Predicate Devices

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Reference Devices

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AI/ML (Artificial Intelligence / Machine Learning)

Unknown
The document mentions "MSIsensor" which is a software component used for microsatellite instability analysis. While "sensor" could imply some form of algorithmic processing, the summary does not explicitly state or provide details suggesting the use of AI or ML techniques within this software or any other part of the device's analysis pipeline. The description of the cutoff establishment and validation is based on concordance with established methods and statistical analysis, not explicitly on AI/ML model training or inference.

Therapeutic

No
The device is described as a qualitative in vitro diagnostic test for detecting tumor gene alterations and microsatellite instability, which provides information for healthcare professionals but is not deemed conclusive or prescriptive for any specific therapeutic product.

Diagnostic

Yes

The "Intended Use / Indications for Use" explicitly states that "The MSK-IMPACT assay is a qualitative in vitro diagnostic test."

SaMD (Software as a Medical Device)

No

The device description explicitly mentions "required equipment, software, reagents, vendors, and storage conditions," indicating that it is not solely software but relies on physical components and processes for its function as an in vitro diagnostic test.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use/Indications for Use: The very first sentence explicitly states: "The MSK-IMPACT assay is a qualitative in vitro diagnostic test..." This is the most direct confirmation.
Nature of the Test: The test analyzes biological specimens (formalin-fixed paraffin-embedded tumor tissue matched with normal specimens) to detect specific markers (tumor gene alterations, somatic mutations, microsatellite instability) for the purpose of providing information for use by healthcare professionals in accordance with professional guidelines. This aligns perfectly with the definition of an in vitro diagnostic device, which is used to examine specimens taken from the human body to provide information for diagnosis, monitoring, or screening.
Device Description: While it describes equipment, software, and reagents, the overall context is that these components are used to perform the diagnostic test on the biological samples.
Anatomical Site: The input is human genomic DNA obtained from tumor tissue, which is a biological specimen.
Intended User/Care Setting: The test is intended for use by "qualified health care professionals," which is typical for IVDs used in a clinical setting.
Performance Studies: The detailed performance studies (Precision, Analytical Sensitivity, Method Comparison) are standard types of studies conducted to validate the performance of an IVD.

The document clearly and repeatedly identifies the MSK-IMPACT assay as an in vitro diagnostic test and describes its use in a manner consistent with IVD regulations and definitions.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The MSK-IMPACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and microsatellite instability for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. MSK-IMPACT is a single-site assay performed at Memorial Sloan Kettering Cancer Center.

Product codes

PZM

Device Description

A description of required equipment, software, reagents, vendors, and storage conditions were provided, and are described in the product labeling (MSK-IMPACT manual). MSK assumes responsibility for the device.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Formalin-fixed paraffin-embedded tumor tissue

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Qualified health care professionals

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

The cutoff was first established using a training specimen dataset consisting of 138 colorectal cancer (CRC) and 40 endometrial carcinoma (EC) specimens with matched normal and having MSI status results from a validated MSI-PCR or MMR IHC test. MSIsensor scores ranged from 0 to 47.7 for CRC and 0 to 43.7 for EC. Based on concordance to either mismatch repair immunohistochemistry (MMR IHC) for MLH1, MSH2, MSH6 and PMS2 expression, or a commercially available PCR assay that detects 5 mononucleotide microsatellite loci including MR-21, BAT-25, MONO-27, NR-24 and BAT-26, a MSIsensor cut-off of 10 was established to delineate microsatellite stable (MMS) from high microsatellite instability (MSI-H).

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

A separate data set was obtained to validate this cut-off. A retrospectiveprospective chart review of 135 CRC patients was conducted to identify cases that had both MSK-IMPACT MSI results and results by a validated IHC panel (MLH1, MSH2, MSH6 and PMS2). A total of 66 specimens had both sets of results. Of these, there were two discordant cases.

To evaluate the ability of the MSIsensor to determine MSI status in cancer types other than CRC or EC cancer types, 119 unique non-CRC and non-EC tumornormal pair samples covering 25 tumor types were assessed for MSI by both MSIsensor and a validated MSI-PCR test.

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

Precision Studies:

Objective: To assess between-run and within-run precision.
Between-run: Extracted DNA was run once per day for 3 days using different barcodes (n=3 inter-day replicates).
Within-run: For one run, a sample was run in triplicates (n=3 intra-day replicates).
Precision Panel: 10 samples (9 FFPE specimens and one commercial cell line) representing different tumor types, mutation types, and mutant allele frequencies.
Results:
- Panel-Wide Reproducibility: 69 mutations in clinical specimens and 13 mutations in cell line (total 82 mutations). All mutations had 100% concordance in all replicates except for 4 mutations in clinical specimens (AR SNV pQ64K, AR frameshift mutation pL54fs, ARID1B insertion pA445_P446insP, PTEN frameshift mutation pT319Kfs*24) and 3 mutations in commercial sample (IDH1 exon4 R132H; BRAF exon15 V600M; EGFR exon19 E746 A750del). Discordances were due to poor mapping quality in repetitive regions or low frequencies near 2%.
- Coefficient of Variation (%CV) for MAF: 34 (45) of 69 mutations in clinical specimens had %CV ≤10%, 17/69 were 10-20% and 7/69 were >21%.
- Per Specimen Precision:
  - M15-22924 (5 unique mutations): 100.0% PPA (25/25)
  - M15-3038 (3 unique mutations): 100.0% PPA (15/15)
  - M16-19000 (10 unique mutations): 98.0% PPA (49/50)
  - M1688-5C (18 unique mutations): 95.6% PPA (86/90)
  - M-1698-A9 (5 unique mutations): 100.0% PPA (25/25)
  - M-1654-CA (6 unique mutations): 100.0% PPA (30/30)
  - M-1612-28 (4 unique mutations): 100.0% PPA (20/20)
  - M1648-D5 (10 unique mutations): 100.0% PPA (50/50)
  - M-1707-12 (5 unique mutations): 80.0% PPA (20/25)
  - Commercial sample (13 unique mutations): 90.8% PPA (59/65)
- Well-characterized reference material (HapMap cell line NA20810): 23 replicates. Zygosity results 100% concordant, absolute difference between expected and mean observed mutation frequencies = 0.09%±0.45%.
- Microsatellite Instability (MSI): 12 specimens (6 MSI-H, 6 MSS), 3 inter- and 3 intra-run replicates. All samples had 100% agreement between calls.

