← Product Code PQP · P200006

# FoundationOne Liquid CDx (F1 Liquid CDx) (P200006)

_Foundation Medicine, Inc. · PQP · Oct 26, 2020 · Pathology · APCB_

**Canonical URL:** https://fda.innolitics.com/device/P200006

## Device Facts

- **Applicant:** Foundation Medicine, Inc.
- **Product Code:** PQP
- **Decision Date:** Oct 26, 2020
- **Decision:** APCB
- **Device Class:** Class 3
- **Review Panel:** Pathology

## 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 in three (3) genes, and copy number alterations in three (3) genes. 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 to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. 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.

## Device Story

FoundationOne® Liquid CDx is a single-site, laboratory-based NGS assay. It processes cfDNA isolated from plasma derived from peripheral whole blood. The workflow involves cfDNA extraction, library construction, hybrid-capture of 324 cancer-related genes, and sequencing on the Illumina NovaSeq 6000. Proprietary software analyzes sequence data to detect genomic alterations (substitutions, indels, rearrangements, copy number variants). The output is a report identifying specific genomic alterations, which qualified healthcare professionals use to determine patient eligibility for targeted therapies (e.g., rucaparib, alectinib, alpelisib) or for general tumor mutation profiling. The device aids clinical decision-making by identifying actionable biomarkers, potentially benefiting patients by matching them to appropriate targeted therapies. If plasma results are negative for companion diagnostic biomarkers, reflex testing to an FDA-approved tumor tissue test is recommended.

## Clinical Evidence

Clinical evidence based on three bridging studies using residual plasma from clinical trials (SOLAR-1 for PIK3CA/alpelisib, BFAST for ALK/alectinib, ARIEL2 for BRCA1/2/rucaparib). Studies demonstrated concordance between FoundationOne® Liquid CDx and clinical trial assays (CTA). For PIK3CA, PPA 71.7%, NPA 100%; HR 0.46 for PFS. For ALK, PPA 84.0%, NPA 100%; ORR 88.9%. For BRCA1/2, PPA 100% (primary efficacy population), ORR 53.8%. Analytical validation included >7,000 replicates, >31,000 variants, and >30 tumor types. Post-market studies required to confirm performance.

## Technological Characteristics

NGS-based in vitro diagnostic using targeted high-throughput hybridization-based capture. Targets 311 genes (309 coding, 2 non-coding). Materials: cfDNA isolated from plasma using KingFisher Flex Magnetic Particle Processor. Sequencing: Illumina NovaSeq 6000 (SBS chemistry). Connectivity: Single-site assay performed at Foundation Medicine, Inc. Software: Proprietary analysis pipeline (BWA, Samtools, Picard, Biopython). Sterilization: Not applicable (laboratory service).

## 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

- cobas® EGFR Mutation Test v2
- Guardant360 CDx
- therascreen PIK3CA RGQ PCR test
- FoundationACT (FACT)
- FoundationOne® Liquid LDT
- VENTANA ALK (D5F3) IHC test

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
> Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a PMA](https://innolitics.com/services/regulatory/), [a 510(k)](https://innolitics.com/services/510ks/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/).

{0}

# SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

## I. GENERAL INFORMATION

Device Generic Name: Next generation sequencing oncology panel, somatic or germline variant detection system

Device Trade Name: FoundationOne® Liquid CDx (F1 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: P200006

Date of FDA Notice of Approval: October 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.

The FoundationOne® Liquid CDx was approved on August 26, 2020 as a companion diagnostic for BRCA1 and BRCA2 alterations in metastatic castration-resistant prostate cancer (mCRPC) patients who may benefit from treatment with RUBRACA® (rucaparib) and EGFR activating mutations (Exon 19 deletions and L858R substitution mutation) in patients with advanced and metastatic non-small cell lung cancer (NSCLC) who may benefit from treatment with IRESSA® (gefitinib), TAGRISSO® (osimertinib), and TARCEVA® (erlotinib).

The current PMA was submitted to include the intended use of FoundationOne® Liquid CDx as a companion diagnostic for the indications listed in the table below:

New Indications Being Sought in this PMA submission.

|  Biomarker(s) Detected | Therapy | Tumor Type  |
| --- | --- | --- |
|  BRCA1 and BRCA2 alterations | RUBRACA® (rucaparib) | Ovarian Cancer  |
|  ALK Rearrangements | ALECENSA® (alectinib) | NSCLC  |
|  PIK3CA mutations | PIQRAY® (alpelisib) | Breast Cancer  |

PMA P200006: FDA Summary of Safety and Effectiveness Data

{1}

# 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 in three (3) genes, and copy number alterations in three (3) genes. 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.

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)  |
|   |  ALK Rearrangements | ALECENSA® (alectinib)  |
|  Prostate cancer | BRCA1, BRCA2 alterations | RUBRACA® (rucaparib)  |
|  Ovarian Cancer | BRCA1, BRCA2 alterations | RUBRACA® (rucaparib)  |
|  Breast Cancer | PIK3CA mutations C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y | PIQRAY® (alpelisib)  |

Additionally, FoundationOne Liquid CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms.

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

PMA P200006: FDA Summary of Safety and Effectiveness Data

{2}

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-approved 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 three genes (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 genes, copy number variants in three genes, and genomic rearrangements in three genes. A subset of targeted regions in 75 genes is baited for increased sensitivity.

Table 2: Genomic Regions in which Variants are Reported by FoundationOne® Liquid¹

|  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  |

PMA P200006: FDA Summary of Safety and Effectiveness Data
3 of 64

{3}

PMA P200006: FDA Summary of Safety and Effectiveness Data
4 of 64

|  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
[Exons 3, 4, 6, 7, 10, 14, 15, 17] | TGFBR2  |
|  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  |

{4}

|  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 |   |

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.

The reporting of rearrangements and copy number alterations are restricted to those genes included in Table 3, below.

Table 3: Genes Containing Copy Number Alterations and Rearrangements Detected and Reported by the FoundationOne® Liquid CDx

|  Alteration Type | Genes  |
| --- | --- |
|  Copy Number Alterations | BRCA1, BRCA2, ERBB2  |
|  Rearrangements | ALK, BRCA1, BRCA2  |

The test report includes variants reported in the following categories; see Table 4:

Table 4. Category Definitions

PMA P200006: FDA Summary of Safety and Effectiveness Data

{5}

|  Category | FoundationOne® Liquid CDx |   |   | Comments  |
| --- | --- | --- | --- | --- |
|   |  Prescriptive use for a Therapeutic Product | Clinical Performance | Analytical Performance  |   |
|  Category 1: Companion Diagnostic (CDx) | Yes | Yes | Yes | ctDNA biomarkers linked to the safe and effective use of the corresponding therapeutic product, for which FoundationOne® Liquid CDx has demonstrated clinical performance shown to support therapeutic efficacy and strong analytical performance for the biomarker.  |
|  Category 2: ctDNA Biomarkers with Strong Evidence of Clinical Significance in ctDNA | No | No | Yes | ctDNA biomarkers with strong evidence of clinical significance presented by other FDA-approved liquid biopsy companion diagnostics for which FoundationOne® Liquid CDx has demonstrated analytical reliability but not clinical performance.  |
|  Category 3A: Biomarkers with Evidence of Clinical Significance in tissue supported by strong analytical validation using ctDNA | No | No | Yes | ctDNA biomarkers with evidence of clinical significance presented by tissue-based FDA-approved companion diagnostics or professional guidelines for which FoundationOne® Liquid CDx has demonstrated analytical performance including analytical accuracy, and concordance of blood-based testing to tissue-based testing for the biomarker.  |
|  Category 3B: Biomarkers with Evidence of Clinical Significance in tissue supported by analytical validation using ctDNA | No | No | Yes | ctDNA biomarkers with evidence of clinical significance presented by tissue-based FDA-approved companion diagnostics or professional guidelines for which FoundationOne® Liquid has demonstrated minimum analytical performance including analytical accuracy.  |
|  Category 4: Other Biomarkers with Potential Clinical Significance | No | No | Yes | ctDNA biomarkers with emergent evidence based on peer-reviewed publications for genes/variants in tissue, variant information from well-curated public databases, or in-vitro pre-clinical models, for which FoundationOne® Liquid CDx has demonstrated minimum analytical performance.  |

