← Product Code [PQP](/productcode/PQP) · P200010S001

# Guardant360 CDx (P200010S001)

_Guardant Health, Inc. · PQP · May 21, 2021 · Pathology · APPR_

**Canonical URL:** https://fda-staging.innolitics.com/device/P200010S001

## Device Facts

- **Applicant:** Guardant Health, Inc.
- **Product Code:** [PQP](/productcode/PQP.md)
- **Decision Date:** May 21, 2021
- **Decision:** APPR
- **Device Class:** Class 3
- **Review Panel:** Pathology
- **Attributes:** Real-World Evidence

## Real-World Evidence

| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
| --- | --- | --- | --- | --- | --- |
| P200010S001 · May 21, 2021 | Guardant360 CDx | Guardant Health, Inc. | Archived plasma samples from the CHRYSALIS clinical trial (NCT02609776); Archived plasma samples from the NILE clinical study (NCT03615443); Guardant Health Biobank clinical samples | Retrospective analysis of archived clinical trial plasma samples was used to bridge the Guardant360 CDx assay to the clinical trial enrollment assays, demonstrating concordance and clinical efficacy for identifying EGFR exon 20 insertion positive patients for treatment with amivantamab-vmjw. | Clinical bridging study; Retrospective analysis; Archived clinical samples; Concordance analysis |

### Clinical Evidence

| Study Design | Population | Comparator | Key Endpoints |
| --- | --- | --- | --- |
| Guardant360 CDx Clinical Bridging Study; Retrospective clinical bridging study; Follow-up/Duration: Median 9.7 months from onset of response; Study Period: Retrospective analysis of archived samples | NSCLC patients with EGFR exon 20 insertions who progressed on or after platinum-based chemotherapy (from CHRYSALIS study); Sample Size: 78 patients from the primary registration population; 261 total samples in agreement analysis; Number of Sites: Multicenter | Clinical trial enrollment assays (local testing) and central tissue NGS/PCR testing | Overall Response Rate (ORR) by RECIST 1.1, concordance (PPA/NPA) |

## Indications for Use

Guardant360® CDx is a qualitative next generation sequencing-based in vitro diagnostic device that uses targeted high throughput hybridization-based capture technology for detection of single nucleotide variants (SNVs), insertions and deletions (indels) in 55 genes, copy number amplifications (CNAs) in two (2) genes, and fusions in four (4) genes. Guardant360® CDx utilizes circulating cell-free DNA (cfDNA) from plasma of peripheral whole blood collected in Streck Cell-Free DNA Blood Collection Tubes (BCTs). The test is intended to be used as a companion diagnostic to identify non-small cell lung cancer (NSCLC) patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. A negative result from a plasma specimen does not assure that the patient's tumor is negative for genomic findings. NSCLC patients who are negative for the biomarkers listed in Table 1 should be reflexed to tissue biopsy testing for Table 1 biomarkers using an FDA approved tumor tissue test, if feasible. Additionally, the test is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for cancer patients with any solid malignant neoplasms. The test is for use with patients previously diagnosed with cancer and in conjunction with other laboratory and clinical findings. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product. Guardant360® CDx is a single-site assay performed at Guardant Health, Inc.

## Device Story

Guardant360® CDx is a single-site NGS-based liquid biopsy test performed at Guardant Health, Inc. It processes 5-30 ng of cfDNA isolated from peripheral whole blood collected in Streck BCTs. The workflow involves library construction, hybrid capture enrichment for 74 genes (reporting on 55), and paired-end sequencing on the Illumina NextSeq 550. A proprietary bioinformatics pipeline reconstructs digitized signals using molecular barcodes to call SNVs, indels, CNAs, and fusions. The output is a qualitative report identifying genomic alterations, used by oncologists to guide targeted therapy selection (e.g., EGFR-directed therapies in NSCLC). It serves as a companion diagnostic; negative plasma results require reflex to tissue biopsy. The device provides tumor mutation profiling for solid malignant neoplasms, aiding clinical decision-making in conjunction with other findings. It benefits patients by enabling non-invasive genomic testing when tissue biopsy is difficult or unavailable.

## Clinical Evidence

Clinical bridging study using retrospective analysis of 78 plasma samples from the CHRYSALIS clinical trial (NCT02609776). Primary efficacy population (n=81) treated with amivantamab-vmjw. Guardant360® CDx demonstrated 80.4% PPA and 100% NPA relative to local enrollment testing. BICR-assessed ORR in the Guardant360® CDx-positive efficacy set (gCEAS, n=62) was 38.7% (95% CI: 26.6%, 51.9%), exceeding the benchmark ORR of 14%.

## Technological Characteristics

NGS-based in vitro diagnostic; uses hybrid capture technology. Analyzes cfDNA from plasma. Instruments: Agilent 4200 TapeStation, Hamilton Microlab STAR/STARlet, Illumina NextSeq 550, Veriti 96-Well Thermal Cycler. Sequencing depth >2,700 unique molecules. Bioinformatics pipeline maps to hg19. Single-site assay performed at Guardant Health, Inc.

## Regulatory Identification

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

## Reference Devices

- cobas® EGFR Mutation Test v2 (Roche Molecular Systems, Inc.)
- FoundationOne® Liquid CDx (Foundation Medicine, Inc.)
- Oncomine Dx Target Test
- FoundationOne® CDx

## Submission Summary (Full Text)

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

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# SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

## I. GENERAL INFORMATION

|  Device Generic Name: | Next Generation Sequencing Oncology Panel, Somatic or Germline Variant Detection System  |
| --- | --- |
|  Device Trade Name: | Guardant360® CDx  |
|  Device Procode: | PQP  |
|  Applicant’s Name and Address: | Guardant Health, Inc. 505 Penobscot Drive Redwood City, CA 94306 USA  |
|  Date(s) of Panel Recommendation: | None  |
|  Premarket Approval Application (PMA) Number: | P200010/S001  |
|  Date of FDA Notice of Approval: | May 21, 2021  |

The original PMA (P200010) for Guardant360® CDx was approved on August 7, 2020 for the detection of genetic alterations in circulating cell-free DNA (cfDNA) from plasma of peripheral whole blood derived from patients who may benefit from one of the FDA-approved therapies for non-small cell lung cancer (NSCLC). The SSED to support the previously approved indication is available on the CDRH website.

The current panel-track supplement was submitted to expand the intended use and indications for use of Guardant360® CDx to include a companion diagnostic indication for the detection of epidermal growth factor receptor (*EGFR*) exon 20 insertion mutations in NSCLC patients who may benefit from treatment with RYBREVANT™ (amivantamab-vmjw).

## II. INDICATIONS FOR USE

Guardant360® CDx is a qualitative next generation sequencing-based *in vitro* diagnostic device that uses targeted high throughput hybridization-based capture technology for detection of single nucleotide variants (SNVs), insertions and deletions (indels) in 55 genes, copy number amplifications (CNAs) in two (2) genes, and fusions in four (4) genes. Guardant360® CDx utilizes circulating cell-free DNA (cfDNA) from plasma of peripheral whole blood collected in Streck Cell-Free DNA Blood Collection Tubes (BCTs). The test is intended to be used as a companion diagnostic to identify non-small cell lung cancer (NSCLC) patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling.

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Table 1. Companion Diagnostic Indications

|  Indication | Biomarker | Therapy  |
| --- | --- | --- |
|  Non-small cell lung cancer (NSCLC) | EGFR exon 19 deletions, L858R, and T790M* | TAGRISSO® (osimertinib)  |
|   |  EGFR exon 20 insertions | RYBREVANT™ (amivantamab-vmjw)  |

A negative result from a plasma specimen does not assure that the patient's tumor is negative for genomic findings. NSCLC patients who are negative for the biomarkers listed in Table 1 should be reflexed to tissue biopsy testing for Table 1 biomarkers using an FDA approved tumor tissue test, if feasible.

*The efficacy of TAGRISSO® (osimertinib) has not been established in the EGFR T790M plasma-positive, tissue-negative or unknown population and clinical data for T790M plasma-positive patients are limited; therefore, testing using plasma specimens is most appropriate for consideration in patients from whom a tumor biopsy cannot be obtained.

Additionally, the test is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for cancer patients with any solid malignant neoplasms. The test is for use with patients previously diagnosed with cancer and in conjunction with other laboratory and clinical findings.

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

Guardant360® CDx is a single-site assay performed at Guardant Health, Inc.

### III. CONTRAINDICATIONS

There are no known contraindications.

### IV. WARNINGS AND PRECAUTIONS

The warnings/precautions and limitations are included in the Guardant360® CDx assay labeling.

### V. DEVICE DESCRIPTION

Guardant360® CDx is a single-site test performed at Guardant Health, Inc. The test includes reagents, software, and procedures for testing cfDNA from whole blood samples. The test uses 5-30 ng of cfDNA for library construction and next generation sequencing. Sequencing data is processed using a customized bioinformatics pipeline designed to detect several classes of genomic alterations, including nucleotide substitutions, indels, CNA, and genomic fusions / rearrangements. The device is designed to sequence 74 genes, but only report pre-defined and de novo alterations within the 55

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genes outlined in Table 2. The test’s reportable range for SNVs and indels covers approximately 46,000 bases.

**Table 2. Genes Containing Alterations Detected by the Guardant360® CDx**

|  Alteration Type | Genes  |
| --- | --- |
|  Single Nucleotide Variants (SNVs) | *AKT1, ALK, APC, AR, ARAF, ATM*, BRAF, BRCA1**, BRCA2**, CCND1, CDH1, CDK4, CDK6, CDK12*, CDKN2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, GATA3, GNA11, GNAQ, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MLH1, MTOR, MYC, NF1, NFE2L2, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, PTEN, RAF1, RET, RHEB, ROS1, SMAD4, SMO, STK11, TERT, TSC1, VHL*  |
|  Indels | *AKT1, ALK, APC, ATM*, BRAF, BRCA1**, BRCA2**, CDH1, CDK12*, CDKN2A, EGFR, ERBB2, ESR1, FGFR2, GATA3, HNF1A, HRAS, KIT, KRAS, MET, MLH1, NF1, PDGFRA, PIK3CA, PTEN, RET, ROS1, STK11, TSC1, VHL*  |
|  Copy Number Amplifications (CNAs) | *ERBB2, MET*  |
|  Fusions / Rearrangements | *ALK, NTRK1, RET, ROS1*  |

*Reporting is enabled for pathogenic germline alterations only. Somatic alterations will not be reported.