Analytical Sensitivity Limit of Detection (LoD):

Part 1 (Dilution Series): Identified lowest reliable mutant fraction for 5 validation exons with lowest coverage (ERBB2 exon 20 (V777L), PDGFRA exon 18 (D842V), PIK3CA exon 10 (E545K), EGFR exon 19 (indel), KIT exon 9 (indel)) and 5 validation exons with highest coverage (BRAF exon 15 (V600E), KRAS exon 2 (G12D), PIK3CA exon 2 (R88Q), KIT exon 11 (indel), EGFR exon 20 (indel)). All results were called at the lowest dilution except for PIK3CA.
Part 2 (Confirmation of LoD): 5 replicates tested for 3 deletions, 4 insertions, and 6 SNVs at 5% minor allele frequency. All variants had 100% positive call rates except for one replicate for a deletion on PTEN exon 6 (failed mutation read depth and below estimated LoD of 5%).
Microsatellite instability (MSI): Minimum tumor proportion required for MSIsensor score robustness was 25% with 200X average coverage. Qualitatively reproducible to 8% tumor proportion.
DNA-Input: Optimized and recommended DNA concentration is 250ng, with acceptable range 50-250ng. Sequencing failures increased as DNA input decreased (78% success for 50-100ng vs 97% for 250ng).

Analytical Specificity:

Maintained by paired tumor/matched normal sequencing.

Accuracy (Method Comparison):

Sample Size: 433 FFPE tumor specimens tested for 267 unique mutations in 48 exons of 20 genes.
Key Results:
- 418 of 433 specimens met ≥200X coverage criteria.
- Known mutation successfully detected in 432 out of 433 cases (99.8% PPA (98.7%, 100.0% CI)).
- One discordant case observed in M-1994-BC-T (EGFR exon 20 insertion) due to incorrect filtering by the calling algorithm, which was subsequently modified.
- PPA for SNV/MNVs by Gene: All genes (AKT1, ALK, BRAF, EGFR, ERBB2, FGFR2, FGFR3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, TP53) showed 100.0% PPA.
- PPA for insertions by gene: EGFR (93.8%), ERBB2 (100.0%), FGFR3 (100.0%), KIT (100.0%), PIK3CA (100.0%), TP53 (100.0%).
- PPA for deletions by gene: EGFR (100.0%), IDH2 (100.0%), KIT (100.0%), MET (100.0%), PIK3CA (100.0%), TP53 (100.0%).
Supplemental Method Comparison Study for Wildtype Calls:
- Sample Size: 95 specimens for 33 "hotspots" in 10 genes.
- Key Results: 109 mutations and 3026 wild-type calls.
- PPA: 100% (96.7%, 100.0% CI) for mutations.
- NPA: 100% (99.9%, 100.0% CI) for wild-type locations.
Method Comparison of the MSK-IMPACT MSIsensor:
- CRC patients: 66 specimens compared to IHC.
  - PPV: 92.3% (12/13) (64.0%-99.8% CI).
  - NPV: 98.1%. (52/53) (90.0%, 100.0% CI).
- Non-CRC and non-EC cancer types: 119 samples (25 tumor types) compared to MSI-PCR.
  - Excluding missing specimens: PPV = 93.9% (46/49) (83.1%, 98.7% CI), NPV = 96.7% (58/60) (88.5%, 99.6% CI).
  - Accounting for missing specimens: PPV = 78.0% (46/59) (65.3%, 87.7% CI), NPV = 96.7% (58/60) (88.5%, 99.6% CI).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Precision Studies:

Concordance: All mutations have 100% concordance in all replicates except for 4 mutations in clinical specimens and 3 mutations in the commercial sample.
Coefficient of Variation (%CV) for MAF: 34 (45) of the 69 mutations in the clinical specimens had %CV ≤10%, 17/69 were between 10 and 20% and 7/69 were >21%.
Positive Call Rate for individual specimens: Ranges from 80.0% to 100.0%.
MSIsensor Score Precision: MSIsensor scores greater than 0.5. Coefficient of variance (%CV) for MSI-H specimens ranged from 1.7% to 10.7%; for MSS specimens ranged from 15.3% to 223.6%.
MSIsensor Positive Call Rate (two-sided 95% CI): 100% (47.8%, 100.0%) for all MSIsensor scores.

Analytical Sensitivity Limit of Detection (LoD):

Positive Call Rate (Part 2): All variants have 100% positive call rates except for one replicate for a deletion on PTEN exon 6 (80.0% positive call rate, 7-19 AD, 0.036-0.079 MAF, 0.31-0.48 NormDP).