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

PMA P200006: FDA Summary of Safety and Effectiveness Data

{6}

PMA P200006: FDA Summary of Safety and Effectiveness Data
7 of 64

# Instruments

The FoundationOne® Liquid CDx assay is intended to be performed with the serial number-controlled instruments indicated in Table 5, below. All instruments are qualified by Foundation Medicine, Inc. (Foundation Medicine or FMI) under Foundation Medicine's Quality System.

Table 5: 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 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

{7}

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 from 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, 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 flow cell (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.

PMA P200006: FDA Summary of Safety and Effectiveness Data
8 of 64

{8}

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.

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.

PMA P200006: FDA Summary of Safety and Effectiveness Data
9 of 64

{9}

PMA P200006: FDA Summary of Safety and Effectiveness Data
10 of 64

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.

I. CDx Classification Criteria

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

The CDx classification criteria and the list of BRCA1/BRCA2 missense mutations for rucaparib, based on the trial prespecifications are described in Table 6 and Table 7; however, not all of the missense mutations listed below were observed in the TRITON2, ARIEL2, and Study 10 clinical studies.

Table 6: 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  |

{10}

|  Large protein truncating rearrangements | Sequence analysis identifies protein truncating rearrangements  |
| --- | --- |
|  Deleterious missense mutations | Curated list (Table 7)  |

Table 7: 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 |  |  |  |   |

2. CDx classification criteria for EGFR alterations:
- Base substitutions resulting in EGFR L858R
- In-frame deletions occurring within EGFR Exon 19

3. ALK rearrangements to identify patients eligible for treatment with ALECENSA® (alectinib):
CDx positivity for an ALK rearrangement is based on the following variant classification criteria:
- The ALK rearrangement must have pathogenic driver status (FMI driver status of "known" or "likely")
- AND the disease type must be NSCLC
- AND one of the following two conditions must hold:
1. The partner gene is EML4, or
2. The ALK breakpoint occurs within ALK intron 19

VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are FDA-approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using cfDNA isolated from plasma samples, as listed in Table 1 of the FoundationOne® Liquid CDx intended use statement. The approved CDx tests are listed in Table 8, below; for additional details see FDA List of Cleared or Approved

PMA P200006: FDA Summary of Safety and Effectiveness Data

{11}

Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.

Table 8: FDA-approved companion diagnostic (CDx) alternatives

|  Biomarker(s) Detected | Device | Company | Technology | Therapy | Indication  |
| --- | --- | --- | --- | --- | --- |
|  EGFR: Exon 19 deletions & L858R substitution | cobas® EGFR Mutation Test v2 | Roche Molecular Systems, Inc. | Polymerase Chain Reaction (PCR) | TARCEVA® (erlotinib), TAGRISSO® (osimertinib), and IRESSA® (gefitinib) | NSCLC  |
|   |  FoundationOne® Liquid CDx | Foundation Medicine, Inc. | Next-Generation Sequencing (NGS)  |   |   |
|   |  Guardant360 CDx | Guardant Health, Inc. | NGS | TAGRISSO® (osimertinib)  |   |
|  BRCA1/BRCA2 | FoundationOne® Liquid CDx | Foundation Medicine, Inc. | NGS | RUBRACA® (rucaparib). | metastatic castration-resistant prostate cancer (mCRPC)  |
|  PIK3CA: C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y | therascreen PIK3CA RGQ PCR test | QIAGEN, Inc. | PCR | PIQRAY® (alpelisib) | Breast Cancer  |

There are no FDA-approved CDx alternatives for the detection of genomic alterations of BRCA1 or BRCA2 for the identification of ovarian cancer patients eligible for treatment with RUBRACA® (rucaparib) nor for the identification of ALK rearrangements in patients with metastatic NSCLC for treatment with Alecensa® (alectinib).

## 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 was approved on August 26, 2020 for the detection of genomic alterations of BRCA1 or BRCA2 for the identification of prostate cancer patients eligible for treatment with RUBRACA® (rucaparib) and the detection of EGFR Exon 19 deletions (Exon 19del) and L858R substitutions in plasma obtained from patients with advanced and metastatic NSCLC for treatment with TARCEVA® (erlotinib), TAGRISSO® (osimertinib), and IRESSA® (gefitinib). The FoundationOne®

PMA P200006: FDA Summary of Safety and Effectiveness Data

{12}

Liquid CDx assay was also approved for 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.

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 FDA approved package inserts for RUBRACA® (rucaparib); ALECENSA® (alectinib); and PIQRAY® (alpelisib) 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 and performed as described in the Summary of Safety and Effectiveness Data for P190032. Table 9 below provides 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.

Due to the lack of sufficient volume of clinical specimens, some of the studies used contrived samples, which consisted of enzymatically sheared cell line DNA spiked into human plasma and diluted with cfDNA isolated from healthy donor plasma. A contrived sample functional characterization (CSFC) study (Section IX.A.1) was conducted to demonstrate comparable performance of sheared cell line DNA samples as compared to cfDNA isolated from plasma specimens obtained from cancer positive patient specimens. Clinical specimens were used to assess analytical accuracy, precision and confirmation of the estimated limit of detection (LoD), and evaluate sample stability.

PMA P200006: FDA Summary of Safety and Effectiveness Data

{13}

The validation studies included >7,000 sample replicates, >31,000 unique variants, >30 tumor types, representing all 311 genes targeted by the assay. Please refer to the Summary of Safety and Effectiveness (SSED) for P190032 for the representation of tumor types and variants included in the original device approval.

Table 9: Representation of tumor types and variants* across validation studies

|  Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # of Unique Genes | # of Unique  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |  Subs | Indels | Rearrang. | Copy Number Amplif. | Copy Number Losses  |
|  Contrived Sample Functional Characterization (CSFC) Study | Breast cancer Colorectal cancer Lung cancer Contrived samples | 13 | 1843 | 228 | 563 | 81 | 11 | 1 | 1  |
|  FoundationOne Liquid CDx to Validated NGS Tumor Tissue Test Concordance: BRCA1 and BRCA2 Variants | Prostate cancer Ovarian cancer | 279 | N/A | 2 | 100 | 87 | 9 | 0 | 2  |
|  FoundationOne Liquid CDx to Validated NGS cfDNA Assay Concordance: PIK3CA mutations | Breast cancer | 412 | N/A | 1 | 32 | 5 | 0 | 0 | 0  |
|  Orthogonal Concordance | 23 cancer types Contrived samples | 278 | N/A | 64 | 541 | 12 | 11 | 3 | 0  |
|  LoD Estimation | Prostate Contrived samples | 10 | 877 | 286 | 1490 | 247 | 32 | 13 | 3  |
|  LoB | Healthy Donors | 28 | 79 | 322 | 26134 | 4482 | 911 | 222 | 42  |
|  Potentially Interfering Substances | Contrived samples | 9 | 336 | 18 | 16 | 11 | 11 | 1 | 2  |
|  Hybrid Capture Bait Specificity | 25 cancer types Contrived samples | 3546 | N/A | 324 | N/A | N/A | N/A | N/A | N/A  |
|  Reagent Stability | Contrived samples | 8 | 142 | 279 | 1090 | 215 | 32 | 17 | 2  |
|  Reagent Interchangeability | Contrived samples | 8 | 192 | 20 | 15 | 11 | 11 | 1 | 1  |
|  Precision study 1 | Breast cancer Colon cancer Lung cancer Ovarian cancer Prostate cancer Skin cancer Contrived samples | 47 | 1121 | 280 | 900 | 229 | 63 | 49 | 5  |

