NYU Langone Genome PACT (Genome Profiling of Actionable Cancer Targets)

K202304 · Nyu Langone Medical Center · PZM · Jul 14, 2021 · Pathology

Device Facts

• Record ID: K202304
• Device Name: NYU Langone Genome PACT (Genome Profiling of Actionable Cancer Targets)
• Applicant: Nyu Langone Medical Center
• Product Code: PZM · Pathology
• Decision Date: Jul 14, 2021
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6080
• Device Class: Class 2
• Attributes: Real-World Evidence, 3rd-Party Reviewed

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K202304 · Jul 14, 2021	NYU Langone Genome PACT (Genome Profiling of Actionable Cancer Targets)	Nyu Langone Medical Center	Retrospective clinical FFPE tumor samples; Orthogonal clinical testing results	Retrospective clinical samples were used to assess the accuracy of the NYU Langone Genome PACT assay by comparing its performance against results obtained from previously validated orthogonal methods.	Retrospective study; Accuracy cohort; FFPE tumor samples; Method comparison

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Accuracy cohort; Retrospective method comparison	455 unique FFPE cancer samples from 19 different cancer types; Sample Size: 455; Number of Sites: 1	Validated orthogonal NGS methods	Positive percent agreement (PPA) and negative percent agreement (NPA) for SNVs, insertions, and deletions

Indications for Use

The NYU Langone Genome PACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens with solid malignant neoplasms to detect tumor gene alterations in a 607-gene panel. The test is intended to provide information on somatic mutations and small insertions and deletions) 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. NYU Langone Genome PACT is a single-site assay performed at NYU Langone Health.

Device Story

NYU Langone Genome PACT is a targeted next-generation sequencing (NGS) assay; analyzes FFPE tumor tissue matched with normal specimens; detects somatic mutations and small insertions/deletions in 607 genes. Performed as a single-site assay at NYU Langone Health. Input: tumor/normal tissue samples; Output: genomic alteration report. Used by oncologists/pathologists to inform clinical decision-making regarding cancer treatment; provides actionable genomic information; not prescriptive for specific therapies.

Clinical Evidence

Clinical evidence based on an accuracy cohort of 409 tumor specimens (777 unique mutations) across 19 cancer types. Comparison against orthogonal methods showed 99.1% PPA (770/777 detected). Precision studies (intra- and inter-assay) demonstrated high reproducibility (99.6% positive call rate across 270 replicates). Analytical sensitivity (LoD) established at 5% VAF with >200X coverage and 100ng DNA input. Specificity established via HapMap negative controls (<2% false positive rate).

Technological Characteristics

Targeted next-generation sequencing (NGS) assay; 607-gene panel; utilizes FFPE tumor tissue and matched normal specimens; qualitative detection of somatic mutations and small insertions/deletions.

Indications for Use

Indicated for patients with solid malignant neoplasms. Qualitative detection of somatic mutations and small insertions/deletions in a 607-gene panel using FFPE tumor tissue matched with normal specimens. For use by qualified healthcare professionals; not conclusive or prescriptive for specific therapeutic products.

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.

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I. Background Information:

### A. 510(k) Number:

K202304

### B. Applicant

NYU Langone Health (NYU)

### C. Proprietary and Established Names:

NYU Langone Genome PACT (Profiling of Actionable Cancer Targets)

### D. Regulatory Information

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PZM | Class II (Special Controls) | 21 CFR 866.6080 | 88 Pathology |

## II. Submission/Device Overview:

### A. Purpose for Submission:

New device

### B. Measurand:

Somatic single nucleotide variants, and insertions, and deletions (indels) smaller than 35 bp, in human genomic DNA obtained from formalin-fixed, paraffin-embedded tumor tissue.

Refer to Appendix 1 for complete list of genes included in this assay.

### C. Type of Test:

Next generation sequencing tumor profiling test

## III. Intended Use/Indications for Use:

### A. Indications for Use:

The NYU Langone Genome PACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded (FFPE) matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene

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alterations in a 607-gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) 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. NYU Langone Genome PACT is a single-site assay performed at NYU Langone Health.

### B. Special conditions for use statement(s):

Rx - For prescription use.

For in vitro diagnostic use.

### C. Special instrument requirements:

Illumina NextSeq 500/550 Sequencers (qualified by NYU Langone Health).

## IV. Device/System Characteristics:

### A. Device Description:

A description of required equipment, software, reagents, vendors, and storage conditions were provided, and are described in the product labeling (NYU Langone Genome PACT Standard Operating Procedure manual). NYU Langone Health assumes responsibility for the device.

#### 1. Sample Preparation:

The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve the necessary quality performance are shown in the Table 1 below:

Table 1. Specimen Handling and Processing for Validated Specimen Types

| Tissue Type | Volume | Minimum Tumor Proportion | Macro-dissection requirements (Based on tumor proportion) | Limitations | Storage |
| --- | --- | --- | --- | --- | --- |
| FFPE sections | 10-20 unstained sections, 5 microns thick | More than 10% of viable tumor cells; | Yes, macrodissection to minimize the % of non-neoplastic tissue in the sample | Archival paraffin-embedded material subjected to acid decalcification is unsuitable for analysis because acid decalcification severely damages nucleic acids. | Room temp |

Genomic DNA is extracted from tissue specimens per protocol. DNA is quantified and concentrated if necessary. The amount of DNA required to perform the test is 100-250ng. Tumor and Normal DNA sample is run in singlicate.

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# 2. Library Preparation:

Sequence libraries are prepared using KAPA Biosystems HyperPlus Reagents by first enzymatically fragmenting DNA at 37C for 20 minutes. This product immediately undergoes end-repair via production of blunt-ended, 5'-phosphorylated fragments. To the 3' ends of the dsDNA library fragments, dAMP is added (A-tailing). Next, dsDNA adapters with 3'dTMP is ligated to the A-tailed library fragments. Library fragments with appropriate adapter sequences are amplified via ligation-mediated pre-capture PCR. A quality control check on the amplified DNA libraries is performed: Samples should be a smear; average fragment size of ~291bp; and concentration >1.6ng/μL (to ensure adequate hybridization for capture.

# 3. Hybrid Capture NGS:

Library capture is conducted using xGen Lockdown Capture reagents. Pooled sequencing libraries are hybridized to the vendor oligo pool. Capture beads are used to pull down the complex of capture oligos and genomic DNA fragments. Unbound fragments are washed away. The enriched fragment pool is amplified by ligation-mediated PCR. The success of the enrichment is measured as a quality control step: Samples should be a smear, average fragment size with the peak at ~313bp; the molarity of the amplified DNA pool should be ≥2nM. Reactions can be stored at 4-20°C until ready for purification, up to 1 week.

# 4. Sequencing and Data Analysis:

Sequencing is conducted with the Illumina NextSeq 500/550 Sequencing Instruments and reagents and PhiX Control v3. The sequencing process uses multiple quality checks. Bioinformatics tools are summarized in the Table 2.

a) Data Management System (DMS): Automated sample tracking and archival of run-associated metadata (barcode, run name, samples accession number, patient medical record number, source (class), specimen type, and panel version) is conducted with the following key functions: Tracking sample status through various stages of data analysis; tracking iterations of analysis applied to a given sample; recording versions of databases and algorithms used in analysis; archival of selected pipeline output files (FASTQ, BAM, VCF) and sequencing run statistics (% of undetermined reads per lane, quality scores, reads per sample, number of targets with 0X and <50X coverage, average and median coverage of the Tumor and Normal sample).
b) Demultiplexing and FASTQ generation: The analysis pipeline uses software provided by Illumina. Two FASTQ files are generated per samples corresponding to full length forward and reverse reads. Demultiplexing quality control includes quality metrics for per-base sequence quality, sequence content, GC content and sequence length distribution, relative percentages of unmatched indices.
c) Read alignment and BAM generation: Spurious adapter sequences are trimmed prior to read alignment. Reads are aligned in paired-end mode to the hg19 b37 version of the human genome. Aligned reads are written to a Sequence Alignment Map (SAM) file, which is then converted into Binary Alignment Map (BAM) format. PCR duplicates are removed. Each base within a read is assigned a base quality score by the sequencing software, which reflects the probability an error was made with the

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base call. To account for systemic biases that may not accurately reflect the actual error probabilities observed empirically, the analysis pipeline uses another tool to adjust the reported quality scores based on the selected covariates. Reassigned quality scores are subject to a threshold of 20, corresponding to a 1/100 chance of error.

Table 2. Summary overview of bioinformatics tools (qualified by Langone)

| Bioinformatics step |
| --- |
| Adapter and low-quality bases trimming |
| 1st pass alignment |
| Removing duplicate reads |
| Realigning and recalibrating |
| Fragment size distribution, capture efficiency, depth of coverage |
| Analyzing Tumor and Normal samples separately for SNV, insertions and deletions |
| Analyzing matched Tumor – Normal pairs for SNV, insertions and deletions |

d) Sample QC checks: The unique combination of homozygous SNPs specific to a given sample serves as a 'fingerprint' for the identity of the corresponding patient and serves to identify potential sample mix-ups and contamination between samples and barcodes. QC checks involving the use of these 'fingerprint' SNPs are detailed below:

i. Sample mix-up check: The analysis pipeline identifies the homozygous SNPs in all Tumor and Normal samples. All Tumor-Normal pairs are compared for the matching homozygous variants. The fidelity QC filter is set to show only homozygous variants that fit the fidelity criteria: >200 coverage, VAF >0.98. The Tumor and Normal from the same patient should show >90% overlap of these homozygous SNP variants. Conversely, the concordance between samples from different patients should be low (<25%). Pairs of samples from the same patient with >10% discordance ("unexpected mismatches") can be due to hypermutant tumors with thousands of new mutations due to sun exposure (melanoma) or alkylating chemotherapy agents (glioblastoma). However, these samples are relatively rare, mutations are usually heterozygous, and in case that a clinical run includes such sample, all other Tumor-Normal pairs should still show high fidelity.

ii. Check for presence of tumor in normal: Normal samples are expected to be free of known pathogenic hotspot SNVs and insertions and deletions (indels) that are commonly (somatically) recurrent in tumor samples. As a first pass check, the pipeline annotates normal samples at known 'hotspot' locations derived from somatic mutation catalogs. If a known pathogenic tumor-specific mutation (i.e. BRAF V600E, EGFR L858R) is detected with mutation frequency > 1.8% (established false positive rate) in a normal sample, the normal sample is flagged for review and possible exclusion from analysis. If the normal sample shows tumor contamination, the Tumor-Normal pair is excluded from further analysis and DNA from a Tumor sample with matched Normal control is extracted and the analysis repeated.

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e) Mutation calling – SNVs and Indels: The analysis pipeline identifies two classes of mutations: (1) single nucleotide variants (SNVs) and (2) small insertions and deletions (indels). Paired sample mutation calling is performed on tumor samples and their respective matched normal controls. In this test, a normal DNA is always required for comparison. Filtering is performed to remove low quality sequence data, sources of sequencing artifacts, and germline results.

i. Analysis of positive and negative controls: data from controls is used to confirm lack of contamination as well as analytical sensitivity and specificity.
ii. Filters on sample coverage: A sequence coverage ≥ 100X is required to achieve 95% power to detect mutations with underlying variant frequency of 10% or greater. To ensure that at least 98% of targeted exons meet this coverage, per sample mean coverage has been conservatively set at ≥ 300X. A lower coverage threshold for the matched normal is set at 100X.
iii. Filtering for high confidence mutations: Raw SNV and indel calls are subjected to a series of filtering steps to ensure only high-confidence calls are admitted to the final step of manual review. These parameters include (1) evidence of it being a somatic mutation i.e., the mutation has to be present in ≥ 5% of Tumor reads and less than <1.8% of Normal reads. Mutation has to have coverage of ≥ 200 reads (≥10 mutant reads); in the Normal sample the coverage of the site should be ≥ 100x and <1.8% of Normal reads should show the mutation (<2 reads with mutation) resulting in ration between the Tumor and Normal samples to be >/= 5.0, (2) technical characteristics that use coverage depth (Depth), number of mutant reads, variant allele frequency (VAF).
iv. Mutation functional and site annotation: Predicted functional effect and site for each mutation is curated by automated software using information from ANNOVAR.
v. Confirmation of variants: Variants identified in genes in which less than 20 somatic variants of that type have previously identified are confirmed by Sanger sequencing.
vi. Reporting: Somatic variants are reported into two FDA recommended categories. These include Level 2: “Cancer Mutations with Evidence of Clinical Significance” and Level 3: “Cancer Mutations with Potential Clinical Significance”. Somatic Tier I variants listed in Oncokb as level 1 and 2 are being reported as Cancer Mutations with Evidence of Clinical Significance. Other Tier I, and all Tier II are being reported as Cancer Mutations with Potential Clinical Significance. The test does not report variants of unknown clinical significance, benign or likely benign variants. The filtering scheme and threshold are shown in Figure 1 below. The threshold values for the filtering criteria were established based on paired-sample mutation analysis on replicates of normal FFPE samples and optimized to accept high confidence mutation calls reject false positive calls.