** Reporting is enabled for both germline and somatic alterations.

### Test Output

The test report includes variants reported in the following categories; see Table 3:

**Table 3. Category Definitions**

|  Category | Guardant360® 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 Guardant360® 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 Guardant360® CDx has demonstrated analytical reliability but not clinical performance.  |
|  Category 3A: Biomarkers with Evidence of Clinical Significance in | No | No | **Yes** | ctDNA biomarkers with evidence of clinical significance presented by tissue-based FDA-approved companion diagnostics or professional guidelines for  |

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|  Category | Guardant360^{®} CDx |   |   | Comments  |
| --- | --- | --- | --- | --- |
|   |  Prescriptive use for a Therapeutic Product | Clinical Performance | Analytical Performance  |   |
|  tissue supported by: strong analytical validation using ctDNA |  |  |  | which Guardant360^{®} 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 Guardant360^{®} CDx 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 Guardant360^{®} CDx has demonstrated minimum analytical performance.  |

### Test Kit Contents

The test includes the Guardant360$^{®}$ CDx Blood Collection Kit (BCK), which is sent to ordering laboratories. Each BCK contains two blood collection tubes. The BCK also contains supporting packaging materials, instructions for use and a return shipping label. The BCK contains the following components:

- Streck blood collection tubes for specimen collection, stabilization, and transport of cfDNA; 2 per kit.
- Cushioning materials to prevent breakage of the blood collection tubes; 2 per kit
- Foam tray for protection of collection tubes during transport
- Absorbent sheet to be used during specimen shipping
- Biohazard specimen bag for protection during specimen transport
- Return shipping label for return of specimen to Guardant Health
- Barcodes for specimen identification and shipping instructions
- Instructions for Use for blood draw
- Patient welcome brochure which contains an overview of the test
- Test requisition form to complete to order Guardant360$^{®}$ CDx for a patient.

The test also includes the Guardant360$^{®}$ CDx Sample Preparation Kit (SPK), which is used in the Guardant Health Clinical Laboratory. The SPK contains reagents for library preparation, library enhancement, and cfDNA quantification/qualification. The kit is assembled into six (6) different boxes (referred to as box 1, 2, 3, 4a, 4b, and 4c) based on

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the usage of the reagents. The division of reagents amongst the boxes reflects different storage conditions and/or locations (e.g. different laboratory spaces).

## Instruments

Guardant360® CDx is intended to be performed with serial number-controlled instruments as indicated in Table 4. All instruments are qualified by Guardant Health, Inc. under the Guardant Health Quality System.

Table 4. Serial Number Controlled Instruments for use with the Guardant360® CDx assay

|  Instrument  |
| --- |
|  Agilent Technologies 4200 TapeStation Instrument  |
|  Hamilton Company Microlab STAR  |
|  Hamilton Company Microlab STARlet  |
|  Illumina NextSeq 550 Sequencer  |
|  Veriti 96-Well Thermal Cycler  |

## Test Process

### A. Whole Blood Collection and Shipping

The Guardant360® CDx Blood Collection Kit is used by ordering laboratories / physicians to collect whole blood specimens and ship them to the Guardant Health Clinical Laboratory. A minimum of 5 mL whole blood must be received in order to achieve optimal performance for the Guardant360® CDx assay. Underfilling of tubes with less than 5 mL of blood may lead to incorrect analytical results or poor product performance.

### B. Plasma Isolation and cfDNA Extraction

Whole blood specimens are processed in the Guardant Health Clinical Laboratory within 7 days of blood collection. Plasma is isolated from both tubes of whole blood via centrifugation. One tube of plasma is stored, while the second tube is used for cfDNA extraction using the QIAGEN QIAsymphony SP Instrument and reagent system. The resulting cfDNA is quantified using the 4200 TapeStation. Input amounts ranging from 5 to 30 ng of cfDNA are further processed for each sample.

### C. Library Preparation and Enrichment

Reagents from the Guardant360® CDx Sample Preparation Kit are used during library preparation, enrichment, enrichment wash, and quantitation steps using the Veriti 96-Well Thermal Cycler, Microlab STAR and STARlet, and 4200 TapeStation Instruments. During library preparation, cfDNA fragment ends are repaired and library adapters containing inline barcodes are attached using blunt-end ligation. The resulting DNA is amplified by PCR to create libraries suitable for enrichment.

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Amplified libraries are enriched for genes of interest using hybrid target capture with custom biotinylated RNA probes. Each enriched library is amplified by PCR using a unique index primer that also contains a sequencing flow cell attachment sequence. Amplified enriched libraries are pooled in equimolar amounts, denatured, and diluted to appropriate concentration for sequencing.

### D. DNA Sequencing

Paired-end sequencing by synthesis is performed with the Illumina NextSeq 550 Sequencing system. The amplified cfDNA is analyzed by parallel sequencing of amplified target genes to an average depth of coverage of greater than 2,700 unique molecules.

### E. Data Analysis and Reporting

The Guardant360® CDx Software uses a custom-developed analysis bioinformatics pipeline (BIP) software module. The BIP software module uses the raw data (output) from the targeted sequencing, partitions the data based on the sample index sequence (barcode) of each read to separate reads originating from individual samples, and executes a proprietary algorithmic reconstruction of the digitized sequencing signals based on molecular barcodes for high-fidelity molecule-based alteration calling downstream. The sequence data then undergoes an alignment process where it is mapped to the human genome (hg19) and an analysis of sequence alteration data is performed.

Alteration detection is conducted according to alteration calling metrics derived from clinical sample analysis. All alterations must pass alteration calling metrics as described in Table 5.

The SNV and indel cut-offs are defined in terms of mutant allele fraction (MAF) estimate, number and type of molecules supporting the alteration, pseudo-gene assessment, and likelihood ratio (LLR) score. The MAF estimate describes the calculated allelic fraction of an SNV or indel. The number of molecules describes the observed number of molecules meeting requirements for a particular alteration call. The LLR score is a calculated number that reflects how much observed support for the mutation exceeds expectations based on PCR and sequencing induced artifacts.

Table 5. Alteration Analytical Calling Threshold/Cut-Off Metrics

|  SNV Calling Property | Metric  |
| --- | --- |
|  DNA Molecule Support | ≥ 2  |
|  MAF Estimate | ≥ 0.001%  |
|  Log Likelihood Ratio | ≥ 0  |
|  Indel Calling Property | Metric  |
|  DNA Molecule Support | ≥ 2  |
|  MAF Estimate | ≥ 0.01%  |
|  Log Likelihood Ratio | ≥ 10  |

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|  CNA Calling Property | Metric  |
| --- | --- |
|  ERBB2 copy number | ≥ 2.18  |
|  ERBB2 Z-score | ≥ 10  |
|  ERBB2 amplification is not associated with chromosome-arm aneuploidy | TRUE  |
|  MET copy number | ≥ 2.16  |
|  MET Z-score | ≥ 10  |
|  MET amplification is not associated with chromosome-arm aneuploidy | TRUE  |
|  Fusion Calling Property | Metric  |
|  MAPQ score of supporting molecule to fusion sequence | > 30  |
|  Number of unique fusion molecules | ≥ 2  |
|  Number of unique fusion reads | > 2  |

The laboratory and physician receive a qualitative alteration-level result. A sample will receive an overall “Failed” result when any QC metric is failed. Samples failing any QC metric are automatically held and not released. The laboratory may attempt to rerun a patient sample that has failed a QC metric by using stored plasma or intermediate products.

Results from samples passing all QC metrics are formatted onto an IVD results report with CDx relevant information (Category 1) and all other biomarkers (Categories 2-4) within the LIMS system. The IVD results report will be populated with patient-specific information and may be merged with additional information provided by Guardant Health as a professional service prior to approval and release by the laboratory director or designee.

### F. Quality Control Measures

The Guardant360® CDx Sample Preparation Kit includes the Variant Control, which is engineered to contain known positive and negative alterations and is treated as a sample. Additionally, a no template negative control (NTC) is run in parallel with patient samples.

The Variant Control consists of a mixture of cfDNA from multiple human cancer cell lines containing all four alteration types, SNVs, indels, CNAs and fusions. The control is treated as a sample and processed starting from 15 ng cfDNA input through sequencing where it is analyzed for the presence and absence of the specific alterations.

Although the Variant Control does not contain all the alterations that the test is capable of detecting, concordant detection of alterations targeted in the Variant Control indicates that assay is performing as expected across the panel.

In addition to assessing Variant Control performance within a batch, the test is assessing multiple per-sample in-process and post-sequencing analytical metrics for

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each of the patient samples tested. These metrics provide in depth analytical QC information that complements Variant Control performance data and is specific and informative to that sample performance.

The NTC samples are absent of a DNA template, so cfDNA extraction, library preparation, and enrichment steps are expected to result in background level metrics.

## **VI. ALTERNATIVE PRACTICES AND PROCEDURES**

There is an FDA approved companion diagnostic (CDx) alternative for the detection of *EGFR* exon 19 deletions, *EGFR* L858R, and *EGFR* T790M genetic alterations using cfDNA, for the TAGRISSO® (osimertinib) therapeutic. The approved CDx test is detailed below: for additional details see FDA List of Cleared or Approved Companion Diagnostic Devices at https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm301431.htm?source=govdelivery

- cobas® EGFR Mutation Test v2 (Roche Molecular Systems, Inc.)
  - Technology: Polymerase Chain Reaction (PCR)
  - Therapy: TAGRISSO® (osimertinib)
  - Indication: Non-small cell lung cancer (NSCLC)
- FoundationOne® Liquid CDx (F1 Liquid CDx) (Foundation Medicine, Inc.)
  - Technology: Next Generation Sequencing (NGS)
  - Therapy: TAGRISSO® (osimertinib)
  - Indication: Non-small cell lung cancer (NSCLC)

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.

There is no FDA approved CDx alternative for the detection of *EGFR* exon 20 insertions in patients with NSCLC using cfDNA for the RYBREVANT™ (amivantamab-vmjw) therapeutic.

## **VII. MARKETING HISTORY**

Guardant Health, Inc. initially designed and developed the Guardant360 laboratory developed test (Guardant360 LDT), and the first commercial sample was tested in 2012 to detect the presence of genomic alterations in plasma isolated from whole blood. The Guardant360® CDx was FDA-approved on August 7, 2020 and subsequently commercialized in the USA.

Guardant360® CDx has not been marketed in 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 test results, and subsequently, may lead to inappropriate patient

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management decisions. Patients with false positive results may undergo treatment with the therapies listed in the intended use statement without clinical benefit and may experience adverse reactions associated with the therapies. Patients with false negative results may not be considered for treatment with the indicated therapies. There is also a risk of delayed results, which may lead to delay of treatment with indicated therapy.

For the specific adverse events that occurred in the clinical studies, please see the RYBREVANT™ (amivantamab-vmjw) FDA approved package insert which is available at Drugs@FDA.