PMA P200006: FDA Summary of Safety and Effectiveness Data

{14}

|  Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # of Unique Genes | # of Unique  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |  Subs | Indels | Rearrang. | Copy Number Amplif. | Copy Number Losses  |
|  Precision study 2 | Lung cancer Prostate cancer Stomach cancer Colorectal cancer Bile duct cancer Breast cancer | 10 | 230 | 6 | 6 | 4 | 0 | 0 | 0  |
|  DNA Extraction | Colorectal cancer Prostate cancer Breast cancer Lung cancer Skin cancer | 6 | 72 | 161 | 265 | 53 | 2 | 0 | 0  |
|  Whole Blood Sample Stability | Lung cancer Colorectal cancer Prostate cancer Breast cancer | 11 | 22 | 66 | 75 | 15 | 1 | 0 | 0  |
|  Inverted Tube Whole Blood Sample Stability | Lung cancer Colorectal cancer Breast cancer Ovarian cancer Prostate cancer | 130 | 260 | 237 | 594 | 91 | 5 | 5 | 0  |
|  Cross Contamination | Contrived samples | 5 | 376 | 39 | 9 | 5 | 4 | 21 | 1  |
|  Guard Banding | Contrived samples | 10 | 375 | 20 | 17 | 12 | 12 | 1 | 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 | 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 | 0 | 1  |
|  Clinical validation study for detection of deleterious alterations in BRCA1 and BRCA2 in ovarian | Ovarian cancer | 217 | N/A | 2 | 48 | 49 | 3 | 0 | 0  |

PMA P200006: FDA Summary of Safety and Effectiveness Data
15 of 64

{15}

|  Study Title | Cancer Types Represented | # Unique Samples | # of Sample Replicates | # of Unique Genes | # of Unique  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  |  |  |  | Subs | Indels | Rearrang. | Copy Number Amplif. | Copy Number Losses  |
|  cancer |  |  |  |  |  |  |  |  |   |
|  Clinical validation study for detection of PIK3CA mutations in breast cancer | Breast | 359 | N/A | 1 | 28 | 4 | 0 | 0 | 0  |
|  Clinical validation study for ALK rearrangements in NSCLC | Lung cancer | 249 | N/A | 1 | 13 | 1 | 11 | 1 | 0  |
|  Blood Collection Tube Equivalence | Ovarian cancer
Breast cancer
Colorectal cancer
Prostate cancer
Lung cancer
Skin cancer
Stomach cancer | 60 | 192 | 116 | 135 | 39 | 13 | 5 | 0  |
|  Automation Line Equivalence | Contrived samples | 8 | 187 | 303 | 1926 | 337 | 63 | 61 | 4  |
|  Variant Report Curation | Breast cancer
Colorectal cancer
Lung cancer
Prostate cancer
Skin cancer | 19 | 57 | 183 | 300 | 104 | 15 | 11 | 2  |
|  Pan-tumor performance (includes historical analysis) | 20 cancer types | 19868 | N/A | 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 | N/A  |
|  FoundationOne Liquid LDT to FoundationOne Liquid CDx Concordance | 25 cancer types | 927 | N/A | 73 | 1815 | 376 | 109 | 46 | N/A  |

*Variant result totals may include variants classified as variants of unknown significance (VUS) or benign.
# FoundationOne Liquid LDT to FoundationOne® Liquid CDx concordance.

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

PMA P200006: FDA Summary of Safety and Effectiveness Data

{16}

sample availability, a postmarket study is planned to confirm the performance of the FoundationOne® Liquid CDx test using intended use clinical specimens.

Actionable alterations were identified in the 39 contrived samples representing 17 genes and included 2 ALK rearrangements, 2 BRCA1 (positive for 2 indels and 1 substitutions), and 3 BRCA2 samples (positive for 5 indels), 5 PIK3CA substitutions, and 2 ERBB2 copy number amplifications. These samples were used to supplement the samples used to support the performance of the ALK, BRCA1, BRCA2, and PIK3CA CDx indications listed in Table 1 as well as the tumor mutation profiling claims which include those genes listed in Table 3.

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. The study was conducted as described in the Summary of Safety and Effectiveness Data for P190032.

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.

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, rearrangements, gene fusions, and copy number alterations (See Section XIII).

2. Analytical Accuracy/Concordance with an Orthogonal Method:

a. Concordance data for CDx-associated alterations:
i. Comparison with Validated NGS Plasma-Based Assay:
Additional data was provided to that included in the Summary of Safety and Effectiveness Data for P190032 for the assessment of analytical accuracy/concordance with a validated NGS plasma-based assay for rearrangements, including gene fusions, and copy number alterations. 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. The study included samples selected from clinical FoundationOne® Liquid testing and contrived samples to represent rare alterations.

For assessment of alterations that would be classified as rearrangements, FoundationOne® Liquid CDx was compared with the orthogonal method.

PMA P200006: FDA Summary of Safety and Effectiveness Data
17 of 64

{17}

The samples included 3 ALK rearrangements of which one was discordant and not called by the orthogonal method and one was excluded/filtered for either the gene or the partner not being included in the region interrogated by comparator assay. Twelve ERBB2 amplified clinical samples and one contrived sample were also compared and found to be concordant with the orthogonal NGS test. A summary of the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) results are shown in the table below.

Table 10. Concordance of CDx alterations called between FoundationOne® Liquid CDx and the comparator assay (n = 74)

|  Targeted Alteration | N | PPA (95% CI) | NPA (95% CI)  |
| --- | --- | --- | --- |
|  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%)  |
|  PIK3CA substitutions | 49 | 100% (92.7%-100.0%) | 100% (99.9%-100.0%)  |
|  ALK rearrangements | 1 | 100% (2.5%-100.0%) | 99.9% (99.7%-100.0%)  |

Since adequate samples from all CDx indications were not represented in the above study either due to sample limitations or due to inadequate coverage of panel space in the orthogonal method. Data from an additional analytical accuracy study will be provided post-market as a condition of approval study (See Section XIII).

ii. Concordance with Orthogonal cfDNA-based NGS Method #2

An additional analytical accuracy study was conducted for breast cancer patients with samples harboring PIK3CA mutations with residual plasma samples from the SOLAR-1 clinical study. Of the 549 residual plasma samples, 542 were previously tested with the externally-validated NGS (evNGS) method and produced valid results. Of the 459 plasma samples available for testing, only 445 samples had sufficient volume ($\geq 2.5\mathrm{mL}$) for testing with the FoundationOne® Liquid CDx test. Of those, 20 were determined to not meet the minimum assay requirement for cfDNA after extraction resulting in 425 evaluable samples.