Accuracy (Method Comparison):

Percent Positive Agreement (PPA):
- Known mutation detection in accuracy study: 99.8% (432 out of 433 cases) with two-sided 95% CI of (98.7%, 100.0%).
- SNV/MNVs by Gene: All genes showed 100.0% PPA.
- Insertions by gene: EGFR (93.8% (69.8%, 100.0% CI)), others 100.0%.
- Deletions by gene: All genes showed 100.0% PPA.
- Supplemental Method Comparison (mutations): 100% (96.7%, 100.0% CI).
Negative Percent Agreement (NPA):
- Supplemental Method Comparison (wild-type locations): 100% (99.9%, 100.0% CI).
MSIsensor Results Compared to IHC MMR for CRC:
- Positive Predictive Value (PPV): 92.3% (12/13) with two-sided 95% CI of 64.0%-99.8%.
- Negative Predictive Value (NPV): 98.1%. (52/53) with two-sided 95% CI of 90.0%, 100.0%.
MSIsensor Results Compared to PCR 5 Loci MSI Panel for Other Cancer Types:
- Excluding missing specimens with 95%CI: PPV is 93.9% (46/49) (83.1%, 98.7%). NPV is 96.7% (58/60) (88.5%, 99.6%).
- Accounting for missing specimens with 95%CI: PPV=78.0% (46/59) (65.3%, 87.7%). NPV= 96.7% (58/60) (88.5%, 99.6%).

Predicate Device(s)

Not Found

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

To support the continuous implementation of process improvements to the existing 468 gene panel, protocols with specific procedures and acceptance criteria for modifications that could be anticipated at the time of submission were provided, reviewed by FDA, and cleared as part of this marketing authorization. Future modifications by MSK for the specified types of changes below that are made in accordance with the applicable validation strategy and the pre-specified success criteria would not require a new 510(k) submission. Significant changes such as adding new genes or variant types to the panel would require a new submission with appropriate validation.

Type of change: New pre-analytical protocol, kits or reagents

Validation Strategy: Sequence at least 10 specimens with known mutations. Measure sequence coverage distribution, and call somatic mutations in all samples.
Pre-specified success criteria: For cases sequenced to >200x, ensure that 95% of exons are covered to 100x or more. Concordance for known mutations should be >95%.

Type of change: New library preparation protocol, kits, or reagents

Validation Strategy: Sequence at least 40 DNA specimens (tumor / normal pairs) or three pools previously sequenced by MSK-IMPACT. Measure sequence coverage distribution, and call somatic mutations in all samples.
Pre-specified success criteria: For cases sequenced to >200x, ensure that 95% of exons are covered to 100x or more. Concordance for calling somatic mutations with variant allele fraction >10% should be >98%.

Type of change: Changes to probes for already analytically validated genes

Validation Strategy: Re-capture existing sequence libraries from at least 3 runs (at least 40 samples) with new probes, sequence, and analyze.
Pre-specified success criteria: For cases sequenced to >200x, ensure that 95% of exons in analytically validated genes are covered to 100x or more. Concordance for calling somatic mutations with variant allele fraction >10% should be >98%.

Type of change: New sequencing instrument or reagents using similar chemistry and technology, and the sequence depth and read length are not changed from previous platform.

Validation Strategy: Re-sequence existing captured libraries from at least 3 runs, and call somatic mutations in all samples.
Pre-specified success criteria: Sequence coverage distribution and GC bias across targeted regions should be within 5% of prior sequencing runs. Concordance for calling somatic mutations with variant allele fraction >10% should be >98%.

Type of change: Bioinformatics pipeline - Update to underlying annotation database or transcript isoforms

Validation Strategy: Reanalyze FASTQ files (raw sequencing reads) from at least 3 runs (at least 40 samples). Compare variants calls between the clinical analysis results and the current modified results
Pre-specified success criteria: Confirm the changes do not change the variant call results. Confirm the annotations for the unaffected transcripts do not change. Confirm the annotations for the affected transcripts are modified as expected.

Type of change: Bioinformatics pipeline - Update to data management system and system database

Validation Strategy: Reanalyze FASTQ files (raw sequencing reads) from at least 3 runs (at least 40 samples) in production mode. Compare variants calls between the clinical analysis results and the current modified results
Pre-specified success criteria: Ensure that all previously called mutations are recovered and the variants in the database of results are concordant with the variants in the pipeline output files

Type of change: Bioinformatics pipeline - Modification to an existing component of the analysis pipeline (e.g., tool or algorithm) where the underlying algorithm or main parameter settings (e.g. minimal coverage/VAF threshold for SNV/indel calling; MSIsensor score cut-off for MSI-H calling, etc.) are not changed.

Validation Strategy: Reanalyze FASTQ files (raw sequencing reads) from at least 3 runs (at least 40 samples). Compare variants calls between the clinical analysis results and the current modified results
Pre-specified success criteria: Ensure that all previously called mutations are recovered and that newly detected mutations can be explained by pipeline modifications.

Regulation Number and Section

§ 866.6080 Next generation sequencing based tumor profiling test.

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

Summary

0

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets)

DECISION SUMMARY

A. DEN Number:

DEN170058

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation for the MSK-IMPACT

C. Measurand:

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

Refer to Appendix 1a for complete list of hotspot mutations and Appendix 1b for complete list of genes included in this assay.

D. Type of Test:

Next generation sequencing tumor profiling test

E. Applicant:

Memorial Sloan Kettering (MSK)

F. Proprietary and Established Names:

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

G. Regulatory Information:

1. Regulation section:

21 CFR 866.6080

2. Classification:

Class II

3. Product code:

PZM

1

4. Panel:

Pathology

H. Indications for Use:

1. Indications for Use:

The MSK-IMPACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and microsatellite instability for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. MSK-IMPACT is a single-site assay performed at Memorial Sloan Kettering Cancer Center.

2. Special conditions for use statement(s):

For prescription use.

For in vitro diagnostic use.

