Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a standard NGS assay and its analytical performance characteristics. There is no mention of AI or ML in the intended use, device description, or performance studies. The analysis focuses on traditional metrics like sensitivity, specificity, and concordance with orthogonal methods.

Therapeutic

No

Explanation: The device is an in vitro diagnostic (IVD) device used for tumor mutation profiling, not for treatment. Its intended use states it provides information "not conclusive or prescriptive for labeled use of any specific therapeutic product."

Diagnostic

Yes.

The "Intended Use" section explicitly states that the PGDx elio™ tissue complete assay is a "qualitative in vitro diagnostic device." It is designed to provide tumor mutation profiling information for use by healthcare professionals for previously diagnosed cancer patients, which directly aligns with the function of a diagnostic device.

SaMD (Software as a Medical Device)

No

The device is described as an "in vitro diagnostic assay" that uses "targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue". This indicates it involves physical components for sample preparation, sequencing, and analysis, not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicit Statement: The "Intended Use / Indications for Use" section explicitly states: "The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device..."
Device Description: The "Device Description" section also refers to it as an "in vitro diagnostic assay".
Nature of the Test: The device analyzes DNA isolated from patient tissue samples (formalin-fixed, paraffin-embedded tumor tissue) to detect genetic alterations. This is a classic example of an in vitro diagnostic test, as it is performed outside of the living body on biological specimens.
Intended Use: The intended use is to provide tumor mutation profiling information for use by qualified healthcare professionals in oncology, which is a diagnostic purpose.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel.

PGDx elio tissue complete is intended to provide tumor mutation profiling information on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients, and is not conclusive or prescriptive for labeled use of any specific therapeutic product.

Product codes (comma separated list FDA assigned to the subject device)

PZM

Device Description

PGDx elio tissue complete is an in vitro diagnostic assay that uses NGS to detect turnor gene alterations in genomic DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue from a variety of tumor types, using a targeted panel (505 genes). The assay takes less than 7 days from DNA to report and provides information on single nucleotide variants (SNVs) in a range of GC content and genomic contexts, insertion/ deletions (indels), 1 amplification as well as 4 translocations. It also identifies microsatellite instability based on select mononucleotide tracts and signatures of sequence mutations. The PGDx elio tissue complete assay utilizes a ~1.3 Mb region of interest (ROI) to calculate tumor mutation burden (TMB). Figure 1.1 describes components of the assay. A complete list of components, equipment and materials can be found in Part 2 (User Guide) of the PGDx elio tissue complete Manual (MN-ETC-03). The panel gene list is provided in Table 1.1.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms.

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Qualified healthcare professionals in oncology for previously diagnosed cancer patients.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Studies:

Specificity:
- Limit of Blank (LoB): Evaluated Non-cancerous FFPE tissues and two NIST reference standards (NA24531, NA24385) with 100ng DNA input. Specificity was 100% with no unverified mutations. False positive rate for Variants with Evidence of Clinical Significance =95% of replicates are detected. Evaluated through dilution series using cell lines and confirmed with clinical specimens. 10 unique clinical cases (SNVs, insertions, deletions) tested with 10 replicates across 2 kit lots (n=20 per specimen), total 200 observations. Additional evaluations used FFPE clinical specimens (11 SNVs, 3 insertions, 5 deletions from 5 clinical FFPE specimens, 5 replicates per dilution level). Call rate >= 95% at 5.9-12.6% MAF. Cell lines used to establish LoD MAF range for 451 SNVs and 31 indels (150 observations).
  - Hotspot SNVs: 3.1% to 5.4% MAF Range.
  - Non-hotspot SNVs: 6.3% to 17.8% MAF Range.
  - Indels at homopolymer context (>=5 bp repeat): 13.7% to 17.5% MAF Range.
  - Indels at non-homopolymer context: 6.1% to 10.9% MAF Range.
- LoD ERBB2, ALK, RET, NTRK2, NTRK3 and MSI: Confirmed by testing 7 clinical FFPE cases diluted with normal FFPE DNA. Each case confirmed at >=95% call rate at 1 tumor purity level with 10 replicates per kit lot across 2 unique lots for translocations and amplifications. For MSI-H, 3 cases confirmed at 1 tumor purity level with 10 replicates each.
  - MSI-H: 18.1% LoD Tumor Purity.
  - ERBB2 amplifications: 4.4% LoD Tumor Purity.
  - ALK translocations: 5.6% LoD Tumor Purity.
  - NTRK2 translocations: 30% LoD Tumor Purity (in silico down sampling suggests 3%).
  - NTRK3 translocations: 11.5% LoD Tumor Purity.
  - RET translocations: 12.8% LoD Tumor Purity.
- TMB and Tumor Purity: Minimum tumor purity for robust TMB reporting established using 8 clinical FFPE cases. Samples serially diluted with replicates ranging from 18 to 50 per sample. PGDx elio tissue complete TMB performance consistent across tumor purities at or above 15%.
- DNA Extraction: 3 FFPE specimens and 1 cell line extracted in duplicate by 2 operators using 3 different methods, total 48 samples processed in duplicate for 96 observations. Overall pass rate for FFPE samples was 93.1% (67/72). %CV for TMB 97.2% and NPA > 99.9% for all variants. TMB MAPE ranged from 0% to 6.0%.
- Endogenous Interference: Impact of necrosis (0-75%) and FFPE block age (0-253 months) on pass rates assessed in 521 samples. No correlation between necrosis and pass rate. Correlation between overall pass rate and age of block (as age increases, pass rate decreases).
Assay Acceptance Rates:
- Overall Clinical FFPE Sample Acceptance Rate: Aggregated data from >40 tumor types (2874 unique clinical cases). First Pass Rate: 81.8% (2352/2874). Overall Pass Rate: 92.9% (2671/2874).
- Pan-Tumor Type/Tissue Comparability: Invalid rates for >18 FFPE tumor types (total 521 samples). Overall invalid rate: 8.1% (42/521).
Accuracy - Concordance to Orthogonal Methods:
- General: Study with 582 samples having PGDx elio tissue complete data and orthogonal data (pre-screened for variant enrichment).
- SNVs and Indels:
  - SNVs with Evidence of Clinical Significance: PPA – 97.2% (35/36), NPA - 99.9% (3994/3996).
  - Hotspot SNVs: PPA - 97.1% (132/136), NPA - 99.9% (35845/35850).
  - Non-hotspot SNVs: PPA – 85.1% (516/606), NPA - 99.9% (178513452/178513618).
  - Hotspot deletions: PPA – 100% (20/20), NPA – 99.9% (2064/2067).
  - Hotspot insertions: PPA – 100% (1/1), NPA - 100% (2015/2015).
  - Non-hotspot indels: PPA - 81.4% (79/97), NPA – 99.9% (67104842/67104857).
- ERBB2 Amplifications: Compared to ERBB2 FISH (147 cases). PPA 75.0% (42/56), NPA 96.7% (88/91). Excluding borderline FISH cases (120 cases): PPA 87.0% (40/46), NPA 95.9% (71/74).
- ALK Translocations: Compared to ALK FISH (71 cases). PPA 92.9% (13/14), NPA 98.2% (56/57). In silico analysis (410 observations from 10 clinical samples): 88% positive call rate at 20% positive nuclei by FISH.
- RET Translocations: Compared to RET FISH (27 cases). PPA 55.6% (5/9), NPA 100% (18/18).
- Accuracy TMB: Compared to matched tumor-normal whole exome sequencing across 8 tumor types (118 cases). Spearman correlation coefficient of 0.903.
- Accuracy - MSI: Assessed in 283 samples (18 tumor types). Compared to MSI PCR.
  - All cases: Excluding failed/indeterminate specimens: PPA 98.8% (79/80), NPA 99.3% (142/143), PPV 98.8% (79/80), NPV 99.3% (142/143).
  - CRC and Endometrial cases (84 total): PPA 100% (51/51), NPA 100% (33/33), PPV 100% (51/51), NPV 100% (33/33).
  - Non-CRC and Non-Endometrial cases (199 total): Excluding failed/indeterminate specimens: PPA 96.6% (28/29), NPA 99.1% (109/110), PPV 96.6% (28/29), NPV 99.1% (109/110).
- Method Comparison Study for Wild Type Calls: 112 specimens assessed for accuracy of 75 hotspot loci within 20 genes compared to 2 orthogonal methods. Overall variant-level concordance: PPA 96.4%, NPA 99.9%.
Reproducibility:
- Interlaboratory Reproducibility: Across 3 different sites, using DNA extracted from 13 FFPE tissue specimens and 1 cell line. Each of 14 samples tested in duplicate by 2 operators on 12 sequencing runs across 3 non-consecutive days at each of 3 sites (total 504 replicates). First pass rate 90.3% (455/504), overall pass rate 98.2% (495/504).
  - Positive call rate across all variants: 86.2%. For SNVs: 88.8%, insertions: 82.8%, deletions: 80.5%.
  - APA and ANA across all 3 sites > 92% for all variant types. No differences across sources of imprecision.
  - Precision of MSI evaluated across 8 MSS and 6 MSI-H samples. Positive call rates for MSI cases: 94.4% to 100%.
  - Precision of TMB evaluated across 11 samples (TMB scores > 7.2 Muts/Mb).
- Lot to Lot Precision: Assessed across 3 unique kit lots. 5 test cases in triplicate (total 45 observations). Overall pass rate 100% (45/45).
  - APA for all variants > 86%.
  - %CV for TMB analyses