## IX. SUMMARY OF NONCLINICAL STUDIES

### A. Laboratory Studies

The primary evidence for supporting the performance of Guardant360® CDx in detecting *EGFR* exon 20 insertion mutations was from the data presented using intended use specimens across all validation studies. In addition to the existing platform-level *EGFR* exon 20 insertion validation results (P200010), analytical accuracy/concordance, limit of detection (LoD), and precision at LoD studies were conducted to support the indication for *EGFR* exon 20 insertion mutations. Further, results from exogenous interference and guardbanding studies that were completed to fulfil the conditions of approval for the original PMA P200010 and that utilized samples carrying *EGFR* exon 20 insertion mutations, were also included to support this PMA supplement.

For Guardant360® CDx platform-level validation (P200010), performance characteristics were established using plasma-derived cfDNA samples from a wide range of cancer types. Each study included CDx variants as well as a broad range of representative alteration types (substitution, indels, CNAs, rearrangements) in various genomic contexts across several genes. The platform validation studies included NSCLC samples with *EGFR* exon 20 insertions. These results from the platform-level validation (P200010) have been leveraged to support Guardant360® CDx detection of *EGFR* exon 20 insertions. (please see Section IX.A.10, Table 33 in Summary of Safety and Effectiveness Data P200010).

Additional validation studies to support expansion of the intended use to include *EGFR* exon 20 insertions are described below.

#### 1. Analytical Accuracy/Concordance

An analytical accuracy study was performed with available plasma clinical specimens (56 *EGFR* exon 20 insertion positive patients and 23 *EGFR* exon 20 insertion negative patients) from NSCLC patients enrolled in the CHRYSALIS clinical trial (refer to Section X.A for study details) to demonstrate the concordance between Guardant360® CDx and an externally validated orthogonal NGS assay for the detection of *EGFR* exon 20 insertions. The clinical trial samples accounted for 76% of the *EGFR* exon 20 insertions observed in the CHRYSALIS clinical study. Due to the low prevalence of some *EGFR* exon 20 insertions and small size of biomarker negative samples from the CHRYSALIS clinical trial, samples from Guardant Health’s biobank (128 *EGFR* exon 20

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insertion positive samples and 70 *EGFR* exon 20 insertion negative samples) were included in the accuracy study.

A total of 293 patient samples were originally selected for the accuracy study. Of the 293 samples, 13 samples were excluded due to insufficient material for testing and 3 samples were excluded due to issues with inclusion criteria to the CHRYSALIS study or since they did not meet the diagnostic study inclusion criteria. Among the remaining 277 samples, five samples were excluded from the study; four samples failed testing with the comparator assay due to sequencing failures, while one sample failed testing with Guardant360$^{®}$ CDx due to enrichment QC metrics.

A summary of positive percent agreement (PPA) and negative percent agreement (NPA) and corresponding 95% two-sided exact confidence intervals (CIs) is provided in Table 6. Since the samples were selected from different sources based on different assays, the unadjusted agreements in Table 6 may be subject to potential bias. Positive agreement was 96.30% while negative agreement was 86.91% for detection of *EGFR* exon 20 insertions in the concordance analysis. It was noted that the discordance was due to sensitivity differences between Guardant360$^{®}$ CDx and the comparator assay.

**Table 6. Summary of Concordance Between Guardant360$^{®}$ CDx and NGS Comparator for *EGFR* Exon 20 Insertions**

|  Alteration Type | Guardant360 CDx(+), Comparator #2 (+) | Guardant360 CDx(+), Comparator #2 (-) | Guardant360 CDx(-), Comparator #2 (+) | Guardant360 CDx(-), Comparator #2 (-) | Patients (n) | PPA (95% CI) | NPA (95% CI) | PPV (95% CI) | NPV (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  *EGFR* exon 20 insertions | 78 | 25 | 3 | 166 | 272 | 96.30% (89.56%, 99.23%) | 86.91% (81.29%, 91.35%) | 75.73% (66.29%, 83.64%) | 98.22% (94.9%, 99.63%)  |

PPA, Positive Percent Agreement; NPA, Negative Percent Agreement; PPV, Positive Predictive Value; NPV, Negative Predictive Value

To further differentiate between comparator assay false negatives and Guardant360$^{®}$ CDx false positives as the origin for the 25 Guardant360$^{®}$ CDx positive comparator negative samples, the agreement between Guardant360$^{®}$ CDx and the comparator assay was calculated for each sample source independently (Table 7). All Guardant360$^{®}$ CDx positive comparator negative samples were found in the Guardant Health (GH) Biobank-Pos and Clinical Validation (CV) Local Test cohorts, which demonstrated challenged NPA values of 83.1% (54/65) and 63.2% (24/38), respectively. As the CV LocalTest cohort was predominantly composed of samples with tissue testing results, it was expected that some of

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these could have lower circulating tumor amounts resulting in more stochastic detections by the comparator reducing the NPA relative to the GH-Biobank-Pos cohort. These NPA values were in marked contrast to the GH-Biobank-Unselected and CV_Sensitivity populations, which comprised no Guardant360® CDx positive comparator negative samples and demonstrated an NPA of 100% (68/68) and 100% (20/20), respectively. The CIs for GH-Biobank-Pos and CV_LocalTest overlapped and were below the CI for GH-Biobank-Unselected. The analysis showed that NPA was similar between the GH-Biobank-Pos and CV_LocalTest cohorts, which had significantly lower NPA than the GH-Biobank-Unselected and CV_Sensitivity cohorts.

Table 7. Summary of Concordance Between Guardant360® CDx and NGS Comparator for EGFR Exon 20 Insertions by Data Cohort

|  Sample Cohort | Guardant360 CDx(+), Comparator #2(+) | Guardant360 CDx(+), Comparator #2 (-) | Guardant360 CDx(-), Comparator #2 (+) | Guardant360 CDx(-), Comparator #2(-) | Patients (n) | PPA (95% CI) | NPA (95% CI) | PPV (95% CI) | NPV (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Guardant Health Biobank-Pos | 61 | 11 | 1 | 54 | 127 | 98.39% (91.34%, 99.96%) | 83.08% (71.73%, 91.24%) | 84.72% (74.31%, 92.12%) | 98.18% (90.28%, 99.95%)  |
|  CV_Local Test | 16 | 14 | 2 | 24 | 56 | 88.89% (65.29%, 98.62%) | 63.16% (45.99%, 78.19%) | 53.33% (34.33%, 71.66%) | 92.31% (74.87%, 99.05%)  |
|  CV_Sensitivity | 1 | 0 | 0 | 20 | 21 | 100% (2.5%, 100%) | 100% (83.16%, 100%) | 100% (2.5%, 100%) | 100% (83.16%, 100%)  |
|  Guardant Health Biobank-Unselected | 0 | 0 | 0 | 68 | 68 | NaN% (0%, 100%) | 100% (94.72%, 100%) | NaN% (0%, 100%) | 100% (94.72%, 100%)  |

NaN, Not a Number

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## 2. Analytical Sensitivity

### a. Limit of Blank (LoB)

Please refer to the Summary of Safety and Effectiveness Data P200010 (Section IX.A.3.a) for Guardant360® CDx platform-level analytical sensitivity data for LoB. There were no false positives for *EGFR* exon 20 insertions.

### b. Limit of Detection (LoD)

The LoD for *EGFR* exon 20 insertions was established panel-wide as 0.8% and 0.2% MAF for 5 ng and 30 ng cfDNA input levels, respectively, using a single *EGFR* exon 20 insertions variant, p.A767_V769dup. This result was included in P200010 for Guardant360® CDx (refer to P200010 SSED Section IX.A.3 for details). Because the LoD for *EGFR* exon 20 insertion, A767_V769dup was established using pools of cfDNA from clinical plasma samples from multiple cancer types, the LoD was confirmed in NSCLC patient samples for this indication.

To supplement the existing LoD study, Guardant Health, Inc. performed additional LoD establishment and confirmation studies using NSCLC patient samples that were representative of varying prevalence and insertions lengths of *EGFR* exon 20 insertions as observed in the CHRYSALIS clinical study (refer to Section X for clinical study details). Three sample pools harboring three *EGFR* exon 20 insertions; *EGFR* p.A767_V769dup (9 bp insertion, Pool 1), *EGFR* p.N771_H773dup (9 bp insertion, Pool 2), and *EGFR* p.H773dup (3 bp insertion, Pool 3) were prepared from NSCLC clinical sample cfDNA identified from Guardant Health’s Biobank. These variants accounted for 39% of the *EGFR* exon 20 insertions observed in the CHRYSALIS clinical trial. Pools 2 and 3 were used for LoD establishment, while Pool 1 was used for the LoD confirmation study. The presence of *EGFR* exon 20 insertions in each source pool and corresponding mutant allele frequencies (MAFs) were confirmed by a validated NGS comparator method. Patient cfDNA samples positive for selected *EGFR* exon 20 insertions were diluted with mutation-negative cfDNA derived from NSCLC clinical samples (“WT cfDNA”) to target the highest MAF level of the source pool. Pool 2 was subsequently diluted serially to form a titration series consisting of 5 different MAF levels at 5 and 30 ng cfDNA inputs. Pool 3 LoD estimation was also done similar to Pool 2 except the study included only 5 ng cfDNA input level. The LoDs were established using 21 replicates for 5 ng cfDNA input and 14 replicates for 30 ng cfDNA input at each titration point. LoD confirmation for *EGFR* p.A767_V769dup was performed using 21 and 14 replicates at target MAFs of 1.4% for 5 ng input and 0.3% for 30 ng input, respectively.

The LoD results for selected *EGFR* exon 20 insertions varied between 0.8% to 1.8% MAF (median 1.2% MAF) for 5 ng cfDNA input, while they were between 0.2% to 0.3% MAF (median 0.3% MAF) for 30 ng cfDNA input level. LoD establishment and confirmation results are summarized in Table 8.

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Table 8. Summary of Established and Confirmed LoDs for EGFR Exon 20 Insertions in NSCLC Clinical Samples

|  Alteration | Alteration Type | LoD, 5ng input (MAF) | LoD, 30 ng input (MAF)  |
| --- | --- | --- | --- |
|  EGFR exon 20 insertions | H773dup (3 bp) | 0.9% MAF | N.D.  |
|   |  A767_V769dup (9 bp)* | 1.4% MAF | 0.3% MAF  |
|   |  N771_H773dup (9 bp) | 1.8% MAF | 0.3% MAF  |
|   |  A767_V769dup (9 bp)** | 0.8% MAF | 0.2% MAF  |

N.D.: not determined, * Confirmed LoD, ** Established LoD from mixed cancer types.

Given that not all representative EGFR exon 20 insertion mutations that had a high prevalence in the clinical study were evaluated in the LoD study, a post-market study is planned with additional samples harboring 6 bp and 12 bp EGFR exon 20 insertions (see section XIII).