Of the remaining 425 samples, 7 failed genomic curation due to noise resulting in 418 samples. Of those, only 415 generated valid FoundationOne® Liquid CDx results. Three (3) were excluded after it being determined they were identified as not having been processed on the evNGS method. samples resulting in a final total of 412 samples having produced valid results on both assays. One hundred ninety-two (192) positive variants were detected across 188 patients, with four patients possessing two positive variants each. The distribution of counts per positive variant is listed in Table 11.

PMA P200006: FDA Summary of Safety and Effectiveness Data
18 of 64

{18}

Table 11: Distribution of Variants Detected with FoundationOne® Liquid CDx evaluable samples.

|  Protein Effect in PIK3CA | # Variant Calls (188 Positive Samples)  |
| --- | --- |
|  C420R | 3  |
|  E542K | 25  |
|  E545A | 1  |
|  E545G | 2  |
|  E545K | 50  |
|  H1047L | 9  |
|  H1047R | 100  |
|  H1047Y | 1  |
|  Q546R | 1  |
|  Total | 192  |

A total of 412 valid samples generated valid results with both assays. The primary analysis using comparator assay as the reference assay achieved a PPA (95% CI) of 97.1% (93.3%, 99.0%), and an NPA (95% CI) of 91.7% (87.5%, 94.9%) and Overall Percent Agreement (OPA) (95% CI) of 93.9% (91.2%, 96.0%). The contingency table for this comparison is provided in Table 12 below, with counts representing number of samples (versus number of variant calls).

Table 12: Contingency Table Comparing FoundationOne® Liquid CDx with the Reference Assay, Primary Analysis with 412 Cases

|  FoundationOne® Liquid CDx | NGS Comparator #2  |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   |  Positive | Negative | Non-evaluable | Missing | Total  |
|  Positive | 165 | 20 | 2 | 1 | 188  |
|  Negative | 5 | 222 | 1 | 2 | 230  |
|  Invalid | 0 | 7 | 0 | 0 | 7  |
|  Total | 170 | 249 | 3 | 3 | 425  |

The agreement calculations, relative to the orthogonal method were:
- PPA: 97.1% (93.3%, 99.0%)
- NPA: 91.7% (87.5%, 94.9%)
- OPA: 93.9% (91.2%, 96.0%)

3. Analytical Sensitivity:

a. Limit of Blank (LoB):

See Summary of Safety and Effectiveness Data for P190032.

PMA P200006: FDA Summary of Safety and Effectiveness Data

{19}

A post-market LoB study will be conducted to confirm the results in accordance with the FoundationOne® Liquid CDx assay workflow (See Section XIII).

b. Limit of Detection (LoD):

The LoD study was performed as described in the Summary of Safety and Effectiveness Data for P190032. The LoD by hit rate was defined as the variant allelic fraction (VAF) value (for short variants and rearrangements) or mean tumor fraction (TF) 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.

The median estimated LoD for CDx alterations are presented in Table 13. The estimated LoD for ERBB2 copy number amplification included under the tumor profiling claim was 19.8% TF. The median LoD for targeted short variant, rearrangement, and copy number alterations were consistent with the platform LoD.

Table 13. LoD estimation for CDx alterations

|  Gene | Alteration Subtype | # Samples Evaluated | Median LoD^{1}  |
| --- | --- | --- | --- |
|  BRCA1 | Substitutions | 8 | 0.34% VAF  |
|   |  Indels | 1 | 0.38% 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  |
|  ALK | ALK-EML4 Rearrangement^{3} | 1 | 0.24% VAF  |
|   |  NPM1-ALK Rearrangement | 1 | 0.94% VAF  |
|  PIK3CA | Substitutions | 6 | 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 15 and 16 for confirmed LoD values).

1 The accuracy of %VAF/%TF has not been analytically validated.
2 The LoD for these alterations were 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

PMA P200006: FDA Summary of Safety and Effectiveness Data
20 of 64

{20}

region. The median LoD for rearrangements was estimated to be 0.37% for the enhanced sensitivity region and 0.9% for the standard sensitivity region.

4. Analytical Specificity:
a. Potentially Interfering Substances:
See Summary of Safety and Effectiveness Data for P190032.
b. Hybrid Capture Bait Specificity:
See Summary of Safety and Effectiveness Data for P190032.

5. Carryover/Cross-Contamination:
See Summary of Safety and Effectiveness Data for P190032.

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. Also see the Summary of Safety and Effectiveness Data for P190032. The samples representing CDx alterations that are the subject of this PMA are summarized in Table 14.

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 8 BRCA1 or BRCA2 alterations, 1 ALK rearrangements, and 3 PIK3CA mutations to equal, 7 substitutions and indels, 2 rearrangements, and 2 copy number alterations (gains and losses). One lung sample included an ALK-EML4 rearrangement and 3 of the breast cancer specimens included 3 independent PIK3CA mutations (E542K, E545K, and H1047R). The remaining samples included multiple other actionable genes and variant types. The samples representing the CDx alterations are summarized in the table below:

Table 14: CDx Precision Sample Set

PMA P200006: FDA Summary of Safety and Effectiveness Data
21 of 64

{21}

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

The repeatability of CDx alterations is summarized in Table 15 and the reproducibility of CDx alterations is summarized in Table 16.

Table 15: Repeatability of CDx alterations targeted in precision study at  $\geq  1\mathrm{x}$  LoD*

|  Variant Type | Alteration1 | Concordant Pairs | Repeatability (%) | 95% CIs (%) | Level Tested2 | X LoD  |
| --- | --- | --- | --- | --- | --- | --- |
|  Short variant | BRCA1 2338C>T | 12/12 | 100 | (73.5, 100.0) | 1.11% VAF | 3.3  |
|  Short variant | BRCA1 2475delC | 12/12 | 100 | (73.5, 100.0) | 0.61% VAF | 1.6  |
|  Short variant | BRCA1 2475delC | 11/11 | 100 | (71.5, 100.0) | 1.26% VAF | 3.3  |
|  Short variant | BRCA2 5351delA | 12/12 | 100 | (73.5, 100.0) | 1.22% VAF | 3.2  |
|  Short variant | BRCA2 5351delA | 12/12 | 100 | (73.5, 100.0) | 1.85% VAF | 4.9  |
|  Short variant | BRCA2 5351delA | 11/11 | 100 | (71.5, 100.0) | 1.07% VAF | 2.8  |
|  Short variant | BRCA2 5351delA | 12/12 | 100 | (73.5, 100.0) | 2.24% VAF | 5.9  |
|  Short variant | BRCA2 5465_5466insA | 12/12 | 100 | (73.5, 100.0) | 0.92% VAF | 2.4  |
|  Short variant | BRCA2 5465_5466insA | 11/11 | 100 | (71.5, 100.0) | 1.19% VAF | 3.1  |
|  Short variant | BRCA2 8961_8964delGAGT | 12/12 | 100 | (73.5, 100.0) | 1.07% VAF | 2.8  |
|  Short variant | BRCA2 c.799G>T | 10/12 | 83.3 | (51.6, 97.9) | 0.5% VAF | 1.5  |
|  Short variant | BRCA2 c.9097_9098insA | 6/11 | 54.6 | (23.4, 83.3) | 0.71% VAF | 1.9  |