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Figure 1. Summary of mutation filtering scheme


### 5. Controls:

a) Matched normal control: Genomic DNA is extracted from patient-matched normal peripheral blood for use as a matched normal control.
b) Positive control (PC): A commercially available, multiplexed mixture of biosynthetic DNA targets precisely blended at 10% each with a single, well-characterized genomic background, is qualified and used by Langone as positive control material. This control is used to assess the performance of the NGS-based somatic mutation assay across a range of genes and mutation types including SNVs, insertions and deletion in 28 genes. The assay tracks 35 SNVs and indels that showed consistent performance across 8 consecutive test runs (Table 4A and 4B).

For each clinical run, the VAF and coverage of Positive Control mutations are reviewed to ensure that the mutations are detected above the coverage and VAF thresholds. Furthermore, the assay tracks the VAF and coverage of PC mutations from run to run and compares them to the pool of previously profiled PC to ensure that they are within a 2 SD range of the pool. The detection of PC mutations must stay within the established sensitivity of the assay. The Positive Control with expected variant frequency (VAF) prior to pooling are shown in Table 3.

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Table 3. Positive Controls and Expected Mutation Frequencies

| Gene ID | COSMIC Identifier | Mutation Type | HGVS Nomenclature | Amino Acid | Target AF |
| --- | --- | --- | --- | --- | --- |
| AKT1 | COSM33765 | Substitution | c.49G>A | p.E17K | 10% |
| APC | COSM13127 | Substitution | c.4348C>T | p.R1450* | 10% |
| APC | COSM18561 | Insertion in HP 7N | c.4666_4667insA | p.T1556fs*3 | 10% |
| ATM | COSM21924 | Deletion | c.1058_1059delGT | p.C353fs*5 | 10% |
| ERBB2 | COSM682 / 20959 | Insertion | c.2324_2325ins12 | p.A775_G776insY VMA | 10% |
| GNA11 | COSM52969 | Substitution | c.626A>T | p.Q209L | 10% |
| GNAQ | COSM28758 | SNV in HP 3N | c.626A>C | p.Q209P | 10% |
| KIT | COSM1314 | Substitution | c.2447A>T | p.D816V | 10% |
| MPL | COSM18918 | Substitution | c.1544G>T | p.W515L | 10% |
| PDGFRA | COSM736 | Substitution | c.2525A>T | p.D842V | 10% |
| PIK3CA | COSM763 | Substitution | c.1633G>A | p.E545K | 10% |
| SMAD4 | COSM14105 | Insertion | c.1394_1395insT | p.A466fs*28 | 10% |
| CTNNB1 | COSM5664 | Substitution | c.121A>G | p.T41A | 10% |
| EGFR | COSM6224 | SNV in 3N | c.2573T>G | p.L858R | 10% |
| GNAS | COSM27887 | Substitution | c.601C>T | p.R201C | 10% |
| JAK2 | COSM12600 | SNV in HP 3N | c.1849G>T | p.V617F | 10% |
| KRAS | COSM521 | Substitution | c.35G>A | p.G12D | 10% |
| NPM1 | COSM17559 | Insertion | c.863_864insTCTG | p.W288fs*12 | 10% |
| NRAS | COSM584 | Substitution | c.182A>G | p.Q61R | 10% |
| PTEN | COSM4986 | Insertion | c.741_742insA | p.P248fs*5 | 10% |
| PTEN | COSM5809 | Deletion 6N > 5N | c.800delA | p.K267fs*9 | 10% |
| TP53 | COSM10648 | Substitution | c.524G>A | p.R175H | 10% |
| TP53 | COSM10660 | Substitution | c.818G>A | p.R273H | 10% |
| TP53 | COSM10662 | Substitution | c.743G>A | p.R248Q | 10% |
| TP53 | COSM6530 | Deletion | c.723delC | p.C242fs*5 | 10% |
| BRAF | COSM476 | Substitution | c.1799T>A | p.V600E | 10% |
| EGFR | COSM12378 | Insertion | c.2310_2311insGGT | p.D770_N771insG | 10% |
| EGFR | COSM6225 | Deletion | c.2236_2250del15 | p.E746_A750delE LREA | 10% |
| EGFR | COSM6240 | Substitution | c.2369C>T | p.T790M | 10% |
| FGFR3 | COSM715 | Substitution | c.746C>G | p.S249C | 10% |
| FLT3 | COSM783 | Substitution | c.2503G>T | p.D835Y | 10% |
| PDGFRA | COSM28053 | Insertion | c.1694_1695insA | p.S566fs*6 | 10% |
| PIK3CA | COSM12464 | Insertion | c.3204_3205insA | p.N1068fs*4 | 10% |
| PIK3CA | COSM775 | Substitution | c.3140A>G | p.H1047R | 10% |
| RET | COSM965 | Substitution | c.2753T>C | p.M918T | 10% |

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Table 4A. Summary of VAF precision for variants in the positive controls, for n consecutive runs (events) within 2 SD.

| Variant ID | n | Mean AF | Median AF | Min. AF | Max AF | 2SD AF | 2SD AF min | 2SD AF max | n within 2SD | % within 2SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AKT1 p.E17K | 8 | 0.10 | 0.10 | 0.07 | 0.12 | 0.04 | 0.06 | 0.14 | 8 | 100 |
| APC p.R1450* | 8 | 0.10 | 0.09 | 0.09 | 0.11 | 0.02 | 0.08 | 0.12 | 8 | 100 |
| APC p.T1556fs*3 | 8 | 0.08 | 0.08 | 0.06 | 0.10 | 0.02 | 0.06 | 0.10 | 8 | 100 |
| ATM p.C353fs*5 | 8 | 0.09 | 0.10 | 0.08 | 0.11 | 0.02 | 0.08 | 0.11 | 8 | 100 |
| BRAF p.V600E | 8 | 0.10 | 0.10 | 0.08 | 0.12 | 0.02 | 0.08 | 0.13 | 8 | 100 |
| CTNNB1 p.T41A | 8 | 0.09 | 0.09 | 0.06 | 0.12 | 0.04 | 0.05 | 0.13 | 8 | 100 |
| EGFR p.D770_N771insG | 8 | 0.08 | 0.08 | 0.07 | 0.09 | 0.01 | 0.07 | 0.09 | 8 | 100 |
| EGFR p.E746_A750delEL REA | 8 | 0.06 | 0.06 | 0.04 | 0.07 | 0.02 | 0.04 | 0.08 | 7 | 87.5 |
| EGFR p.L858R | 8 | 0.09 | 0.09 | 0.08 | 0.11 | 0.02 | 0.07 | 0.10 | 7 | 87.5 |
| EGFR p.T790M | 8 | 0.09 | 0.09 | 0.07 | 0.11 | 0.03 | 0.07 | 0.12 | 8 | 100 |
| ERBB2 p.A775_G776insYV MA | 8 | 0.05 | 0.05 | 0.04 | 0.07 | 0.02 | 0.03 | 0.07 | 7 | 87.5 |
| FGFR3 p.S249C | 8 | 0.08 | 0.08 | 0.05 | 0.09 | 0.03 | 0.05 | 0.11 | 7 | 87.5 |
| FLT3 p.D835Y | 8 | 0.10 | 0.09 | 0.08 | 0.11 | 0.02 | 0.08 | 0.11 | 8 | 100 |
| GNA11 p.Q209L | 8 | 0.09 | 0.10 | 0.06 | 0.11 | 0.04 | 0.05 | 0.13 | 8 | 100 |
| GNAQ p.Q209P | 8 | 0.09 | 0.09 | 0.08 | 0.10 | 0.02 | 0.07 | 0.11 | 8 | 100 |
| GNAS p.R201C | 8 | 0.10 | 0.10 | 0.08 | 0.11 | 0.02 | 0.08 | 0.12 | 7 | 87.5 |
| JAK2 p.V617F | 8 | 0.09 | 0.09 | 0.09 | 0.10 | 0.01 | 0.08 | 0.10 | 8 | 100 |
| KIT p.D816V | 8 | 0.10 | 0.10 | 0.07 | 0.12 | 0.03 | 0.07 | 0.13 | 8 | 100 |
| KRAS p.G12D | 8 | 0.10 | 0.10 | 0.07 | 0.11 | 0.03 | 0.07 | 0.12 | 8 | 100 |
| MPL p.W515L | 8 | 0.10 | 0.10 | 0.09 | 0.11 | 0.01 | 0.09 | 0.12 | 8 | 100 |
| NPM1 p.W288fs*12 | 8 | 0.07 | 0.07 | 0.05 | 0.08 | 0.02 | 0.04 | 0.09 | 8 | 100 |
| NRAS p.Q61R | 8 | 0.09 | 0.09 | 0.06 | 0.10 | 0.02 | 0.06 | 0.11 | 8 | 100 |
| PDGFRA p.D842V | 8 | 0.10 | 0.10 | 0.08 | 0.11 | 0.02 | 0.08 | 0.12 | 8 | 100 |
| PDGFRA p.S566fs*6 | 8 | 0.10 | 0.10 | 0.08 | 0.11 | 0.02 | 0.08 | 0.12 | 8 | 100 |
| PIK3CA p.E545K | 8 | 0.08 | 0.08 | 0.08 | 0.09 | 0.01 | 0.07 | 0.10 | 8 | 100 |
| PIK3CA p.H1047R | 8 | 0.09 | 0.09 | 0.08 | 0.10 | 0.01 | 0.07 | 0.10 | 8 | 100 |
| PIK3CA p.N1068fs*4 | 8 | 0.09 | 0.09 | 0.08 | 0.11 | 0.02 | 0.07 | 0.11 | 8 | 100 |
| PTEN p.K267fs*9 | 8 | 0.10 | 0.10 | 0.07 | 0.13 | 0.04 | 0.06 | 0.14 | 8 | 100 |
| PTEN p.P248fs*5 | 8 | 0.09 | 0.09 | 0.07 | 0.11 | 0.02 | 0.07 | 0.12 | 8 | 100 |
| RET p.M918T | 8 | 0.10 | 0.10 | 0.08 | 0.12 | 0.03 | 0.07 | 0.13 | 8 | 100 |
| SMAD4 p.A466fs*28 | 8 | 0.09 | 0.09 | 0.07 | 0.11 | 0.02 | 0.07 | 0.12 | 8 | 100 |
| TP53 p.C242fs*5 | 8 | 0.09 | 0.09 | 0.07 | 0.11 | 0.03 | 0.06 | 0.12 | 8 | 100 |
| TP53 p.R175H | 8 | 0.09 | 0.09 | 0.07 | 0.10 | 0.02 | 0.06 | 0.11 | 8 | 100 |
| TP53 p.R248Q | 8 | 0.09 | 0.09 | 0.06 | 0.11 | 0.03 | 0.06 | 0.11 | 8 | 100 |
| TP53 p.R273H | 8 | 0.08 | 0.08 | 0.06 | 0.09 | 0.02 | 0.06 | 0.10 | 8 | 100 |

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Table 4B. Summary of Depth (DP) for positive controls across 8 consecutive runs.

