Who is the manufacturer of PGDx elio tissue complete?

Personal Genome Diagnostics filed the 510(k) submission for PGDx elio tissue complete (K192063).

What is the FDA product code for PGDx elio tissue complete?

PZM. It is classified under 21 CFR 866.6080 as a Class 2 device in the Pathology panel.

What is the regulatory pathway for PGDx elio tissue complete?

510(k) clearance (submission type: Traditional).

Who can help prepare an FDA 510(k) submission like PGDx elio tissue complete?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA 510(k) submissions. See https://innolitics.com/services/510ks/ or contact https://innolitics.com/contact.

PGDx elio tissue complete

K192063 · Personal Genome Diagnostics · PZM · Apr 24, 2020 · Pathology

Device Facts

Record ID	K192063
Device Name	PGDx elio tissue complete
Applicant	Personal Genome Diagnostics
Product Code	PZM · Pathology
Decision Date	Apr 24, 2020
Decision	SESE
Submission Type	Traditional
Regulation	21 CFR 866.6080
Device Class	Class 2

Intended 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 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.

Device Story

Assay uses targeted NGS on DNA from FFPE tumor tissue; requires 100ng DNA input; workflow includes mechanical shearing, end-repair, adapter ligation, PCR enrichment, and hybrid capture using biotinylated RNA baits. Sequencing performed on NextSeq 550Dx. PGDx elio platform software processes FASTQ files, aligns to reference genome, and performs variant calling for SNVs, indels, amplifications, translocations, MSI, and TMB. Used in clinical laboratories by qualified professionals. Output provides tumor mutation profiling information to assist healthcare providers in oncology decision-making; not prescriptive for specific therapies. Benefits include comprehensive genomic characterization of solid tumors to guide clinical management.

Clinical Evidence

Bench-only validation. Accuracy assessed against orthogonal methods (NGS panels, PCR, FISH, whole exome sequencing) across 582 samples. SNV/indel PPA/NPA >80% and >99% respectively. ERBB2 amplification concordance with FISH PPA 75-87%. ALK translocation PPA 92.9%. TMB Spearman correlation 0.903 vs. whole exome sequencing. MSI performance PPA 98.8% (excluding failed/indeterminate). Reproducibility assessed across 3 sites with 14 samples; APA/ANA >92% for most variants. Analytical sensitivity (LoD) established for SNVs, indels, amplifications, and translocations.

Technological Characteristics

Targeted NGS hybrid capture assay. Materials: biotinylated RNA baits, magnetic streptavidin beads. Energy: electrical (NextSeq 550Dx). Connectivity: PGDx elio platform software (server-based). Sterilization: N/A (reagent kit). Software: automated bioinformatic pipeline for variant calling and TMB/MSI quantification. Panel: 505 genes. DNA input: 100ng recommended.

Indications for Use

Indicated for previously diagnosed cancer patients with solid malignant neoplasms. Used for qualitative detection of somatic tumor gene alterations (SNVs, small indels, ERBB2 amplification, ALK/RET/NTRK2/NTRK3 translocations), MSI, and TMB from FFPE tumor tissue.

Regulatory Classification

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.

Special Controls

*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.

Predicate Devices

MSK-IMPACT (DEN170058)

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Submission Summary (Full Text)