Regulation Number and Section

§ 866.6080 Next generation sequencing based tumor profiling test.

(a)
Identification. A next generation sequencing (NGS) based tumor profiling test is a qualitative in vitro diagnostic test intended for NGS analysis of tissue specimens from malignant solid neoplasms to detect somatic mutations in a broad panel of targeted genes to aid in the management of previously diagnosed cancer patients by qualified health care professionals.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) A detailed description of all somatic mutations that are intended to be detected by the test and that are adequately supported in accordance with paragraph (b)(1)(v) of this section and reported in the test results in accordance with paragraph (b)(2)(iv) of this section, including:
(A) A listing of mutations that are cancer mutations with evidence of clinical significance.
(B) As appropriate, a listing of mutations that are cancer mutations with potential clinical significance.
(ii) The indications for use must specify the following:
(A) The test is indicated for previously diagnosed cancer patients.
(B) The intended specimen type(s) and matrix (
e.g., formalin-fixed, paraffin-embedded tumor tissue).(C) The mutation types (
e.g., single nucleotide variant, insertion, deletion, copy number variation or gene rearrangement) for which validation data has been provided.(D) The name of the testing facility or facilities, as applicable.
(iii) A detailed device description including the following:
(A) A description of the test in terms of genomic coverage, as follows:
(
1 ) Tabulated summary of all mutations reported, grouped according to gene and target region within each gene, along with the specific cDNA and amino acid positions for each mutation.(
2 ) A description of any within-gene targeted regions that cannot be reported and the data behind such conclusion.(B) Specifications for specimen requirements including any specimen collection devices and preservatives, specimen volume, minimum tumor content, specimen handling, DNA extraction, and criteria for DNA quality and quantity metrics that are prerequisite to performing the assay.
(C) A detailed description of all test components, reagents, instrumentation, and software required. Detailed documentation of the device software including but not limited to, software applications and hardware-based devices that incorporate software.
(D) A detailed description of the methodology and protocols for each step of the test, including description of the quality metrics, thresholds, and filters at each step of the test that are implemented for final result reporting and a description of the metrics for run-failures, specimen-failures, invalids, as applicable.
(E) A list of links provided by the device to the user or accessed by the device for internal or external information (
e.g., decision rules or databases) supporting clinical significance of test results for the panel or its elements in accordance with paragraphs (b)(1)(v) and (b)(2)(vi) of this section.(F) A description of internal and external controls that are recommended or provided and control procedures. The description must identify those control elements that are incorporated into the testing procedure.
(iv) Information demonstrating analytical validity of the device according to analytical performance characteristics, evaluated either specifically for each gene/mutation or, when clinically and practically justified, using a representative approach based on other mutations of the same type, including:
(A) Data that adequately supports the intended specimen type (
e.g., formalin-fixed, paraffin-embedded tumor tissue), specimen handling protocol, and nucleic acid purification for specific tumor types or for a pan-tumor claim.(B) A summary of the empirical evidence obtained to demonstrate how the analytical quality metrics and thresholds were optimized.
(C) Device precision data using clinical samples to adequately evaluate intra-run, inter-run, and total variability. The samples must cover all mutation types tested (both positive and negative samples) and include samples near the limit of detection of the device. Precision must be assessed by agreement within replicates on the assay final result for each representative mutation, as applicable, and also supported by sequencing quality metrics for targeted regions across the panel.
(D) Description of the protocols and/or data adequately demonstrating the interchangeability of reagent lots and multiplexing barcodes.
(E) A description of the nucleic acid assay input concentration range and the evidence to adequately support the range.
(F) A description of the data adequately supporting the limit of detection of the device.
(G) A description of the data to adequately support device accuracy using clinical specimens representing the intended specimen type and range of tumor types, as applicable.
(
1 ) Clinical specimens tested to support device accuracy must adequately represent the list of cancer mutations with evidence of clinical significance to be detected by the device.(
2 ) For mutations that are designated as cancer mutations with evidence of clinical significance and that are based on evidence established in the intended specimen type (e.g., tumor tissues) but for a different analyte type (e.g., protein, RNA) and/or a measurement (e.g., incorporating a score or copy number) and/or with an alternative technology (e.g., IHC, RT-qPCR, FISH), evidence of accuracy must include clinically adequate concordance between results for the mutation and the medically established biomarker test (e.g., evidence generated from an appropriately sized method comparison study using clinical specimens from the target population).(
3 ) For qualitative DNA mutations not described in paragraph (b)(1)(iv)(G)(2 ) of this section, accuracy studies must include both mutation-positive and wild-type results.(H) Adequate device stability information.
(v) Information that adequately supports the clinical significance of the panel must include:
(A) Criteria established on what types and levels of evidence will clinically validate a mutation as a cancer mutation with evidence of clinical significance versus a cancer mutation with potential clinical significance.
(B) For representative mutations of those designated as cancer mutations with evidence of clinical significance, a description of the clinical evidence associated with such mutations, such as clinical evidence presented in professional guidelines, as appropriate, with method comparison performance data as described in paragraph (b)(1)(iv)(G) of this section.
(C) For all other mutations designated as cancer mutations with potential clinical significance, a description of the rationale for reporting.
(2) The 21 CFR 809.10 compliant labeling and any product information and test report generated, must include the following, as applicable:
(i) The intended use statement must specify the following:
(A) The test is indicated for previously diagnosed cancer patients.
(B) The intended specimen type(s) and matrix (
e.g., formalin-fixed, paraffin-embedded tumor tissue).(C) The mutation types (
e.g., single nucleotide variant, insertion, deletion, copy number variation or gene rearrangement) for which validation data has been provided.(D) The name of the testing facility or facilities, as applicable.
(ii) A description of the device and summary of the results of the performance studies performed in accordance with paragraphs (b)(1)(iii), (b)(1)(iv), and (b)(1)(v) of this section.
(iii) A description of applicable test limitations, including, for device specific mutations validated with method comparison data to a medically established test in the same intended specimen type, appropriate description of the level of evidence and/or the differences between next generation sequencing results and results from the medically established test (
e.g., as described in professional guidelines).(iv) A listing of all somatic mutations that are intended to be detected by the device and that are reported in the test results under the following two categories or equivalent designations, as appropriate: “cancer mutations panel with evidence of clinical significance” or “cancer mutations panel with potential clinical significance.”
(v) For mutations reported under the category of “cancer mutations panel with potential clinical significance,” a limiting statement that states “For the mutations listed in [cancer mutations panel with potential clinical significance or equivalent designation], the clinical significance has not been demonstrated [with adequate clinical evidence (
e.g., by professional guidelines) in accordance with paragraph (b)(1)(v) of this section] or with this test.”(vi) For mutations under the category of “cancer mutations panel with evidence of clinical significance,” or equivalent designation, link(s) for physicians to access internal or external information concerning decision rules or conclusions about the level of evidence for clinical significance that is associated with the marker in accordance with paragraph (b)(1)(v) of this section.