| Variant ID | Mean DP | Median DP | Min DP | Max DP | 2SD DP | 2SD DP min | 2SD DP max | n within 2SD | % within 2SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AKT1 p.E17K | 306 | 303 | 197 | 438 | 167 | 139 | 472 | 8 | 100 |
| APC p.R1450* | 1342 | 1373 | 888 | 1876 | 743 | 599 | 2086 | 8 | 100 |
| APC p.T1556fs*3 | 1323 | 1290 | 744 | 2041 | 939 | 384 | 2262 | 8 | 100 |
| ATM p.C353fs*5 | 1144 | 1001 | 599 | 1933 | 990 | 154 | 2133 | 8 | 100 |
| BRAF p.V600E | 904 | 855 | 472 | 1427 | 687 | 217 | 1591 | 8 | 100 |
| CTNNB1 p.T41A | 623 | 577 | 363 | 938 | 432 | 191 | 1055 | 8 | 100 |
| EGFR p.D770_N771insG | 1156 | 1192 | 846 | 1529 | 527 | 629 | 1684 | 8 | 100 |
| EGFR p.E746_A750delE LREA | 953 | 848 | 588 | 1449 | 620 | 333 | 1573 | 8 | 100 |
| EGFR p.L858R | 1340 | 1361 | 935 | 1837 | 708 | 632 | 2047 | 8 | 100 |
| EGFR p.T790M | 1230 | 1263 | 893 | 1613 | 563 | 667 | 1792 | 8 | 100 |
| ERBB2 p.A775_G776insY VMA | 944 | 930 | 666 | 1371 | 499 | 445 | 1442 | 8 | 100 |
| FGFR3 p.S249C | 467 | 499 | 149 | 821 | 463 | 4 | 930 | 8 | 100 |
| FLT3 p.D835Y | 1359 | 1322 | 860 | 2084 | 891 | 468 | 2250 | 8 | 100 |
| GNA11 p.Q209L | 1397 | 1405 | 934 | 1935 | 786 | 610 | 2183 | 8 | 100 |
| GNAQ p.Q209P | 1118 | 1067 | 602 | 1772 | 814 | 304 | 1933 | 8 | 100 |
| GNAS p.R201C | 1348 | 1313 | 801 | 1996 | 903 | 444 | 2251 | 8 | 100 |
| JAK2 p.V617F | 897 | 809 | 435 | 1459 | 759 | 138 | 1656 | 8 | 100 |
| KIT p.D816V | 733 | 698 | 445 | 1126 | 505 | 228 | 1238 | 8 | 100 |
| KRAS p.G12D | 1534 | 1478 | 862 | 2439 | 1117 | 417 | 2652 | 8 | 100 |
| MPL p.W515L | 865 | 844 | 549 | 1295 | 531 | 334 | 1397 | 8 | 100 |
| NPM1 p.W288fs*12 | 723 | 583 | 363 | 1330 | 722 | 0 | 1445 | 8 | 100 |
| NRAS p.Q61R | 818 | 808 | 493 | 1213 | 510 | 307 | 1328 | 8 | 100 |
| PDGFRA p.D842V | 1275 | 1317 | 853 | 1874 | 796 | 478 | 2071 | 8 | 100 |
| PDGFRA p.S566fs*6 | 1163 | 1087 | 728 | 1786 | 780 | 384 | 1943 | 8 | 100 |
| PIK3CA p.E545K | 943 | 845 | 459 | 1582 | 811 | 132 | 1754 | 8 | 100 |
| PIK3CA p.H1047R | 1135 | 1150 | 636 | 1615 | 774 | 361 | 1909 | 8 | 100 |
| PIK3CA p.N1068fs*4 | 1000 | 1000 | 592 | 1434 | 675 | 325 | 1675 | 8 | 100 |
| PTEN p.K267fs*9 | 1003 | 879 | 568 | 1613 | 790 | 213 | 1794 | 8 | 100 |
| PTEN p.P248fs*5 | 1104 | 1011 | 704 | 1791 | 754 | 351 | 1858 | 8 | 100 |
| RET p.M918T | 985 | 899 | 551 | 1575 | 751 | 235 | 1736 | 8 | 100 |
| SMAD4 p.A466fs*28 | 999 | 948 | 550 | 1544 | 737 | 262 | 1736 | 8 | 100 |
| TP53 p.C242fs*5 | 493 | 477 | 338 | 707 | 251 | 243 | 744 | 8 | 100 |
| TP53 p.R175H | 904 | 898 | 629 | 1245 | 464 | 439 | 1368 | 8 | 100 |
| TP53 p.R248Q | 533 | 530 | 365 | 746 | 257 | 276 | 791 | 8 | 100 |
| TP53 p.R273H | 851 | 843 | 604 | 1224 | 463 | 388 | 1314 | 8 | 100 |

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c) Negative control (NC): DNA from a HapMap cell line (GM12878) is used as a negative control in the assay. The HapMap cell line has been generated from EBV-transformed lymphocytes. This cell line has been previously sequenced and contains germline polymorphisms but no known somatic variants. Thus, this cell line serves as a known reference sequence to compare with the published reference sequence for both sequencing accuracy using the assay and for analytical specificity for the detection of somatic variants. The number of private variants identified in each HapMap sample represents the false positive rate of ~1.75% for all mutations and ~1.68% for nonsynonymous exonic mutations. A HapMap sample from each clinical run is compared to the random pool of 5 HapMap samples from the validation NC pool, thus creating a pool of known expected mutations observed in HapMap samples. The same filtering criteria are applied as for the clinical samples including overall coverage >300X; variant coverage >200X and VAF >0.05. The number of a) all private mutations and b) nonsynonymous exonic mutations private to the HapMap sample in the clinical run is reviewed to ensure <2% false positive rate. All nonsynonymous exonic mutations are screened to ensure that no known actionable cancer mutations are detected. If such mutations are observed in the HapMap sample, (e.g., EGFR exon 19/21 BRAF V600E, etc.), the run will be flagged and repeated using new reagents and controls to eliminate the possibility of cross contamination.

d) PCR reagent control [No Template Control (NTC)]: The NTC control should have a Qubit measurement of < 2.0ng/μL. Sequencing data from the NTC control sample will also be subjected to analysis using the pipeline, to verify that no known hotspot mutations are detected. If a hotspot mutation is detected, any samples containing that mutation in the pool will be reviewed to determine if a re-run is necessary.

### 6. Result Reporting:

- Upon review, results are reported out under two categories: Level 2:"Cancer Mutations with Evidence of Clinical Significance" and Level 3: "Cancer Mutations with Potential Clinical Significance" as described in the FDA Fact Sheet available here https://www.fda.gov/media/109050/download.
The two categories are based on the supporting level of clinical evidence. Refer to the Clinical Performance Section for more information.
- Results are reported for point mutations and small insertions and deletions in protein-coding exons, promoters or splice sites of the 607 gene panel. Refer to Appendix 1b for a list of genes.
- The NYU Langone Genome PACT does not report mutations in 73 exons due to consistently low coverage (<50x) in those exons. Refer to Appendix 1c for a list of excluded exons.
- Reporting takes in account the following quality metrics in the Table 5 below.

Table 5. Sample Level Quality Control Metrics

| QC Metrics | Acceptance Criteria |
| --- | --- |
| After Demultiplexing | <10% Undetermined Reads per LaneBase Quality Scores >30/sample>=10 million total reads/sample |

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| After the Pipeline Analysis | >= 10 million deduplicated mapped reads per sample Average coverage of the tumor sample >300X Average coverage of the Normal Sample >100X |
| --- | --- |
| Coverage uniformity | ≥ 98% target exons above 100X coverage < 1% of targets with 0X and <50X coverage |
| % Cluster passing | The percent cluster passing filter (Cluster PF) > 80% |
| % Reads passing filter | The percent reads passing filter (Reads PF) > 80% |
| Somatic mutation assessment | VAF-Tu / VAF-Norm ≥5 |
| Somatic mutation coverage | ≥ 200X |
| Positive Control | Detection with 94% sensitivity (33 out of 35 variants), VAF within 2SD of expected range |
| Negative Control | <2% of private mutations not observed in HapMap pool (false positive rate) |
| Sample Mix-up QC | The Tumor and Normal from the same patient should show >90% overlap of homozygous SNP variants. Conversely, the concordance between samples from different patients should be low (<25%). <1.8% of known pathogenic variants observed in any Normal Sample. |
| Major Contamination QC | >2% of Mutations identified as somatic in any Tumor present in a Normal sample. |
| Criteria for calling test failure | mean coverage across all exons < 50x |

### D. Test Principle:

The NYU Langone Genome PACT assay is a custom targeted sequencing platform, utilizing solution- phase exon capture and sequencing, to detect somatic alterations (point mutations, small insertions and deletions) in tumor specimens compared to matched normal control. The NYU Langone Genome PACT assay involves hybridization capture and deep sequencing of all protein-coding exons of 607 cancer- associated genes. The assay uses custom DNA probes corresponding to all exons of oncogenes and tumor suppressor genes. Probes are synthesized by a secondary manufacturer and are biotinylated to enable sequence enrichment through capture by streptavidin-conjugated beads. Probes were designed to tile the entire length of each target sequence in an overlapping fashion, typically extending 20-50 base pairs beyond the boundaries of the target.

Genomic DNA is extracted from Tumor and patient-matched blood as a Normal sample. Sequence libraries are prepared through a series of enzymatic steps including shearing of double-stranded DNA, end repair, A-base addition, ligation of barcoded sequence adaptors, and low cycle PCR amplification. Multiple barcoded sequence libraries are pooled and captured using the custom-designed biotinylated probes. Captured DNA fragments are then sequenced on an Illumina NextSeq 500/550 as paired-end reads. Sequence reads are then aligned to the reference human genome. By comparing the identity of bases from the tumor DNA to the matched normal DNA and the reference human genome, somatic alterations are identified in the tumor.

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# V. Substantial Equivalence Information:

# A. Predicate Device Name:

MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets)

# B. Predicate 510(k) Number:

DEN170058

# C. Comparison with Predicate:

| Item | Similarities | |
| --- | --- | --- |
| | New Device | Predicate |
| Indications | The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) 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. | Same |
| Test principle | Custom targeted sequencing platform, utilizing solution- phase exon capture and sequencing | Same |
| Specimens | Matched Tumor and Normal DNA analyzed | Same |
| Tumor tissue type | Formalin fixed paraffin embedded tissue | Same |
| Type of sequencing | Massive parallel sequencing | Same |
| Sequencing chemistry | Illumina Exonic Hybrid Capture Sequencing-By-Synthesis chemistry | Same |
| | | |
| Non-hotspot Mutation threshold | 5% variant allele frequency | Same |
| Optimized and recommended starting DNA input | 250ng | Same |
| Filter for somatic variants (Variant Allele Frequency) | VAF-Tumor / VAF-Norm ≥ 5 | Same |
| Coverage uniformity | ≥ 98% target exons above 100X coverage | Same |
| Cluster passing | > 80% | Same |
| Clinical Evidence Curation Oncopanel results are reported under one of these two categories: “Cancer Mutations with Evidence of | Same | OncoKB knowledge base |

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| Item | Similarities | |
| --- | --- | --- |
| | New Device | Predicate |
| Clinical Significance” or “Cancer Mutations with Potential Clinical Significance.” | | |

| Item | Differences | |
| --- | --- | --- |
| | New Device | Predicate |
| Mutation type detection | Single nucleotide variants (SNV), small insertions and deletions (indels) | Single nucleotide variants (SNV), small insertions and deletions (indels) and Microsatellite Instability (MSI) |
| Sequencing instruments | Illumina NextSeq 500/550 | Illumina HiSeq 2500 Sequencer |
| Hotspot Mutation calling threshold | 5% variant allele frequency | 2% variant allele frequency |
| Tumor analysis only (in the absence of the matched Normal) | No, the test always requires a matched Normal DNA | Yes, in the absence of the Normal, a Tumor can be compared against a pool of normal samples |
| Number of targeted genes | 607 | 468 |
| Average target coverage | >300X | >200X |

## VI. Standard/Guidance Document Referenced (if applicable):

Not applicable

## VII. Performance Characteristics:

### A. Determination of pipeline thresholds:

#### 1. Requirements on exon coverage:

The power analysis to establish the confidence intervals for variants to be identified at the given coverage was performed. The following criteria were applied:

Calculate number of reads needed to determine if a variant is present at 95% confidence (p = 0.05)

- Null Hypothesis: No variant present; background sequencing error rate only
- Alternative Hypothesis: Variant present at the given variant allele frequency (VAF)
- background error rate: 2% (Q20)

Data are summarized in the Tables 6 and 7 below. In summary approximately 100x coverage is necessary to detect mutations present at 10% VAF with 0.95 power and 500x coverage is necessary for 95% confidence for detection of a variant with 5% frequency.

