lines at high input concentrations with known genomic backgrounds. Unique variants of each cell line were characterized by independent control sequencing runs. The samples were arrayed in a checkerboard fashion across four 96-well PCR plates to detect cross-contamination events. A cross-contamination rate of 0.53% (2/376) was observed in this study. These data demonstrate a low probability of cross contamination during the FoundationOne Liquid CDx process.

6. Precision: Repeatability and Reproducibility
Precision was evaluated for alterations associated with CDx claims, as well as tumor mutation profiling 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 reagent lots, two sequencers, and two processing runs.

a. Results for a subset of highly-actionable alterations
A set of 39 unique samples were used to evaluate precision of FoundationOne Liquid CDx for detecting a set of highly-actionable variants, including 8 contrived samples representing various targeted alterations and 31 clinical samples. The samples representing CDx alterations are summarized in Table 17.

The 31 clinical samples consisted of 7 different cancers (10 lung, 6 prostate, 3 colon, 2 melanoma, 4 ovarian, 5 breast, and 1 unknown). The samples included 30 actionable gene alterations including 7 BRCA1 or BRCA2 alterations and 4 EGFR substitutions and indels. The remaining samples included multiple other actionable genes and variant types.

Target alterations were assessed near LoD and/or 2x - 3x LoD. Each sample was divided into 24 aliquots, with 12 duplicates being processed on the same plate under the same conditions. Across 47 samples (31 clinical specimens at

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one dilution level and 8 contrived samples across two dilution levels), a total of 57 unique alterations were evaluated.

Table 17: CDx sample set assessed for precision

| Targeted Alteration | Disease Ontology of Patient from which Sample was Derived |
| --- | --- |
| EGFR Exon 19 deletions and EGFR exon 21 L858R alterations | 5 contrived samples |
| BRCA1, BRCA2 alterations | 6 contrived samples |
| BRCA1 E23fs*17 | Ovary cancer |
| BRCA1 Q780* | Ovary high grade serous carcinoma |
| BRCA1 Rearrangement | Unknown primary malignant neoplasm |
| BRCA2 G267* | Ovary serous carcinoma |
| BRCA2 Loss (15 of 26) | Prostate acinar adenocarcinoma |
| BRCA2 Loss (26 of 26) | Prostate acinar adenocarcinoma |
| BRCA2 S2988fs*12 | Ovary cancer |
| BRCA2- EDA Truncation | Prostate cancer |
| EGFR E746_A750del | Non-small cell lung carcinoma |
| EGFR L858R | Non-small cell lung carcinoma |
| EGFR L858R | Non-small cell lung carcinoma |

The repeatability of CDx alterations is summarized in Table 18 and the reproducibility of CDx alterations is summarized in Table Table 19.

Table 18: Repeatability of CDx alterations targeted in precision study at $> {1x}\mathrm{{LoD}}{}^{ * }$

| Variant Type | Alteration | Concordant Pairs | Repeatability (%) | 95% CIs (%) | Level Tested** |
| --- | --- | --- | --- | --- | --- |
| Short variant | BRCA1_2338C>T | 12/12 | 100 | (73.54, 100) | 1.11% VAF |
| Short variant | BRCA1_2475delC | 12/12 | 100 | (73.54, 100) | 0.61% VAF |
| Short variant | BRCA1_2475delC | 12/12 | 100 | (73.54, 100) | 0.93% VAF |
| Short variant | BRCA1_2612C>TT | 11/11 | 100 | (71.51, 100) | 0.51% VAF |
| Short variant | BRCA1_68_69delAG | 12/12 | 100 | (73.54, 100) | 0.66% VAF |
| Short variant | BRCA1_P871fs*32 | 12/12 | 100 | (73.54, 100) | 1.08% VAF |
| Rearrangement | BRCA1-BRCA1 | 12/12 | 100 | (73.54, 100) | 0.87% VAF |
| Short variant | BRCA2_3599_3600delGT | 12/12 | 100 | (73.54, 100) | 0.58% VAF |
| Short variant | BRCA2_3599_3600delGT | 12/12 | 100 | (73.54, 100) | 0.92% VAF |
| Short variant | BRCA2_4284_4285insT | 12/12 | 100 | (73.54, 100) | 0.94% VAF |
| Short variant | BRCA2_4284_4285insT | 11/11 | 100 | (71.51, 100) | 1.26% VAF |
| Short variant | BRCA2_5351delA | 12/12 | 100 | (73.54, 100) | 1.22% VAF |
| Short variant | BRCA2_5351delA | 12/12 | 100 | (73.54, 100) | 1.85% VAF |
| Short variant | BRCA2_5351delA | 11/11 | 100 | (71.51, 100) | 1.07% VAF |
| Short variant | BRCA2_5351delA | 12/12 | 100 | (73.54, 100) | 2.24% VAF |
| Short variant | BRCA2_5465_5466insA | 12/12 | 100 | (73.54, 100) | 0.92% VAF |
| Short variant | BRCA2_5465_5466insA | 11/11 | 100 | (71.51, 100) | 1.19% VAF |
| Short variant | BRCA2_8961_8964delGAGT | 12/12 | 100 | (73.54, 100) | 1.07% VAF |
| Short variant | BRCA2_c.799G>T | 10/12 | 83.33 | (51.59, 97.91) | 0.5% VAF |