Summary

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April 24, 2020

Image /page/0/Picture/1 description: The image contains the logos of the Department of Health & Human Services and the Food and Drug Administration (FDA). The Department of Health & Human Services logo is on the left, and the FDA logo is on the right. The FDA logo includes the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" and "ADMINISTRATION" in blue text.

Personal Genome Diagnostics Jennifer Dickey, Ph.D., RAC VP, Regulatory and Quality 2809 Boston Street, Suite 503 Baltimore, Maryland 21224

Re: K192063

Trade/Device Name: PGDx™ elio tissue complete Regulation Number: 21 CFR 866.6080 Regulation Name: Next generation sequencing based tumor profiling test Regulatory Class: Class II Product Code: PZM Dated: July 31, 2019 Received: August 1, 2019

Dear Jennifer Dickey:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part

1

801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4. Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Donna Roscoe, Ph.D. Chief Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Ouality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K192063

Device Name PGDx elio™ tissue complete

Indications for Use (Describe)

The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel.

PGDx elio tissue complete is intended to provide tumor mutation on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients, and is not conclusive or prescriptive for labeled use of any specific therapeutic product.

Type of Use (Select one or both, as applicable)

	Prescription Use (Part 21 CFR 801 Subpart D)
	Over-The-Counter Use (21 CFR 801 Subpart C)

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Image /page/3/Picture/0 description: The image shows the logo for Personal Genome Diagnostics (PGDx). The logo consists of the letters "PGD" in blue, with the "D" having an orange accent. To the right of the letters, the words "Personal Genome Diagnostics" are written in a smaller, blue font. The letters PGD are large and bold, making them the most prominent part of the logo.

510(k) Summary

Submission Date: April 24, 2019

Submitter Information:

Submitted By: Personal Genome Diagnostics Inc. 2809 Boston Street, Suite 503 Baltimore, MD 21224

Contact Person: Jennifer S. Dickey PhD, RAC Vice President, Regulatory & Quality Personal Genome Diagnostics Tel: (443) 602-8833 Email: jdickey@pgdx.com

A. Proprietary and Established Names

PGDx elio™ tissue complete

B. 510(k) number

K192063

C. Measurand

Somatic single nucleotide variants, insertions and deletions, select amplifications and translocations, microsatellite instability and tumor mutation burden in human genomic DNA obtained from formalin-fixed, paraffin-embedded tumor tissue.

D. Regulatory Information

1. Regulation section

21 CFR 866.6080

2. Classifications

Class II

3. Product Code

PZM

E. Indications for Use

1. Indications for Use

The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-

CONFIDENTIAL

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Image /page/4/Picture/0 description: The image shows the logo for Personal Genome Diagnostics (PGDx). The logo consists of the letters "PGD" in blue, followed by an orange "X". To the right of the letters is the text "Personal Genome Diagnostics" in blue, with a small "TM" symbol next to the word "Diagnostics".

embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel.

PGDx elio tissue complete is intended to provide tumor mutation profiling information on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients, and is not conclusive or prescriptive for labeled use of any specific therapeutic product.

2. Special conditions for use statement(s):

For prescription use.

For in vitro diagnostic use.

3. Special Instrument Requirements

NextSeq® 550Dx (qualified by PGDx)

F. Substantial Equivalence Information:

1. Predicate device name(s)
  MSK-IMPACT
1. Predicate 510(k) Number
  DEN170058

3. Comparison with predicate

| | PGDx elio tissue complete | MSK-IMPACT
(Predicate Device) |
|---------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| K Number | K192063 | DEN170058 |
| SIMILARITIES | | |
| Assay
Intended Use
/ Indications
for Use | The PGDx elio™ tissue complete
assay is a qualitative in vitro
diagnostic device that uses targeted
next generation sequencing of DNA
isolated from formalin-fixed,
paraffin-embedded tumor tissue
from patients with solid malignant
neoplasms to detect tumor gene | The MSK-IMPACT assay is a
qualitative in vitro diagnostic test
that uses targeted next generation
sequencing of formalin-fixed
paraffin-embedded tumor tissue
matched with normal specimens
from patients with solid malignant
neoplasms to detect tumor gene
alterations in a broad multi gene
panel. The test is intended to provide |
| | PGDx elio tissue complete | MSK-IMPACT
(Predicate Device) |
| | alterations in a broad multi-gene
panel.

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Image /page/5/Picture/0 description: The image is a logo for Personal Genome Diagnostics, or PGDx. The letters "PGD" are in blue, and the "x" is in orange. To the right of the letters is the text "Personal Genome Diagnostics" in a smaller font size. The letters are bolded and the text is in a sans-serif font.

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Image /page/6/Picture/0 description: The image shows the logo for Personal Genome Diagnostics (PGDx). The letters "PGD" are in blue, and the "X" is in orange. To the right of the letters is the text "Personal Genome Diagnostics" in blue. The letters are in a sans-serif font.

G. Summary and Explanation

4. Product Description

PGDx elio tissue complete is an in vitro diagnostic assay that uses NGS to detect turnor gene alterations in genomic DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue from a variety of tumor types, using a targeted panel (505 genes). The assay takes less than 7 days from DNA to report and provides information on single nucleotide variants (SNVs) in a range of GC content and genomic contexts, insertion/ deletions (indels), 1 amplification as well as 4 translocations. It also identifies microsatellite instability based on select mononucleotide tracts and signatures of sequence mutations. The PGDx elio tissue complete assay utilizes a ~1.3 Mb region of interest (ROI) to calculate tumor mutation burden (TMB). Figure 1.1 describes components of the assay. A complete list of components, equipment and materials can be found in Part 2 (User Guide) of the PGDx elio tissue complete Manual (MN-ETC-03). The panel gene list is provided in Table 1.1.

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Image /page/7/Figure/3 description: The image shows the assay components of PGDx elio tissue complete. The left side of the image shows the assay components provided by PGDx, which include PGDx elio tissue complete, PGDx elio server, and PGDx elio platform. The right side of the image shows the assay components provided by the user, which include DNA extraction method, molecular laboratory equipment, NextSeq 550Dx sequencing reagents, and NextSeq 550Dx sequencer.

Figure 1.1 PGDx elio tissue complete assay components. PGDx provided components include reagent kits, software for data analysis, and a server.