{0}------------------------------------------------ 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) | | <input checked="True" type="checkbox"/> Prescription Use (Part 21 CFR 801 Subpart D) | |---------------|--------------------------------------------------------------------------------------| | | <input type="checkbox"/> Over-The-Counter Use (21 CFR 801 Subpart C) | #### CONTINUE ON A SEPARATE PAGE IF NEEDED. This section applies only to requirements of the Paperwork Reduction Act of 1995. #### *DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.* The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to: > Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff@fda.hhs.gov "An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number." {3}------------------------------------------------ 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 {4}------------------------------------------------ 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 - 2. 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. 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. | information on somatic mutations (point mutations and small insertions and deletions) and microsatellite instability for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. MSK-IMPACT is a single-site assay performed at Memorial Sloan Kettering Cancer Center. | | Classification | II | Same | | Product Code | PZM | Same | | Regulation | 21 CFR 866.6080 | Same | | Assay Method | Qualitative | Same | | Sample Type | FFPE tumor tissue from cancer patients with solid malignant neoplasms | Same | | Target Population | Previously diagnosed cancer patients with solid malignant neoplasms | Same | | Mode of Measurement | PCR and Next Generation Sequencing (hybrid capture methodology) | Same | | Calibrators | None | Same | | | PGDx elio tissue complete | MSK-IMPACT (Predicate Device) | | DNA Input | 100 ng | 100-250 ng | | DIFFERENCES | | | | Test Environment | Kit | Single-site assay (performed at Memorial Sloan Kettering Cancer Center) | | Controls | Positive control, negative control, normalized to database of common germline SNPs | Matched normal, positive control and negative control | | Assay Target | SNVs and indels in 505 genes MSI Amplification in ERBB2 Translocations in ALK, RET, NTRK2, and NTRK3 TMB | SNVs and indels in 468 genes MSI | | Instrument | NextSeq 550Dx (qualified by PGDx) | HiSeq 2500 Sequencer (qualified by MSK) | {5}------------------------------------------------ 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. {6}------------------------------------------------ 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. {7}------------------------------------------------ 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 | | | | | | | | {8}------------------------------------------------ Image /page/8/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. The letters "TM" are in superscript. {9}------------------------------------------------ Image /page/9/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 a smaller font size, with a trademark symbol. : - 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. {10}------------------------------------------------ Image /page/10/Picture/0 description: The image is a logo for Personal Genome Diagnostics. The logo has the letters PGD in blue and the letter X in orange. To the right of the letters is the text "Personal Genome Diagnostics" in a smaller font size. The letters PGD are in a bold sans-serif font. 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 {11}------------------------------------------------ Image /page/11/Picture/1 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 a smaller font size. The letters and text are all aligned horizontally. 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 < 2% | Table 1: Summary of PGDx elio tissue complete Quality Control Metrics Postsequencing {12}------------------------------------------------ Image /page/12/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 yellow. To the right of the letters is the text "Personal Genome Diagnostics" in a smaller blue font. The letters are stacked on top of each other. | Quality Metric | Level of Qualification | Passing Criteria | |---------------------------------------------------------------------------|---------------------------|------------------------------------------------------------------------------------| | Select SNVs and Indels with Evidence of Clinical Significance | Analyte-level | Mutant reads $\ge$ 4 MAF > 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 < 5 bp or Homopolymer regions $\ge$ 5 bp with MAF $\ge$ 12% | | ERBB2 Amplifications | Analyte-level | Fold change $\ge$ 2.5 in $\ge$ 25% regions covered | | Translocations (ALK, NTRK2, NTRK3 and RET) | Analyte-level | Fusion reads $\ge$ 3 | ### 12. Analytical Studies ### 13. Specificity #### i. Limit of Blank (LoB) Non-cancerous FFPE tissues were assessed for analytical specificity to confirm the reporting thresholds and quality metrics minimize false positives. Two reference standards from National Institute of Standards and Technology (NIST), NA24531 and NA24385 were evaluated by PGDx elio tissue complete for variants reported at the 100ng DNA input. Specificity was observed at 100% with no unverified mutations reported across 5 replicates for each standard. Unique test cases from normal FFPE samples were processed with the recommended 100 ng DNA input across 2 different lots of the PGDx elio tissue complete assay kit. For Variants with Evidence of Clinical Significance, the rate of false positives is < 0.1% while the false positive rate for hotspot SNVs is < 3.2% (n=2/63). For MSI-H, the false positive rate is < 1.6%. - ii. Cross Reactivity {13}------------------------------------------------ Image /page/13/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, light blue font. An in silico cross-reactivity analysis was performed to evaluate specificity of capture baits based on sequence identity of target baits to the human reference genome. Bait sequences corresponding to gene regions of clinical significance demonstrated specificity to target regions based on mapping quality or unique sequence identity to the human reference genome. Any bait that had some sequence identity to regions other than the target region of interest was further investigated and were shown to be specific. This study demonstrated that the oligonucleotide baits in the PGDx elio tissue complete assay are specific to the target regions of DNA intended to be reported. ### 14. Sensitivity - i. Limit of Detection (LoD) - SNVs and Indels The LoD was assessed by variant type and is defined as the lowest MAF at which ≥95% of replicates are detected. The LoD was evaluated 2 ways, through a dilution series using cell lines to determine the LoD and by confirmation with clinical specimens. Ten unique clinical cases were selected for SNVs, insertions and deletions and diluted with normal DNA derived from FFPE tissues. Each unique clinical case was tested with 10 replicates across 2 kit lots (n=20 per specimen) for a total of 200 observations (Error! Reference source not found.). Data was aggregated across 2 reagent kit lots when possible, otherwise the lot with the higher MAF LoD is displayed. Additional evaluations of analytical sensitivity performance used dilution series of FFPE clinical specimens. The positive call rates were assessed for a total of 11 SNVs, 3 insertions, and 5 deletions from 5 clinical FFPE specimens with 5 replicates per dilution level. A range of 5.9-12.6% MAF was observed using the lowest average MAF where the positive call rates was ≥ 95%. Cell lines were used to establish the LoD MAF range for 451 SNVs and 31 indels across the panel. A total of 150 observations were generated (3 samples with 10 replicates at 5 dilution levels). The established ranges were then confirmed with a ≥ 95% call rate with FFPE clinical cases on a per variant level (Error! Reference source not found.) and for the entire panel (Table 1.2). A summary of the LoD by variant type across all replicates is shown in the table below. | Variant | MAF Range | Cell Line Variants Evaluated to Establish MAF Range | Number of Variants in Clinical Cases in the Established Range | |-----------------------------------|----------------|--------------------------------------------------------------|---------------------------------------------------------------------| | Hotspot SNVs | 3.1% to 5.4% | 8 | 2 | | Non-hotspot SNVs | 6.3% to 17.8% | 443 | 176 | | Indels at homopolymer context1 | 13.7% to 17.5% | 10 | 9 | Table 1.2 Analytical Sensitivity (LoD MAF) for Representative SNVs and Indels {14}------------------------------------------------ Image /page/14/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. | Indels at non-homopolymer context | 6.1% to 10.9% | 19 | 4 | |-----------------------------------|---------------|----|---| |-----------------------------------|---------------|----|---| 1 Greater than or equal to 5 bp repeat - ii. LoD ERBB2, ALK, RET, NTRK2, NTRK3 and MSI Analytical sensitivity of ERBB2, ALK, RET, NTRK3, and MSI was confirmed by testing 7 clinical FFPE cases diluted with normal FFPE DNA to achieve targeted detection levels. Each unique case