PMA P200006: FDA Summary of Safety and Effectiveness Data

{22}

Table 16: Reproducibility of CDx alterations targeted in precision study at  $\geq 1\mathbf{x}$  LoD*

|  Variant Type | Alteration | Concordant Replicates | Reproducibility (%) | 95% CIs (%) | Level Tested** | xLoD  |
| --- | --- | --- | --- | --- | --- | --- |
|  Short variant | BRCA1 2338C>T | 24/24 | 100 | (85.8, 100.0) | 1.11% VAF | 3.3  |
|  Short variant | BRCA1 2475delC | 24/24 | 100 | (85.8, 100.0) | 0.61% VAF | 1.6  |
|  Short variant | BRCA1 2475delC | 24/24 | 100 | (85.8, 100.0) | 0.93% VAF | 2.4  |
|  Short variant | BRCA1 2612C>TT | 23/23 | 100 | (85.2, 100.0) | 0.51% VAF | 1.3  |
|  Short variant | BRCA1 68_69delAG | 24/24 | 100 | (85.8, 100.0) | 0.66% VAF | 1.7  |
|  Short variant | BRCA1 P871fs*32 | 24/24 | 100 | (85.8, 100.0) | 1.08% VAF | 2.8  |
|  Rearrangement | BRCA1-BRCA1 | 24/24 | 100 | (85.8, 100.0) | 0.87% VAF | 1.0  |
|  Short variant | BRCA2 3599_3600delGT | 24/24 | 100 | (85.8, 100.0) | 0.58% VAF | 1.6  |
|  Short variant | BRCA2 3599_3600delGT | 24/24 | 100 | (85.8, 100.0) | 0.92% VAF | 2.6  |
|  Short variant | BRCA2 4284_4285insT | 24/24 | 100 | (85.8, 100.0) | 0.94% VAF | 2.6  |
|  Short variant | BRCA2 4284_4285insT | 23/23 | 100 | (85.2, 100.0) | 1.26% VAF | 3.5  |
|  Short variant | BRCA2 5351delA | 24/24 | 100 | (85.8, 100.0) | 1.22% VAF | 3.4  |
|  Short variant | BRCA2 5351delA | 24/24 | 100 | (85.8, 100.0) | 1.85% VAF | 5.1  |
|  Short variant | BRCA2 5351delA | 23/23 | 100 | (85.2, 100.0) | 1.07% VAF | 3.0  |
|  Short variant | BRCA2 5351delA | 24/24 | 100 | (85.8, 100.0) | 2.24% VAF | 6.2  |
|  Short variant | BRCA2 5465_5466insA | 24/24 | 100 | (85.8, 100.0) | 0.92% VAF | 2.6  |
|  Short variant | BRCA2 5465_5466insA | 23/23 | 100 | (85.2, 100.0) | 1.19% VAF | 3.3  |
|  Short variant | BRCA2 799G>T | 22/24 | 91.7 | (73.0, 99.0) | 0.5% VAF | 1.4  |

*Several clinical samples were mostly tested at  $2\mathrm{x} - 3\mathrm{x}$  LoD rather than  $1\mathrm{x} - 1.5\mathrm{x}$  LoD
See Table 14 for BRCA1/BRCA2 sample source type
2 The accuracy of  $\% \mathrm{VAF} / \% \mathrm{TF}$  has not been analytically validated.

PMA P200006: FDA Summary of Safety and Effectiveness Data

{23}

|  Variant Type | Alteration | Concordant Replicates | Reproducibility (%) | 95% CIs (%) | Level Tested** | xLoD  |
| --- | --- | --- | --- | --- | --- | --- |
|  Short variant | BRCA2 8961_8964delGAGT | 24/24 | 100 | (85.8, 100.0) | 1.07% VAF | 3.0  |
|  Short variant | BRCA2 9097_9098insA | 22/24 | 91.7 | (73.0, 99.0) | 1.03% VAF | 2.9  |
|  Short variant | BRCA2 c.799G>T | 22/24 | 91.7 | (73.0, 99.0) | 0.5% VAF | 1.4  |
|  Short variant | BRCA2 c.9097_9098insA | 5/23 | 21.7 | (7.5, 43.7) | 0.71% VAF | 2.0  |
|  Short variant | BRCA2 c.9097_9098insA | 22/24 | 91.7 | (73.0, 99.0) | 1.03% VAF | 2.9  |
|  Copy Number Loss | BRCA2 loss | 21/24 | 87.5 | (67.6, 97.3) | 39.43% TF | 0.8  |
|  Rearrangement | BRCA2-EDA Truncation | 23/23 | 100 | (85.8, 100.0) | 0.48% VAF | 1.0  |
|  Rearrangement | ALK-EML4 | 24/24 | 100 | (85.8, 100.0) | 0.64% VAF | 2.7  |
|  Rearrangement | ALK-EML4 | 23/23 | 100 | (85.8, 100.0) | 0.89% VAF | 3.7  |
|  Rearrangement | ALK-EML4 | 24/24 | 100 | (85.8, 100.0) | 1.39% VAF | 5.8  |
|  Rearrangement | ALK-NPM1 | 24/24 | 100 | (85.8, 100.0) | 0.64% VAF | 0.7  |
|  Rearrangement | ALK-NPM1 | 18/23 | 78.3 | (56.3, 92.5) | 0.4% VAF | 0.4  |
|  Short variant | PIK3CA E542K | 24/24 | 100 | (85.8, 100.0) | 0.89% VAF | 2.6  |
|  Short variant | PIK3CA E545K | 24/24 | 100 | (85.8, 100.0) | 0.45% VAF | 1.3  |
|  Short variant | PIK3CA E545K (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.66% VAF | 1.9  |
|  Short variant | PIK3CA E545K (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.5% VAF | 1.5  |
|  Short variant | PIK3CA E545A (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.52% VAF | 1.5  |
|  Short variant | PIK3CA E545A (contrived) | 23/23 | 100 | (85.2, 100.0) | 0.70% VAF | 2.1  |
|  Short variant | PIK3CA Q546R (contrived) | 22/23 | 95.7 | (78.1, 99.9) | 0.49% VAF | 1.4  |
|  Short variant | PIK3CA Q546R (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.92% VAF | 2.7  |
|  Short variant | PIK3CA D549N (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.48% VAF | 1.4  |
|  Short variant | PIK3CA D549N (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.73% VAF | 2.1  |
|  Short variant | PIK3CA H1047R | 23/23 | 100 | (85.2, 100.0) | 0.41% VAF | 1.2  |
|  Short variant | PIK3CA H1047R (contrived) | 24/24 | 100 | (85.8, 100.0) | 0.76% VAF | 2.2  |
|  Short variant | PIK3CA H1047R (contrived) | 24/24 | 100 | (85.8, 100.0) | 1.04% VAF | 3.1  |

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

For repeatability, of the ALK fusion/rearrangements and PIK3CA samples assessed in this PMA, 93.8% (15/16) samples demonstrated 100% repeatability. Four BRCA2 samples demonstrated repeatability below 95% (54.6% - 91.7%). The BRCA2 loss was tested at an 39.4% TF below the estimated LoD of 48.1% TF and used a cfDNA input below the recommended cfDNA input of 30 ng. Of the remaining 3 poorly performing samples, only one was at a % VAF (0.5% VAF) near the estimated LoD (0.37% VAF), while the remaining 2 were tested at levels higher than the estimated LoDs for each sample. Therefore, the reason for the observed performance is not clear. One PIK3CA Q546R sample (tested at 1.4x LoD) demonstrated a repeatability of 90.9%. All 3 ERBB2 amplified samples, 1 contrived (tested at 35.8%, and 39.8% TF) and 1 clinical (tested at 61.7% TF) demonstrated 100% reproducibility.