3. Special instrument requirements:

Illumina HiSeq™ 2500 Sequencer (qualified by MSK)

I. Device Description:

A description of required equipment, software, reagents, vendors, and storage conditions were provided, and are described in the product labeling (MSK-IMPACT manual). MSK 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:

| Tissue
Type | Volume | Minimum Tumor
Proportion | Macrodissection
requirements
(Based on tumor
proportion) | Limitations | Storage |
|------------------|----------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|
| FFPE
sections | 5-20
unstained
sections,
10
microns
thick | More than 10% of tumor
cells;
sections containing

20% viable tumor are
preferred.
For MSI testing, >25%
tumor cells. | Yes,
macrodissection
to obtain non-
neoplastic tissue
for analysis | Archival paraffin-
embedded material
subjected to acid
decalcification is
unsuitable for analysis
because acid
decalcification severely
damage nucleic acids. | Room
temp |

Table 1. Specimen Handling and Processing for Validated Specimen Types

2

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. DNA is run in singlicate. DNA shearing is conducted per protocol and a quality control check is performed. Average fragment size should be ~200bp. Sheared DNA is stored at -20°C if not proceeding directly to Library Preparation. The DNA can be stored at 37°C for 10-20 minutes, stored at 2-8°C for 24 hours, or at -20°C for longer periods.

2. Library Preparation:

Sequence libraries are prepared using KAPA Biosystems Library Preparation Reagents by first producing 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 with the peak at ~200bp; and concentration between 5-300ng/uL to ensure adequate hybridization for capture.

3. Hybrid Capture NGS:

Library capture is conducted using NimbleGen 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 ~300bp; the concentration of the amplified DNA library should be 5-45ng/uL; the LM-PCR yield should be ≥ 250ng. Reactions can be stored at 4°C until ready for purification, up to 72 hours.

4. Sequencing and Data Analysis:

Sequencing is conducted with the Illumina HiSeq2500 Sequencing Instruments and reagents and PhiX Control v3. The sequencing process uses multiple quality checks.

a) Data Management System (DMS): Automated sample tracking and archival of runassociated 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 (FASTO, BAM, VCF) and sequencing run statistics (e.g., cluster density, %clusters passing filter, unassigned read indices).
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.

3

c) Indexing OC check: The potential for index contamination is managed by demultiplexing all sequencing reads for all possible barcodes. If the number of reads > 15,000 for any unused barcodes, then those reads are analyzed with the pipeline and the fingerprint SNPs are used to identify which of the barcodes used in the pool could be causing the appearance of extra reads.
d) 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 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.
e) Sample QC checks: The baits used for hybridization capture include custom intergenic and intronic probes targeting >1000 regions throughout the genome containing common single nucleotide polymorphisms (SNPs). The unique combination of 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. OC checks involving the use of these 'fingerprint' SNPs are detailed below:
- i. Sample mix-up check: The analysis pipeline computes the 'percent discordance' between a reference and query sample, defined as the percent of homozygous sites in the reference sample that are homozygous for the alternate allele in the query sample. The expected discordance between tumors and their respective matched normal should be low ( 5% discordance ("unexpected mismatches") and from different patients with 1% in a normal sample, the normal sample is flagged for review and possible exclusion from analysis. Tumor

4

samples with matched normal controls excluded due to possible tumor contamination will be considered as unmatched tumor samples for subsequent analyses.

f) Mutation calling SNVs and Indels: The analysis pipeline identifies two classes of mutations: (1) single nucleotide variants (SNVs) and (2) indels. Paired sample mutation calling is performed on tumor samples and their respective matched normal controls. In instances where a matched normal sample is unavailable, or where the matched normal sample was sequenced with low 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, a per sample coverage requirement has been conservatively set at ≥ 200X. A lower coverage threshold for the matched normal is set at 50X.
- 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., ratio between mutation frequencies in the tumor and normal samples to be > 5.0); (2) whether the mutation is a known hotspot mutation (refer to Appendix 1a for details); (3) reference on in house 'standard normal' based on common artifacts; (4) technical characteristics that use coverage depth (DP), number of mutant reads (AD), mutation frequency (VF).

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 reject all false positive SNVs and almost all false positive indel calls from the reference dataset.

5

Image /page/5/Figure/0 description: This image shows a flowchart of the filtering process for SNVs and Indels. The raw pipeline output for SNVs is MuTect, and for Indels, it is SomaticIndelDetector. The standard filter for somatic variants is VFtumor/VFnormal ≥ 5X, AD ≥ 5, and VF ≥ 1%. After annotation with AnnoVar, a two-tiered filtering scheme is applied based on whether the variant is in a hotspot, with different DP, AD, and VF thresholds for each case, and the process ends with manual review.

Figure 1. Summary of mutation filtering scheme

g) Mutation annotation: Predicted functional effect and clinical interpretation for each mutation is curated by automated software using information from several databases.
h) Microsatellite Instability (MSI) status calling: The somatic MSI status is inferred by interrogating all available genomic microsatellites covered by MSK-IMPACT within tumor samples against the matched normal DNA using the program MSIsensor (Nui B et al. 2014). Essentially, the sequencing results are analyzed via MSIsensor to assess the number and length of homo-polymers / microsatellites within the targeted regions of tumor-normal sample pair. This results in a continuous rather than categorical MSI score assignment for the tumor sample. Loci are considered unstable (somatic) if k-mer distributions are significantly different between the tumor and matched normal using a standard multiple testing correction of x2 p-values. The percentage fraction of unstable sites is reported as the MSIsensor score. The assay uses a MSIsensor score threshold of 10 or greater to define MSI-H by MSIsensor.