### 3. Analytical Specificity

Please refer to the Summary of Safety and Effectiveness Data P200010 (Section IX.A.3) for platform-level validation of analytical specificity, including endogenous and microbial interfering substance and in silico hybrid capture bait specificity for Guardant360® CDx.

#### a. Exogenous Interfering Substances

The purpose of the study was to evaluate the effect of cfDNA extraction kit wash buffer carry-over on the performance of Guardant360® CDx assay.

Two cfDNA clinical sample pools derived from a total of 74 clinical cfDNA samples were used in the study. The pools were prepared using proportions for 5 ng cfDNA input. The sample pools were comprised of clinical cfDNA (pool 1) and a mixture of clinical cfDNA and cell line-derived DNA (pool 2) with 10 known variants diluted in WT clinical cfDNA to 1 – 2x LoD (Table 9). The sample pools were spiked with 10% (v/v) of the wash buffer. In the study, a total of 24 replicates (6 replicates/pool x 2 experimental combinations x 2 sample pools), 1 reagent lot, 3 instrument combinations, and 2 operators were used.

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Table 9. The characteristics of the 10 variants used in the Exogenous Interference study

|  Pool | Variant Type | Variant | Adequacy of MAF / CN level in relationship to the LoD (5ng), as assessed by Guardant360 CDx  |   |   |
| --- | --- | --- | --- | --- | --- |
|   |   |   |  Mean of Reference Condition (MAF% or copy number) | LoD (MAF% or copy number) | Fold over LoD  |
|  Pool 1 | SNVs | EGFR L858R | 3.00 | 1.5 | 2.0  |
|   |   |  EGFR T790M | 2.03 | 1.4 | 1.4  |
|   |   |  KRAS G12C | 2.88 | 1.8 | 1.6  |
|   |  Indels | EGFR E746_A750del | 2.43 | 1.5 | 1.6  |
|   |   |  EGFR A767_V769dup | 0.79 | 0.8 | 1.0  |
|   |  CNA | MET | 2.87 | 2.4 | 2.2  |
|  Pool 2 | SNVs | PIK3CA E545A | 4.11 | 2.4 | 1.7  |
|   |   |  PIK3CA H1047R | 3.10 | 1.7 | 1.8  |
|   |  Fusions | EML4-ALK | 2.77 | 1.4 | 2.0  |
|   |   |  TPM3-NTRK1 | 2.86 | 0.9 | 3.2  |

The study result was considered valid if the pre-specific acceptance criteria for Qualitative Detection Rate (QDR) and NPA were met for test and reference conditions. For each test condition, equivalency was concluded if either QDR for the test condition was not significantly lower than the corresponding reference condition by a two-sample test for equality of proportions using a chi-square statistic (one-sided, alpha = 0.05) or the lower limit of the Exact 95% CI for QDR exceeded 80%. For each condition (test and reference), the acceptance criteria for NPA at targeted variant sites was 100%.

Reference condition showed a QDR of 98.3%, with one variant (EGFR exon 20, A767_V769dup) missing in one sample. The test condition showed a 100% QDR. For each condition, the lower limits of the 95% confidence interval were higher than 80% meeting the acceptance criteria. The chi-square test also showed a non-significant difference of QDRs (p-value = 0.49) between the test condition and reference condition. Per-sample NPA was 100% for both conditions. All acceptance criteria were met. The exogenous interfering substance study results are summarized in Tables 10 and 11.

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**Table 10. Summary of QDR analysis results for exogenous interfering substance study for Guardant360® CDx**

|  QDR* |   | Reference | Test  |
| --- | --- | --- | --- |
|  Exogenous Interference (Wash Buffer) | Positive detected variants across samples | 59 | 64  |
|   |  QDR | 59/60 = 98.33% | 64/64 = 100%  |
|   |  95% CI | [91.06%, 99.96%] | [94.40%, 100%]  |

*The Quantitative Detection Rate (QDR) is defined as the number of positively detected targeted variants tested across eligible samples (D) divided by the total number of targeted variants tested across eligible samples (N) expressed as a percentage (100 * D / N).

**Table 11. Per-sample NPA analysis results for exogenous interfering substance study for Guardant360® CDx**

|  Study | Condition | per-sample NPA  |
| --- | --- | --- |
|  Exogenous Interference (Wash Buffer) | Reference | 12/12 = 100%  |
|   |  Test | 13/13 = 100%  |

The exogenous interfering substance study results fulfilled the condition of approval #3 in Section XIII of P200010.

#### 4. Precision

Please see the Summary of Safety and Effectiveness Data for P200010 (Section IX.A.5) for Guardant360® CDx platform-level validation of precision, including precision from cfDNA pools and precision from plasma extraction.

##### a. Precision from mutation positive samples

The purpose of the precision study was to demonstrate the repeatability and within-site reproducibility of Guardant360® CDx through closeness of agreement between measured qualitative output obtained in replicate testing using different combinations of three reagent lots, two instruments, four operators, and more than three testing days for detection of *EGFR* exon 20 insertions using NSCLC clinical sample pools. Variant source pools were prepared by diluting NSCLC patient cfDNA samples positive for selected *EGFR* exon 20 insertions with mutation-negative cfDNA derived from NSCLC clinical samples. The *EGFR* exon 20 insertions used in the precision study are listed in Table 12. Each insertion was tested across six precision combinations at 5 ng input at MAF levels indicated in Table 12.

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All CDx alterations demonstrated acceptable precision with PPA ranging from 97.6% to 100.0%. The combined precision for EGFR exon 20 insertion variant category was 98.4% with 95% CI of 94.3% - 99.8% (Table 13).

Table 12. EGFR Exon 20 Insertions with LoD Values Represented in Precision Study

|  EGFR Variant | Insertion Length | LoD at 5 ng | Precision (fold change to target)  |
| --- | --- | --- | --- |
|  A767_V769dup | 9 bp | 1.4% MAF | 1.4% MAF (1X)  |
|  H773dup | 3 bp | 0.9% MAF | 0.9% MAF (1X)  |
|  N771_H773dup | 9 bp | 1.8% MAF | 1.8% MAF (1X)  |
|   |   |  1.8% MAF | 1.25% MAF (0.7X)  |

Table 13. Precision Study Summary

|  EGFR Exon 20 Insertion | Number Positive / Number Expected | PPA (95% CI)  |
| --- | --- | --- |
|  A767_V769dup | 41 / 42 | 97.6% [87.4% - 99.9%]  |
|  N771_H773dup | 41 / 41 | 100% [91.4% - 100%]  |
|  H773dup | 41 / 42 | 97.6% [87.4% - 99.9%]  |
|  Overall [95% CI] | 123 / 125 | 98.4% [94.3% - 99.8%]  |

### b. Precision from mutation negative samples

Samples from healthy donors were pre-screened by an externally validated orthogonal method. Mutation negative samples by the orthogonal method were tested by Guardant360® CDx in three reproducibility conditions (i.e., different reagent lots, operators, instruments, and days). Four replicates from each donor were tested with Guardant360® CDx across the different reproducibility conditions, with a total of 240 replicates evaluated for the study. Within-condition and between-condition Average Negative Agreement (ANA) values were 100.0% for EGFR exon 20 insertions.

### 5. Carryover/Cross-Contamination

Please refer to the Summary of Safety and Effectiveness Data of P200010 (Section IX.A.6) for platform-level carryover/cross-contamination data for Guardant360® CDx.

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## 6. Reagent Lot Interchangeability

Please refer to the Summary of Safety and Effectiveness Data P200010 (Section IX.A.7) for platform-level reagent lot interchangeability data for Guardant360® CDx.

## 7. Stability

Please refer to the Summary of Safety and Effectiveness Data of P200010 (Section IX.A.8) for platform-level stability data including reagent stability, whole blood stability, plasma stability, cfDNA stability, and intermediate sample stability for Guardant360® CDx.

## 8. Guardbanding/Robustness

The study established the tolerance of Guardant360® CDx to variation in critical parameters; namely, cfDNA input, adapter volume, hybridization time, and hybridization wash buffer temperature guardbanding (Table 14). A total of 75 clinical cfDNA samples and two cancer cell lines were used to create the 2 sample pools. The sample pools included clinical cfDNA (Pool 1) and a mixture of clinical cfDNA and cell line-derived cfDNA (Pool 2) with 10 known variants diluted in WT clinical cfDNA targeted at 1 – 2x LoD for 5ng and 30ng input cfDNA amounts (Table 15). The study included a total of 126 replicates (6 replicates/pool x 8 experimental combinations x 2 sample pools and 5 replicates/pool x 3 experimental combinations x 2 sample pools), 2 reagent lots, 7 instrument combinations, and 3 operators. The QDR and NPA for test and reference conditions were estimated.

Guardbanding conditions were evaluated based on the rate of positive agreement for detection of variants in Pool 1 and 2, expressed as the QDR. The study results were considered valid if the pre-specific acceptance criteria for QDR and NPA were met for test and reference conditions. The pre-specified QDR was 95.0% (95% CI: 83.0% - 99.4%). The QDR analysis was performed using a chi-square statistic (one-sided, alpha = 0.05) or the lower limit of the Exact 95% CI for QDR greater than 80%. The NPA at targeted variant sites was set as 100%.

Table 14. Guardbanding Study Overview for Guardant360® CDx

|  Guardbanding Condition | Reference condition | Condition 1 | Condition 2  |
| --- | --- | --- | --- |
|  cfDNA Input amount | 5 ng | 2.5 ng | 4 ng  |
|  cfDNA Input amount | 30 ng | 36 ng | 45 ng  |
|  Adapter volume | 18.0 μL | 16.2 μL | 19.8 μL  |
|  Hybridization Time | 12 hours | 24 hours | N/A  |

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|  Wash Buffer Temperature | 71°C | 70°C | 72°C  |
| --- | --- | --- | --- |

N/A, Not Applicable.