The established >300X average coverage enables a detection of mutations present at 6% VAF with 0.95 power.

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Table 6. Number of reads required to detect variant with variant allele frequency (VAF) for given power level ranging from 0.8 to 0.99.

| VAF | 0.8 | 0.9 | 0.95 | 0.98 | 0.99 |
| --- | --- | --- | --- | --- | --- |
| 0.02 | Inf | Inf | Inf | Inf | Inf |
| 0.03 | 1983 | 2606 | 3185 | 3905 | 4426 |
| 0.04 | 603 | 784 | 950 | 1157 | 1306 |
| 0.05 | 315 | 406 | 489 | 593 | 667 |
| 0.06 | 203 | 260 | 312 | 377 | 423 |
| 0.07 | 146 | 186 | 223 | 269 | 301 |
| 0.08 | 113 | 143 | 171 | 206 | 230 |
| 0.09 | 91 | 115 | 137 | 165 | 184 |
| 0.10 | 76 | 96 | 114 | 137 | 153 |
| 0.11 | 65 | 82 | 97 | 116 | 130 |
| 0.12 | 56 | 71 | 84 | 101 | 112 |
| 0.13 | 50 | 63 | 74 | 89 | 99 |
| 0.14 | 45 | 56 | 66 | 79 | 88 |
| 0.15 | 40 | 50 | 60 | 71 | 79 |
| 0.16 | 37 | 46 | 54 | 65 | 72 |
| 0.17 | 34 | 42 | 50 | 59 | 66 |
| 0.18 | 31 | 39 | 46 | 55 | 61 |
| 0.19 | 29 | 36 | 42 | 51 | 56 |
| 0.20 | 27 | 33 | 40 | 47 | 52 |

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**Table 7. 95% Confidence intervals for various VAF's at given coverages**

| VAF | Coverage | | | | |
| --- | --- | --- | --- | --- | --- |
| | 50 | 100 | 200 | 500 | 1000 |
| 0.02 | (0.002, 0.092) | (0.004, 0.064) | (0.007, 0.047) | (0.01, 0.035) | (0.013, 0.03) |
| 0.03 | (0.004, 0.107) | (0.008, 0.078) | (0.012, 0.061) | (0.01, 8 0.048) | (0.021, 0.042) |
| 0.04 | (0.007, 0.121) | (0.013, 0.092) | (0.019, 0.074) | (0.025, 0.06) | (0.029, 0.053) |
| 0.05 | (0.011, 0.135) | (0.019, 0.105) | (0.026, 0.086) | (0.033, 0.072) | (0.038, 0.065) |
| 0.06 | (0.016, 0.149) | (0.025, 0.118) | (0.033, 0.099) | (0.041, 0.083) | (0.046, 0.076) |
| 0.07 | (0.0, 2 0.162) | (0.031, 0.131) | (0.04, 0.111) | (0.05, 0.095) | (0.05, 5 0.087) |
| 0.08 | (0.026, 0.175) | (0.037, 0.144) | (0.048, 0.123) | (0.05, 8 0.106) | (0.06, 4 0.098) |
| 0.09 | (0.031, 0.188) | (0.044, 0.156) | (0.055, 0.135) | (0.06, 7 0.117) | (0.073, 0.109) |
| 0.10 | (0.037, 0.201) | (0.051, 0.168) | (0.063, 0.146) | (0.076, 0.128) | (0.082, 0.12) |
| 0.11 | (0.043, 0.213) | (0.058, 0 18) | (0.071, 0.158) | (0 .084 , 0 .139) | (0.092 , 0.13) |
| 0.12 | (0.049, 0.226) | (0.066, 0.192) | (0.08, 0.169) | (0.09, 3 0.15) | (0.1,01 0.141) |
| 0.13 | (0.055, 0.238) | (0.073, 0.204) | (0.088, 0.181) | (010,2 0.161) | (0.1, 1 0.152) |
| 0.14 | (0.062, 0.25) | (0.081, 0.215) | (0.096, 0.192) | (0.11, 1 0.172) | (0.11, 9 0.162) |
| 0.15 | (0.068, 0.262) | (0.088, 0.227) | (0.105, 0.203) | (0.12, 0.183) | (0.129, 0.173) |
| 0.16 | (0.075, 0.274) | (0.096, 0.238) | (0.113, 0.214) | (0.129, 0.194) | (0.138, 0.183) |
| 0.17 | (0.082, 0.285) | (0.104, 0.25) | (0.122, 0.225) | (0.139, 0.204) | (0.147, 0.194) |
| 0.18 | (0.089, 0.297) | (0.112, 0.261) | (0.13, 0.236) | (0.148, 0.215) | (0.157, 0.204) |
| 0.19 | (0.096, 0.308) | (0.12, 0.272) | (0.139, 0.247) | (0.15, 7 0.226) | (0.16, 6 0.215) |
| 0.20 | (0.103, 0.32) | (0.128, 0.283) | (0.148, 0.258) | (0.16, 6 0.236) | (0176, 0.225) |

The Average sample coverage of NYU Langone Genome PACT is >700X and Median sample coverage is >600X (Table 8), representing approximately 0.99 power for detection of variants at 0.05 allele frequency on average (coverage 667X, Table 6). However, regions with <500x coverage would be below 95% confidence interval for VAF 5% detection and if no mutation was identified they can represent a false negative finding.

**Table 8. Summary of sample coverage across ~410 samples**

| Metric | Cumulative Average* | Cumulative Median** | Range |
| --- | --- | --- | --- |
| **Tumor and Normal samples** | | | |
| Average sample coverage | 720 | 711 | 286-1307 |
| Median sample coverage | 620 | 613 | 172-1000 |
| **Positive Control (Commercially available reference material qualified by Langone)** | | | |
| Average sample coverage | 788 | 799 | 612-940 |
| Median sample coverage | 780 | 791 | 605-938 |
| **Negative Control (HapMap)** | | | |

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| Metric | Cumulative Average* | Cumulative Median** | Range |
| --- | --- | --- | --- |
| Average sample coverage | 851 | 847 | 729-1006 |
| Median sample coverage | 823 | 828 | 676-982 |
| | | | |
| No template control (water) | | | |
| Average sample coverage | 0.545 | 0.545 | 0.46-0.63 |
| Median sample coverage | 1 | 1 | 1-1 |

* Cumulative Average coverage (Average of all samples' Averages)

**Cumulative Median coverage (Median of all the samples' Medians)

These estimates were confirmed by profiling 325 unique mutations and SNPs for which VAF was available from an orthogonal study with expected frequencies as low as 5% and average coverage highlighted in Table 7.

Figure 3. Observed vs. Expected (shown by orthogonal NGS method) Variant Frequency, Pearson correlation coefficient: 0.974


### 2. Requirements on sample coverage:

Coverage analysis data from 10 consecutive runs and 238 samples, including samples, as well as positive and negative control samples included in the runs, shows that approximately \(\sim 99.6\%\) of cases had on average a coverage \(>300\mathrm{X}\) (only 0.42 had coverage \(< 300\mathrm{X}\)) and \(98.3\%\) of cases had Median coverage \(>300\mathrm{X}\), see Figure 3 below. Therefore, we established \(300\mathrm{X}\) as Average coverage cutoff for a sample to be acceptable for analysis.

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**Figure 3: Cumulative presentation of average and median coverage below coverage cutoffs. X axis indicates the coverage, Y axis represents % of cases <the cutoff. N = 238 samples. >98% of the samples have Average**


There were exons that presented with consistently low coverage values. None of the exons of the clinically validated genes are among those with consistently low coverage. To assess the effect of GC content on the metrics and the quality of coverage, correlation analysis from 10 independent runs was carried out. Correlation between coverage and GC content shows that poor coverage is associated with both high and low GC content. The coverage is optimal for target regions of moderate GC content (35 to 65 %).

Based on the validation, >99% of exons can be expected to be sequenced with average and median coverage greater than 100X. A 100X minimum coverage threshold per exon is required based on the power analysis calculations, which showed 100X coverage was necessary to call mutations with true underlying mutation frequency 10% or greater, with 95% power at an alpha level of 0.05). To be conservative, a threshold of 300X on mean and median sample coverage is used to determine if a sample is sequenced to sufficient depth for subsequent analysis. A sample is flagged as being at increased risk of false negatives if its mean coverage is below 300X.

3. Requirements on mutation coverage, DNA input and variant allele frequency for positive calls: Based on the validation, requirements on mutation coverage are ≥ 200X with ≥ 5% mutant reads with Normal coverage at minimum of ≥100X and <2% mutant reads. This results in ratio between the Tumor and Normal sample mutant reads to be ≥ 5.0. Calls with <50X coverage are not reported. Mutation calls with coverage between 50X and 200X need to be validated by an independent method if Level 2, see Table 9 for summary.

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Table 9. Summary of coverage criteria for reporting

| Coverage and VAF of the somatic mutation | Result |
| --- | --- |
| <50x, any finding | Fail |
| Somatic mutations identified with VAF > 0.05; coverage 50X-199X | Indeterminate |
| Somatic mutations identified with VAF > 0.05 coverage >200X | Pass |
| <500X, no mutations identified | No mutation reported. |

### B. Pre-Analytical performance:

Minimum DNA requirements were established by measuring assay performance based on different inputs and validated on precision studies. DNA was extracted from FFPE samples. The % of targets below the coverage cutoff did not vary between different sources of DNA. DNA samples are normalized to yield 100 – 250 ng input and maximized to 35 ul prior to library preparation. 100 ng is the minimal required input. The normalization and DNA quantification are performed.

Based on the established clinical VAF cutoff of 5%, samples with <10% tumor cell content are insufficient to detect any driver heterozygous mutations (estimated normalized VAF of ~50%). Therefore, only samples with >10% tumor cell content are accepted for the analysis. From the same sample a section with higher tumor cell content is preferred. When feasible, tumor content can be enriched by scalpel macrodissection. The data demonstrated that the DNA extraction has been optimized across tumor types to reasonably conclude that the analytical performance presented is representative across FFPE tumor types (Table 10).

Table 10. Pre-analytical performance summary

| Tumor Type | Original samples (N) | DNA Pass (>2.9ng/uL) % | Pass Library Qubit (>2ng/μL) % | Frag Pass (>70%) % | All failed unique samples* | Fail % | Final Pass samples |
| --- | --- | --- | --- | --- | --- | --- | --- |
| All Samples (%) | 455 | 96.3 | 97.5 | 95.1 | 46 | 10.11 | 409 |
| Brain tumors | 77 | 96.1 | 98.6 | 92.2 | 6 | 7.8 | 71 |
| Lung Non-small cell carcinoma (Adenocarcinoma and Squamous Cell Carcinoma) | 98 | 95.9 | 98.9 | 96.7 | 8 | 8.1 | 90 |
| Melanoma | 59 | 94.9 | 98.2 | 89.8 | 6 | 10.2 | 53 |
| Colorectal Adenocarcinoma | 54 | 100.0 | 100.0 | 96.3 | 2 | 3.7 | 52 |
| Breast carcinoma | 26 | 100.0 | 100.0 | 88.5 | 3 | 11.5 | 23 |
| Prostate adenocarcinoma | 12 | 91.7 | 90.9 | 83.3 | 2 | 16.7 | 10 |
| Urothelial carcinoma | 9 | 88.9 | 100.0 | 77.8 | 2 | 22.2 | 7 |
| Testicular stromal tumor | 1 | 100.0 | 100.0 | 100.0 | 0 | 0.0 | 1 |
| Small intestine carcinoma | 1 | 100.0 | 100.0 | 100.0 | 0 | 0.0 | 1 |
| Soft tissue tumors (Sarcoma and GIST) | 10 | 100.0 | 80 | 75 | 4 | 40 | 6 |
| Pancreatic adenocarcinoma | 11 | 81.8 | 88.9 | 63.6 | 4 | 36.4 | 7 |
| Ovarian stromal tumor | 19 | 94.7 | 94.4 | 89.5 | 2 | 10.5 | 17 |
| GYN carcinomas (Ovarian and Uterine) | 30 | 96.6 | 93.1 | 96.3 | 4 | 13.3 | 26 |

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| Tumor Type | Original samples (N) | DNA Pass (>2.9ng/uL) % | Pass Library Qubit (>2ng/μL) % | Frag Pass (>70%) % | All failed unique samples* | Fail % | Final Pass samples |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Kidney tumor | 40 | 100.0 | 97.5 | 97.5 | 1 | 2.5 | 39 |
| Hepato-biliary carcinoma | 7 | 85.7 | 100.0 | 71.4 | 2 | 28.6 | 5 |
| Large Cell neuroendocrine | 1 | 100.0 | 100.0 | 100.0 | 0 | 0.0 | 1 |

### C. Analytical Performance:

The hybridization-capture-based targeted re-sequencing assay is designed to detect point mutations [single nucleotide variants (SNVs)] as well as small insertions/deletions (indels) < 35bp in length in the coding exons of 607 genes (Appendix 1). A total of 10,577 exons are sequenced, 94 targets were excluded during assay development due to low sequence coverage and high GC content (Appendix 2). A paired-sample analysis pipeline (tumor vs. matched normal) is used to identify somatic mutations in the targeted exons. NYU took a representative approach to validation of the SNVs and indels targeted in this panel, which is appropriate for variants of this type.