5. Controls

a) Matched normal control: Genomic DNA is extracted from patient-matched normal tissue (when available) or peripheral blood, for use as a matched normal control. In the event a matched normal is unavailable, or where the matched normal sample was sequenced with low coverage ( 200X |
| Coverage Uniformity | ≥ 98% target exons above 100X coverage |
| Base Quality | > 80% of bases with QS above > Q30 |
| % Cluster passing | The percent cluster passing filter (Cluster PF) > 80% |
| % Reads passing
filter | The percent reads passing filter (Reads PF) > 80% |
| | Mutation Coverage (DP) ≥ 20, |
| Hotspot Mutation*
calling threshold | Number of Mutant Reads (AD) ≥ 8, |
| | Mutation Frequency (VF) ≥ 2% |
| Non-hotspot
Mutation**
threshold | DP > 20, AD ≥ 10, VF ≥ 5% |
| Indels | Fewer than 20% of samples in an established 'standard
normal'database |
| Positive Run Control | The difference between the observed and expected frequencies for the
known mutations should be within 5%. |
| Negative Run
Control | The correlation between expected and observed mutation frequencies
should be 0.9 or higher |
| Sample-Mix up QC | Check over 1000 custom intergenic/intronic "fingerprint" SNPs.
Flagged if pairs of samples from the same patient with > 5%
discordance and from different patients with 0.9 as a QC metric for the whole pool analyzed.

Image /page/9/Figure/2 description: The image is a boxplot showing the relationship between true VAF (variant allele frequency) and observed VAF. The x-axis represents the true VAF, ranging from 0.05 to 1.0 in increments of 0.05. The y-axis represents the observed VAF, ranging from 0.0 to 1.0. Each boxplot shows the distribution of observed VAF values for a given true VAF, and the observed VAF generally increases as the true VAF increases.

Figure 2. Observed vs. Expected Variant Frequency

b) Requirements on sample coverage: Ten normal (diploid) FFPE samples were profiled in duplicate using the IMPACT assay (total = 20 replicates) to generate summary statistics across all targeted exons. The mean coverage across all targeted exons for the normal samples was 571X (SD = 373X). Summary statistics were also computed on coverage values per exon normalized by per-sample coverage. There were exons that presented with consistently low coverage values. None of the exons of the genes in the clinical validation are among those with consistently low coverage. It was determined the low coverage was due to sequence similarity with other loci, and high GC content. The exons were removed from the MSK-IMPACT assay. Of the remaining exons across all genes, 99.5% were sequenced to a depth of 100X or greater while 98.6% were sequenced to a depth of 250X or greater. This analysis of normal samples indicates that with a mean sample coverage of 571X, 98% of exons are sequenced with coverage greater than 306X, or with normalized coverage greater

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than 0.54. (The 'mean-normalized coverage' is the coverage of the mutation divided by the mean coverage across all exons; it serves as a measure of how deeply the validation exon was sequenced relative to the overall coverage of the sample. A mean-normalized coverage below 1 indicates the exon coverage is below average; conversely if greater than 1, it indicates above average coverage.) The data are shown in Figures 3 and Figure 4

Figure 3. Distribution of mean coverage values for targeted exons. Dashed line indicates coverage at 100X.

Image /page/10/Figure/2 description: The image is a histogram showing the frequency of coverage depth. The x-axis represents the coverage depth, ranging from 0 to 1500. The y-axis represents the frequency, with the highest frequency around a coverage depth of 500. A vertical dashed line is present at a coverage depth of approximately 100.

Figure 4. Distribution of mean coverage for targeted exons, normalized by persample coverage. Dashed line indicates 20% of mean sample coverage.

Image /page/10/Figure/4 description: The image is a histogram showing the distribution of normalized coverage depth. The x-axis represents the normalized coverage depth, ranging from 0.0 to 2.5. The y-axis represents the frequency, ranging from 0 to 400. The histogram shows a bell-shaped distribution, with a peak around a normalized coverage depth of 1.0.

Based on the calculations, 98% of exons can be expected to be sequenced to coverage greater than 100X, when mean sample coverage is 185X (0.54* 185X = 100X). (A 100X minimum coverage threshold per exon is required based on the power

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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 200X on mean 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 200X.

To provide empirical data for these requirements, MSK utilizes the pool normal sample with known expected single nucleotide mutations (n = 2436) and the underlying mutation allele fractions (MAF). In silico downsampling analysis was conducted with a pool normal mix down to 45% where the sample coverage decreased from 452X to 203X. At this coverage level, 94% of the mutations with expected underlying VAF of 10% were called.

c) Requirements on mutation coverage, allele depth and frequency for positive calls: Permissive standard filters were used to intentionally generate false positives to identify suitable thresholds for parameters such as mutation coverage (DP), alternate allele depth (AD) and mutation frequency (VF) to optimize specificity. The following criteria allows optimal rejection of false positive SNVs (stratified by whether they are hotspots or not) and indel calls, while maintaining ability to detect true positive events with underlying frequency of 10% (5-17.6% observable). Potential strand-bias is also evaluated in the standard somatic mutation calling pipeline. An example of the number of false positive events detected pre and post filtering for coverage depth(DP), number of mutant reads (AD) and variant frequency (VF) is shown in Table 4.

	Mutations -Cosmic database		Mutations
Filter criteria	DP ≥ 20X, AD ≥ 8,VF ≥ 2%		DP ≥ 20X, AD ≥ 10,VF ≥ 5%
	SNVs	Indels	SNVs	Indels
Pre-filter	1	24	342	40,793
Post-filter	0	0	0	8
Rejection Rate	1.00	1.00	1.00	0.999

Table 4. Sample error correction by DP/AD/VF filter
-----------------------------------------------------	--	--	--

1. Pre-Analytical performance:
  Minimum DNA requirements were established by measuring assay performance based on different inputs from normal blood and FFPE tumor samples. DNA samples are normalized to yield 50 - 250 ng input and maximized to 55 ul prior to shearing. The normalization and DNA quantification are performed.