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Table 19: Reproducibility of CDx alterations targeted in precision study at $>1\mathrm{x}$ LoD*

| Variant Type | Alteration | Concordant Replicates | Reproducibility (%) | 95% CIs (%) | VAF/TF** Level Tested |
| --- | --- | --- | --- | --- | --- |
| Short variant | BRCA1_2338C>T | 24/24 | 100 | (85.75, 100) | 1.11% |
| Short variant | BRCA1_2475delC | 24/24 | 100 | (85.75, 100) | 0.61% |
| Short variant | BRCA1_2475delC | 24/24 | 100 | (85.75, 100) | 0.93% |
| Short variant | BRCA1_2612C>TT | 23/23 | 100 | (85.18, 100) | 0.51% |
| Short variant | BRCA1_68_69delAG | 24/24 | 100 | (85.75, 100) | 0.66% |
| Short variant | BRCA1_P871fs*32 | 24/24 | 100 | (85.75, 100) | 1.08% |
| Rearrangement | BRCA1-BRCA1 | 24/24 | 100 | (85.75, 100) | 0.87% |
| Short variant | BRCA2_3599_3600delGT | 24/24 | 100 | (85.75, 100) | 0.58% |
| Short variant | BRCA2_3599_3600delGT | 24/24 | 100 | (85.75, 100) | 0.92% |
| Short variant | BRCA2_4284_4285insT | 24/24 | 100 | (85.75, 100) | 0.94% |
| Short variant | BRCA2_4284_4285insT | 23/23 | 100 | (85.18, 100) | 1.26% |
| Short variant | BRCA2_5351delA | 24/24 | 100 | (85.75, 100) | 1.22% |
| Short variant | BRCA2_5351delA | 24/24 | 100 | (85.75, 100) | 1.85% |
| Short variant | BRCA2_5351delA | 23/23 | 100 | (85.18, 100) | 1.07% |
| Short variant | BRCA2_5351delA | 24/24 | 100 | (85.75, 100) | 2.24% |
| Short variant | BRCA2_5465_5466insA | 24/24 | 100 | (85.75, 100) | 0.92% |
| Short variant | BRCA2_5465_5466insA | 23/23 | 100 | (85.18, 100) | 1.19% |
| Short variant | BRCA2_5465_5466insA | 24/24 | 100 | (85.75, 100) | 1.25% |
| Short variant | BRCA2_5465_5466insA | 23/23 | 100 | (85.18, 100) | 1.22% |

*Clinical samples were mostly tested at 2x - 3x LoD rather than 1x - 1.5x LoD
**The accuracy of %VAF/%TF have not been analytically validated.