Table 15. The Characteristics of the 10 Variants Used in the Guardbanding Study for Guardant360® CDx

|  Pool | Variant Type | Variant | Adequacy of MAF / CN level in relationship to the LoD (5ng), as assessed by Guardant360 CDx |   |   | Adequacy of MAF / CN level in relationship to the LoD (30ng), as assessed by Guardant360 CDx  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  Observed Mean (MAF% or copy number) in Reference | LoD (MAF% or copy number) | Fold change over LoD | Observed Mean (MAF% or copy number) in Reference | LoD (MAF% or copy number) | Fold change over LoD  |
|  Pool 1 | SNVs | EGFR L858R | 3.27 | 1.5 | 2.2 | 0.31 | 0.2 | 1.5  |
|   |   |  EGFR T790M | 2.40 | 1.4 | 1.7 | 0.49 | 0.2 | 2.4  |
|   |   |  KRAS G12C | 3.56 | 1.8 | 2.0 | 0.84 | 0.5 | 1.7  |
|   |  Indels | EGFR E746_A750del | 2.96 | 1.5 | 2.0 | 0.36 | 0.2 | 1.8  |
|   |   |  EGFR A767_V769dup | 1.05 | 0.8 | 1.3 | 0.28 | 0.2 | 1.4  |
|   |  CNA | MET | 3.10 | 2.4 | 2.8 | 3.16 | 2.4 | 2.9  |
|  Pool 2 | SNVs | PIK3CA E545A | 3.45 | 2.4 | 1.4 | 0.33 | 0.4 | 0.8  |
|   |   |  PIK3CA H1047R | 3.26 | 1.7 | 1.9 | 0.42 | 0.3 | 1.4  |
|   |  Fusions | EML4-ALK | 3.00 | 1.4 | 2.1 | 0.46 | 0.2 | 2.3  |
|   |   |  TPM3-NTRK1 | 1.88 | 0.9 | 2.1 | 0.5 | 0.2 | 2.5  |

For each tested guardbanding condition, all the lower limits of 95% CI were higher than 80%, meeting the acceptance criteria. The chi-square test was performed only between the 4 ng condition and reference condition regarding guard banding testing of cfDNA input at 5 ng, and it showed a non-significant difference of QDRs (p-value = 0.59) between the 4 ng input condition and reference condition (Table 16). The per-sample NPA at targeted variants across all conditions were 100% (Table 17).

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**Table 16. Summary of Guardbanding Study using QDR Analysis**

|  QDR |   | Reference (18.0 µL) | Guardbanding Condition 1 (16.2 µL) | Guardbanding Condition 2 (19.8 µL)  |
| --- | --- | --- | --- | --- |
|  Adaptor Volume | Positive detected variants across samples | 56 | 60 | 50  |
|   |  QDR | 56/56 = 100% | 60/60 = 100% | 50/50 = 100%  |
|   |  95% CI | [93.62%, 100%] | [94.04%, 100%] | [92.89%, 100%]  |
|  QDR |   | Reference (30 ng) | Guardbanding Condition 1 (36 ng) | Guardbanding Condition 2 (45 ng)  |
|  cfDNA input at 30 ng | Positive detected variants across samples | 50 | 46 | 50  |
|   |  QDR | 50/50 = 100% | 46/46 = 100% | 50/50 = 100%  |
|   |  95% CI | [92.89%, 100%] | [92.29%, 100%] | [92.89%, 100%]  |
|  QDR |   | Reference (5ng) | Guardbanding Condition 1 (4 ng) |   |
|  cfDNA input at 5 ng | Positive detected variants across samples | 56 | 35 | N/A  |
|   |  QDR | 56/56 = 100% | 35/36 = 97.22% | N/A  |
|   |  95% CI | [93.62%, 100%] | [85.47%, 99.93%] | N/A  |
|  QDR |   | Reference (12 hours) | Guardbanding Condition 1 (24hours) |   |
|  Hybridization Time | Positive detected variants across samples | 56 | 60 | N/A  |
|   |  QDR | 56/56 = 100% | 60/60 = 100% | N/A  |
|   |  95% CI | [93.62%, 100%] | [94.04%, 100%] | N/A  |
|  QDR |   | Reference (71°C) | Guardbanding Condition 1 (70°C) | Guardbanding Condition 2 (72°C)  |
|  Wash Buffer Temperature | Positive detected variants across samples | 56 | 60 | 60  |
|   |  QDR | 56/56 = 100% | 60/60 = 100% | 60/60 = 100%  |
|   |  95% CI | [93.62%, 100%] | [94.04%, 100%] | [94.04%, 100%]  |

N/A, Not Applicable

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Table 17. Summary of Guardbanding Study using NPA Analysis per sample

|  Study | Condition | per-sample NPA  |
| --- | --- | --- |
|  Adaptor Volume | Reference (18.0 μL) | 11/11 = 100%  |
|   |  Condition 1 (16.2 μL) | 12/12 = 100%  |
|   |  Condition 2 (19.8 μL) | 10/10 = 100%  |
|  cfDNA input at 30 ng | Reference (30 ng) | 10/10 = 100%  |
|   |  Condition 1 (36 ng) | 9/9 = 100%  |
|   |  Condition 2 (45 ng) | 10/10 = 100%  |
|  cfDNA input at 5ng | Reference (5 ng) | 11/11 = 100%  |
|   |  Condition (4 ng) | 6/6 = 100%  |
|  Hybridization Time | Reference (12 hours) | 11/11 = 100%  |
|   |  Test condition (24 hours) | 12/12 = 100%  |
|  Wash Buffer Temperature | Reference (71°C) | 11/11 = 100%  |
|   |  Condition 1 (70°C) | 12/12 = 100%  |
|   |  Condition 2 (72°C) | 12/12 = 100%  |

The results of the guardbanding study showed robustness of Guardant360® CDx to variations in the device's workflow and therefore fulfilled the condition of approval #2 in Section XIII for P200010.

### 9. General Lab Equipment and Reagent Evaluation

#### a. cfDNA Extraction

Please refer to the Summary of Safety and Effectiveness Data of P200010 (Section IX.A.9.a) for cfDNA extraction performance data for Guardant360® CDx.

#### b. Other Instruments and Reagents

Please refer to the Summary of Safety and Effectiveness Data of P200010 (Section IX.A.9.b) for Other Instruments and Reagents data for Guardant360® CDx.

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## **B. Animal Studies**

No animal studies were conducted using Guardant360® CDx.

## **C. Additional Studies**

None.

## **X. SUMMARY OF PRIMARY CLINICAL STUDIES**

The clinical performance of Guardant360® CDx for detecting *EGFR* exon 20 insertions in NSCLC patients who may benefit from treatment with RYBREVANT™ (amivantamab-vmjw) (Table 1), was demonstrated through a clinical bridging study using specimens from patients screened for enrollment into the CHRYSALIS (Janssen EDI1001 or 61186372EDI1001 or NCT02609776) study.

### **A. Guardant360 CDx Clinical Bridging Study for *EGFR* Exon 20 Insertions**

The safety and effectiveness of Guardant360® CDx for detecting *EGFR* exon 20 insertions in NSCLC patients who may benefit from treatment with amivantamab-vmjw was demonstrated in a retrospective analysis of plasma specimens from patients enrolled in the CHRYSALIS study. A bridging study was conducted to assess: 1) the concordance between *EGFR* exon 20 insertion status (biomarker positive or negative) tested with the clinical trial enrollment assays and Guardant360® CDx in the intent-to-test population and 2) the clinical efficacy of Guardant360® CDx in identifying *EGFR* exon 20 insertion positive patients for treatment with amivantamab-vmjw monotherapy.

#### **1. Therapeutic Study Design**

The CHRYSALIS clinical study is a first-in-human, open-label, multicenter, 2-part, Phase 1 study in patients with metastatic NSCLC having an in-frame base pair insertion mutation in *EGFR* exon 20 whose disease has progressed on or after platinum-based chemotherapy. The primary amivantamab-vmjw registration population comprised *EGFR* exon 20 insertion mutation-positive subjects from the CHRYSALIS clinical study whose disease progressed on or after platinum-based chemotherapy and who were treated with the recommended phase 2 dose (RP2D) of amivantamab-vmjw. Patients were enrolled based on the presence of *EGFR* exon 20 insertions in their tumor or plasma specimens as determined by CLIA-certified local laboratory testing. This clinical study was used to support the approval of RYBREVANT™ (amivantamab-vmjw) under BLA 761210.

The primary efficacy endpoint of the study was overall response rate (ORR) with 95% 2-sided exact CI using Response Evaluation Criteria in Solid Tumors (RECIST v1.1), based on Investigator and Blinded Independent Central Review (BICR) assessments. The secondary endpoints were defined as clinical benefit rate (CBR; confirmed complete response (CR) + confirmed partial response (PR) + stable disease (SD) for at least 11 weeks), duration of response (DOR), progression-free survival (PFS), time to treatment failure (TTF), and overall survival (OS).

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The primary efficacy population set to support the BLA 761210 application included 81 subjects. All ongoing BICR assessed responders were followed up for at least 6 months with a median of 9.7 months from the onset of response.

2. Guardant360® CDx EGFR Exon 20 Insertions Bridging Study Design

The aim of this bridging study was to determine the concordance between EGFR exon 20 insertion results from the clinical trial enrollment assays generated at the time of patient screening for CHRYSALIS and the EGFR exon 20 insertion results generated using Guardant360® CDx. The study was also conducted to establish the clinical utility of the Guardant360® CDx assay in identifying EGFR exon 20 insertion positive patients for treatment with amivantamab-vmjw monotherapy.

Pre-treatment plasma samples from 78 CHRYSALIS clinical study patients (78/81, 96.3% of the primary registration population) were retrospectively tested with Guardant360® CDx (see sPMA cohort accountability, below). No plasma from the CHRYSALIS clinical study patients negative for EGFR exon 20 insertions by local testing was available to represent the local test-negative portion of the Guardant360® CDx-positive intended use population. Therefore, supplemental matched tissue and plasma samples from the CHRYSALIS clinical study screen fail population (non-EGFR exon 20 insertion cohorts) and the Noninvasive vs. Invasive Lung Evaluation clinical study (the NILE study, NCT03615443) were used to estimate the prevalence of biomarker-negative patients by local testing to evaluate the potential impact of this population on clinical efficacy.

2.1. Clinical Bridging Study Inclusion and Exclusion Criteria

The criteria for inclusion into the clinical bridging study are summarized below:

- Inclusion criteria for plasma samples from the CHRYSALIS clinical study
  - Subject enrolled in the CHRYSALIS clinical study with informed consent for blood sample use for further research.
  - Subject part of the primary amivantamab-vmjw registration population.
  - Adequate pre-treatment plasma sample available for Guardant360® CDx testing or a previously generated Guardant360® CDx test result from the CHRYSALIS clinical study.
- Inclusion criteria for plasma samples from the CHRYSALIS clinical study for the diagnostic study sensitivity analysis prevalence sub-study
  - Subject failed screening for the CHRYSALIS clinical study with informed consent for blood sample use for further research.
  - Pre-treatment plasma sample available for testing with Guardant360® CDx or a Guardant360® CDx test result previously generated under the Guardant Health CHRYSALIS clinical study protocol.

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○ Availability of previously generated CHRYSALIS clinical study central tissue testing results.
• Inclusion criteria for samples from the NILE clinical study
○ Subjects enrolled in the NILE clinical study with documented informed consent.
○ A valid Guardant360® CDx test result previously generated from a pre-treatment plasma sample.
○ Previously generated valid test result from cobas® EGFR Mutation Test v2 testing on tissue slides and/or a tissue block of formalin-fixed paraffin-embedded tissue with sufficient tumor content and quantity for testing as defined by central laboratory testing requirements.