DNA extraction method was validated based on the invalid rates across multiple tumor types obtained from historical data. The data demonstrated that the DNA extraction has been optimized across tumor types to reasonably conclude that the analytical

12

performance presented is representative across FFPE tumor types. Table 5 shows the historical data for invalid rates from a retrospective chart review of >10,000 specimens tested with MSK-IMPACT. The range of invalid rates was 7.2% to 18.4%. The data shows that interference effects from different specimens are not significant across different tumor types supporting the performance of the pan-cancer specimen handling.

| | | | Pre-Run
Invalids | Pre-Run
Invalids | Post-Run
Invalids | |
|---------------------------------|------------------|--------------------|--------------------------------------------|---------------------------------------------|---------------------------------------------|---------------------|
| Tumor Type | Specimen
Type | Number
of Tests | Tumor
Insufficient
(Tumor %
C | G12A | ~7% |
| Lung
Adenocarcinoma | INS | ERBB2
exon 20 | 2310_2311ins
GCATACGTGATG | E770_A771insAYVM | ~15% |
| Lung
Adenocarcinoma | SNV | EGFR
exon 21 | 2573T>G | L858R | ~20% |

Table 6: Summary of the Specimens and Allele Frequencies in the Precision Studies

1 For complex structural variations, such as genomic rearrangements (fusions) and copy number variations (CNVs), the expectation is that the representative approach should be demonstrated at the gene level.

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Tissue type	Mutation type	Gene/ exon	cDNA change	Amino acid change	Mutation frequency
CRC	SNV	KRAS exon 2	C34G>T	G12C	~30%
Lung Adenocarcinoma	DEL (15bp)	EGFR exon 19	2236_2250delGAATTAA GAGAAGCA	E746_A750del	~30%
CRC	SNV	BRAF exon 15	c.1799T>A	V600E	~40%
GIST	DEL (6bp)	Kit exon 11	1667_1672delAGTGGA	Q556_K558del	~50%
FFPE Cell Line	DEL, SNV	Hotspot mutations in BRAF, EGFR, FLT3, GNA11, IDH1, KRAS, NRAS and PIK3CA genes			~2%-15%

ii. Precision- Panel-Wide Reproducibility: The precision analysis was performed for the known mutations (as listed in Table 6), and also performed for all additional mutations identified in each specimen in any of the test replicates. A total of 69 mutations in the clinical specimens and 13 mutations in the cell line were detected for a total of 82 mutations. In addition to SNV/MNVs, there were 9 deletions and 8 insertions.
The results showed that all mutations have 100% concordance in all replicates except for 4 mutations in the clinical specimens and 3 mutations in the commercial sample. In the clinical specimen discordance was observed for an SNV (pQ64K) and a frameshift mutation (pL54fs) in AR exon1, an insertion (pA445_P446insP) ARID1B exon1; and a frameshift mutation (pT319Kfs*24) in PTEN exon 8. The discordance on AR and ARID1B mutations were due to poor mapping quality in the highly repetitive regions.

The 3 mutations from the commercial control sample that were discordant were 2 SNVs and one deletion (IDH1 exon4 R132H; BRAF exon15 V600M; EGFR exon19 E746 A750del). These 3 mutations were believed to be discordant because they have low frequencies near 2%.

The coefficient of variation (%CV) for the mutation allele frequency was also calculated for all 5 replicates. Thirty-four (45) of the 69 mutations in the clinical specimens had %CV ≤10%, 17/69 were between 10 and 20% and 7/69 were >21%. All results are summarized in Table 7. Each specimen is separated by a dark gray line. Known mutation within each specimen are in bold. Discordant cases are denoted in light grey. All runs passed the quality metrics criteria.

Table 7. Panel-wide precision summary for all 5 replicates Abbreviations: NC (normalized coverage); MAF (Mutant allele frequency)