Table	1.1
-------	-----

PGDx elio tissue complete Gene List‡
ABL1	ABL2	ACVR1	ACVR1B	ADORA2A	AKT1	AKT2	AKT3
ALK*	ALOX12B	AMER1	APC	AR	ARAF	ARFRP1	ARID1A
ARID1B	ARID2	ARID5B	ASXL1	ASXL2	ATM	ATR	ATRX
AURKA	AURKB	AXIN1	AXIN2	AXL	B2M	BAP1	BARD1
BBC3	BCL2	BCL2L1	BCL2L11	BCL2L2	BCL6	BCOR	BCORL1
BCR	BIRC2	BLM	BMPR1A	BRAF	BRCA1	BRCA2	BRD4
BRIP1	BTG1	BTG2	BTK	BUB1B	C11ORF30	CALR	CARD11
CASP8	CBFB	CBL	CCND1	CCND2	CCND3	CCNE1	CD22
CD274	CD276	CD70	CD79A	CD79B	CDC73	CDH1	CDK12
CDK4	CDK6	CDK8	CDKN1A	CDKN1B	CDKN1C	CDKN2A	CDKN2B
CDKN2C	CEBPA	CHD2	CHD4	CHEK1	CHEK2	CIC	CREBBP
CRKL	CSF1	CSF1R	CSF2	CSF3	CSF3R	CTCF	CTLA4
CTNNA1	CTNNB1	CUL3	CUL4A	CXCR2	CXCR4	CYLD	CYP17A1
DAXX	DCUN1D1	DDB2	DDR1	DDR2	DICER1	DIS3	DNMT1
DNMT3
A	DNMT3
B	DOT1L	E2F3	EED	EGFL7	EGFR	EIF1AX
EP300	EPAS1	EPCAM	EPHA2	EPHA3	EPHA5	EPHA7	EPHB1
EPHB4	ERBB2#	ERBB3	ERBB4	ERCC1	ERCC2	ERCC3	ERCC4
ERCC5	ERCC6	ERCC8	ERG	ERRFI1	ESR1	ETV1	ETV4
ETV5	ETV6	EWSR1	EXT1	EXT2	EZH2	FAM175	FAM46CA
FANCA	FANCB	FANCC	FANCD2	FANCE	FANCF	FANCG	FANCI
FANCL	FANCM	FAS	FAT1	FBXW7	FGF10	FGF12	FGF14
FGF19	FGF23	FGF3	FGF4	FGF6	FGFR1	FGFR2	FGFR3
FGFR4	FH	FLCN	FLT1	FLT3	FLT4	FOXA1	FOXL2
FOXP1	FRS2	FUBP1	GABRA6	GATA1	GATA2	GATA3	GATA4
GATA6	GID4	GLI1	GNA11	GNA13	GNAQ	GNAS	GPC3
GPR124	GREM1	GRIN2A	GRM3	GSK3B	H3F3A	H3F3B	H3F3C
HDAC1	HDAC2	HDAC6	HGF	HIST1H1C	HIST1H2BD	HIST1H3B	HNF1A
HRAS	HSD3B1	HSP90AA1	HSP90AB1	ICOSLG	ID3	IDH1	IDH2
IFNGR1	IGF1	IGF1R	IGF2	IGF2R	IKBKE	IKZF1	IL10
IL7R	INHBA	INPP4A	INPP4B	INSR	IRF2	IRF4	IRS1
IRS2	JAK1	JAK2	JAK3	JUN	KAT6A	KDM5A	KDM5C
KDM6A	KDR	KEAP1	KEL	KIT	KLF4	KLHL6	KMT2A
KMT2C	KMT2D	KRAS	LATS1	LATS2	LMO1	LRP1B	LTK
LYN	LZTR1	MAF	MAGI2	MAML1	MAP2K1	MAP2K2	MAP2K4
MAP3K1	MAP3K13	MAPK1	MAX	MCL1	MDC1	MDM2	MDM4
MED12	MEF2B	MEN1	MERTK	MET	MITF	MKNK1	MLH1
MLH3	MPL	MRE11A	MSH2	MSH3	MSH6	MST1R	MTAP
MTOR	MUTYH	MYB	MYC	MYCL	MYCN	MYD88	MYOD1
NBN	NCOA3	NCOR1	NF1	NF2	NFE2L2	NFKBIA	NKX2-1
NKX3-1	NOTCH1	NOTCH2	NOTCH3	NOTCH4	NPM1	NRAS	NSD1
NT5C2	NTRK1	NTRK2*	NTRK3*	NUP93	NUTM1	PAK1	PAK3
PAK7	PALB2	PARK2	PARP1	PARP2	PARP3	PAX5	PAX8
PBRM1	PDCD1	PDCD1LG2	PDGFRA	PDGFRB	PDK1	PDPK1	PHOX2B
PIK3C2B	PIK3C2G	PIK3C3	PIK3CA	PIK3CB	PIK3CD	PIK3CG	PIK3R1
PIK3R2	PIK3R3	PIM1	PLCG2	PLK2	PMAIP1	PMS1	PMS2
PNRC1	POLD1	POLE	POLH	POT1	PPARG	PPP2R1A	PPP2R2A
PRDM1	PREX2	PRKAR1
A	PRKCI	PRKDC	PRSS1	PRSS8	PTCH1
PTEN	PTK2	PTPN11	PTPRD	PTPRO	PTPRS	PTPRT	QKI
RAC1	RAD21	RAD50	RAD51	RAD51B	RAD51C	RAD51D	RAD52
RAD54B	RAD54L	RAF1	RANBP2	RARA	RASA1	RB1	RBM10
RECQL4	REL	RET*	RFWD2	RHOA	RICTOR	RIT1	RNF43
ROS1	RPA1	RPS6KA4	RPS6KB2	RPTOR	RUNX1	RUNX1
T1	RYBP
SBDS	SDHA	SDHAF2	SDHB	SDHC	SDHD	SETD2	SF3B1
SGK1	SH2D1A	SHQ1	SLIT2	SLX4	SMAD2	SMAD3	SMAD4
SMARC
A4	SMARC
B1	SMARC
D1	SMO	SNCAIP	SOCS1	SOX10	SOX17
SOX2	SOX9	SPEN	SPOP	SPTA1	SRC	STAG2	STAT3
STAT4	STK11	STK40	SUFU	SUZ12	SYK	TAF1	TBX3
TEK	TERC	TERT	TET1	TET2	TGFBR1	TGFBR2	TIPARP
TLR4	TLR7	TLR8	TLR9	TMEM127	TMPRSS2	TNFAIP3	TNFRSF14
TOP1	TOP2A	TP53	TP53BP1	TP63	TRAF7	TSC1	TSC2
TSHR	TYRO3	U2AF1	VEGFA	VHL	VTCN1	WAS	WEE1
WHSC1	WHSC1
L1	WISP3	WRN	WT1	XIAP	XPA	XPC
XPO1	XRCC1	XRCC2	XRCC3	YAP1	YES1	ZBTB2	ZNF217
ZNF703

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: - All genes listed contribute to TMB score and SNV/indel reporting: * - Translocations reported for this gene: # - Amplifications reported for this gene

5. Sample Preparation

The PGDx elio tissue complete assay requires genomic DNA isolated from FFPE tissue specimens using commercially available DNA extraction methods. The assay is validated for use with DNA recovered from tissue with a minimum of 20% viable tumor nuclei. If less than 100% of the tissue section contains ≥20% tumor purity, the tissue should be macro-dissected to select as much viable tumor as possible and minimize the amount of adjacent non-tumor tissue. The recommended DNA input for the assay is 100 ng at a minimum concentration of 1 ng/μL; results can be obtained with inputs down to 50 ng.

6. Library Preparation

The PGDx elio tissue complete assay workflow begins with genomic DNA. Genomic DNA is quantified using a fluorometer. DNA molecules are mechanically sheared to a target size of 200 bp and subjected to a magnetic bead purification step to remove smaller fragments and perform an exchange of buffer.

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Fragmented DNA is end-repaired, phosphorylated, and adenylated. Indexed adapters are then ligated to the A-tailed DNA molecules. Unincorporated adapters and reagents are removed by magnetic bead purification. Adapter-ligated DNA is enriched by PCR amplification. Primer dimers and residual reagents are removed by magnetic bead purification. Library quality is assessed using a DNA fragment analyzer prior to hybrid capture.

7. Hybrid Capture NGS

The adapter-ligated library is hybridized with biotinylated RNA library baits, and targeted regions are captured using magnetic streptavidin coated beads. Captured libraries are purified to remove baits and incompletely hybridized DNA fragments. Captured libraries are enriched by PCR amplification. Primer dimers and residual reagents are removed by magnetic bead purification. Final library quality is assessed using a DNA fragment analyzer prior to sequencing.

8. Sequencing

Sample libraries are quantified and normalized into a sequencing pool of up to 15 samples and the external control. Pooled sample libraries are fluorometrically quantified, loaded on a sequencing flow cell and sequenced using a NextSeq® 550Dx instrument which has been pre-qualified by PGDx.

9. Data Analysis

Sequence data is processed using the PGDx elio platform software. The software contains a user interface that tracks sample status from sequencing through analysis and reporting. Users configure sequencing runs, and an automated pipeline of software for bioinformatic analysis identifies and reports genomic alterations. After processing, the software generates FASTQ files containing sequences and quality scores for each sample. The FASTQ files are then aligned to a reference genome to generate BAM files, which are processed for variant calling of different alteration types (SNVs, indels, amplifications, translocations, and MSI). SNVs and indels are then used to determine TMB scores reported as mutations per megabase.