# 2.2. Diagnostic Study Objective and Endpoints

The primary objective of the diagnostic study was to demonstrate the comparability of single-agent amivantamab-vmjw efficacy in primary amivantamab-vmjw registration population subjects who are positive for EGFR exon 20 insertions by Guardant360® CDx to the size-adjusted null hypothesis efficacy cited in the CHRYSALIS clinical study protocol. The primary endpoint was ORR by RECIST 1.1 as assessed by BICR.

The possible influence of local test-negative Guardant360® CDx-positive patients not represented in the CHRYSALIS clinical study was assessed through sensitivity analysis. As no plasma samples from CHRYSALIS clinical study patients negative for EGFR mutations by local testing were available to represent the local test-negative portion of the Guardant360® CDx-positive intended use population, samples from non-EGFR exon 20 cohorts of CHRYSALIS clinical study screen fail population and the NILE clinical study were tested with Guardant360® CDx and either central tissue test (CHRYSALIS clinical study screen fail population) or cobas® EGFR Mutation Test v2, using tissue samples (NILE clinical study population) to calculate the prevalence of this population for the sensitivity analysis.

# B. Accountability of PMA Cohort for the Guardant360® CDx Clinical Bridging Study for EGFR Exon 20 Insertions

The Guardant360® CDx diagnostic study efficacy analysis included 78 of the total 81 (96.3%) patients from the primary amivantamab-vmjw registration population (Figure 1). Of these, 62 patients (76.5% of the primary amivantamab-vmjw registration population) tested positive by Guardant360® CDx while 16 (19.8%) tested negative. Three (3/81, 3.7%) were not tested due to unavailability of plasma specimen for testing, and none failed testing.

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Figure 1. Guardant360® CDx EGFR Exon 20 Insertions Bridging Study Efficacy Analysis Patient Accountability and Analysis Set Definitions

![img-0.jpeg](img-0.jpeg)

The Guardant360® CDx diagnostic study assay agreement analysis originally included 268 patients tested with Guardant360® CDx and other test results from both the CHRYSALIS and NILE clinical studies (Figure 2). The agreement analysis set included 97 patients with local test results (9 with plasma testing results, 87 with tissue testing results, 1 with test results using an unknown analyte), 83 screen-fail patients with central tissue test results from other cohorts of CHRYSALIS, and 88 with cobas® EGFR Mutation v2 PCR tissue test results from the NILE study. The additional 19 samples (19/97) included in the positive agreement analysis had the same inclusion criteria as the primary registration population except that these began treatment after the clinical cutoff date and therefore did not have 3 post-baseline disease assessment at the clinical cutoff. The negative agreement analysis cohort did not include samples from the primary registration population, but the 83 samples were screen fails from other arms of the clinical study (non-EGFR exon 20 insertion cohorts of CHRYSALIS). Of the 83 screen-fail samples and the 88 samples from the NILE study, 4 and 3 samples, respectively, had EGFR exon 20 insertion mutations identified; and, therefore excluded from the negative agreement analysis. The remaining 164 samples were used for negative agreement analysis. The final number of samples used in the agreement analysis was 261.

Central testing for the screen fail samples utilized two different tissue-based NGS tests (69% with FoundationOne® CDx and 31% with Oncomine Dx Target Test) while samples from the NILE study were selected using the tissue-based cobas® EGFR Mutation v2 PCR Test. Overall, the combination of the NILE clinical study and CHRYSALIS non-registration cohorts closely represents the local testing distribution used to enroll the registration population, both in terms of general test methodology (i.e. the registration population 40% PCR, 55% NGS; the supplemental cohorts 51% PCR, 49% NGS) and specific test methodology (i.e. the registration population enrolled by NGS with 35% Oncomine Dx Target Test, 65% FoundationOne® CDx; the supplemental cohorts with 31% and 69% respectively).

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Figure 2. Guardant360® CDx EGFR Exon 20 Insertions Bridging Study Assay Agreement Analysis Patient Accountability and Analysis Set Definitions. Assay agreement subgroups (AAAS-L, AAAS-C, and AAAS-P) shaded in green.

![img-1.jpeg](img-1.jpeg)

*The CHRYSALIS subjects not included in the assay agreement analysis refers to subjects without the requisite central testing data, from the dose escalation series (non-R2PD-treated), without previous chemotherapy exposure, from additional expansion cohorts, and/or without adequate treatment and/or follow-up.

Abbreviations: AAAS-L, Assay agreement analysis set – Local testing; AAAS-C, Assay agreement analysis set – Central NGS tissue testing; AAAS-P, Assay agreement analysis set – PCR testing

### C. Study Population Demographics and Baseline Parameters for the Guardant360® CDx Clinical Bridging Study for EGFR Exon 20 Insertions

Demographics and baseline clinical characteristics of subjects enrolled in CHRYSALIS clinical study were categorized relative to the diagnostic study populations as defined by Guardant360® CDx results. As shown in Tables 18 and 19, the diagnostic study efficacy population (gCEAS) demographics and baseline clinical characteristics closely resemble those of the overall primary amivantamab-vmjw registration population (FAS).

Table 18. Baseline Demographics

|   | FAS | gAS | gNT | gCEAS | gAS- | gAS-F | gAS-Unk (gAS-F +gNT) | p Value gAS vs gAS-Unk  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Analysis set: | 81 | 78 | 3 | 62 | 16 | - | 3 |   |
|  Age, years  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.914  |
|  Mean (SD) | 62.3 (9.96) | 62.3 (10.04) | 61.7 (9.29) | 62.5 (10.03) | 61.6 (10.40) | - | 61.7 (9.29) |   |
|  Median | 62.0 | 62.0 | 59.0 | 62.0 | 62.0 | - | 59.0 |   |
|  Range | (42; 84) | (42; 84) | (54; 72) | (42; 84) | (46; 76) | - | (54; 72) |   |

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|   | FAS | gAS | gNT | gCEAS | gAS- | gAS-F | gAS-Unk (gAS-F +gNT) | p Value gAS vs gAS- Unk  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  <65 | 48 (59.3%) | 46 (59.0%) | 2 (66.7%) | 38 (61.3%) | 8 (50.0%) | - | 2 (66.7%) |   |
|  >=65 | 33 (40.7%) | 32 (41.0%) | 1 (33.3%) | 24 (38.7%) | 8 (50.0%) | - | 1 (33.3%) |   |
|  <75 | 74 (91.4%) | 71 (91.0%) | 3 (100.0%) | 56 (90.3%) | 15 (93.8%) | - | 3 (100.0%) |   |
|  >=75 | 7 (8.6%) | 7 (9.0%) | 0 | 6 (9.7%) | 1 (6.3%) | - | 0 |   |
|  Sex |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 1.000  |
|  Female | 48 (59.3%) | 46 (59.0%) | 2 (66.7%) | 40 (64.5%) | 6 (37.5%) | - | 2 (66.7%) |   |
|  Male | 33 (40.7%) | 32 (41.0%) | 1 (33.3%) | 22 (35.5%) | 10 (62.5%) | - | 1 (33.3%) |   |
|  Race |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.104  |
|  Asian | 40 (49.4%) | 39 (50.0%) | 1 (33.3%) | 34 (54.8%) | 5 (31.3%) | - | 1 (33.3%) |   |
|  Black or African American | 2 (2.5%) | 1 (1.3%) | 1 (33.3%) | 1 (1.6%) | 0 | - | 1 (33.3%) |   |
|  White | 30 (37.0%) | 29 (37.2%) | 1 (33.3%) | 21 (33.9%) | 8 (50.0%) | - | 1 (33.3%) |   |
|  Not reported | 9 (11.1%) | 9 (11.5%) | 0 | 6 (9.7%) | 3 (18.8%) | - | 0 |   |
|  Ethnicity |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 1.000  |
|  Hispanic or Latino | 3 (3.7%) | 3 (3.8%) | 0 | 3 (4.8%) | 0 | - | 0 |   |
|  Not Hispanic or Latino | 68 (84.0%) | 65 (83.3%) | 3 (100.0%) | 53 (85.5%) | 12 (75.0%) | - | 3 (100.0%) |   |
|  Not reported | 10 (12.3%) | 10 (12.8%) | 0 | 6 (9.7%) | 4 (25.0%) | - | 0 |   |
|  Weight, kg |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.563  |
|  Mean (SD) | 67.49 (16.784) | 67.28 (16.407) | 73.03 (29.258) | 65.20 (16.149) | 75.34 (15.297) | - | 73.03 (29.258) |   |
|  Median | 62.50 | 62.95 | 57.10 | 61.60 | 73.60 | - | 57.10 |   |
|  Range | (35.4; 115.0) | (35.4; 115.0) | (55.2; 106.8) | (35.4; 106.2) | (52.0; 115.0) | - | (55.2; 106.8) |   |
|  Height, cm |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.504  |
|  Mean (SD) | 163.71 (9.020) | 163.84 (9.044) | 160.27 (9.295) | 163.12 (9.406) | 166.66 (7.034) | - | 160.27 (9.295) |   |
|  Median | 162.60 | 162.75 | 154.90 | 160.05 | 165.65 | - | 154.90 |   |
|  Range | (144.5; 192.0) | (144.5; 192.0) | (154.9; 171.0) | (144.5; 192.0) | (150.0; 176.6) | - | (154.9; 171.0) |   |
|  Body mass index, kg/m^{2} |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.320  |
|  Mean (SD) | 24.993 (4.9047) | 24.886 (4.8151) | 27.776 (7.5866) | 24.330 (4.7289) | 27.043 (4.6727) | - | 27.776 (7.5866) |   |
|  Median | 24.250 | 24.508 | 23.798 | 23.455 | 25.858 | - | 23.798 |   |
|  Range | (14.00; 36.87) | (14.00; 36.87) | (23.01; 36.52) | (14.00; 36.72) | (19.57; 36.87) | - | (23.01; 36.52) |   |
|  Underweight ht <18.5 | 4 (4.9%) | 4 (5.1%) | 0 | 4 (6.5%) | 0 | - | 0 |   |
|  Normal 18.5-<25 | 43 (53.1%) | 41 (52.6%) | 2 (66.7%) | 35 (56.5%) | 6 (37.5%) | - | 2 (66.7%) |   |
|  Overweight t 25-<30 | 21 (25.9%) | 21 (26.9%) | 0 | 16 (25.8%) | 5 (31.3%) | - | 0 |   |
|  Obese >=30 | 13 (16.0%) | 12 (15.4%) | 1 (33.3%) | 7 (11.3%) | 5 (31.3%) | - | 1 (33.3%) |   |
|  Local Test Type* |  |  |  |  |  |  |  |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.803  |
|  NGS (Blood) | 4 (4.9%) | 4 (5.1%) | 0 | 3 (4.8%) | 1 (6.3%) | - | 0 |   |
|  NGS (Tissue) | 34 (42.0%) | 33 (42.3%) | 1 (33.3%) | 24 (38.7%) | 9 (56.3%) | - | 1 (33.3%) |   |

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|   | FAS | gAS | gNT | gCEAS | gAS- | gAS-F | gAS-Unk (gAS-F +gNT) | p Value gAS vs gAS- Unk  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  OTHER (Blood) | 1 (1.2%) | 1 (1.3%) | 0 | 1 (1.6%) | 0 | - | 0 |   |
|  OTHER (Tissue) | 7 (8.6%) | 7 (9.0%) | 0 | 7 (11.3%) | 0 | - | 0 |   |
|  PCR (Blood) | 1 (1.2%) | 1 (1.3%) | 0 | 1 (1.6%) | 0 | - | 0 |   |
|  PCR (Tissue) | 30 (37.0%) | 28 (35.9%) | 2 (66.7%) | 23 (37.1%) | 5 (31.3%) | - | 2 (66.7%) |   |
|  UNKNOWN N (Tissue) | 4 (4.9%) | 4 (5.1%) | 0 | 3 (4.8%) | 1 (6.3%) | - | 0 |   |

Abbreviations: FAS, Full Analysis Set; gAS, Guardant360® CDx analysis set; gNT, Guardant360® CDx not tested set; gCEAS, Guardant360® CDx primary clinical efficacy analysis set; gAS-, Guardant360® CDx analysis set- negative; gAS-F, Guardant360® CDx analysis set- failed; gAS-Unk, Guardant360® CDx unknown set. SD, standard deviation. All percentages calculated using N as denominator.