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| Gene
Exon | Mutation
(cDNA/Protein
Changes) | NC
range | MAF range | MAF
mean | MAF
median | MAF
(SD) | MAF
(%CV) | Positive
/Total
Calls | Positive Call Rate
(two-sided 95% CI) |
|-------------------|---------------------------------------------------------------|-------------|-------------|-------------|---------------|-------------|--------------|-----------------------------|------------------------------------------|
| EGFR
exon19 | c.2236_2250delG
AATTAAGAGA
AGCA
746_750del | 0.84-1 | 0.311-0.342 | 0.323 | 0.316 | 0.013 | 4.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| PTEN
exon2 | c.T83G I28S | 0.62-0.73 | 0.502-0.569 | 0.543 | 0.544 | 0.027 | 5.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| TET2
exon3 | c.C311G S104C | 1.04-1.32 | 0.085-0.103 | 0.098 | 0.102 | 0.008 | 8.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| TP53
exon7 | c.C742T R248W | 0.97-1.22 | 0.648-0.664 | 0.66 | 0.663 | 0.007 | 1.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| BRAF
exon15 | c.T1799A V600E | 1.26-1.44 | 0.415-0.454 | 0.431 | 0.425 | 0.015 | 3.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| BRCA2
exon14 | c.A7388G N2463S | 0.84-0.96 | 0.19-0.23 | 0.209 | 0.21 | 0.015 | 7.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| BRD4
exon19 | c.G3922A A1308T | 0.44-0.56 | 0.5-0.636 | 0.553 | 0.54 | 0.054 | 9.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| FBXW7
exon9 | c.G1268T G423V | 0.91-1.05 | 0.369-0.418 | 0.395 | 0.391 | 0.02 | 5.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| GRIN2A
exon7 | c.C1514A A505E | 0.92-1.1 | 0.194-0.211 | 0.202 | 0.203 | 0.006 | 3.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| PTPRD
exon12 | c.G10A V4I | 0.5-0.63 | 0.281-0.361 | 0.336 | 0.35 | 0.034 | 10.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| RUNX1
exon9 | c.806-1G>A NA | 1.01-1.23 | 0.185-0.21 | 0.202 | 0.207 | 0.01 | 5.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| SPEN
exon12 | c.C10445T
P3482L | 0.94-1.03 | 0.189-0.235 | 0.208 | 0.2 | 0.018 | 8.7% | 5/5 | 100.0% (47.8%, 100.0%) |
| SYK
exon13 | c.C1768T R590W | 1.13-1.22 | 0.233-0.292 | 0.273 | 0.279 | 0.023 | 8.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| TP53
exon6 | c.G610T E204X | 0.9-1.01 | 0.525-0.56 | 0.547 | 0.551 | 0.013 | 2.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| APC
exon16 | c.G3856T E1286X | 0.8-1.05 | 0.326-0.39 | 0.351 | 0.349 | 0.026 | 7.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| | | | | | | | | | |
| APC exon7 | c.C646T R216X | 0.87-1.06 | 0.148-0.185 | 0.162 | 0.16 | 0.015 | 9.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| CREBBP
exon29 | c.G4837A
V1613M | 1-1.19 | 0.159-0.196 | 0.178 | 0.18 | 0.017 | 9.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| KRAS
exon2 | c.G34T G12C | 1.13-1.31 | 0.289-0.352 | 0.314 | 0.305 | 0.024 | 7.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| NOTCH1
exon34 | c.7541dupC
P2514fs | 1.28-1.5 | 0.144-0.211 | 0.184 | 0.189 | 0.025 | 13.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| SMAD4
exon11 | c.C1333T R445X | 0.76-0.95 | 0.206-0.238 | 0.223 | 0.229 | 0.014 | 6.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| ALOX12B
exon11 | c.G1406A R469Q | 1.03-1.31 | 0.333-0.377 | 0.355 | 0.356 | 0.016 | 4.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| ARID1B
exon1 | c.1333_1334insCG
C A445_P446insP | 0.2-0.2 | 0.2-0.2 | 0.2 | 0.2 | NA | NA | 1/5 | 20.0% (0.5%, 71.6%) |
| CDK8
exon10 | c.C1014A D338E | 0.59-0.7 | 0.256-0.336 | 0.303 | 0.315 | 0.032 | 10.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| DNMT1
exon36 | c.T4380G H1460Q | 1.18-1.51 | 0.51-0.558 | 0.534 | 0.53 | 0.017 | 3.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| ERBB2
exon2 | c.G140A R47H | 1.16-1.59 | 0.596-0.712 | 0.656 | 0.666 | 0.045 | 6.9% | 5/5 | 100.0% (47.8%, 100.0%) |
| ERBB2
exon20 | c.2310_2311insG
CATACGTGAT
G
E770_A771insAY
VM | 1.02-1.38 | 0.142-0.199 | 0.173 | 0.171 | 0.023 | 13.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| ERCC2
exon21 | c.C1904T A635V | 1.19-1.47 | 0.363-0.466 | 0.409 | 0.423 | 0.045 | 11.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| IRS1 exon1 | c.C3639A S1213R | 0.42-0.49 | 0.384-0.494 | 0.449 | 0.455 | 0.04 | 8.9% | 5/5 | 100.0% (47.8%, 100.0%) |
| MED12
exon37 | c.5258_5282delCT
CCTACCCTGCT
AGAGCCTGAGA
A A1753fs | 1.08-1.36 | 0.141-0.187 | 0.164 | 0.17 | 0.019 | 11.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| MED12
exon43 | c.6339_6340insCA
GCAACACCAG
Q2113_Q2114ins
QQHQ | 0.96-1.43 | 0.37-0.422 | 0.4 | 0.399 | 0.021 | 5.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| NF1
exon51 | c.C7595T A2532V | 0.92-1.04 | 0.627-0.68 | 0.664 | 0.676 | 0.022 | 3.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| NTRK1
exon1 | c.G53A G18E | 0.28-0.55 | 0.6-0.668 | 0.631 | 0.63 | 0.027 | 4.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| PDGFRB
exon7 | c.G946A V316M | 0.73-1.14 | 0.615-0.681 | 0.646 | 0.642 | 0.026 | 4.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| PIK3CB
exon15 | c.A2150G N717S | 0.67-0.85 | 0.273-0.317 | 0.299 | 0.308 | 0.018 | 6.0% | 5/5 | 100.0% (47.8%, 100.0%) |
| PTPRS
exon32 | c.C4822T