#### 1. Precision Studies

##### a) Intra-assay reproducibility

The intra-assay reproducibility was demonstrated by analyzing a set of 9 patient samples in triplicates within the same run. Three (3) samples were selected to represent each variant type: SNV, Insertion, and Deletion. The results, samples selection and critical parameters (quality, depth of coverage and variant allele frequency) and statistical analysis are summarized in Table 11 and 12, respectively. All replicates were performed with 100 ng DNA input (lowest required input).

Table 11. Intra-assay reproducibility: SNV, insertions, and deletions samples

| Sample-replicate | Gene coding mutation amino acid change | Qual | Depth | Freq |
| --- | --- | --- | --- | --- |
| 1-1 | NPM1 c.859_860insTCTG:p.L287fs | 359 | 215 | 0.08 |
| 1-2 | | 727 | 214 | 0.107 |
| 1-3 | | 375 | 161 | 0.081 |
| 2-1 | ERBB2 c.2265_2266insGCATACGTGATG:p.E755delinsEAYVM | 3756 | 659 | 0.162 |
| 2-2 | | 3229 | 622 | 0.15 |
| 2-3 | | 2796 | 664 | 0.123 |
| 3-1 | EGFR c.2300_2301insCAGCGTGGA:p.A767delinsASVD | 1970 | 456 | 0.129 |
| 3-2 | | 2130 | 452 | 0.142 |
| 3-3 | | 1630 | 470 | 0.106 |
| 4-1 | EGFR c.2236_2250del:p.746_750del | 1134 | 612 | 0.074 |
| 4-2 | | 567 | 386 | 0.057 |
| 4-3 | | 1044 | 734 | 0.056 |
| 5-1 | EGFR c.2309_2310insCAACCCCCACGG:p.D770delinsDNPHG | 1695 | 610 | 0.084 |
| 5-2 | | 945 | 621 | 0.055 |
| 5-3 | | 1639 | 555 | 0.088 |
| 6-1 | BRAF c.1798_1799delGTinsAA:p.V600K | 1072 | 236 | 0.208 |
| 6-2 | | 2028 | 387 | 0.23 |

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| Sample-replicate | Gene coding mutation amino acid change | Qual | Depth | Freq |
| --- | --- | --- | --- | --- |
| 6-3 | | 1172 | 350 | 0.157 |
| 7-1 | TP53 c.G197A:p.G66D | 404 | 466 | 0.056 |
| 7-2 | | 305 | 255 | 0.071 |
| 7-3 | | 459 | 506 | 0.059 |
| 8-1 | PIK3CA c.G1633A:p.E545K | 390 | 291 | 0.076 |
| 8-2 | | 156 | 255 | 0.059 |
| 8-3 | | 268 | 280 | 0.061 |
| 9-1 | ATM c.A5071C:p.S1691R | 3359 | 293 | 0.478 |
| 9-2 | | 3334 | 328 | 0.448 |
| 9-3 | | 4417 | 364 | 0.508 |

Table 12. Intra-assay reproducibility: SNV, insertions, and deletions statistical analysis

| | mean | sd | median | minimum | maximum | positive call | positive call rate(95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| NPM1.c.859_860insTCTG.p.L287fs | 0.09 | 0.02 | 0.08 | 0.08 | 0.11 | 3 out of 3 | 100%(31%,100%) |
| ERBB2.c.2265_2266insGCATACGTGATG.p.E755delinsEAYVM | 0.15 | 0.02 | 0.15 | 0.12 | 0.16 | 3 out of 3 | 100%(31%,100%) |
| EGFR.c.2300_2301insCAGCGTGGA.p.A767delinsASVD | 0.13 | 0.02 | 0.13 | 0.11 | 0.14 | 3 out of 3 | 100%(31%,100%) |
| EGFR.c.2236_2250del.p.746_750del | 0.06 | 0.01 | 0.06 | 0.06 | 0.07 | 3 out of 3 | 100%(31%,100%) |
| EGFR.c.2309_2310insCAACCCCACGG.p.D770delinsDNPHG | 0.08 | 0.02 | 0.08 | 0.06 | 0.09 | 3 out of 3 | 100%(31%,100%) |
| BRAF.c.1798_1799delGTinsAA.p.V600K | 0.20 | 0.04 | 0.21 | 0.16 | 0.23 | 3 out of 3 | 100%(31%,100%) |
| TP53.c.G197A.p.G66D | 0.06 | 0.01 | 0.06 | 0.06 | 0.07 | 3 out of 3 | 100%(31%,100%) |
| PIK3CA.c.G1633A.p.E545K | 0.07 | 0.01 | 0.06 | 0.06 | 0.08 | 3 out of 3 | 100%(31%,100%) |
| ATM.c.A5071C.p.S1691R | 0.48 | 0.03 | 0.48 | 0.45 | 0.51 | 3 out of 3 | 100%(31%,100%) |

# b) Inter-assay panel-wide reproducibility: SNVs, insertions, deletions

The SNV, indel inter-assay reproducibility was assessed testing 16 patients selected for 28 previously known unique mutations detected with orthogonal NGS methods (Table 13). All sixteen samples were FFPE derived. All reproducibility samples were prepared independently for each run and carried a different molecular barcode to account for the reproducibility of all steps of the procedure. All replicates were performed with 100 ng DNA input (lowest required input). Critical parameters including variant allele frequency (VAF), depth of coverage and quality between runs were assessed and correlated. All runs passed QC metric criteria. All expected mutations showed 100% Positive call rate, (Table 14A).

Table 13. Inter-assay panel-wide reproducibility (16 unique patients) – select variants

| Tumor type | Gene | Coding | AA change | Variant type |
| --- | --- | --- | --- | --- |
| Colon adenocarcinoma | ATM | c.A5071C | p.S1691R | nonsynonymous SNV |
| Colon adenocarcinoma | KRAS | c.G34T | p.G12C | nonsynonymous SNV |
| Lung | SAMD9 | c.1800_1801del | p.E600fs | frameshift deletion |

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| Tumor type | Gene | Coding | AA change | Variant type |
| --- | --- | --- | --- | --- |
| adenocarcinoma | | | | |
| Lung adenocarcinoma | ERBB2 | c.2220_2221insGCATACGTGATG | p.E740delinsEA YVM | nonframeshift insertion |
| Colon adenocarcinoma | MET | c.T1085C | p.M362T | nonsynonymous SNV |
| Colon adenocarcinoma | NRAS | c.G34T | p.G12C | nonsynonymous SNV |
| Colon adenocarcinoma | KMT2D | c.11750_11758del:p.3917_3920del | p.3917_3920del | nonframeshift deletion |
| Colon adenocarcinoma | HRAS | c.488_507del | p.L163fs | frameshift deletion |
| Pancreatic adenocarcinoma | PIK3CA | c.G1633A | p.E545K | nonsynonymous SNV |
| Pancreatic adenocarcinoma | KRAS | c.G35T | p.G12V | nonsynonymous SNV |
| Pancreatic adenocarcinoma | SMAD4 | c.1239_1241del:p.413_414del | p.413_414del | nonframeshift deletion |
| PNET | HSP90AA1 | c.1108_1109del | p.E370fs | frameshift deletion |
| Medulloblastoma | AURKC | c.20_27del | p.T7fs | frameshift deletion |
| Medulloblastoma | KMT2B | c.1126_1128del:p.376_376del | p.376_376del | nonframeshift deletion |
| Pediatric glioma | SPEN | c.535_561del:p.179_187del | p.179_187del | nonframeshift deletion |
| Pediatric glioma | HIST1H1C | c.590_604del:p.197_202del | p.197_202del | nonframeshift deletion |
| Pediatric glioma | TAF1L | c.2927_2928del | p.T976fs | frameshift deletion |
| Lung adenocarcinoma | EGFR | c.2309_2310insCAACCCCCA CGG | p.D770delinsDN PHG | nonframeshift insertion |
| Glioblastoma | EPHA7 | c.72_73del | p.T24fs | frameshift deletion |
| Glioblastoma | FANCD2 | c.1278_1278del | p.L426fs | frameshift deletion |
| Glioblastoma | ICK | c.1106_1117del:p.369_373del | p.369_373del | nonframeshift deletion |
| Lung adenocarcinoma | EGFR | c.2236_2250del:p.746_750del | p.746_750del | nonframeshift deletion |
| Melanoma | BRAF | c.G1798_1799delinsAA | p.V600K | nonsynonymous DNV |
| Glioblastoma | PTEN | c.1649_1652del | p.Y550fs | frameshift deletion |
| Lung adenocarcinoma | SDHA | c.1944_1945del | p.T648fs | frameshift deletion |
| Lung adenocarcinoma | EGFR | c.2300_2301insCAGCGTGGA | p.A767delinsAS VD | nonframeshift insertion |
| Lung adenocarcinoma | CYP2D6 | c.775delA | p.R259fs | frameshift deletion |

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**Table 14A. Inter-assay reproducibility SNV -variant allele frequency**

| Gene | AA Change | Mean | SD | Median | Minimum | Maximum | Positive Call | Positive Call Rate (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| BRAF | p.V600K | 0.33 | 0.02 | 0.33 | 0.29 | 0.35 | 5 out of 5 | 100% (46.3%, 100%) |
| KRAS | p.G12V | 0.07 | 0.01 | 0.07 | 0.06 | 0.09 | 5 out of 5 | 100% (46.3%, 100%) |
| PIK3CA | p.E545K | 0.08 | 0.02 | 0.08 | 0.06 | 0.11 | 5 out of 5 | 100% (46.3%, 100%) |
| MET | p.M362T | 0.45 | 0.01 | 0.45 | 0.44 | 0.46 | 5 out of 5 | 100% (46.3%, 100%) |
| NRAS | p.G12C | 0.14 | 0.01 | 0.14 | 0.12 | 0.15 | 5 out of 5 | 100% (46.3%, 100%) |
| ATM | p.S1691R | 0.50 | 0.02 | 0.50 | 0.47 | 0.53 | 5 out of 5 | 100% (46.3%, 100%) |
| KRAS | p.G12C | 0.06 | 0.01 | 0.06 | 0.06 | 0.07 | 5 out of 5 | 100% (46.3%, 100%) |