Reproducibility of 100% was observed in 16/18 (88.9%) alterations.

PMA P200006: FDA Summary of Safety and Effectiveness Data

{24}

A single contrived ALK-NPM1 fusion/rearrangement demonstrated poor repeatability at 54.6% and reproducibility of 78.3% due to the sample being tested below the estimated VAF of 0.94%. at 0.4% VAF which is below the estimated LoD of 0.94%. Six BRCA2 samples demonstrated reproducibility below 95% (21.7 – 91.7). The BRCA2 loss was tested at an %TF below the estimated LoD and used a cfDNA input below the recommended cfDNA input of 30 ng. Of the remaining 5 poorly performing samples, only one was at a %VAF (0.5% VAF) near the estimated LoD (0.37% VAF), while the remaining 4 were tested at levels higher than the estimated LoDs for each sample. Therefore, the reason for the observed performance is not clear. All 3 ERBB2 amplified samples, 1 contrived (tested at 35.8%, and 39.8%TF) and 1 clinical (tested at 61.7% TF) demonstrated 100% reproducibility.

b. Confirmation of LoD and Precision in Clinical Specimens: The combined confirmation of LoD and precision study was performed as described in the Summy of Safety and Effectiveness for P190032. In this study, 29 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 loss that had 87.5% reproducibility and 91.7% repeatability. This sample had cfDNA input below the recommended minimum input. The other sample harbored a BRCA2 substitution (c.799G>T) with 91.7% reproducibility and 83.3% repeatability. The average VAF of this variant was 0.5% across replicates, which is near the LoD for this variant type (LoD of 0.37% VAF). A summary of the Confirmation of LoD and precision results for a subset of highly-actionable alterations are provided in Table 17.

Table 17: Confirmation of LoD and precision in clinical specimens

|  Target Alteration | LoD | Mean Level Tested | Reproducibility (95% CI) | 95% CIs (%)  |
| --- | --- | --- | --- | --- |
|  ALK-EML4 Rearrangement | 0.24% VAF | 1.39 %VAF | 100 | (85.8, 100.0)  |
|  BRCA1 E23fs*17 | 0.38% VAF | 0.66% VAF | 100 | (85.8, 100.0)  |
|  BRCA1 Q780* | 0.34% VAF | 1.11%VAF | 100 | (85.8, 100.0)  |
|  BRCA1 Rearrangement | 0.26%-.47% VAF¹ | 0.87% VAF | 100 | (85.8, 100.0)  |
|  BRCA2 S2988fs*12 | 0.36% VAF | 1.07% VAF | 100 | (85.8, 100.0)  |
|  BRCA2- EDA Truncation | 0.26%-.47% VAF¹ | 0.48% VAF | 100 | (85.2, 100.0)  |
|  PIK3CA E542K | 0.34% VAF | 0.89% VAF | 100 | (85.8, 100.0)  |
|  PIK3CA E545K | 0.34% VAF | 0.5% VAF | 100 | (85.8, 100.0)  |
|  PIK3CA H1047R | 0.34% VAF | 1.04% VAF | 100 | (85.8, 100.0)  |
|  ERBB2 CNA | 19.8% TF | 61.73% TF | 100 | (85.8, 100.0)  |

¹ Estimated LoD levels reported in Table 13.
² The accuracy of %VAF/%TF has not been analytically validated

In general, most of the targeted variants were tested at levels higher (or lower) than estimated near LoD (1x); therefore, the tested LoD level values (%VAF/%TF) are considered to be the confirmed LoD. A post-market study

PMA P200006: FDA Summary of Safety and Effectiveness Data
25 of 64

{25}

is planned to demonstrate precision using samples at near the estimated LoD for those tested above or below the estimated LoD (See Section XIII).

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 tested across 24 replicates. Six samples were included in the primary analysis for samples with  $\geq 30$  ng DNA input. Two BRCA substitutions showed  $100\%$  repeatability and reproducibility, one BRCA2 indel (BRCA2 5351_5352insA) had repeatability of  $75.0\%$  and an  $87.7\%$  reproducibility, the PIK3CA Q546R SNV had repeatability of  $8.3\%$  and a  $91.7\%$  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 18.

Table 18: Confirmation of LoD and precision in clinical specimens for CDx alterations

|  Targeted Alteration | Mean Input Mass (ng) | Concordant /Total | Repeatability (%) | Concordant/ Total | Reproducibility (%) | Average VAF (%) | Estimated LoD (VAF %)  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  BRCA1 1395T>A | 32.8 | 12/12 | 100% (75.8%, 100%) | 24/24 | 100% (86.2%, 100%) | 0.51% | 0.34%  |
|  BRCA2 5351_5352insA | 36.6 | 9/12 | 75% (46.8%, 91.1%) | 21/24 | 87.5% (69.0%, 95.7%) | 0.34% | 0.36%  |
|  BRCA2 8524C>T | 31.5 | 11/11 | 100% (74.1%, 100%) | 23/23 | 100% (85.7%, 100%) | 0.59% | 0.49%2  |
|  PIK3CA Q546R | 37.5 | 10/12 | 83.3% (55.2%, 95.3%) | 22/24 | 91.7%3 (74.2%, 97.7%) | 0.44% | 0.34%  |

1 The accuracy of  $\% \mathrm{VAF} / \% \mathrm{TF}$  has not been analytically validated.

As summarized in Table 18 above, both CDx variants with  $\geq 30$  ng DNA input had reproducibility  $\geq 95\%$  with the exception of one variant (BRCA2 5351_5352insA) which was tested at a  $\%$  VAF just below the LoD.

# c. Tumor Mutation Profiling Variants:

Across 39 unique samples, including 8 contrived samples, and 31 clinical samples, a total of 1,240 variants were evaluated with variant types including 898 substitutions, 228 indels, 60 rearrangements, 49 copy number amplifications, and 5 copy number losses. The overall repeatability for all variants was  $99.5\%$  with  $95\%$  2-sided exact CIs  $(99.5\%, 99.5\%)$ . The repeatability result for each variant type are summarized in Table 19.

Table 19: 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)  |

PMA P200006: FDA Summary of Safety and Effectiveness Data

{26}

|  Variant Type | # Concordant Pairs | # Total Pairs | Repeatability (%) | 95% CIs (%)  |
| --- | --- | --- | --- | --- |
|  Rearrangements | 33105 | 33480 | 98.88 | (98.76, 98.99)  |
|  Copy Number Alterations | 29880 | 30132 | 99.16 | (99.05, 99.26)  |

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

The overall reproducibility results were 99.59% with the 95% 2-sided exact CIs (99.58%, 99.60%). The reproducibility result for each variant type are summarized Table 20.

Table 20: 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)  |
|  Rearrangements | 66723 | 67260 | 99.20 | (99.13, 99.27)  |
|  Copy Number Alterations | 60115 | 60534 | 99.31 | (99.24, 99.37)  |

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

d. Reagent Lot-to-Lot Reproducibility:

See Summary of Safety and Effectiveness Data for P190032.

e. Instrument-to-Instrument Reproducibility:

See Summary of Safety and Effectiveness Data for P190032.

f. Reagent Lot Interchangeability:

See Summary of Safety and Effectiveness Data for P190032.

g. Curator Precision:

See Summary of Safety and Effectiveness Data for P190032.

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 and the FoundationACT (FACT), each of which includes only a subset of the genes included in FoundationOne® Liquid CDx. A summary of this study is found in the Summary of Safety and Effectiveness Data for P190032.