Table 19. Baseline Clinical Characteristics

|   | FAS | gAS | gNT | gCEAS | gAS- | gAS-F | gAS-Unk | p Value gAS vs gAS- Unk  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Analysis set: | 81 | 78 | 3 | 62 | 16 | - | 3 |   |
|  Initial diagnosis  |   |   |   |   |   |   |   |   |
|  NSCLC subtype  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.922  |
|  Adeno- carcinoma | 77 (95.1%) | 74 (94.9%) | 3 (100.0%) | 59 (95.2%) | 15 (93.8%) | - | 3 (100.0%) |   |
|  Large cell carcinoma | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  Squamous cell carcinoma | 3 (3.7%) | 3 (3.8%) | 0 | 2 (3.2%) | 1 (6.3%) | - | 0 |   |
|  Other | 1 (1.2%) | 1 (1.3%) | 0 | 1 (1.6%) | 0 | - | 0 |   |
|  Not reported | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  Histology grade at initial diagnosis  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.708  |
|  Moderately differentiat ed | 18 (22.2%) | 17 (21.8%) | 1 (33.3%) | 16 (25.8%) | 1 (6.3%) | - | 1 (33.3%) |   |
|  Poorly differentiat ed | 12 (14.8%) | 11 (14.1%) | 1 (33.3%) | 8 (12.9%) | 3 (18.8%) | - | 1 (33.3%) |   |
|  Well differentiat ed | 5 (6.2%) | 5 (6.4%) | 0 | 5 (8.1%) | 0 | - | 0 |   |
|  Other | 46 (56.8%) | 45 (57.7%) | 1 (33.3%) | 33 (53.2%) | 12 (75.0%) | - | 1 (33.3%) |   |
|  Not reported | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  Cancer stage at initial diagnosis  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.078  |
|  0 | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  IA | 6 (7.4%) | 6 (7.7%) | 0 | 4 (6.5%) | 2 (12.5%) | - | 0 |   |
|  IB | 1 (1.2%) | 1 (1.3%) | 0 | 1 (1.6%) | 0 | - | 0 |   |
|  IIA | 1 (1.2%) | 1 (1.3%) | 0 | 1 (1.6%) | 0 | - | 0 |   |
|  IIB | 4 (4.9%) | 3 (3.8%) | 1 (33.3%) | 3 (4.8%) | 0 | - | 1 (33.3%) |   |
|  IIIA | 4 (4.9%) | 3 (3.8%) | 1 (33.3%) | 2 (3.2%) | 1 (6.3%) | - | 1 (33.3%) |   |
|  IIIB | 4 (4.9%) | 4 (5.1%) | 0 | 3 (4.8%) | 1 (6.3%) | - | 0 |   |
|  IV | 61 (75.3%) | 60 (76.9%) | 1 (33.3%) | 48 (77.4%) | 12 (75.0%) | - | 1 (33.3%) |   |
|  Not reported | 0 | 0 | 0 | 0 | 0 | - | 0 |   |

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|   | FAS | gAS | gNT | gCEAS | gAS- | gAS-F | gAS-Unk | p Value gAS vs gAS-Unk  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Location of metastasis^{a}  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.598  |
|  Bone | 34 (42.0%) | 33 (42.3%) | 1 (33.3%) | 30 (48.4%) | 3 (18.8%) | - | 1 (33.3%) |   |
|  Liver | 7 (8.6%) | 7 (9.0%) | 0 | 5 (8.1%) | 2 (12.5%) | - | 0 |   |
|  Brain | 18 (22.2%) | 17 (21.8%) | 1 (33.3%) | 14 (22.6%) | 3 (18.8%) | - | 1 (33.3%) |   |
|  Lymph Node | 43 (53.1%) | 43 (55.1%) | 0 | 38 (61.3%) | 5 (31.3%) | - | 0 |   |
|  Adrenal Gland | 3 (3.7%) | 3 (3.8%) | 0 | 3 (4.8%) | 0 | - | 0 |   |
|  Other | 45 (55.6%) | 42 (53.8%) | 3 (100.0%) | 31 (50.0%) | 11 (68.8%) | - | 3 (100.0%) |   |
|  Not reported | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  Time from initial diagnosis of cancer to first dose (months)  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.881  |
|  Mean (SD) | 22.905 (21.1901) | 22.835 (21.3828) | 24.717 (18.7773) | 23.972 (22.8978) | 18.427 (13.7407) | - | 24.717 (18.7773) |   |
|  Median | 17.018 (1.45; | 16.986 (1.45; | 26.021 | 16.789 (2.86; | 18.431 | - | 26.021 |   |
|  Range | 130.10) | 130.10) | (5.32; 42.81) | 130.10) | (1.45; 45.37) | - | (5.32; 42.81) |   |
|  Time from metastatic disease diagnosis to first dose (months)  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.401  |
|  Mean (SD) | 18.071 (16.4424) | 18.374 (16.6647) | 10.185 (5.0347) | 18.886 (17.4686) | 16.388 (13.3918) | - | 10.185 (5.0347) |   |
|  Median | 14.160 (0.69; | 14.883 (0.69; | 9.856 | 14.883 (0.69; | 14.850 | - | 9.856 |   |
|  Range | 116.40) | 116.40) | (5.32; 15.38) | 116.40) | (1.35; 45.37) | - | (5.32; 15.38) |   |
|  Number of prior lines of therapy  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.614  |
|  Mean (SD) | 2.3 (1.41) | 2.2 (1.40) | 2.7 (2.08) | 2.3 (1.47) | 1.9 (1.06) | - | 2.7 (2.08) |   |
|  Median | 2.0 (1; 7) | 2.0 (1; 7) | 2.0 (1; 5) | 2.0 (1; 7) | 2.0 (1; 4) | - | 2.0 (1; 5) |   |
|  Range |  |  |  |  |  |  |  |   |
|  ECOG performance status  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.980  |
|  0 | 26 (32.1%) | 25 (32.1%) | 1 (33.3%) | 19 (30.6%) | 6 (37.5%) | - | 1 (33.3%) |   |
|  1 | 54 (66.7%) | 52 (66.7%) | 2 (66.7%) | 42 (67.7%) | 10 (62.5%) | - | 2 (66.7%) |   |
|  2 | 1 (1.2%) | 1 (1.3%) | 0 | 1 (1.6%) | 0 | - | 0 |   |
|  >2 | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  Not reported | 0 | 0 | 0 | 0 | 0 | - | 0 |   |
|  History of smoking  |   |   |   |   |   |   |   |   |
|  N | 81 | 78 | 3 | 62 | 16 | 0 | 3 | 0.631  |
|  Yes | 38 (46.9%) | 37 (47.4%) | 1 (33.3%) | 25 (40.3%) | 12 (75.0%) | - | 1 (33.3%) |   |
|  No | 43 (53.1%) | 41 (52.6%) | 2 (66.7%) | 37 (59.7%) | 4 (25.0%) | - | 2 (66.7%) |   |
|  Unknown | 0 | 0 | 0 | 0 | 0 | - | 0 |   |

Abbreviations: FAS, Full Analysis Set; gAS, Guardant360 CDx analysis set; gNT, Guardant360® CDx not tested set; gCEAS, Guardant360® CDx primary clinical efficacy analysis set; gAS-, Guardant360® CDx

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analysis set- negative; gAS-F, Guardant360® CDx analysis set- failed; gAS-Unk, Guardant360® CDx unknown set; ECOG, Eastern Cooperative Oncology Group; SD, standard deviation. All percentages calculated using N as denominator.

### D. Safety and Effectiveness Results for the Guardant360® CDx Clinical Bridging Study for EGFR Exon 20 Insertions

#### 1. Safety Results

The safety with respect to treatment with amivantamab-vmjw was addressed during the review of the BLA and is not addressed in this SSED. Please refer to Drugs@FDA for complete safety information on RYBREVANT™ (amivantamab-vmjw). No adverse events were reported in the conduct of the diagnostic studies used to support this PMA supplement as these involved retrospective testing of banked specimens only.

#### 2. Effectiveness Results

##### a. Concordance Results

Concordance between Guardant360® CDx and predominantly tissue testing in the assay analysis agreement set (AAAS) population, which included subjects with CHRYSALIS clinical study local enrolling test results (for biomarker-positives) to determine the positive percent agreement (PPA) and with central tissue NGS test results and NILE clinical study central tissue PCR test results (representing biomarker-negatives) to determine the negative percent agreement (NPA), is shown in Table 20. Guardant360® CDx demonstrated NPA of 100% (95% CI, 97.7% – 100%), PPA of 80.4, (95% CI, 71.4% – 87.1%) relative to local testing results.