10. Controls

i. Negative Control: A no template control (NTC) can be processed to serve as a negative control to validate the acceptability of all the test samples processed through library preparation and capture steps by testing for sample or reagent contamination. The NTC is not included on the sequencing run.
ii. Positive Control: An external control that is provided in the PGDx elio tissue complete assay reagent kit consists of cell line derived-DNA with multiple verified sequence mutations. The external control is processed from library preparation through sequencing to serve as an end to end control to demonstrate assay

CONFIDENTIAL

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performance. The external control is checked for quality during library preparation and after sequencing. Failure of the external control to meet the pre-defined quality metrics will result in all test samples on the run being reported as 'No result.'

11. Results Reporting

PGDx elio tissue complete reports SNVs and indels in protein coding regions across all genes in the panel. In addition, amplifications are reported for ERBB2 as well as translocations for ALK, RET, NTRK2, and NTRK3. The assay also reports on 2 genomic signatures, MSI and TMB.

The variants listed in the section Variants with Evidence of Clinical Significance are determined based on the selected tumor type. Only variants clinically associated with the tested tumor type will appear in the Variants with Evidence of Clinical Significance section. Any remaining detected variants will appear as the Variants with Potential Clinical Significance. A qualified healthcare professional can select the appropriate tumor type, and depending on the tumor type selected, variants will be reported as Variants with Evidence of Clinical Significance by PGDx elio tissue complete. Any variants clinically associated with tumor types other than the one selected will be reported in the section labeled 'Variants with Potential Clinical Significance. A list of all genes is provided in Appendix A.

SNVs and indels results are also presemted in terms of somatic hotspot or non-hotspot. PGDx defines hotspots as > 25 exact hits in COSMIC version 72. A lower minimum MAF is used when reporting these hotspot mutations.

Quality Metric	Level of Qualification	Passing Criteria
Cluster Density	Batch-level	Sequencer Cluster Density $≥$ 130
Q30 Reads	Batch-level	%Q30 (Read1 and Read4) $≥$ 80%
%Q30 (Read2 and Read3) $≥$ 85%
External Control	Batch-level	All expected sequence mutations are detected and passes all other quality criteria
Percent Regions Covered	Sample-level	$≥$ 90% exons with > 100x Median Distinct Coverage
Percent Reads Identified	Sample-level	Percent Reads Identified 15%-35%
Contamination QC	Sample-level	Estimated contamination levels 0.4%
Hotspot SNVs
and Indels	Analyte-level	Mutant reads $\ge$ 4
MAF > 2%
Non-hotspot
SNVs	Analyte-level	Mutant reads $\ge$ 6
MAF with lower bound 95% CI $\ge$ 5%
Non-hotspot
Indels	Analyte-level	Mutant reads $\ge$ 6
MAF > 5%
Homopolymer
Indels	Analyte-level	Homopolymer regions 97.2%) and NPA (>99.9%) for all variants. The TMB mean absolute percent error (MAPE) ranged from 0% to 6.0% across conditions. The results show minimal risk to assay performance from interfering exogenous substances.

Substance	Amount in Excess of Standard Conditions
Proteinase K	2X and 3X
Indexed adapters	15% and 30%
Melanin	0.2 $ \mu $ g/mL and 1.6 $ \mu $ g/mL
Ethanol	2.5% and 5%

Table 1.7 Exogenous Interfering Substances Tested
---------------------------------------------------	--	--	--

ii. Endogenous Interference

The impact of necrosis and FFPE block age on the performance of PGDx elio tissue complete was evaluated by assessing the first pass and overall pass rates of samples processed in the accuracy study (see Accuracy section below). Of 521 samples enrolled for accuracy, 448 were evaluated for necrosis over a range of 0-75%, and 378 were evaluated for age of block over a range of 0-253 months. The data indicated there is no

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correlation between necrosis and pass rate. A logistic regression analysis was performed to establish the probability of sample pass/fail according to FFPE block age as shown in Figure 1.3. There is a correlation between overall pass rate and the age of block; as the age of block increases, the overall pass rate decreases. The probability of samples passing from blocks aged roughly 175 months (or 14.5 years) is approximately 75%. The results show minimal risk to assay performance from interfering endogenous factors.

Image /page/18/Figure/4 description: The image contains two scatter plots that show the probability of success as a function of FFPE Block Age. The left plot shows the probability of first pass success, while the right plot shows the probability of overall pass. In both plots, the x-axis represents the FFPE Block Age in months, and the y-axis represents the probability of success, ranging from 0 to 1. Both plots show a decreasing trend in the probability of success as the FFPE Block Age increases.

Figure 1.3 Logistic Regression Graphs of First Pass and Overall Acceptability vs FFPE Block Age. The orange line represents a regression line, and orange shading represents the 95% confidence interval. The blue dots represent individual samples assessed.

16. Assay Acceptance Rates

Multiple factors can influence overall robustness and performance of complex molecular tests, including pre-analytical factors and overall sample quality. If key in-process or automated data quality metrics are not met. PGDx elio tissue complete supports repeating samples through the workflow. Performance throughout verification and validation of the device was tracked and a summary of the rates for first pass (no repeat) and overall pass (allowing for a single repeat) are presented below.

i. Overall Clinical FFPE Sample Acceptance Rate
Data were aggregated for unique clinical cases from >40 tumor types assessed during verification and validation of PGDx elio tissue complete. Resulting pass rates for clinical samples are presented in Table 1.8. The data indicate that results are obtained on a high percentage of FFPE samples after processing through the PGDx elio tissue complete assay.

Table 1.8 PGDx elio tissue complete Acceptability Rates

First Pass Rate (n/N) (2-sided 95% CI)	Overall Pass Rate (n/N) (2-sided 95% CI)
81.8% (2352/2874) (80.4%, 83.2%)	92.9% (2671/2874) (91.9%, 93.8%)

ii. Pan-Tumor Type/Tissue Comparability

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Invalid rates for the different tumor types assessed in the analytical accuracy study are provided in Table 1.9 below.

Tumor Type	Passing Samples	Total Samples	Invalid Rate (%)
Bladder	6	7	14.3
Brain	10	10	0
Breast	60	72	16.7
Colorectal	91	97	6.2
Endometrial	27	27	0
Gastric	25	31	19.4
Glioma	4	4	0
Head and Neck	5	6	16.7
Lung - NOS1	64	68	5.9
Melanoma	34	36	5.6
NOS1	8	8	0
NSCLC1	85	92	7.6
Other2	21	22	4.5
Ovarian	8	9	11.1
Pediatric Glioma	9	9	0
Prostate	7	8	12.5
Skin	4	4	0
Triple Negative Breast	11	11	0
Total	479	521	8.1

	Table 1.9 Specimen Invalid Rates for >18 FFPE Tumor Types

1 NOS: not otherwise specified; NSCLC: non-small cell lung cancer.

20ther (n ≤ 3 cases per tumor type): cervical, cholangiocarcinoma, gallbladder, pancreatic, rhabdomyosarcoma, trachea, esophageal, fallopian tube, liver, mediastinum, peritoneal, renal, and thyroid.

17. Accuracy - Concordance to Orthogonal Methods

In order to demonstrate the accuracy of PGDx elio tissue complete as a tumor profiling device, a study was performed with 582 samples that had both PGDx elio tissue complete data and orthogonal data. Due to the rarity of specific variants in solid tumor FFPE samples, most samples selected for this study were pre-screened, resulting in enrichment of certain variants relative to real-world clinical prevalence. Data were

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April 24, 2020

PGDx elio™ tissue complete

aggregated at the variant level for SNVs, insertions, and deletions, gene level for amplifications and translocations, and case level for MSI and TMB.