PMA P200006: FDA Summary of Safety and Effectiveness Data

{27}

Additional data was provided for the second analysis that was performed to evaluate the concordance between the FoundationOne® Liquid LDT, FACT, and the FoundationOne® Liquid CDx assays based on the concordance of unique samples processed on both the FoundationOne® Liquid LDT and FoundationOne® Liquid CDx assays positive for additional gene rearrangements. The concordance analysis using FoundationOne® Liquid LDT or FoundationOne® Liquid CDx as the reference assay is summarized by variant category in Table 21.

Samples, sequence, and variant data were drawn from different clinical studies being used to support the approval of the FoundationOne® Liquid CDx assay. Only those regions commonly baited between the assays were included in the analysis. All comparisons were performed using FoundationOne® Liquid LDT results, which have been analyzed using the latest version of the that test's analysis pipeline. As with the study above, for samples processed using the FoundationOne® Liquid LDT and FACT assays, only those regions commonly baited between the respective version of the FoundationOne® Liquid LDT and the bait set used by FoundationOne® Liquid CDx were included in the analysis (and thus the variants contained therein). Copy number losses are not called by the FoundationOne® Liquid LDT and therefore were not considered in the analysis.

Table 21: Concordance* between FoundationOne® Liquid LDT and FoundationOne® Liquid CDx

|  Variant*/ Mutation Type | CDx(+)/ LDT(+) | CDx(-)/ LDT(+) | CDx(+)/ LDT(-) | CDx(-)/ LDT(-) | PPA (95% CI) | NPA (95% CI) | OPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Including VUS Results  |   |   |   |   |   |   |   |
|  All Short Variants | 2871 | 123 | 32 | 1171180 | 95.9% (95.1%, 96.6%) | >99.9% (>99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Base Substitutions | 2415 | 104 | 31 | 999032 | 95.9% (95.0%, 96.6%) | >99.9% (>99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Indels | 456 | 19 | 1 | 172148 | 96.0% (93.8%, 97.6%) | >99.9% (>99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Copy Number Alterations (gains) | 173 | 32 | 110 | 59463 | 84.4% (78.7%, 89.1%) | 99.8% (99.8%, 99.8%) | 99.8% (99.7%-99.8%)  |
|  Rearrangements | 147 | 20 | 24 | 59587 | 88.0% (82.1%, 92.5%) | >99.9% (>99.9%, 100.0%) | 99.9% (99.9%, 99.9%)  |
|  Total | 3191 | 175 | 166 | 1290230 | 94.8% (94.0%, 95.5%) | >99.9% (>99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Excluding VUS Results:  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All Short Variants | 1635 | 66 | 28 | 534382 | 96.1% (95.1%, 97.0%) | >99.9% >99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Base Substitutions | 1264 | 49 | 27 | 400278 | 96.3% (95.1%, 97.2%) | >99.9% >99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Indels | 371 | 17 | 1 | 134104 | 95.6% (93.1%, 97.4%) | >99.9% >99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |
|  Copy Number Alterations (gains) | 155 | 18 | 69 | 59536 | 89.6% (84.1%, 93.7%) | 99.9% (99.9%, 99.9%) | 99.9% (99.8%, 99.9%)  |
|  Rearrangements | 100 | 13 | 16 | 59649 | 88.5% | >99.9% | >99.9%  |

PMA P200006: FDA Summary of Safety and Effectiveness Data

{28}

|  Variant*/ Mutation Type | CDx(+) / LDT(+) | CDx(-) / LDT(+) | CDx(+) / LDT(-) | CDx(-) / LDT(-) | PPA (95% CI) | NPA (95% CI) | OPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  |  |  |  | (81.1%, 93.7%) | >99.9%, 100.0%) | (99.9%, 100.0%)  |
|  Totals | 1890 | 97 | 113 | 653567 | 95.1% (94.1%, 96.0%) | >99.9% >99.9%, 100.0%) | >99.9% (>99.9%, 100.0%)  |

* Concordance was assessed between two versions of the F1 Liquid LDT and F1 Liquid CDx. Only those regions that are commonly baited between the 3 tests were included in the analyses.

The overall PPA between FoundationOne® Liquid LDT and FoundationOne® Liquid CDx assays, with FoundationOne® Liquid LDT as the reference assay, was 95.1% with a 95% two-sided CI of (94.1%, 96.0%). The respective short variant, CNAs, and rearrangement PPA values (excluding VUS results), with 95% two-sided CI, were: 96.1% (95.1%, 97.0%), 89.6% (84.1%, 93.7%), and 88.5% (81.1%, 93.7%). The PPA values when VUS results were included were relatively similar. Despite the study only including regions that were commonly baited between the tests discordances were noted, including those that were identified as being uniquely identified by either test. For short variants (substitutions and small indels), discordant results were due primarily to lower VAF values when tested with the FoundationOne® Liquid CDx. For copy number alterations, the 32 calls reported on FoundationOne® Liquid LDT but not on FoundationOne® Liquid CDx test was primarily due to copy number events that were observed by the analysis pipeline and were near to, but did not meet the ploidy-based copy number threshold. For rearrangements, discordant calls unique to the FoundationOne® Liquid LDT test tended to have lower VAF values and those variant calls with lower VAFs that were closer to the LoD for each assay tended to demonstrate lower concordance. The results from this study support the agreement between FoundationOne® Liquid LDT and FoundationOne® Liquid CDx and the applicability of the tumor comparability analysis performed using historical FoundationOne® Liquid data.

8. Stability:

a. Reagent Stability:

See Summary of Safety and Effectiveness Data for P190032.

b. Stability of cfDNA and Plasma Samples:

See Summary of Safety and Effectiveness Data for P190032.

c. Whole Blood Specimen Stability and Inverted Tube Stability:

See Summary of Safety and Effectiveness Data for P190032.

9. Guard-banding and Robustness:

PMA P200006: FDA Summary of Safety and Effectiveness Data
29 of 64

{29}

a. DNA Extraction:

DNA extraction evaluated 72 samples across five cancer types: lung cancer (including NSCLC), CRC, prostate cancer, breast cancer, and skin cancer (melanoma, sarcoma), using three reagent lots and two KingFisher Magnetic Particle processors.

Reproducibility of the FoundationOne® Liquid CDx DNA extraction process across King Fisher instruments and extraction reagent lots were analyzed utilizing a factorial design (3 reagent lots × 2 KingFisher instruments × 2 replicates). The success rate of the DNA extraction (DNAx) yield for three reagent lots range from 95.8% to 100.0% and two KingFisher instruments ranged from 97.2% to 100.0%.

Variant calls included in the concordance analysis were identified based on the majority call across all 12 replicates for a given disease ontology. Agreements were computed across the replicates for each somatic alteration for each sample, and aggregated by variant type (deletion, insertion, rearrangement, and substitution) for variants at ≥1x LoD. The percent agreements by disease ontologies were from 90.3% to 99.8% for PPA, and 99.1% to 100.0% for NPA (Table 22). The percent agreement results across all variant types (deletion, insertion, rearrangement and substitution) evaluated at ≥1x LoD were from 90.6% to 96.8% for PPA and 98.9% to 100.0% for NPA (Table 23).