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| Variant Type | Alteration | Concordant Replicates | Reproducibility (%) | 95% CIs (%) | VAF/TF** Level Tested |
| --- | --- | --- | --- | --- | --- |
| Short variant | BRCA2_799G>T | 22/24 | 91.67 | (73.0, 98.97) | 0.5% |
| Short variant | BRCA2_8961_8964delGAGT | 24/24 | 100 | (85.75, 100) | 1.07% |
| Short variant | BRCA2_9097_9098insA | 22/24 | 91.67 | (73.0, 98.97) | 1.03% |
| Short variant | BRCA2_c.799G>T | 22/24 | 91.67 | (73.0, 98.97) | 0.5% |
| Short variant | BRCA2_c.9097_9098insA | 5/23 | 21.74 | (7.46, 43.7) | 0.71% |
| Short variant | BRCA2_c.9097_9098insA | 22/24 | 91.67 | (73.0, 98.97) | 1.03% |
| Copy Number Loss | BRCA2 loss | 21/24 | 87.5 | (67.64, 97.34) | 39.43% TF |
| Rearrangement | BRCA2-EDA | 23/23 | 100 | (85.18, 100) | 0.48% |
| Short variant | EGFR_2369C>T | 24/24 | 100 | (85.75, 100) | 0.44% |
| Short variant | EGFR_2369C>T | 24/24 | 100 | (85.75, 100) | 0.66% |
| Short variant | EGFR_2369C>T | 23/23 | 100 | (85.18, 100) | 0.36% |
| Short variant | EGFR_2369C>T | 24/24 | 100 | (85.75, 100) | 0.65% |
| Short variant | EGFR_2369C>T | 24/24 | 100 | (85.75, 100) | 1.26% |
| Short variant | EGFR_2573T>G | 24/24 | 100 | (85.75, 100) | 0.46% |
| Short variant | EGFR_2573T>G | 24/24 | 100 | (85.75, 100) | 0.68% |
| Short variant | EGFR_2573T>G | 24/24 | 100 | (85.75, 100) | 0.68% |
| Short variant | EGFR_2573T>G | 23/23 | 100 | (85.18, 100) | 0.95% |
| Short variant | EGFR_2573T>G | 24/24 | 100 | (85.75, 100) | 0.64% |
| Short variant | EGFR_2573T>G | 24/24 | 100 | (85.75, 100) | 1.64% |
| Short variant | EGFR_E746_A750del | 24/24 | 100 | (85.75, 100) | 0.51% |
| Short variant | EGFR_E746_A750del | 24/24 | 100 | (85.75, 100) | 0.74% |
| Short variant | EGFR_E746_A750del | 24/24 | 100 | (85.75, 100) | 0.93% |
| Short variant | EGFR_E746_A750del | 23/23 | 100 | (85.18, 100) | 1.2% |
| Short variant | EGFR_E746_A750del | 23/23 | 100 | (85.18, 100) | 0.51% |
| Short variant | EGFR_E746_A750del | 24/24 | 100 | (85.75, 100) | 1.01% |
| Short variant | EGFR_E746_A750del | 22/22 | 100 | (84.56, 100) | 0.34% |

*Clinical samples were mostly tested at 2x – 3x LoD rather than 1x – 1.5x LoD
**The accuracy of %VAF/%TF have not been analytically validated.

For repeatability, 42 samples with 53 targeted alterations were evaluated. Of the 53 alterations that were targeted, 43 alterations demonstrated 100% repeatability. Within the targeted CDx variants assessed, the overall repeatability was 96.39% (95.28%, 97.30%).

Reproducibility of 100% was observed in 42 of 55 (76.4%) alterations. These results demonstrate the robustness of FoundationOne Liquid CDx assay, as 55 targeted alterations passed the acceptance criterion for reproducibility. For the targeted CDx variants assessed, the overall reproducibility was 97.33% (96.67%, 97.89%).

b. Confirmation of LoD and Precision in Clinical Specimens:

Twenty-nine clinical cfDNA samples targeting variants at 1-1.5x LoD were evaluated to confirm LoD and precision in clinical specimens. Twenty-six had 100% reproducibility, one had 95.8% reproducibility, and two samples had reproducibility below 90%. Of these two samples, one contained a BRCA2

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loss that had 87.5% reproducibility and 91.67% repeatability. This sample had cfDNA input below the recommended minimum. The other sample harbored a BRCA2 substitution (c.799G>T) with 91.67% reproducibility and 83.33% repeatability. The average VAF of this variant was 0.5% across replicates, which is near the LoD for this variant type (LoD of 0.4% VAF). A summary of the Confirmation of LoD and precision results for a subset of highly-actionable alterations are provided in Table 20.

Table 20: Confirmation of LoD* and precision in clinical specimens for CDx alterations

| Target Alteration | LoD^{1} | Mean Level Tested^{2} | Reproducibility (95% CI) | 95% CIs (%) |
| --- | --- | --- | --- | --- |
| BRCA1 E23fs*17 | 0.38% VAF | 0.66% VAF | 100 | (85.75, 100) |
| BRCA1 Q780* | 0.34% VAF | 1.11%VAF | 100 | (85.75, 100) |
| BRCA1 Rearrangement | 0.87% VAF | 0.87% VAF | 100 | (85.75, 100) |
| BRCA2 799G>T | 0.40% VAF | 0.50% VAF | 91.6 | (73.0, 98.97) |
| BRCA2 Loss | 48.1% TF | 39.43%TF | 87.5 | (67.64, 97.34) |
| BRCA2 S2988fs*12 | 0.36% VAF | 1.07% VAF | 100 | (85.75, 100) |
| BRCA2- EDA Truncation | 0.48% VAF | 0.48% VAF | 100 | (85.18, 100) |
| EGFR E746_A750del | 0.27% VAF | 0.34% VAF | 100 | (84.56, 100) |
| EGFR L858R | 0.34% VAF | 1.64% VAF | 100 | (85.75, 100) |
| EGFR L858R | 0.34% VAF | 0.64% VAF | 100 | (85.75, 100) |