The results summarized in Table 1.10 indicate that the assay accurately detects SNVs and indels.

| Variant | Orthogonal
Method(s) | Performance (n/N) (2-sided 95% CI) |
|-------------------------------------------------------|----------------------------------------|---------------------------------------------------------------------------------------------|
| SNVs with Evidence
of Clinical
Significance | 2 NGS
targeted
panels | PPA – 97.2% (35/36) (85.8%, 99.5%)
NPA - 99.9% (3994/3996) (99.8%, 99.9%) |
| Hotspot SNVs | 2 NGS
targeted
panels and
PCR | PPA - 97.1% (132/136) (92.7%, 98.9%)
NPA - 99.9% (35845/35850) (99.9%,
99.9%) |
| Non-hotspot SNVs | 2 NGS
targeted
panels | PPA – 85.1% (516/606) (82.1%, 87.8%)
NPA - 99.9% (178513452/178513618)
(99.9%, 99.9%) |
| SNVs with Potential
Clinical Significance | 2 NGS
targeted
panels | PPA – 86.4% (591/684) (83.6%, 88.8%)
NPA - 99.9% (178513372/178513540)
(99.9%, 99.9%) |
| Hotspot deletions | 2 NGS
targeted
panels and
PCR | PPA – 100% (20/20) (84.5%, 100%)
NPA – 99.9% (2064/2067) (99.6%, 99.9%) |
| Hotspot insertions | 2 NGS
targeted
panels | PPA – 100% (1/1) (20.7%, 100%)
NPA - 100% (2015/2015) (99.8%, 100%) |
| Non-hotspot indels | NGS
targeted
panel | PPA - 81.4% (79/97) (72.6%, 87.9%)
NPA – 99.9% (67104842/67104857)
(99.9%, 99.9%) |
| Non-hotspot
insertions | NGS
targeted
panel | PPA – 80.8% (21/26) (62.1%, 91.5%)
NPA - 99.9% (67104926/67104928) |
| Non-hotspot
deletions | NGS
targeted
panel | PPA - 81.7% (58/71) (71.2%, 89.0%)
NPA - 99.9% (67104870/67104883) |
| Insertions with
Potential Clinical
Significance | NGS
targeted
panel | PPA - 80.8% (21/26) (62.1%, 91.5%)
NPA - 99.9% (67497962/67497964)
(99.9%, 99.9%) |
| Deletions with
Potential Clinical
Significance | NGS
targeted
panel | PPA – 82.7% (62/75) (72.6%, 89.6%) |
| | | NPA – 99.9% (67497902/67497915) |
| | | (99.9%, 99.9%) |

Table 1.10 Accuracy – SNVs and Indels

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a) Accuracy for ERBB2 Amplifications
Concordance for ERBB2 Amplifications was assessed by comparing PGDx elio tissue complete to ERBB2 FISH (Table 1.11 and Table 1.12). The PPA was 75.0% (95% CI: 62.3%, 84.5%) for all cases and 87.0% (95% CI: 74.3%, 93.9%) when excluding borderline FISH cases (defined as HER2/CEP17 ratio between 1.5 and 2.5). The NPA was 96.7% (95% CI: 90.8%, 98.9%) for all cases and 95.9% (95% CI: 88.7%, 98.6%) when excluding borderline FISH cases.

Table 1.11 Summary of Concordance between PGDx elio tissue complete and ERBB2 FISH Including Borderline Cases

	ERBB2 FISH
PGDx elio tissue complete	ERBB2 Positive	ERBB2 Negative	Total
ERBB2 Positive	42	3	45
ERBB2 Negative	14	88	102
Total	56	91	147

Table 1.12 Summary of Concordance between PGDx elio tissue complete and ERBB2 FISH Excluding Borderline Cases

	ERBB2 FISH
PGDx elio tissue
complete	ERBB2 Positive	ERBB2 Negative	Total
ERBB2 Positive	40	3	43
ERBB2 Negative	6	71	77
Total	46	74	120

b) Accuracy for ALK Translocations

Concordance for ALK translocations was assessed by comparing PGDx elio tissue complete to ALK FISH (Table 1.13). The PPA was 92.9% (95% CI: 68.5%, 98.7%), and NPA was 98.2% (95% CI: 90.7%, 99.7%).

Table 1.13 Summary of Concordance between PGDx elio tissue complete and ALK

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FISH

	ALK FISH
PGDx elio tissue
complete	ALK Positive	ALK Negative	Total
ALK Positive	13	1	14
ALK Negative	1	56	57
Total	14	57	71

ALK translocations were additionally assessed in silico due to limited availability of clinical cases close to the ALK FISH equivocal zone (10%-50% rearrangement positive nuclei). A total of 410 observations were generated for ALK by down-sampling 10 clinical samples from analytical accuracy to 4 tumor purity dilution levels with 10 replicates per level, to mimic samples in the FISH equivocal zone. For example, if the undiluted sample had a FISH score of 50% from analytical accuracy, the sample was diluted with wild type reads by a factor of 0.8 to get to a 40% positive nuclei FISH score. These data demonstrate an 88% positive call rate at 20% positive nuclei by FISH (Table 1.14).

| FISH (%Positive
Nuclei) | FISH
Call | PGDx elio tissue complete Positive Call Rate (%) (n/N)
(95% CI) |
|----------------------------|--------------|--------------------------------------------------------------------|
| 50 - 88 | + | 100% (10/10) (72%, 100%) |
| 40 | + | 98% (98/100) (93%, 99%) |
| 30 | + | 95% (95/100) (89%, 98%) |
| 20 | + | 88% (88/100) (80%, 93%) |
| 10 | - | 80% (80/100) (71%, 87%) |

c) Accuracy for RET Translocations

Concordance for RET translocations was assessed by comparing PGDx elio tissue complete to RET FISH (Table 1.15). The PPA was 55.6% (95% CI: 26.7%, 81.1%), and NPA was 100% (95% CI: 82.4%, 100%).

Table 1.15 Summary of Concordance between PGDx elio tissue complete and RET FISH

	RET FISH
PGDx elio	RET Positive	RET Negative	Total
RET Positive	5	0	5
RET Negative	41	18	22

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Total	9	18	27
-------	---	----	----

1No read data supporting RET translocations was found in the raw data for the discrepant cases. In addition to the clinical samples processed to assess RET translocations, 3 RET translocation-positive cell lines were also tested with PGDx elio tissue complete. All 3 cell lines were positive for a fusion either by a validated assay performed by the cell line provider, or via literature. PGDx elio tissue complete detected all 3 fusions in these cell lines.

ii. Accuracy TMB
The PGDx elio tissue complete assay reports a TMB score comprised of sequence mutations detected across the entire coding region of interest per sample. The ability of PGDx elio tissue complete to accurately identify TMB in multiple solid tissue FFPE tumor types was assessed by comparing to matched tumor-normal whole exome sequencing results. Across 8 tumor types (non-small cell lung carcinoma (NSCLC), melanoma, renal, bladder, endometrial, triple negative breast, head and neck, lung-NOS (not otherwise specified)), 118 cases were enrolled covering a dynamic range of 1.5-118.5 Muts/Mb. Of those, 31 fell below the established LoB (≤ 7.2 Muts/Mb). The Spearman correlation coefficient was used to determine the relationship between the 2 assays. Assessment of all 118 cases resulted in a Spearman correlation coefficient of 0.903. The results in Figure 1.4 show strong concordance between PGDx elio tissue complete TMB scores and tumor-normal whole exome sequencing.

Image /page/23/Figure/7 description: The image is a scatter plot that compares PGDx elio TMB (Muts/Mb) on the y-axis to Exome TMB (Muts/Mb) on the x-axis. The data points are clustered, showing a positive correlation between the two variables. A dashed line represents the regression fit, with the equation y=1.56*x + 6.09, and the Spearman correlation is 0.903, indicating a strong positive relationship.

Figure 1.4 PGDx elio tissue complete TMB score vs. Matched Tumor-Normal Exome Sequencing.

iii. Accuracy - MSI

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Table 1.16 contains MSI performance for all 283 samples including PGDx elio tissue complete and PCR failures and indeterminates. This cohort is further divided into performance for colorectal (CRC) and endometrial cases (Table 1.17) and non-CRC and non-endometrial cases (Table 1.18). MSI accuracy was assessed in 18 tumor types: ampulla (1), bladder (7), breast (21), colorectal (66), endometrial (18), esophagus (1), fallopian tube (1), gall bladder (1), gastric (40), lung (39), kidney (3), omentum (1), ovarian (2), prostate (4), sarcoma (3), skin (8), thyroid (2), and cancer of unknown primary (5).