Table 22: Concordance summary by disease ontology at ≥1x LoD for cfDNA extraction study

|  Disease Ontology | Positive Detected/ Positive Total* | PPA (95% CI) | Negative Detected/ Negative Total* | NPA (95% CI) | Overall Detected/ Total* | OPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  Breast Cancer | 347/348 | 99.7% (98.4%,100.0%) | 3144/3144 | 100.0% (99.9%,100.0%) | 3491/3492 | 100.0% (99.8%,100.0%)  |
|  CRC | 1122/1188 | 94.4% (93.0%,95.7%) | 2284/2304 | 99.1% (98.7%,99.5%) | 3406/3492 | 97.5% (97.0%,98.0%)  |
|  Lung Cancer | 431/432 | 99.8% (98.7%,100.0%) | 3053/3060 | 99.8% (99.5%,99.9%) | 3484/3492 | 99.8% (99.5%,99.9%)  |
|  NSCLC | 600/612 | 98.0% (96.6%,99.0%) | 2878/2880 | 99.9% (99.7%,100.0%) | 3478/3492 | 99.6% (99.3%,99.8%)  |
|  Prostate Cancer | 486/492 | 98.8% (97.4%,99.6%) | 2987/3000 | 99.6% (99.3%,99.8%) | 3473/3492 | 99.5% (99.2%,99.7%)  |
|  Skin Cancer | 455/504 | 90.3% (87.4%,92.7%) | 2987/2988 | 100.0% (99.8%,100.0%) | 3442/3492 | 98.6% (98.1%,98.9%)  |

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

Table 23: Concordance summary by variant type at ≥1x LoD for cfDNA extraction study

PMA P200006: FDA Summary of Safety and Effectiveness Data
30 of 64

{30}

|  Variant Type | Positive Detected/ Positive Total* | PPA (95% CI) | Negative Detected/ Negative Total* | NPA (95% CI) | Overall Detected/ Total* | OPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- |
|  Deletions | 386/408 | 94.6% (91.9%, 96.6%) | 2036/2040 | 99.8% (99.5%, 99.9%) | 2422/2448 | 98.9% (98.4%, 99.3%)  |
|  Insertions | 163/180 | 90.6% (85.3%, 94.4%) | 819/828 | 98.9% (97.9%, 99.5%) | 982/1008 | 97.4% (96.2%,98.3%)  |
|  Rearrangements | 23/24 | 95.8% (78.9%, 99.9%) | 120/120 | 100.0% (97.0%, 100.0%) | 143/144 | 99.3% (96.2%, 100.0%)  |
|  Substitutions | 2869/2964 | 96.8% (96.1%, 97.4%) | 14358/14388 | 99.8% (99.7%, 99.9%) | 17227/17352 | 99.3% (99.1%, 99.4%)  |

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

These results demonstrate robustness of the FoundationOne® Liquid CDx DNA extraction process across KingFisher instruments, extraction reagent lots, and cancer types.

b. cfDNA Input:

See the Summary of Safety and Effectiveness Data for P190032.

c. Molecular Index Barcode Performance:

See the Summary of Safety and Effectiveness Data for P190032.

d. Automation Line Equivalence:

See the Summary of Safety and Effectiveness Data for P190032.

## B. Animal Studies

Not Applicable.

## C. Additional Studies

Foundation Medicine performed additional studies, including Blood Collection Tube Equivalence, Whole Blood Stability, and Stability of cfDNA and Plasma Samples to support of the clinical validation studies. These studies are described in the Summary of Safety and Effectiveness Data for P190032.

## X. SUMMARY OF PRIMARY CLINICAL STUDIES

Foundation Medicine performed three separate clinical bridging studies to establish a reasonable assurance of safety and effectiveness of the FoundationOne® Liquid CDx for the three new CDx indications being sought. Data from this clinical study were the basis for the PMA approval decision. A summary of the clinical studies are presented below.

PMA P200006: FDA Summary of Safety and Effectiveness Data

{31}

PMA P200006: FDA Summary of Safety and Effectiveness Data
32 of 64

# A. Clinical Bridging Study: Detection of PIK3CA mutations to Determine Eligibility for Treatment with Alpelisib

Clinical validity of using FoundationOne® Liquid CDx to identify breast cancer patients harboring PIK3CA mutations eligible for treatment with alpelisib was assessed through retrospective testing of plasma samples collected prior to study treatment from advanced or metastatic breast cancer patients enrolled in clinical trial CBYL719C2301 (SOLAR-1). Alpelisib was approved under NDA 212526 on May 24, 2019.

# 1. Study Design

SOLAR-1 was a randomized, double-blind, placebo-controlled phase III clinical trial to evaluate the safety and efficacy of alpelisib in combination with fulvestrant for men and postmenopausal women with hormone receptor positive, HER2-negative advanced breast cancer which progressed on or after aromatase inhibitor treatment.

In the device bridging study, all available plasma samples from patients collected at baseline prior to randomization into the Novartis SOLAR-1 clinical trial were tested with FoundationOne® Liquid CDx.

a. Bridging Study Inclusion and Exclusion Criteria

A bridging study was conducted to evaluate: 1) the concordance between PIK3CA mutation status by the clinical trial assays (CTA) and FoundationOne® Liquid CDx, and 2) the clinical efficacy of alpelisib treatment in patients that would be eligible for therapy based on PIK3CA mutation status as determined by FoundationOne® Liquid CDx.

The sample inclusion and exclusion criteria for the retrospective testing of the clinical bridging study were:

Sample inclusion criteria:
- Samples from all randomized patients in the SOLAR-1 trial collected prior to start of SOLAR-1 study treatment
- Availability of adequate sample to generate a CDx test result including ≥ 2.5 mL plasma volume

Sample exclusion criteria:
- Lack of clear subject identification or label on stored patient sample
- Obvious physical damage of stored patient sample
- Insufficient sample (< 2.5 mL)

b. Clinical Endpoints

The primary endpoint for the study was progression-free survival (PFS) using Response Evaluation Criteria in Solid Tumors (RECIST v1.1), based on investigator assessment in advanced or metastatic breast cancer patients

{32}

enrolled with a PIK3CA alteration. Safety and tolerability were evaluated by assessment of type, frequency, and severity of adverse events and laboratory toxicities per Common Terminology Criteria for Adverse Events (CTCAE) v4.03.

## 2. Accountability of PMA Cohort

Of the 572 SOLAR-1 randomized patients [341 PIK3CA-positive and 231 PIK3CA-negative, as determined by the enrolling clinical trial assays (CTA1 or CTA2)], all had either been prospectively enrolled by CTA1 (n = 395) or were enrolled by CTA2 (n = 177). All 395 CTA1 enrolled samples were retrospectively tested with CTA2. Baseline samples from 432 of the 572 patients enrolled in SOLAR-1 were tested using FoundationOne® Liquid CDx, among which 375 (including 12 FoundationOne® Liquid CDx invalids and 4 CTA2 invalids) were tested with DNA input ≥ 30 ng. An additional 57 samples were tested with DNA input ≥ 20 ng and < 30 ng (52 valid results and 5 invalids). Sample accountability for this clinical bridging study is summarized in Table 24.

Table 24: Sample accountability for alpelisib clinical bridging study

|  Description | # Samples  |
| --- | --- |
|  Patients enrolled in SOLAR-1 study | 572  |
|  Available baseline…

---

**Source:** [https://fda.innolitics.com/device/P200006](https://fda.innolitics.com/device/P200006)

**Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices. If you're preparing [a PMA](https://innolitics.com/services/regulatory/), [a 510(k)](https://innolitics.com/services/510ks/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/), [get in touch](https://innolitics.com/contact).

**Cite:** Innolitics at https://innolitics.com