1 Estimated LoD levels reported in Table 14.
2 The accuracy of %VAF/%TF have not been analytically validated

As observed in the tables above (19, 20, and 21), several BRCA2 positive samples (c.799G>T and c.9097_9098insA, and a BRCA2 loss) demonstrated poor performance for both repeatability and reproducibility. For the BRCA2 specimen harboring the c.799G>T, upon investigation the average %VAF was determined to be 0.5% near the LoD of 0.4% for this variant type. The BRCA2 c.9097_9098insA variant had a 93% hit rate at the highest level tested in the LoD study, 1.16% VAF, indicating that the levels evaluated in this precision analysis were below the LoD for this variant. This variant is an insertion of an A in a highly repetitive homopolymer region of eight As, which impacts sensitivity. The replicates for the clinical sample harboring the BRCA2 loss were processed at 24 ng cfDNA input, below the minimum cfDNA input of 30 ng.

In general most of the targeted variants were tested at levels higher than near 1x LoD; therefore the tested LoD level values (%VAF/%TF) are actually considered to be the confirmed LoD. A post-market study is planned to demonstrate precision using samples at near the estimated LoD.

A second study with 10 samples targeting variants at 1-1.5x LoD was performed to confirm LoD and precision in clinical specimens. Similar to above, each sample was divided into 24 aliquots, with 12 duplicates being processed on the same plate under the same conditions. Each sample was

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tested across 24 replicates Six samples were included in the primary analysis for samples with ≥30 ng DNA input. Three had 100% reproducibility, one had 95.7% reproducibility, one had 91.7% reproducibility, and one had 91.3% reproducibility. The other four samples had a majority of sample replicates with DNA input <30 ng. A summary of the Confirmation of LoD and Precision results for CDx alterations are provided in Table 21.

Table 21: Confirmation of LoD and precision in clinical specimens for CDx alterations

| Target Alteration | LoD | Mean Level Tested^{1} | Reproducibility (95% CI) | 95% CIs (%) |
| --- | --- | --- | --- | --- |
| BRCA1 1395T>A | 0.34% | 0.51% | 100% | [86.2%, 100%] |
| BRCA2 5351_5352insA | 0.36% | 0.34% | 87.5% | [69.0%, 95.7%] |
| EGFR 2235_2249del | 0.27% | 0.45% | 95.7% | [79.0%, 99.2%] |

1 The accuracy of %VAF/%TF have not been analytically validated.

As summarized in Table 21 above, both CDx variants with ≥30 ng DNA input had reproducibility ≥95% with the exception of one variant (BRCA2 5351_5352insA) which was tested at a variant allele fraction below the LoD.

c. Tumor Mutation Profiling Variants:

Across 39 unique samples, including 8 contrived samples, and 31 clinical samples, a total of 1,126 variants were evaluated with variant types including substitutions and indels. The number of variants in each variant bin are summarized in Table 22.

Table 22: Number of each variant type

| Variant Category | N |
| --- | --- |
| Substitutions | 898 |
| Indels | 228 |
| Total | 1126 |

The overall repeatability for all short variants was 99.51% with 95% 2-sided exact CIs (99.49%, 99.53%). The repeatability result for each variant type are summarized in Table 23.

Table 23: Assessment of repeatability of tumor mutation profiling variants* per type

| Variant Type | # Concordant Pairs | # Total Pairs | Repeatability (%) | 95% CIs (%) |
| --- | --- | --- | --- | --- |
| Substitution | 498765 | 501084 | 99.54 | (99.52, 99.56) |
| Indels | 126475 | 127224 | 99.41 | (99.37, 99.45) |

*Variant result totals include variants classified as VUS or benign.

The overall reproducibility results were 99.62% with the 95% 2-sided exact CIs (99.61%, 99.63%). The reproducibility result for each variant type are summarized Table 24.