# **a) Deletions**

| Gene | AA change | Mean | SD | Median | Min | Max | Positive Call | Positive Call Rate(95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| FANCD2 | p.L426fs | 0.35 | 0.02 | 0.35 | 0.32 | 0.38 | 5 out of 5 | 100% (46.3%, 100%) |
| CYP2D6 | p.R259fs | 0.21 | 0.01 | 0.21 | 0.19 | 0.22 | 5 out of 5 | 100% (46.3%, 100%) |
| SDHA | p.T648fs | 0.15 | 0.01 | 0.15 | 0.14 | 0.17 | 5 out of 5 | 100% (46.3%, 100%) |
| PTEN | p.Y550fs | 0.46 | 0.03 | 0.45 | 0.42 | 0.50 | 5 out of 5 | 100% (46.3%, 100%) |
| EGFR | p.746_750del | 0.09 | 0.01 | 0.10 | 0.08 | 0.10 | 5 out of 5 | 100% (46.3%, 100%) |
| ICK | p.369_373del | 0.34 | 0.05 | 0.36 | 0.26 | 0.38 | 5 out of 5 | 100% (46.3%, 100%) |
| EPHA7 | p.T24fs | 0.50 | 0.03 | 0.49 | 0.47 | 0.54 | 5 out of 5 | 100% (46.3%, 100%) |
| HIST1H1C | p.197_202del | 0.27 | 0.02 | 0.27 | 0.25 | 0.30 | 5 out of 5 | 100% (46.3%, 100%) |
| TAF1L | p.T976fs | 0.44 | 0.04 | 0.43 | 0.38 | 0.49 | 5 out of 5 | 100% (46.3%, 100%) |
| SPEN | p.179_187del | 0.19 | 0.01 | 0.19 | 0.18 | 0.19 | 5 out of 5 | 100% (46.3%, 100%) |
| AURKC | p.T7fs | 0.51 | 0.03 | 0.50 | 0.47 | 0.55 | 5 out of 5 | 100% (46.3%, 100%) |
| KMT2B | p.376_376del | 0.64 | 0.02 | 0.63 | 0.62 | 0.67 | 5 out of 5 | 100% (46.3%, 100%) |
| HSP90AA1 | p.E370fs | 0.48 | 0.02 | 0.48 | 0.44 | 0.50 | 5 out of 5 | 100% (46.3%, 100%) |
| SMAD4 | p.413_414del | 0.17 | 0.02 | 0.17 | 0.14 | 0.19 | 5 out of 5 | 100% (46.3%, 100%) |
| HRAS | p.L163fs | 0.36 | 0.02 | 0.35 | 0.34 | 0.39 | 5 out of 5 | 100% (46.3%, 100%) |
| KMT2D | p.3917_3920del | 0.36 | 0.02 | 0.37 | 0.34 | 0.38 | 5 out of 5 | 100% (46.3%, 100%) |
| SAMD9 | p.E600fs | 0.44 | 0.02 | 0.44 | 0.42 | 0.48 | 5 out of 5 | 100% (46.3%, 100%) |

# **b) Insertions**

| Gene | AA Change | Mean | SD | Median | Min | Max | Positive Call | Positive Call Rate(95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| NPM1 | p.L287fs | 0.13 | 0.02 | 0.14 | 0.11 | 0.16 | 5 out of 5 | 100% (46.3%, 100%) |
| EGFR | p.D770delinsDNPHG | 0.07 | 0.01 | 0.06 | 0.06 | 0.08 | 5 out of 5 | 100% (46.3%, 100%) |
| EGFR | p.A767delinsASVD | 0.14 | 0.01 | 0.15 | 0.13 | 0.16 | 5 out of 5 | 100% (46.3%, 100%) |
| ERBB2 | p.E740delinsEAYVM | 0.17 | 0.01 | 0.17 | 0.16 | 0.19 | 5 out of 5 | 100% (46.3%, 100%) |

# **c) Panel-wide Run-to-Run Precision:**

The precision analysis was performed for the known mutations, and also performed for all additional mutations identified in each specimen in any of the test replicates. Testing identified 26 additional somatic mutations that were not known before the

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start of the precision studies, likely due to the size of the panel/s used. All were subsequently confirmed by orthogonal testing. Mutations showed reproducibility across all replicates with only one deletion, which was missed on one of the replicates, likely due to low VAF (median VAF: 0.06). In total, 54 mutations were tested with a positive call rate 269 out of 270 replicates. Precision per specimen across all mutation types, and precision per type of mutation are summarized in Table 14B. One discordant was observed for AFF3 deletion. All runs passed the quality metrics criteria.

Table 14B. Panel-wide precision summary for all 5 replicates for 16 unique patient samples

| Gene | Mutation Type | Exon, coding, amino acid change | Median Coverage (range) | VAF mean | SD | Median | Min | Max | Call rate |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Colon adenocarcinoma | | | | | | | | | |
| ATM | nonsynonymous SNV | exon34, c.A5071C, p.S1691R | 853 (762-1417) | 0.5 | 0.02 | 0.5 | 0.47 | 0.53 | 5 / 5 |
| KRAS | nonsynonymous SNV | exon2, c.G34T, p.G12C | 1211 (1211-1855) | 0.06 | 0.01 | 0.06 | 0.06 | 0.07 | 5 / 5 |
| APC | nonsynonymous SNV | exon16, c.T3920A, p.I1307K | 492 (377-661) | 0.45 | 0.05 | 0.47 | 0.4 | 0.49 | 5 / 5 |
| SDHA | frameshift deletion | exon15, c.1944_1945del, p.L649Efs*4 | 1442 (1185-1774) | 0.15 | 0.01 | 0.15 | 0.14 | 0.17 | 5 / 5 |
| Lung adenocarcinoma | | | | | | | | | |
| SAMD9 | frameshift deletion | exon3, c.1800_1801del, p.E600fs | 228 (202-297) | 0.44 | 0.02 | 0.44 | 0.42 | 0.48 | 5 / 5 |
| ERBB2 | nonframeshift deletion | exon20, c.2220_2221insGCATAC GTGATG, p.E740delinsEAYVM | 1112 (804-1379) | 0.17 | 0.01 | 0.17 | 0.16 | 0.19 | 5 / 5 |
| TP53 | nonsynonymous SNV | exon4, c.G314A, p.G105D | 363 (357-396) | 0.41 | 0.04 | 0.42 | 0.36 | 0.45 | 5 / 5 |
| Colon adenocarcinoma | | | | | | | | | |
| MET | nonsynonymous SNV | exon14, c.T1085C, p.M362T | 926 (663-952) | 0.45 | 0.01 | 0.45 | 0.44 | 0.46 | 5 / 5 |
| NRAS | nonsynonymous SNV | exon2, c.G34T, p.G12C | 720 (472-769) | 0.14 | 0.01 | 0.14 | 0.12 | 0.15 | 5 / 5 |
| HRAS | frameshift deletion | exon5, c.488_507del, p.L163fs | 818 (445-1084) | 0.36 | 0.02 | 0.35 | 0.34 | 0.39 | 5 / 5 |
| KMT2D | nonframeshift deletion | exon39, c.11750_11758del, p.3917_3920del | 1593 (1238-1876) | 0.36 | 0.02 | 0.37 | 0.34 | 0.38 | 5 / 5 |
| HNF1A | nonsynonymous SNV | exon1, c.G92A, p.G31D | 570 (406-721) | 0.16 | 0.02 | 0.17 | 0.14 | 0.2 | 5 / 5 |
| MLH3 | nonsynonymous SNV | exon2, c.G2221T, p.V741F | 1067 (883-1756) | 0.16 | 0.01 | 0.17 | 0.15 | 0.17 | 5 / 5 |
| CHD7 | nonframeshift insertion | exon3, c.2049_2050insAAAGCA, p.A685_K686dup | 923 (857-1380) | 0.41 | 0.02 | 0.42 | 0.39 | 0.42 | 5 / 5 |
| SETD2 | frameshift | exon18, c.7330dupT, | 824 | 0.15 | 0.02 | 0.15 | 0.14 | 0.17 | 5 / 5 |

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| Gene | Mutation Type | Exon, coding, amino acid change | Median Coverage (range) | VAF mean | SD | Median | Min | Max | Call rate |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | insertion | p.Y2444Lfs*2 | (726-1159) | | | | | | |
| CREB3L2 | nonframeshift deletion | exon2, c.299_301del, p.100_101del | 458 (352-597) | 0.3 | 0.03 | 0.29 | 0.26 | 0.34 | 5 / 5 |
| Pancreatic adenocarcinoma | | | | | | | | | |
| PIK3CA | nonsynonymous SNV | exon10, c.G1633A, p.E545K | 638 (503-972) | 0.08 | 0.02 | 0.08 | 0.06 | 0.11 | 5 / 5 |
| KRAS | nonsynonymous SNV | exon2, c.G35T, p.G12V | 823 (726-1295) | 0.07 | 0.01 | 0.07 | 0.06 | 0.09 | 5 / 5 |
| SMAD4 | nonframeshift deletion | exon10, c.1239_1241del, p.413_414del | 511 (455-841) | 0.17 | 0.02 | 0.17 | 0.14 | 0.19 | 5 / 5 |
| AR | nonframeshift deletion | exon1, c.171_179del, p.57_60del | 696 (243-821) | 0.1 | 0.04 | 0.09 | 0.06 | 0.15 | 5 / 5 |
| PNET | | | | | | | | | |
| HSP90AA1 | frameshift deletion | exon6, c.1108_1109del, p.E370fs | 425 (416-711) | 0.48 | 0.02 | 0.48 | 0.44 | 0.5 | 5 / 5 |
| FGFR1 | nonsynonymous SNV | exon13, c.A1729G, p.N577D | 642 (431-799) | 0.44 | 0.02 | 0.45 | 0.41 | 0.46 | 5 / 5 |
| IL7R | nonsynonymous SNV | exon2, c.T197C, p.I66T | 401 (326-567) | 0.46 | 0.04 | 0.48 | 0.41 | 0.49 | 5 / 5 |
| Medulloblastoma | | | | | | | | | |
| AURKC | frameshift deletion | exon3, c.20_27del, p.T7fs | 470 (418-641) | 0.51 | 0.03 | 0.5 | 0.47 | 0.55 | 5 / 5 |
| KMT2B | nonframeshift deletion | exon3, c.1126_1128del, p.376_376del | 1034 (846-1475) | 0.64 | 0.02 | 0.63 | 0.62 | 0.67 | 5 / 5 |
| v | Pediatric glioma | | | | | | | | |
| SPEN | nonframeshift deletion | exon3, c.535_561del, p.Q179_R187del | 1599 (1476-1951) | 0.19 | 0.01 | 0.19 | 0.18 | 0.19 | 5 / 5 |
| IDH1 | nonsynonymous SNV | exon4, c.G395A, p.R132H | 1240 (1138-1918) | 0.18 | 0.01 | 0.19 | 0.17 | 0.2 | 5 / 5 |
| Pediatric glioma | | | | | | | | | |
| HIST1H1C | nonframeshift deletion | exon1, c.590_604del, p.A197_K201del | 1236 (881-1520) | 0.27 | 0.02 | 0.27 | 0.25 | 0.3 | 5 / 5 |
| TAF1L | frameshift deletion | exon1, c.2927_2928del, p.T976fs | 383 (376-622) | 0.44 | 0.04 | 0.43 | 0.38 | 0.49 | 5 / 5 |
| FGFR4 | nonsynonymous SNV | exon9, c.G1162A, p.G388R | 1066 (470-1842) | 0.42 | 0.03 | 0.43 | 0.38 | 0.44 | 5 / 5 |
| Lung adenocarcinoma | | | | | | | | | |
| EGFR | nonframeshift insertion | exon20, c.2309_2310insCAACCC CCACGG, p.D770delinsDNPHG | 1058 (873-1297) | 0.07 | 0.01 | 0.06 | 0.06 | 0.08 | 5 / 5 |
| TP53 | nonsynonymous SNV | exon8, c.A838G, p.R280G | 566 (410-732) | 0.08 | 0.01 | 0.08 | 0.06 | 0.1 | 5 / 5 |
| AFF3 | nonframeshift deletion | exon13, c.1330_1332del, p.444_444del | 1918 (1610-2509) | 0.06 | 0.01 | 0.06 | 0.05 | 0.07 | 4/5 |
| Glioblastoma | | | | | | | | | |
| EPHA7 | frameshift | exon1, c.72_73del, | 518 | 0.5 | 0.03 | 0.49 | 0.47 | 0.54 | 5 / 5 |