was 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 were confirmed at 1 tumor purity level with 10 replicates each. Results summarized in Table 1.3 indicate that the assay is sensitive in detecting specific translocations, amplifications and MSI-H. Table 1.3 Analytical Sensitivity (LoD Tumor Purity) - Translocations, Amplifications and MSI | Variant | LoD Tumor Purity | |----------------------|------------------| | MSI-H | 18.1% | | ERBB2 amplifications | 4.4% | | ALK translocations1 | 5.6% | | NTRK2 translocations | 30%2 | | NTRK3 translocations | 11.5% | | RET translocations | 12.8% | 1 The enrolled ALK case was evaluated with only 17 total replicates due to insufficient DNA quantity. 2 In silico down sampling suggests LoD 3%. iii. TMB and Tumor Purity The minimum tumor purity requirement for input into PGDx elio tissue complete is 20%. The minimum tumor purity required for robust reporting of TMB scores by PGDx elio tissue complete was established using 8 clinical FFPE cases. Samples 1-5 were serially diluted across 3 levels with 5 replicates per level with 3 replicates (18 total), sample 6 was serially diluted across 5 levels at 10 replicates per level (50 total), and samples 7-8 were serially diluted across 5 levels with 5 replicates per level (25 total, 10 of which had a tumor purity ≥15%). The total number of replicates per sample and the %CV of all replicates with ≥15% tumor purity are shown below in Table 1.4. Together these data show PGDx elio tissue complete TMB performance across tumor purities at or above 15%. Figure 1.2 demonstrates the consistency in TMB as a function of tumor purity for each of the specimens assessed across a range of TMB Muts/Mb scores. {15}------------------------------------------------ Image /page/15/Picture/0 description: The image shows the logo for PGDx, which stands for Personal Genome Diagnostics. The letters "PGD" are in blue, while the "x" is in orange. To the right of the letters is the text "Personal Genome Diagnostics" in blue, with a small "TM" symbol next to the word "Diagnostics". | Sample | Reference Undiluted TMB Score | CV of Replicates ≥ 15% Tumor Purity | Number of Replicates ≥ 15% Tumor Purity | |--------|----------------------------------|----------------------------------------|--------------------------------------------| | 1 | 33.4 | 12.5% | 18 | | 2 | 24.8 | 10.5% | 18 | | 3 | 50.8 | 6.3% | 18 | | 4 | 64.7 | 15.9% | 18 | | 5 | 31.5 | 8.0% | 18 | | 6 | 455.4 | 3.3% | 50 | | 7 | 10.0 | 14.8% | 10 | | 8 | 14.6 | 6.0% | 10 | ### Table 1.4 TMB Precision for Samples ≥ 15% Tumor Purity Image /page/15/Figure/5 description: The figure is a plot of mean TMB score in Muts/Mb vs tumor purity. There are 8 samples plotted on the figure, with tumor purity ranging from 0% to 80%. The mean TMB score ranges from 1 to 500 Muts/Mb. The samples are labeled as Sample 1 through Sample 8. Figure 1.2 Linearity of TMB score with tumor purity in PGDx elio tissue complete. The tumor purity is shown on the x-axis and the mean TMB score of the replicates at a specific tumor purity is shown on the y-axis. iv. DNA Extraction PGDx elio tissue complete is compatible with genomic DNA extracted from FFPE samples using any appropriate commercially available FFPE extraction method. Samples (3 FFPE specimens and 1 cell line) were extracted in duplicate by 2 operators using 3 different methods, equaling 48 total samples and processed in duplicate for a total of 96 observations to assess concordance. Data for all variant types, including BRAF V600 ### CONFIDENTIAL {16}------------------------------------------------ Image /page/16/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 a smaller blue font, with a trademark symbol next to the word "Diagnostics". SNV, ALK and NTRK3 translocations and ERBB2 amplification, were aggregated and Positive Percent Agreement (PPA) and Negative Percent (NPA) are presented in Table 1.5. Method 2 (bead-based) and Method 3 (automated) were compared to the reference Method 1 (column-based). The overall pass rate for FFPE samples was 93.1% (67/72). The %CV for TMB in assessed cases was < 12.5%. All DNA extraction methods yielded concordant results in variant calls with PGDx elio tissue complete. | DNA Extraction Method | Concordance in Variant Calls with Method 1 (n/N) (2-sided 95% CI) | |-----------------------|-------------------------------------------------------------------| | Method 2 | PPA - 97.8% (673/688) (96.4%, 98.7%) | | | NPA - 99.9% (71535481/71535520) (99.9%, 100%) | | Method 3 | PPA - 97.5% (624/640) (96.0%, 98.5%) | | | NPA - 99.9% (51416128/51416155) (99.9%, 100%) | ### Table 1.5 DNA Extraction