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Table 24: Assessment of reproducibility of tumor mutation profiling variants* per type

| Variant Type | # of Concordant Replicates | # of Total Replicates | Reproducibility (%) | 95% CIs (%) |
| --- | --- | --- | --- | --- |
| Substitution | 1002981 | 1006658 | 99.63 | (99.62, 99.65) |
| Indels | 254509 | 255588 | 99.58 | (99.55, 99.60) |

*Variant result totals include variants classified as VUS or benign.

d. Reagent Lot-to-Lot Reproducibility:
Three lots of critical reagents were assessed in a factorial design. Reagents were evaluated as internally prepared kits for each process step (LC, HC, sequencing). The variant level pairwise average positive agreement (APA) and average negative agreement (ANA) among three reagent lots and the corresponding 95% confidence interval per sample were calculated. For APA, 43 of 47 samples (91.5%) had APA results above 90%, ranging from 90.11% to 100%. For ANA, 47 of 47 samples (100%) had ANA results above 97%, ranging from 97.5% to 100%.

e. Instrument-to-Instrument Reproducibility:
Two sequencers were assessed in a factorial design. The variant level pairwise APA/ANAs among two sequencers and the corresponding 95% CI per sample were calculated. For APA, 43 of 47 samples (91.5%) had APA results above 90%, ranging from 90.74% to 100%. For ANA, 47 of 47 samples (100%) had ANA results above 97%, ranging from 97.53% to 100%.

f. Reagent Lot Interchangeability:
The interchangeability of critical reagent lots for library construction (LC), hybrid capture (HC) and sequencing within the FoundationOne Liquid CDx assay was evaluated by testing eight (8) contrived samples from either enzymatically fragmented cell line gDNA containing alterations of interest or enzymatically fragmented plasmid DNA. Each of the contrived samples was tested in triplicate using two different lots each of LC, HC, and sequencing reagents. Eight reagent pairings were assessed. A total of eight analyses for each specimen were completed. 192 tests in total were included in this study. Four Master Pool Libraries (MPLs) were evaluated on each of two flowcells on a NovaSeq 6000 sequencer, using two different Sequencing reagent lots. Of the 49 alterations assessed in the sample set, 43 had a percent agreement greater than 90% (39 alterations had percentage agreement equal to 100%, one had percent agreement equal to 95.83%, one had percent agreement equal to 95.65%, and two had percent agreement equal to 91.67%), exceeding the pre-specified acceptance criteria. For the remaining six alterations the observed detection rates for these variants were similar to the predicted detection rate based on the LoD analysis. These results demonstrate the interchangeability of critical reagent lots in the FoundationOne Liquid CDx assay.

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g. Curator Precision:

This study was performed to evaluate the precision of genomic variant call curation, following analysis by the FoundationOne Liquid CDx analysis pipeline. This was established by analyzing targeted alterations, including CDx alterations, and platform-wide alterations within samples used in the FoundationOne Liquid CDx Precision and LoD and Precision Confirmation Study. The study design reflected the intermediate precision design and evaluated curator precision in reporting of targeted and platform alterations. A total of 19 samples were selected for this study. Three curators were chosen randomly amongst all qualified curators to curate variant calls in a set of randomly chosen replicates from each of the 19 samples. The variant calls were generated from each sample per curator. The overall average percent agreement for targeted alterations was 93.3% (95% CI; 83.80%, 98.15%), and for platform genomic alterations was 99.14% (95% CI; 98.47%, 99.57%).

7. Comparability Across Cancer Types:

A large-scale retrospective analysis was performed to demonstrate consistent test performance of FoundationOne Liquid CDx across samples derived from patients with different tumor types based on the performance of two prior versions of the FoundationOne Liquid CDx assay. The FoundationOne Liquid CDx assay was developed based on two versions of the FoundationOne Liquid LDT assay, each of which includes only a subset of the genes included in FoundationOne Liquid CDx. FoundationACT (FACT) included 62 genes where the FoundationOne Liquid LDT included 70 genes. The workflow is substantially similar between the two assays. The test performance of FoundationOne Liquid CDx was evaluated by comparing in-process QC metrics across tumor types using historical data from samples processed in Foundation Medicine's clinical laboratory. In order to support the use of historical data in this study, only those regions commonly baited between the respective versions of the FoundationOne Liquid LDT and the bait set used by FoundationOne Liquid CDx were included in the analysis.

The sample set for this analysis included 19,868 distinct samples from 25 tumor type categories that had previously been tested using the Foundation Medicine FoundationOne Liquid LDT and FACT assays, previous versions of FoundationOne Liquid CDx. Table 25 below provides a summary of the tissue types included in the study. Overall, 98.1% samples yielded ≥25 ng cfDNA, which corresponds to a cfDNA input mass of 20 ng for library construction (LC). A total of 89.1% of samples yielded ≥36 ng of cfDNA which corresponds to…