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| Gene | Mutation Type | Exon, coding, amino acid change | Median Coverage (range) | VAF mean | SD | Median | Min | Max | Call rate |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | deletion | p.T24fs | (476-807) | | | | | | |
| ADGRD1 | frameshift insertion | exon6, c.499dupA, p.V168Sfs*57 | 690 (482-754) | 0.43 | 0.02 | 0.43 | 0.41 | 0.46 | 5 / 5 |
| Glioblastoma | | | | | | | | | |
| FANCD2 | frameshift deletion | exon15, c.1278_1278del, p.L426fs | 931 (871-1571) | 0.35 | 0.02 | 0.35 | 0.32 | 0.38 | 5 / 5 |
| ICK | nonframeshift deletion | exon10, c.1106_1117del, p.369_373del | 249 (209-281) | 0.34 | 0.05 | 0.36 | 0.26 | 0.38 | 5 / 5 |
| PTPN11 | nonsynonymous SNV | exon13, c.G1508T, p.G503V | 799 (576-1006) | 0.31 | 0.02 | 0.31 | 0.28 | 0.33 | 5 / 5 |
| Lung adenocarcinoma | | | | | | | | | |
| EGFR | nonframeshift deletion | exon19, c.2236_2250del, p.746_750del | 1133 (987-1680) | 0.09 | 0.01 | 0.1 | 0.08 | 0.1 | 5 / 5 |
| TNK2 | nonsynonymous SNV | exon13, c.G2864A, p.R955H | 479 (368-833) | 0.14 | 0.03 | 0.14 | 0.1 | 0.17 | 5 / 5 |
| NOTCH2 | frameshift deletion | exon1:c.17_18del:p.P6fs | 750 (341-1963) | 0.13 | 0.02 | 0.13 | 0.11 | 0.15 | 5 / 5 |
| Melanoma | | | | | | | | | |
| BRAF | nonsynonymous DNV | exon15, c.G1798_1799delinsAA, p.V600K | 720 (653-1190) | 0.33 | 0.02 | 0.33 | 0.29 | 0.35 | 5 / 5 |
| JAK3 | nonsynonymous SNV | exon16, c.G2164A, p.V722I | 1579 (1044-2069) | 0.48 | 0.01 | 0.48 | 0.46 | 0.5 | 5 / 5 |
| SDHD | nonsynonymous SNV | exon2, c.G34A, p.G12S | 813 (696-966) | 0.45 | 0.02 | 0.45 | 0.42 | 0.47 | 5 / 5 |
| IRS2 | nonframeshift deletion | exon1, c.82_84del, p.28_28del | 1317 (943-2215) | 0.34 | 0.01 | 0.34 | 0.33 | 0.35 | 5 / 5 |
| KAT6A | nonframeshift insertion | exon17, c.4982_4983insACAGCA GCCACAGCC, p.Q1657_P1661dup | 552 (374-746) | 0.18 | 0.03 | 0.18 | 0.13 | 0.22 | 5 / 5 |
| Glioblastoma | | | | | | | | | |
| PTEN | frameshift deletion | exon10, c.1649_1652del, p.Y550fs | 431 (332-491) | 0.46 | 0.03 | 0.45 | 0.42 | 0.5 | 5 / 5 |
| TERT | nonsynonymous SNV | exon6, c.C2177T, p.T726M | 1363 (1066-1680) | 0.47 | 0.02 | 0.47 | 0.43 | 0.5 | 5 / 5 |
| Lung adenocarcinoma | | | | | | | | | |
| EGFR | nonframeshift insertion | exon20, c.2300_2301insCAGCGT GGA, p.A767delinsASVD | 1228 (1042-1372) | 0.14 | 0.01 | 0.15 | 0.13 | 0.16 | 5 / 5 |
| CYP2D6 | frameshift deletion | exon5, c.775delA, p.R259fs | 1090 (871-1214) | 0.21 | 0.01 | 0.21 | 0.19 | 0.22 | 5 / 5 |
| SDHA | frameshift deletion | exon15, c.1944_1945del, p.T648fs | 1442 (1185-1774) | 0.15 | 0.01 | 0.15 | 0.14 | 0.17 | 5 / 5 |
| MN1 | nonframeshift insertion | exon1, c.1619_1620insGCA, p.Q550dup | 627 (430-782) | 0.16 | 0.02 | 0.17 | 0.14 | 0.18 | 5 / 5 |

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| Gene | Mutation Type | Exon, coding, amino acid change | Median Coverage (range) | VAF mean | SD | Median | Min | Max | Call rate |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sarcoma | | | | | | | | | |
| NPM1 | frameshift insertion | exon11, c.859_860insTCTG, p.L287fs | 795 (629-1095) | 0.13 | 0.02 | 0.14 | 0.11 | 0.16 | 5 / 5 |
| DNMT3A | nonsynonymous SNV | exon16, c.C1903T, p.R635W | 1291 (857-1587) | 0.13 | 0.02 | 0.14 | 0.11 | 0.15 | 5 / 5 |

# d) Per Specimen Precision:

Results of the precision studies were combined and precision across all reportable genes was determined for each specimen. The positive call rate based on the total number of mutations along with the 2-sides 95% confidence interval were calculated (Table 14C).

Table 14C: Precision per specimen across all reportable mutations (N – 5 replicates)

| Tumor type | Total No Unique Mutations detected across all 5 replicates | Positive call rate per mutation | Positive call rate (two- sided 95% CI) |
| --- | --- | --- | --- |
| Colon adenocarcinoma | 4 | 5/5 | 20/20 100% (83.9%, 100.0%) |
| Lung adenocarcinoma | 3 | 5/5 | 15/15 100.0% (79.6%, 100.0%) |
| Colon adenocarcinoma | 9 | 5/5 | 45/45 100.0% (92.1%, 100.0%) |
| Pancreatic adenocarcinoma | 4 | 5/5 | 20/20 100% (83.9%, 100.0%) |
| PNET | 3 | 5/5 | 15/15 100.0% (79.6%, 100.0%) |
| Medulloblastoma | 2 | 5/5 | 10/10 100.0 (72.2%, 100.0%) |
| Pediatric glioma | 2 | 5/5 | 10/10 100.0 (72.2%, 100.0%) |
| Pediatric glioma | 3 | 5/5 | 15/15 100.0% (79.6%, 100.0%) |
| **Lung adenocarcinoma** | **3** | **5/5 for 2 4/5 for 1** | **14/15 93.3% (70.2%, 98.8%)** |
| Glioblastoma | 2 | 5/5 | 10/10 100.0 (72.2%, 100.0%) |
| Glioblastoma | 3 | 5/5 | 15/15 100.0% (79.6%, 100.0%) |
| Lung adenocarcinoma | 3 | 5/5 | 15/15 100.0% (79.6%, 100.0%) |

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| Melanoma | 5 | 5/5 | 25/25 100.0% (86.7%, 100.0%) |
| --- | --- | --- | --- |
| Glioblastoma | 2 | 5/5 | 10/10 100.0 (72.2%, 100.0%) |
| Lung adenocarcinoma | 4 | 5/5 | 20/20 100% (83.9%, 100.0%) |
| Myeloid sarcoma | 2 | 5/5 | 10/10 100.0 (72.2%, 100.0%) |
| **Total mutations** | **5** **4** | **5/5 for 53** **4/5 for 1** | **269/270** **99.6% (97.9%, 99.9%)** |
| **SNV** | 2 2 | 5/5 | 110/110 100.0% (96.6%, 100.0%) |
| **Insertion** | 8 | 5/5 for 7 4/5 for 1 | 39/40 97.5% (87.1%, 99.6%) |
| **Deletion** | 2 4 | 5/5 | 120/120 100.0% (96.9%, 100.0%) |

## 2. Analytical Sensitivity – Limit of Detection (LoD)

The LoD of the NYU Langone PACT assay is defined as the mutant allele fraction at which 95% of replicates across all replicates for a variant type are reliably detected. Studies were conducted to demonstrate a putative LoD for each variant type. Two studies were conducted (1) with commercially available reference materials and (2) with clinical specimens.

### a) Reference Material:

Commercially available reference material consisting of mutant DNA blended in genomic DNA was used to obtain a preliminary LoD. For positive calls. The control material provides a mix of mutations (SNV, insertions and deletions) The list of mutations is shown in Table 15.

Table 15. Limit of Detection - Reference Material DNA Mix

| Gene ID | COSMIC Identifier | Mutation Type | HGVS Nomenclature | Amino Acid | Target AF |
| --- | --- | --- | --- | --- | --- |
| AKT1 | COSM3376 5 | Substitution | c.49G>A | p.E17K | 10% |
| APC | COSM1312 7 | Substitution | c.4348C>T | p.R1450* | 10% |
| APC | COSM1856 1 | Insertion in HP 7N | c.4666_4667insA | p.T1556fs*3 | 10% |
| ATM | COSM2192 4 | Deletion | c.1058_1059delG T | p.C353fs*5 | 10% |
| ERBB2 | COSM6 82/ 20959 | Insertion | c.2324_2325ins12 | p.A775_G776insY VMA | 10% |
| GNA11 | COSM5296 | Substitution | c.626A>T | p.Q209L | 10% |

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| Gene ID | COSMIC Identifier | Mutation Type | HGVS Nomenclature | Amino Acid | Target AF |
| --- | --- | --- | --- | --- | --- |
| | 9 | | | | |
| GNAQ | COSM28758 | SNV in HP 3N | c.626A>C | p.Q209P | 10% |
| KIT | COSM1314 | Substitution | c.2447A>T | p.D816V | 10% |
| MPL | COSM18918 | Substitution | c.1544G>T | p.W515L | 10% |
| PDGFRA | COSM736 | Substitution | c.2525A>T | p.D842V | 10% |
| PIK3CA | COSM763 | Substitution | c.1633G>A | p.E545K | 10% |
| SMAD4 | COSM14105 | Insertion | c.1394_1395insT | p.A466fs*28 | 10% |
| CTNNB1 | COSM5664 | Substitution | c.121A>G | p.T41A | 7% |
| EGFR | COSM6224 | SNV in 3N | c.2573T>G | p.L858R | 7% |
| GNAS | COSM27887 | Substitution | c.601C>T | p.R201C | 7% |
| JAK2 | COSM12600 | SNV in HP 3N | c.1849G>T | p.V617F | 7% |
| KRAS | COSM521 | Substitution | c.35G>A | p.G12D | 7% |
| NPM1 | COSM17559 | Insertion | c.863_864insTCTG | p.W288fs*12 | 7% |
| NRAS/CSDE1 | COSM584 | Substitution | c.182A>G | p.Q61R | 7% |
| PTEN | COSM4986 | Insertion | c.741_742insA | p.P248fs*5 | 7% |
| PTEN | COSM5809 | Deletion 6N > 5N | c.800delA | p.K267fs*9 | 7% |
| TP53 | COSM10648 | Substitution | c.524G>A | p.R175H | 7% |
| TP53 | COSM10660 | Substitution | c.818G>A | p.R273H | 7% |
| TP53 | COSM10662 | Substitution | c.743G>A | p.R248Q | 7% |
| TP53 | COSM6530 | Deletion | c.723delC | p.C242fs*5 | 7% |
| BRAF | COSM476 | Substitution | c.1799T>A | p.V600E | 4% |
| EGFR | COSM12378 | Insertion | c.2310_2311insGGT | p.D770_N771insG | 4% |
| EGFR | COSM6225 | Deletion | c.2236_2250del15 | p.E746_A750delELREA | 4% |
| EGFR | COSM6240 | Substitution | c.2369C>T | p.T790M | 4% |
| FGFR3 | COSM715 | Substitution | c.746C>G | p.S249C | 4% |
| FLT3 | COSM783 | Substitution | c.2503G>T | p.D835Y | 4% |
| PDGFRA | COSM28053 | Insertion | c.1694_1695insA | p.S566fs*6 | 4% |
| PIK3CA | COSM12464 | Insertion | c.3204_3205insA | p.N1068fs*4 | 4% |

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| Gene ID | COSMIC Identifier | Mutation Type | HGVS Nomenclature | Amino Acid | Target AF |
| --- | --- | --- | --- | --- | --- |
| PIK3CA | COSM775 | Substitution | c.3140A>G | p.H1047R | 4% |
| RET | COSM965 | Substitution | c.2753T>C | p.M918T | 4% |

Of note, variant allele frequency (VAF) detected by NYU Langone PACT assay in non-diluted conditions was below the manufacturer's advertised uniform content (Figure 4, No Dilution, 250ng). Control material was diluted at 1:2 and 1:4 ratio with the negative control HapMap DNA. In addition, different DNA inputs (250ng, 100ng and 50ng) for each dilution were tested. There was expected decrease of VAF with increasing sample dilution. However, the VAF remained relatively stable down to 100ng of DNA input in the non-diluted control, particularly for all mutations with VAF>5%. However, 50ng input leads to loss of some mutations, most pronounced when VAF is <5%. The drop out of mutations is most pronounced at the 1:4 Dilution. Variants with low frequency are particularly sensitive to decreasing DNA input while variants with VAF >5% are reliably detected even with low DNA input. Dashed line highlights the 5% VAF threshold established in this study.