Methods Compared to Reference v. DNA Input The recommended DNA input for PGDx elio tissue complete is 100 ng. To evaluate assay performance across a range of DNA inputs, 4 unique FFPE samples with known variants were prepared in triplicate at 10, 25, 50, 100, and 200 ng DNA input levels. The variant calls for these samples were compared to the respective reference DNA input of 100 ng for each case to assess concordance. Table 1.6 describes PPA and NPA for each input level where aggregated variants were analyzed, including SNVs, indels, amplifications, translocations, and MSI. For TMB, the mean absolute percent error rate of 10, 25, 50 and 200 ng DNA input compared to 100 ng were 11.8%, 3.3%, 4.4% and 1.8%, respectively. These data indicate the assay is robust around the recommended 100 ng DNA input. | DNA Input | Variant Call Concordance (n/N) (2-sided 95% CI) | |-----------|---------------------------------------------------------------------------------------| | 10 ng | PPA - 92.2% (177/192) (87.5%, 95.2%) NPA - 99.9% (26825815/26825826) (99.9%, 100%) | | 25 ng | PPA - 94.8% (182/192) (90.7%, 97.1%) NPA - 99.9% (26825815/26825826) (99.9%, 100%) | | 50 ng | PPA - 96.9% (186/192) (93.4%, 98.6%) NPA - 99.9% (26825822/26825826) (99.9%, 100%) | | 200 ng | PPA - 97.4% (187/192) (94.0%, 98.9%) NPA - 99.9% (26825818/26825826) (99.9%, 100%) | Table 1.6 DNA Input Compared to 100 ng Reference vi. Sample Carryover and Cross-contamination {17}------------------------------------------------ Image /page/17/Picture/0 description: The image is a logo for Personal Genome Diagnostics. The logo consists of the letters "PGDx" in blue and yellow. The letters "PG" are in blue, the letter "D" is in yellow, and the letter "x" is in yellow. To the right of the letters is the text "Personal Genome Diagnostics" in blue. The text is stacked on top of each other. Cross-contamination (contamination from one sample to another within the same batch) and sample carryover (contamination from a previous sequencing run when using the same instrument) were assessed by evaluating false positive and false negative variant calls in 29 FFPE samples. Seven of the 29 cases had known positive variants, the remaining samples were known negative samples. All FFPE samples were assessed across 2 batches to test for contamination within and between runs. In batch 1, a checkerboard pattern within a 96-well plate was created by alternating the samples with representative positive variants and known negative samples. Batch 2 contained known negative samples and was pooled and sequenced directly after completion of batch 1 sequencing. following standard instrument cleaning procedures. No positive variant results were observed in known negative samples tested. Therefore, the PGDx elio tissue complete assay workflow presents minimal risk for contamination. ### 15. Interference (Endogenous and Exogenous) - i. Interfering Substances (Exogenous) The impact of exogenous interfering substances on the performance of the PGDx elio tissue complete assay was assessed by processing DNA from FPPE samples tested in the presence of each interfering substance at varying amounts (Table 1.7). The samples were evaluated for concordance of variant calls when compared to samples processed without the interfering substances. Replicates for 5 test cases were analyzed for 8 experimental and 2 baseline conditions. Analysis of all variant types tested (SNVs, indels, translocations, amplifications and MSI) showed high PPA (> 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 {18}------------------------------------------------ Image /page/18/Picture/1 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" symbol. To the right of the letters is the text "Personal Genome Diagnostics" in a smaller font size. The letters and text are aligned horizontally. 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 {19}------------------------------------------------ Image /page/19/Picture/0 description: The image is a logo for Personal Genome Diagnostics. The logo consists of the letters "PGDx" in blue and orange. The "x" is orange and larger than the other letters. To the right of the letters are the words "Personal Genome Diagnostics" in a smaller blue font. The letters are stacked on top of each other. 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 {20}------------------------------------------------ Image /page/20/Picture/0 description: The image is a logo for PGDx, which stands for Personal Genome Diagnostics. 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, also in blue. 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 ta…