		MSI PCR				Total
		MSI	MSS	Failed	Indeterminate
PGDx elio tissue
complete	MSI	79	1	0	11	81
	MSS	1	142	0	52	148
	Failed	4	40	10	0	54
Total		84	183	10	6	283
Excluding failed/
indeterminate
specimens with 95%
CI	PPA		98.8% (79/80) (93.3%, 99.8%)
	NPA		99.3% (142/143) (96.1%, 99.9%)
	PPV		98.8% (79/80) (93.3%, 99.8%)
	NPV		99.3% (142/143) (96.1%, 99.9%)
Accounting for
failed/ indeterminate
specimens with 95%
CI	PPA		94.0% (79/84) (86.8%, 97.4%)
	NPA		77.6% (142/183) (71.0%, 83.0%)
	PPV		97.5% (79/81) (91.4%, 99.3%)
	NPV		95.9% (142/148) (91.4%, 98.1%)

Table 1.16 MSI Performance for All Cases

1This case was MSI-H by PGDx elio, and Promega PCR gave an "Indeterminate" result.

2These 5 cases did not have matching normal DNA to test via Promega PCR.

Table 1.17 MSI Performance for CRC and Endometrial Cases

		MSI PCR				Total
		MSI	MSS	Failed	Indeterminate
PGDx elio tissue complete	MSI	51	0	0	0	51
	MSS	0	33	0	0	33
	Failed	0	0	0	0	0
Total		51	33	0	0	84
Excluding failed/
indeterminate	PPA		100% (51/51) (93.0%, 100%)
	NPA		100% (33/33) (89.6%, 100%)

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Image /page/25/Picture/0 description: The image shows the logo for Personal Genome Diagnostics (PGDx). The logo consists of the letters "PGD" in blue, followed by an orange "X". To the right of the letters is the text "Personal Genome Diagnostics" in blue. The letters are in a sans-serif font.

specimens with 95% CI	PPV	100% (51/51) (93.0%, 100%)
	NPV	100% (33/33) (89.6%, 100%)
Accounting for
failed/
indeterminate
specimens with 95%
CI	PPA	100% (51/51) (93.0%, 100%)
	NPA	100% (33/33) (89.6%, 100%
	PPV	100% (51/51) (93.0%, 100%)
	NPV	100% (33/33) (89.6%, 100%)

Table 1.18 MSI Performance for Non-CRC and Non-Endometrial Cases

		MSI PCR				Total
		MSI	MSS	Failed	Indeterminate
PGDx elio tissue	MSI	28	1	0	11	30
complete	MSS	1	109	0	52	115
	Failed	4	40	10	0	54
Total		33	150	10	6	199
Excluding failed/
indeterminate
specimens with 95%
CI	PPA		96.6% (28/29) (82.8%, 99.4%)
	NPA		99.1% (109/110) (95.0%, 99.8%)
	PPV		96.6% (28/29) (82.8%, 99.4%)
	NPV		99.1% (109/110) (95.0%, 99.8%)
Accounting for
failed/ indeterminate
specimens with 95%
CI	PPA		84.8% (28/33) (69.1%, 93.4%)
	NPA		72.7% (109/150) (65.0%, 79.2%)
	PPV		93.3% (28/30) (78.7%, 98.2%)
	NPV		94.8% (109/115) (89.1%, 97.6%)

1This case was MSI-H by PGDx elio, and Promega PCR gave an "Indeterminate" result.

2These 5 cases did not have matching normal DNA to test via Promega PCR.

iv. Method Comparison Study for Wild Type Calls

A study was conducted to assess accuracy for 75 hotspot loci within 20 genes. A total of 112 specimens were tested, and the accuracy of PGDx elio tissue complete at all 75 positions was compared to 2 orthogonal methods (42 samples using 1 method, and 70 using a second method). Within the 112 specimens, there were 112 mutations across samples and 8,283 wild type calls. Overall variant-level concordance (PPA and NPA) was 96.4% and 99.9% respectively with two-sided 95% confidence intervals of (91.1%, 99.0%) for mutations (PPA), and (99.9%, 99.9%) for wild type locations (NPA).

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Image /page/26/Picture/0 description: The image shows the logo for PGDx, which stands for Personal Genome Diagnostics. The letters "PG" are in blue, while "Dx" is in orange. To the right of the letters is the full name of the company, "Personal Genome Diagnostics TM", in a smaller font size and also in blue.

18. Reproducibility

i. Interlaboratory Reproducibility
Interlaboratory reproducibility of the PGDx elio tissue complete assay was assessed across 3 different sites, using DNA extracted from 13 FFPE tissue specimens and 1 cell line. Together these 14 samples represented a range of SNVs and indels, ERBB2 amplifications, ALK, RET, and NTRK3 translocations, MSI, and TMB. Each of the 14 samples was tested in duplicate by 2 different operators on 12 sequencing runs across 3 non-consecutive days at each of the 3 independent laboratory sites using a single kit lot. The first pass rate was 90.3% (455/504) and the overall pass rate of the study was 98.2% (495/504) allowing a maximum of 1 round of repeat testing. Reproducibility was assessed 3 ways; (1) agreement for each positive variant detected across all replicates is reported (Positive call rate), and (2) Average Positive Agreement (APA) and Average Negative Agreement (ANA) and (3) modal analysis was used for per specimen reproducibility.

The positive call rate across all variants was 86.2%, while for SNVs, insertions, and deletions it was 88.8%, 82.8%, and 80.5%, respectively. Table 1.19 shows the positive call rate stratified by variant type and allele fraction.

| Mutation
Type | MAF
Threshold | Positive All Observed Mutations Variants |
|------------------|-------- | All | MAF≥0 | All | MAF≥5 | All | MAF≥8 | All | MAF≥10 | All | MAF≥15 | All SNVs | MAF≥0 | All SNVs | MAF≥5 | All SNVs | MAF≥8 | All SNVs | MAF≥10 | All SNVs | MAF≥15 | All Insertions | MAF≥0 | All Insertions | MAF≥5 | All Insertions | MAF≥8 | All Insertions | MAF≥10 | All Insertions | MAF≥15 | All Deletions | MAF≥0 | All Deletions | MAF≥5 | All Deletions | MAF≥8 Call Rate Among
| Total Unique
----------|----------------------------------------------------|--------------------------|
| 86.2% (14493/16813) | 474 |
| 88.0% (14483/16458) | 464 |
| 91.9% (13921/15146) | 427 |
| 93.1% (13404/14400) | 406 |
| 96.4% (12387/12846) | 362 |
| 88.4% (10549/11937) | 337 |
| 91.0% (10539/11582) | 327 |
| 95.7% (10070/10519) | 297 |
| 97.7% (9618/9845) | 278 |
| 97.8% (8773/8966) | 253 |
| 82.8% (649/784) | 22 |
| 82.8% (649/784) | 22 |
| 86.9% (619/712) | 20 |
| 86.9% (619/712) | 20 |
| 95.9% (614/640) | 18 |
| 80.5% (3295/4092) | 115 |
| 80.5% (3295/4092) | 115 |
| 82.6% (3232/3915) | 110 |

Table 1.19 Interlaboratory Reproducibility Call Rates

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Image /page/27/Picture/0 description: The image is a logo for Personal Genome Diagnostics, or PGDx. The logo has the letters "PGD" in blue, followed by an orange "X". To the right of the letters is the text "Personal Genome Diagnostics" in a smaller font size. The letters and text are all aligned horizontally.

| Mutation
Type | MAF
Threshold | Positive Call Rate Among
All Observed Mutations | Total Unique
Variants |
|------------------|------------------|----------------------------------------------------|--------------------------|
| All Deletions | MAF≥10 | 82.4% (3167/3843) | 108 |
| All Deletions | MAF≥15 | 92.6% (3000/3240) | 91 |

Agreement was also assessed by evaluating the APA and ANA, which assess the degree of agreement for variants based on the average result (Table 1.20) APA and ANA across all 3 sites were > 92% for all variant types tested.

Reproducibility was also assessed for independent variables (site, operator, day and within-run). No differences across sources of imprecision were observed.