Figure 4: Variant allele frequency and DNA input:


A similar pattern was observed when variant coverage was assessed under these

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conditions. While depth of coverage of the non-diluted sample remains robust (>200X) even at 50 ng of input (with an exception of one SNV), increased dilutions results in a dropout of low level variants Based on this analysis it was concluded that SNVs, insertions and deletions with >5% VAF can be reliably detected with >200X coverage with 100ng of the DNA input, Figures 4 and 5. The data demonstrates that low DNA input produces >200x coverage in samples with no dilution. Samples with high dilution show marked dropout of the coverage of predicted variants. Dashed line highlights 200x coverage threshold established in this study. Table with all VAF and Depth data for all dilutions and DNA inputs is provided in Appendix 5

Figure 5: Depth of coverage and DNA input as a function of DNA input


Based on this analysis it was concluded that SNVs, insertions and deletions with >5% VAF can be reliably detected with >200X coverage with 100ng of the DNA input. All reproducibility studies were performed with 100ng input.

The analysis shows that mutations with at least 5-10% VAF, whether due to a low prevalence of the variant in the tumor OR a highly prevalent mutation but in a sample with a low tumor cell content, can be expected to be sequenced with sufficient coverage to enable confident mutation calling. These lower limits of detection thresholds (5% VAF, 200X coverage and 100 ng DNA input) were

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subsequently tested both in intra- and inter- assay reproducibility studies as detailed above. This analysis correlates with the Accuracy cohort in which~95% of all confirmed mutations with VAF >5% had coverage >200X. Applying these criteria to the dataset, the sensitivity of 94% for SNVs and indels and a specificity of 98% using a VAF of 0.05 and coverage >200X was established. Cutoff values for variant frequency and coverage were selected to minimize false positive rates.

False negative rates for SNV and indels will be minimized by requiring at least 10% estimated tumor content and by manual inspection of all detected actionable somatic nonsynonymous actionable mutations even those with coverage 50X-200X (see section on Coverage).

b) Clinical Specimens: Limit of detection was confirmed by profiling 16 FFPE samples including 6 SNV, 5 insertions and 5 deletions with VAF at the LOD in 5 separate runs (80 replicates total). All replicates were performed with 100 ng DNA input (lowest required input). Mutations were accurately called in 80 out of 80 times, 5 out of 5 per mutation, with 100% Positive call rate see Table 16 below.

Table 16. Inter-assay reproducibility at the LOD for VAF

| Gene | Mutation type | AA change | mean | SD | med | min | max | positive call | positive call rate(95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **SNV** | | | | | | | | | |
| AHI1 | SNV | p.S1123F | 0.08 | 0.02 | 0.07 | 0.06 | 0.10 | 5 out of 5 | 100%(46.3%, 100%) |
| TP53 | SNV | p.R280G | 0.08 | 0.01 | 0.08 | 0.06 | 0.10 | 5 out of 5 | 100%(46.3%, 100%) |
| KRAS | SNV | p.G12V | 0.07 | 0.01 | 0.07 | 0.06 | 0.09 | 5 out of 5 | 100%(46.3%, 100%) |
| PIK3CA | SNV | p.E545K | 0.08 | 0.02 | 0.08 | 0.06 | 0.11 | 5 out of 5 | 100%(46.3%, 100%) |
| KRAS | SNV | p.G12C | 0.06 | 0.01 | 0.06 | 0.06 | 0.07 | 5 out of 5 | 100%(46.3%, 100%) |
| BRAF | SNV | p.V600E | 0.06 | 0.01 | 0.06 | 0.05 | 0.07 | 5 out of 5 | 100%(46.3%, 100%) |
| **Insertions** | | | | | | | | | |
| EGFR | Insertion | p.D770delinsDNPHG | 0.07 | 0.01 | 0.06 | 0.06 | 0.08 | 5 out of 5 | 100%(46.3%, 100%) |
| FOXO3 | frameshift insertion | p.D380fs | 0.08 | 0.01 | 0.08 | 0.06 | 0.08 | 5 out of 5 | 100%(46.3%, 100%) |
| EP400 | nonframeshift insertion | p.R2719delinsRQ | 0.07 | 0.01 | 0.07 | 0.06 | 0.08 | 5 out of 5 | 100%(46.3%, 100%) |
| IL21R | frameshift insertion | p.S383Lfs*5 | 0.07 | 0.01 | 0.07 | 0.06 | 0.09 | 5 out of 5 | 100%(46.3%, 100%) |
| BCR | frameshift insertion | BCR:NM_021574:exon18: | 0.08 | 0.01 | 0.08 | 0.07 | 0.09 | 5 out of 5 | 100%(46.3%, 100%) |
| **Deletions** | | | | | | | | | |
| EGFR | Deletion | p.746_750del | 0.09 | 0.01 | 0.10 | 0.08 | 0.10 | 5 out of 5 | 100%(46.3%, |

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| | | | | | | | | | 100%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| KMT2B | frameshift deletion | p.R965Dfs*21 | 0.08 | 0.01 | 0.07 | 0.07 | 0.082814 | 5 out of 5 | 100%(46.3%, 100%) |
| FIP1L1 | frameshift deletion | FIP1L1:NM_001134938:ex | 0.07 | 0.02 | 0.06 | 0.05 | 0.089783 | 5 out of 5 | 100%(46.3%, 100%) |
| ASXL1 | frameshift deletion | p.G643fs | 0.07 | 0.01 | 0.07 | 0.06 | 0.091612 | 5 out of 5 | 100%(46.3%, 100%) |
| ACVR2A | frameshift deletion | p.K437Rfs*5 | 0.08 | 0.02 | 0.08 | 0.07 | 0.110333 | 5 out of 5 | 100%(46.3%, 100%) |

c) DNA-Input: The validated DNA concentration is the amount at which the average read depth over the exon regions was maintained at the criteria established including (e.g., Number of targets with 0 and <50X coverage < 1%, Average coverage of the sample >300X) and have 100% positive mutation call rate based on reproducibility and sensitivity studies. The recommended DNA concentration for the assay is 250ng with the lowest acceptable DNA input of 100 ng. The DNA input range 50-250ng was assessed for accuracy and sequencing failures as a function of the input DNA concentration, see Figures 4 and 5 above.

### 3. Linearity/assay reportable range:

Not applicable

### 4. Traceability (controls, calibrators, or methods):

The NYU Langone Genome PACT is not traceable to any known standard. Controls and quality metrics are described in the device description section.

### 5. Stability

Reagent stability is based on manufacturer expiration dating, and supported by NYU molecular pathology laboratory verification. Stability of the reagents is monitored through the use of consistent controls.

### 6. Expected values:

The laboratory follows protocols for the use of controls consistent with CLIA regulation. The NYU Langone Genome PACT does not use calibrators; however, the verification of mutant allele frequency is maintained by analysis of a positive control sample with expected allele frequencies.

### 7. Analytical specificity:

High analytical specificity is maintained by paired tumor/matched normal sequencing and was established during assay optimization.

### Interference:

The NYU Langone Genome PACT assay pre-analytic steps are designed to minimize interference. Provided the quality of the FFPE specimen received for testing complies with standard fixation and embedding conditions (e.g.: no degradation, not decalcified), no interfering substances were identified during the validation, using DNA extracted by

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protocol standard procedures.

### 8. Assay cut-off:

The NYU Langone PACT does not report mutations below 5% variant allele frequency.

### 9. Comparison studies: Accuracy studies

#### a) Method comparison:

The NYU Langone Genome PACT assay is designed to detect SNVs and small insertions and deletions in the entire coding sequence of 607 genes. The accuracy of the assay was assessed by comparison of the NYU Langone Genome PACT result to the original results obtained with the validated orthogonal methods. Testing was conducted per protocol on 455 unique FFP cancer samples, see Table 17. A total of 777 unique mutations in 409 tumor specimens that passed QC criteria, from 19 different cancer types were tested and are listed in Table 17 and Figure 6 and 7 below.


Figure 6. Distribution of all tumor types in the Accuracy cohort.

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Figure 7. Distribution of all confirmed SNVs (A), insertions (B) and deletions (C).

Table 17. Unique SNVs, Insertions and Deletions Represented in the Accuracy Summary Per Gene, Exon and AA change

| Gene | Mutation type | Exon | AA change | Number of Samples |
| --- | --- | --- | --- | --- |
| ABL1 | SNV | exon6 | N350D | 1 |
| AKT1 | SNV | exon3 | D46E | 1 |
| AKT1 | SNV | exon3 | E17K | 1 |
| APC | SNV | exon16 | E1317Q | 2 |
| APC | Deletion | exon16 | E1464fs | 2 |
| APC | SNV | exon16 | R1114X | 2 |
| APC | SNV | exon16 | V1125A | 2 |
| APC | SNV | exon6 | R876X | 5 |
| APC | SNV | exon16 | E1309K | 1 |
| APC | SNV | exon16 | E1353X | 1 |
| APC | SNV | exon16 | G131X | 1 |
| APC | SNV | exon16 | I1307K | 1 |
| APC | SNV | exon16 | Q1429X | 1 |
| APC | SNV | exon16 | R1450X | 1 |
| AR | SNV | exon5 | W742C | 1 |
| ARID1A | Deletion | exon1 | D1850fs*4 | 1 |
| ARID2 | Deletion | exon19 | K1716fs | 1 |
| ARID2 | SNV | exon19 | Q1717X | 1 |
| ATM | SNV | exon17 | F858L | 2 |
| ATM | SNV | exon12 | P604S | 2 |
| ATM | SNV | exon34 | S1691R | 2 |

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| Gene | Mutation type | Exon | AA change | Number of Samples |
| --- | --- | --- | --- | --- |
| ATM | SNV | exon63 | G3051X | 1 |
| ATM | SNV | exon56 | N2736T | 1 |
| ATM | SNV | exon63 | R3008C | 1 |
| ATM | SNV | exon23 | G1130X | 1 |
| ATM | SNV | exon22 | W1058X | 1 |
| ATR | SNV | exon32 | E1840Q | 1 |
| ATRX | SNV | exon2 | N12S | 1 |
| BARD1 | SNV | exon4 | M383I | 1 |
| BCL6 | Insertion | exon5 | F497_P498insS | 1 |
| BCL6 | SNV | exon4 | R98Q | 1 |
| BRAF | Insertion | exon15 | V600K | 2 |
| BRAF | Insertion | exon15 | VK600_601EN | 2 |
| BRAF | SNV | exon15 | V600E | 21 |
| BRAF | SNV | exon15 | G596R | 1 |
| BRAF | SNV | exon15 | V600M | 1 |
| BRAF | SNV | exon15 | W604L | 1 |
| CALR | Deletion | exon9 | E364fs | 2 |
| CALR | Deletion | exon9 | Q365fs | 2 |
| CALR | Insertion | exon9 | K385fs | 4 |
| CDK12 | SNV | exon1 | M1K | 1 |
| CDKN2A | Deletion | exon2 | A60fs | 1 |
| CDKN2A | Deletion | exon2 | L62fs | 1 |
| CDKN2A | Insertion | exon2 | R80X | 1 |
| CDKN2A | SNV | exon2 | V59L | 1 |
| CDKN2A | SNV | exon2 | W110X | 1 |
| CDKN2A | SNV | exon2 | G55D | 1 |
| CIC | SNV | exon18 | D1440N | 1 |
| CSF1R | SNV | exon20 | E916K | 1 |
| CTNNB1 | SNV | exon4 | S33F | 4 |
| C…