Table 20. Unadjusted Agreement Between Guardant360® CDx and CHRYSALIS Enrollment Testing, CHRYSALIS Central Testing, or NILE clinical study cohort cobas® EGFR Testing

|   | CHRYSALIS Clinical Study Enrolling or Central Testing or NILE Clinical Study cobas® EGFR Mutation v2 Testing  |   |   |
| --- | --- | --- | --- |
|   |  EGFR exon 20 Insertion+ | EGFR exon 20 Insertion- | Total  |
|  Guardant360® CDx |  |  |   |
|  EGFR exon 20 Insertion+ | 78 | 0 | 78  |
|  EGFR exon 20 Insertion- | 19 | 164 | 183  |
|  Total | 97 | 164 | 261  |
|  PPA (95% CI) | 80.4% (71.4% - 87.1%)  |   |   |
|  NPA (95% CI) | 100.0% (97.7% - 100.0%)  |   |   |

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Due to the enrichment of the AAAS-L population for subjects positive for *EGFR* exon 20 insertions, adjusted agreement was assessed using the PPV = P(local test+ | Guardant360$^{®}$ CDx+) and NPV = P(local test– | Guardant360$^{®}$ CDx–) for the total AAAS population. In this analysis, Guardant360$^{®}$ CDx demonstrated high adjusted PPV of 100% (95% CI, 95.6% - 100%) and NPV of 99.6% (95% CI, 99.5% - 99.8%) relative to local testing. The prevalence estimate P(local test+) used in the adjusted agreement was 1.8%.

# b. Clinical Efficacy Results in the CHRYSALIS *EGFR* Exon 20 insertion Cohort

The BICR-assessed confirmed ORR as of the 8 October 2020 clinical cutoff date in the *EGFR* exon 20 insertion plus prior chemotherapy at RP2D primary efficacy population was 39.5% (95% CI: 28.8%, 51.0%). Sixty-four (79%) subjects experienced tumor shrinkage of greater than 10% and the clinical activity was observed with amivantamab-vmjw independent of *EGFR* exon 20 insertion subtype. In the primary efficacy population, the BICR-assessed median DOR was 11.1 months (95% CI: 6.90, not estimable [NE]) as of the clinical cutoff date, with the longest response reported as 21.7 months. Amivantamab-vmjw efficacy results are summarized in Table 21.

**Table 21. Efficacy Results for CHRYSALIS Clinical Study**

|   | Prior Platinum-based Chemotherapy Treated (N=81)  |
| --- | --- |
|  **Overall Response Rate (95% CI)** | 39.5% (28.8%, 51.0%)  |
|  Complete response (CR) | 3.7%  |
|  Partial response (PR) | 35.8%  |
|  **Duration of Response (DOR)** |   |
|  Median, months (95% CI)^{b} | 11.1 (6.9, NE)  |
|  Patients with DOR ≥6 months, | 63%  |

$^{b}$ Based on Kaplan-Meier estimates.

NE=Not Estimable, CI=confidence interval

As shown in Table 22, in the primary efficacy population, a total of 31 different *EGFR* Exon 20 insertion variants with varying insertion lengths was observed. The most prevalent insertion was *EGFR* A767_V769dup (29.9%) followed by *EGFR* S768_D770dup (16.9%) and *EGFR* H773dup (5.2%).

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Table 22. Prevalence of EGFR Exon 20 Insertions in CHRYSALIS Primary Efficacy Population

|  EGFR exon 20 insertion | Prevalence in CHRYSALIS (%)  |
| --- | --- |
|  A767_V769dup | 29.9  |
|  S768_D770dup | 16.9  |
|  H773dup | 5.2  |
|  D770delinsGY | 3.9  |
|  N771_H773dup | 3.9  |
|  A763_Y764insFQEA | 2.6  |
|  D770_N771insGF | 2.6  |
|  H773_V774insAH | 2.6  |
|  H773_V774insNPH | 2.6  |
|  P772_H773dup | 2.6  |
|  D770_N771delinsASVDN | 1.3  |
|  D770_N771insG | 1.3  |
|  D770_N771insGD | 1.3  |
|  D770_N771insKD | 1.3  |
|  D770_N771insY | 1.3  |
|  D770_P772dup | 1.3  |
|  D771_H773dup | 1.3  |
|  H773_V774dup | 1.3  |
|  H773_V774insPH | 1.3  |
|  H773_V774insPHPH | 1.3  |
|  H773delinsNPY | 1.3  |
|  N771_P772insH | 1.3  |
|  N771_P772insT | 1.3  |
|  N771_P772insV | 1.3  |
|  N771delinsGF | 1.3  |
|  N771delinsGY | 1.3  |
|  N771delinsKG | 1.3  |
|  P772_H773insDNP | 1.3  |
|  P772_H773insPNP | 1.3  |
|  S768_V769delinsIL | 1.3  |
|  V769_D770insGVV | 1.3  |

# c. ORR in Patients Positive by Guardant360® for EGFR Exon 20 Insertions

The clinical validity of Guardant360® CDx for the selection of NSCLC patients with EGFR exon 20 insertions for treatment with amivantamab-vmjw was demonstrated by comparing the efficacy of amivantamab-vmjw monotherapy in the BLA registration population who are positive for EGFR exon 20 insertions by Guardant360® CDx to the null hypothesis efficacy cited in the CHRYSALIS study protocol. Guardant Health's primary objective analysis set included 62 (76.5% of the CHYRSALIS primary efficacy population) clinical trial samples with valid Guardant360® CDx positive results. The efficacy of amivantamab-vmjw as measured by the ORR observed in the primary objective analysis set (gCEAS) by BICR was 38.7% (95% CI, 26.6% – 51.9%, Table 23). The lower limit of the 95% CI of 26.6% showed statistically significant amivantamab-vmjw efficacy relative to the size-adjusted benchmark ORR of 14% (unadjusted benchmark 15% for chemotherapies in second line treatment setting) from the CHRYSALIS clinical study in the

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Guardant360® CDx-positive, local test-positive portion of the intended use population. The study met the prespecified efficacy acceptance criterion. The gCEAS ORR point estimate was also similar to the FAS ORR of 39.5% (95% CI, 28.8% – 51.0%, Table 23). CHRYSALIS efficacy was also assessed by DOR by BICR for the FAS and gCEAS populations. As shown in Table 24, no meaningful differences in DOR by BICR were observed between the populations.

**Table 23. Summary of ORR in the gCEAS and FAS by BICR**

|  Analysis set: Efficacy | gCEAS | FAS  |
| --- | --- | --- |
|  Best Overall Response (N) | 62 | 81  |
|  Complete Response (CR) | 2 (3.2%) | 3 (3.7%)  |
|  Partial Response (PR) | 22 (35.5%) | 29 (35.8%)  |
|  Stable Disease (SD) | 29 (46.8%) | 39 (48.1%)  |
|  Progressive Disease (PD) | 7 (11.3%) | 8 (9.9%)  |
|  Not Evaluable/Unknown | 2 (3.2%) | 2 (2.5%)  |
|  Overall Response Rate (Confirmed CR + Confirmed PR) (95% CI) | 24 (38.7%) (26.6%, 51.9%) | 32 (39.5%) (28.8%, 51.0%)  |
|  Clinical Benefit Rate (Confirmed CR + Confirmed PR+SD) (95% CI) | 43 (69.4%) (56.3%, 80.4%) | 60 (74.1%) (63.1%, 83.2%)  |

**Table 24. Summary of DOR in the gCEAS and FAS by BICR**

|   | gCEAS | FAS  |
| --- | --- | --- |
|  Analysis set: Efficacy (N) | 62 | 81  |
|  Responders | 24 | 32  |
|  Event | 12 (50.0%) | 14 (43.8%)  |
|  Censored | 12 (50.0%) | 18 (56.3%)  |
|  Time to event (months) |  |   |
|  25th percentile (95% CI) | 5.55 (3.94, 8.31) | 5.55 (4.17, 10.84)  |
|  Median (95% CI) | 8.31 (5.55, NE) | 11.14 (6.90, NE)  |
|  75th percentile (95% CI) | 21.65 (8.31, NE) | 21.65 (11.14, NE)  |
|  Range (months) | (1.3+, 21.7) | (1.3+, 21.7)  |

# d. Sensitivity Analysis

The primary objective analysis above demonstrated amivantamab-vmjw efficacy in the Guardant360® CDx-positive, local test-positive subset of the Guardant360® CDx intended use population. Sensitivity analysis was performed to evaluate the potential impact of samples not available for testing and the hypothetical Guardant360® CDx-positive enrolling test-negative population. The sensitivity analysis was done using the lower

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bound estimate of the 95% CI for the Pr(local test+|CDx+), which was 95.6%. Sensitivity analysis modeling efficacy across the entire Guardant360® CDx intended use population using ORR by BICR assessment showed robustness to the contribution of the unrepresented Guardant360® CDx-positive, local test-negative subjects, with estimated ORRs for the overall Guardant360® CDx intended use population highly similar to those observed for both the gCEAS and FAS due to the low observed prevalence (0%) of the Guardant360® CDx-positive, local test-negative population. The lower limits of the 95% CI for the estimated ORRs across all modeled conditions exceeded the size-adjusted benchmark ORR of 14%, which demonstrates statistically-significant amivantamab-vmjw efficacy across the entire Guardant360® CDx intended use population, irrespective of amivantamab efficacy in the modeled hypothetical Guardant360® CDx-positive, local test-negative sub-population.

#### **E. Pediatric Extrapolation**

In this premarket application, existing clinical data was not leveraged to support approval of a pediatric patient population.

#### **F. Financial Disclosure**

The Financial Disclosure by Clinical Investigators regulation (21 CFR 54) requires applicants who submit a marketing application to include certain information concerning the compensation to, and financial interests and arrangement of, any clinical investigator conducting clinical studies covered by the regulation. The pivotal clinical study included one investigator who was a full-time of the sponsor and had disclosable financial interests/arrangements as defined in 21 CFR 54.2(a), (b), (c) and (f) and described below:

- Compensation to the investigator for conducting the study where the value could be influenced by the outcome of the study: [0]
- Significant payment of other sorts: [0]
- Proprietary interest in the product tested held by the investigator: [0]
- Significant equity interest held by investigator in sponsor of covered study: [1]

The applicant has adequately disclosed the financial interest/arrangements with clinical investigators. Statistical analyses were conducted by FDA to determine whether the financial interests/arrangements had any impact on the clinical study outcome. The information provided does not raise any questions about the reliability of the data.

### **XI. SUMMARY OF SUPPLEMENTAL CLINICAL INFORMATION**

Not applicable.

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## XII. PANEL MEETING RECOMMENDATION AND FDA'S POST-PANEL ACTION

In accordance with the provisions of section 515(c)(3) of the act as amended by the Safe Medical Devices Act of 1990, this PMA was not referred to the Molecular and Clinical Genetics Panel, an FDA advisory committee, for review and recommendation because the information in the PMA substantially duplicates information previously reviewed by this panel.

## XIII. CONCLUSIONS DRAWN FROM PRECLINICAL AND CLINICAL STUDIES

### A. Effectiveness Conclusions

For the intended use to identify EGFR exon 20 insertions in NSCLC patients to be treated with amiva…

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**Source:** [https://fda-staging.innolitics.com/device/P200010S001](https://fda-staging.innolitics.com/device/P200010S001)

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