Alteration Type	Metric	Overall (CI)	Alteration Type
MSI	APA	99.1% (98.7%,
99.4%)	ERBB2
amplifications	APA	100% (99.3%,
100%)
	ANA	99.3% (99.0%,
99.5%)		ANA	100% (100%,
100%)
SNVs	APA	97.8% (97.7%,
97.9%)	ALK
translocations	APA	98.6% (97.7%,
99.1%)
	ANA	99.9% (99.9%,
100%)		ANA	99.8% (99.6%,
99.9%)
Insertions	APA	95.6% (95.2%,
96.0%)	NTRK3
translocations	APA	92.7% (90.4%,
94.5%)
	ANA	99.9% (99.9%,
100%)		ANA	99.4% (99.2%,
99.5%)
Deletions	APA	94.4% (94.2%,
94.6%)	RET
translocations	APA	98.7% (97.8%,
99.2%)
	ANA	99.9% (99.9%,
100%)		ANA	99.8% (99.6%,
99.9%)
TMB	%CV	3.5%

Table 1.20 Interlaboratory Reproducibility Performance

The modal positive and negative call rates for sequence mutations (SNVs and indels) in each specimen are summarized in Table 1.21.

Table 1.21 Modal Call Rates for Interlaboratory Reproducibility

| Specimen
n | Total Unique
Mutations
Detected
Across All
Replicates | Modal Positive Call Rate1 (n/N)
(two-sided 95% CI) | Modal Negative Call Rate2
(n/N) (two-sided 95% CI) |
|---------------|-------------------------------------------------------------------|-------------------------------------------------------|-------------------------------------------------------|
| 1 | 10 | 99.6% (251/252) (97.8%,
99.9%) | 97.2% (105/108) (92.2%,
99.1%) |
| 23 | 0 | - | - |

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Image /page/28/Picture/0 description: The image is a logo for Personal Genome Diagnostics, or PGDx. The logo has the letters "PGD" in blue, with the "x" in orange. To the right of the letters is the text "Personal Genome Diagnostics" in a smaller font size. The letters are stacked on top of each other.

3	9	100% (315/315) (98.8%, 100%)	-
4	7	100% (216/216) (98.3%, 100%)	97.2% (35/36) (85.8%, 99.5%)
5	43	99.5% (1462/1470) (98.9%, 99.7%)	91.4% (32/35) (77.6%, 97.0%)
6	20	98.9% (639/646) (97.8%, 99.5%)	88.2% (30/34) (73.4%, 95.3%)
7	26	97.8% (678/693) (96.5%, 98.7%)	95.2% (157/165) (90.7%, 97.5%)
8	81	96.4% (1991/2065) (95.5%, 97.1%)	88.7% (683/770) (86.3%, 90.8%)
9	88	97.8% (2710/2772) (97.1%, 98.3%)	80.3% (318/396) (76.1%, 83.9%)
10	30	99.0% (998/1008) (98.2%, 99.5%)	97.2% (70/72) (90.4%, 99.2%)
11	94	96.1% (2907/3024) (95.4%, 96.8%)	83.1% (299/360) (78.8%, 86.6%)
12	33	99.4% (1109/1116) (98.7%, 99.7%)	97.2% (70/72) (90.4%, 99.2%)
13	9	100% (216/216) (98.3%, 100%)	93.5% (101/108) (87.2%, 96.8%)
14	24	96.8% (732/756) (95.3%, 97.9%)	88.0% (95/108) (80.5%, 92.8%)

1Positive call rate was calculated based on variants with majority call detected as positive. 2 Negative call rate was calculated based on variants detected at least once, but with

majority or equal call as negative. For all other locations, the negative call rates are 100%.

3 Specimen 2 was selected for presence of ALK translocation and had no detected SNVs or indels.

Precision of MSI was evaluated across 8 MSS and 6 MSI-H samples with a range of MSI scores evaluated by PGDx elio tissue complete with positive call rates provided in Table 1.22.

					Table 1.22 MSI Performance in the Interlaboratory Reproducibility Study
--	--	--	--	--	-------------------------------------------------------------------------	--	--	--

Case No.	Modal Status	Total Replicates	Mean MSI Score Value	MSI Score Range	SD	%CV	Positive Call Rate (95% CI)
1	MSS	36	10.5	(4.4, 19.1)	3.7	35.1	100% (90.4%, 100%)
2	MSS	35	13.6	(5.8, 20.4)	3.6	26.8	100% (90.1%, 100%)

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Image /page/29/Picture/0 description: The image shows the logo for Personal Genome Diagnostics (PGDx). The letters "PGD" are in blue, and the "x" is in orange. To the right of the letters is the text "Personal Genome Diagnostics" in a smaller font size, also in blue. The logo is simple and modern, and the use of color helps to make it stand out.

Case	Modal	Total	Mean MSI	MSI Score	SD	%CV	Positive Call Rate
No.	Status	Replicates	Score
Value	Range			(95% CI)
3	MSS	35	13.8	(7.2, 20.2)	3.7	26.8	100% (90.1%,
100%)
4	MSS	36	10.5	(3.9, 19.8)	3.2	30.4	100% (90.4%,
100%)
5	MSI-
H	35	209.7	(203.7, 216.5)	3.6	1.7	100% (90.1%,
100%)
6	MSS	34	9.6	(4.7, 16.0)	3.0	31.3	100% (89.9%,
100%)
7	MSS	33	-21.9	(-29.9, -13.2)	5.0	-22.8	100% (89.6%,
100%)
8	MSI-
H	35	223.5	(213.1, 236.3)	6.0	2.7	100% (90.1%,
100%)
9	MSI-
H	36	271.6	(261.6, 287.2)	5.9	2.2	100% (90.4%,
100%)
10	MSI-
H	36	77.5	(62.6, 102.5)	6.6	8.5	100% (90.4%,
100%)
11	MSI-
H	36	219.0	(212.6, 224.0)	2.9	1.3	100% (90.4%,
100%)
12	MSS	36	-56.1	(-61.5, -41.5)	4.1	-7.3	100% (90.4%,
100%)
13	MSS	36	16.4	(10.4, 25.1)	3.9	23.7	100% (90.4%,
100%)
14	MSI-
H	36	49.3	(36.7, 61.7)	6.3	12.8	94.4% (81.9%,
98.5%)

Precision of TMB was evaluated across 11 samples (with TMB scores above TMB LoB of 7.2 Muts/Mb) with a range of TMB across site, operator, and day provided in Figure 1.7.

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Image /page/30/Picture/0 description: The image shows the logo for Personal Genome Diagnostics (PGDx). The letters "PGD" are in blue, and the "x" is in orange. The words "Personal Genome Diagnostics" are in blue and are to the right of the letters "PGDx".

	Site A Op1	Site A Op2	Site B Op1	Site B Op2	Site C Op1	Site C Op2
100	o	o	o	o	o	o	Sample
	△	△	△	△	△	△
	♦	♦	♦	♦	♦	♦
50	□	□	□	□	□	□
	◇	◇	◇	◇	◇	◇
25	▽	▽	▽	▽	▽	▽
	⊠	⊠	⊠	⊠	⊠	⊠
	*	*	*	*	*	*
	⟡	⟡	⟡	⟡	⟡	⟡
10	x	x	x	x	x	x
	•	•	•	•	•	•
	Day1
Day2
Day3	Day1
Day2
Day3	Day1
Day2
Day3	Day1
Day2
Day3	Day1
Day2
Day3	Day1
Day2
Day3	A
B
C
D
E
F
G
H
I
J
K

Figure 1.5 TMB Performance in the Interlaboratory Reproducibility Study by Site, Operator, and Day

Lot to Lot Precision

Performance of PGDx elio tissue complete was assessed across 3 unique kit lots by determining concordance of variant calls in FFPE tissue samples. The 3 unique kit lots were utilized to process 5 test cases in triplicate for a total of 45 observations. All batches were sequenced on the same instrument. The overall pass rate of the study was 100% (45/45). Table 1.23 lists the APA and ANA used to assess lot to lot performance. APA for all variants is > 86%, and %CV for TMB analyses is