R1608W | 0.79-1.06 | 0.526-0.562 | 0.543 | 0.542 | 0.013 | 2.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| RB1 exon2 | c.138-2A>G
splicing mutation | 0.51-0.75 | 0.231-0.345 | 0.291 | 0.284 | 0.047 | 16.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| TET1
exon4 | c.G3476A R1159Q | 0.86-1.34 | 0.499-0.606 | 0.533 | 0.522 | 0.044 | 8.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| TP53
exon5 | c.G524A R175H | 0.75-1.11 | 0.247-0.344 | 0.314 | 0.337 | 0.04 | 12.7% | 5/5 | 100.0% (47.8%, 100.0%) |
| EGFR
exon21 | c.T2573G L858R | 1.4-1.44 | 0.172-0.225 | 0.199 | 0.203 | 0.02 | 10.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| HNF1A
exon4 | c.C934T L312F | 0.35-0.54 | 0.033-0.077 | 0.057 | 0.059 | 0.016 | 28.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| MLL3
exon42 | c.G9671A R3224H | 1.27-1.4 | 0.089-0.118 | 0.104 | 0.105 | 0.011 | 10.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| NTRK3
exon14 | c.1401delC P467fs | 0.49-0.54 | 0.062-0.086 | 0.074 | 0.077 | 0.01 | 13.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| TP53
exon10 | c.A1051T K351X | 0.74-0.84 | 0.075-0.116 | 0.103 | 0.108 | 0.016 | 15.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| AR exonl | c.161_171delTGC
TGCTGCTG
L54fs | 0.34-0.39 | 0.079-0.097 | 0.088 | 0.087 | 0.009 | 10.2% | 3/5 | 60.0% (14.7%, 94.7.0%) |
| AR exon1 | c.C190A Q64K | 0.25-0.29 | 0.134-0.135 | 0.134 | 0.134 | 0.001 | 0.7% | 2/5 | 40.0% (5.3%, 85.3%) |
| KIT
exon11 | c.1667_1672delA
GTGGA
556_558del | 1.65-1.86 | 0.554-0.595 | 0.569 | 0.566 | 0.016 | 2.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| KIT
exon17 | c.T2467G Y823D | 1.28-1.49 | 0.619-0.658 | 0.646 | 0.655 | 0.016 | 2.5% | 5/5 | 100.0%
(47.8%, 100.0%) |
| RPS6KB2
exon10 | c.G840T K280N | 0.93-1.19 | 0.435-0.473 | 0.462 | 0.468 | 0.015 | 3.2% | 5/5 | 100.0%
(47.8%, 100.0%) |
| | | | | | | | | | |
| CARD11
exon25 | c.3382T>A
p.V1128I | 1.34-1.58 | 0.276-0.293 | 0.284 | 0.278 | 0.009 | 3.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| EGFR
exon20 | c.2290_2310dupT
ACGTGATGGC
CAGCGTGGAC
p.Y764_D770dup | 14.36-15.46 | 0.05-0.06 | 0.055 | 0.055 | 0.004 | 7.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| EGFR
exon7 | c.874G>T
p.V292L | 21.51-21.82 | 0.934-0.939 | 0.937 | 0.939 | 0.002 | 0.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| NOTCH3
exon22 | c.3646G>A
p.A1216T | 1.35-1.52 | 0.247-0.318 | 0.281 | 0.281 | 0.026 | 9.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| PTEN
exon5 | c.395G>C
p.G132A | 0.6-0.72 | 0.605-0.667 | 0.635 | 0.631 | 0.029 | 4.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| RUNX1
exon8 | c.899C>T
p.T300M | 0.81-0.92 | 0.244-0.274 | 0.26 | 0.266 | 0.015 | 5.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| STAG2
exon17 | c.1544_1547delAT
AG p.D515Gfs6 | 0.19-0.27 | 0.677-0.842 | 0.753 | 0.741 | 0.067 | 8.9% | 5/5 | 100.0% (47.8%, 100.0%) |
| TERT
Promoter | g.1295228C>T
non-coding | 0.55-0.67 | 0.388-0.467 | 0.421 | 0.417 | 0.033 | 7.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| AKT3
exon2 | c.134T>G p.V45G | 1.14-1.36 | 0.05-0.078 | 0.066 | 0.067 | 0.012 | 18.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| BRAF
exon15 | c.1798_1799delins
AA p.V600K | 1.04-1.32 | 0.065-0.095 | 0.072 | 0.067 | 0.013 | 18.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| KIT
exon11 | c.1735_1737delG
AT p.D579del | 1.08-1.22 | 0.051-0.056 | 0.053 | 0.054 | 0.002 | 3.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| CTCF
exon3 | c.610dupA
p.T204Nfs26 | 0.68-0.86 | 0.041-0.072 | 0.057 | 0.061 | 0.014 | 24.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| EGFR
exon20 | c.2317_2319dupC
AC p.H773dup | 1.15-1.19 | 0.067-0.093 | 0.078 | 0.079 | 0.011 | 14.1% | 5/5 | 100.0% (47.8%, 100.0%) |
| KDM5C
exon23 | c.3755G>A
p.R1252H | 0.88-1.17 | 0.064-0.13 | 0.088 | 0.084 | 0.026 | 29.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| KRAS
exon2 | c.35G>C p.G12A | 0.78-0.94 | 0.044-0.106 | 0.076 | 0.074 | 0.023 | 30.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| PIK3R1
exon13 | c.1672_1683delG
AAATTGACAAA
p.E558_K561del | 0.43-0.52 | 0.067-0.116 | 0.085 | 0.081 | 0.019 | 22.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| PIK3R1
exon9 | c.1023dupA
p.E342Rfs4 | 0.41-0.58 | 0.056-0.102 | 0.083 | 0.086 | 0.017 | 20.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| PIK3R1
exon9 | c.1024G>T
p.E342 | 0.42-0.59 | 0.064-0.108 | 0.093 | 0.095 | 0.017 | 18.3% | 5/5 | 100.0% (47.8%, 100.0%) |
| PTEN
exon6 | c.493-1G>A
p.X165_splice | 0.53-0.64 | 0.173-0.208 | 0.192 | 0.187 | 0.015 | 7.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| PTEN
exon8 | c.956_959delCTTT
T p.T319Kfs*24 | 0.28-0.48 | 0.006-0.079 | 0.049 | 0.052 | 0.029 | 59.2% | 3/5 | 60.0% (14.7%, 94.7.0%) |
| SOX17
exon1 | c.287C>G p.A96G | 1.16-1.51 | 0.061-0.074 | 0.069 | 0.069 | 0.005 | 7.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| | | | | | | | | | |
| BRAF
exon15 | c.1798G>A
V600M | 0.97-1.06 | 0.016-0.041 | 0.027 | 0.027 | 0.01 | 37.0% | 3/5 | 60.0% (14.7%, 94.7.0%) |
| BRAF
exon15 | c.1799T>A V600E | 0.97-1.06 | 0.051-0.08 | 0.064 | 0.067 | 0.012 | 18.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| EGFR
exon18 | c.2155G>A
G719S | 1.23-1.33 | 0.125-0.179 | 0.158 | 0.164 | 0.022 | 13.9% | 5/5 | 100.0% (47.8%, 100.0%) |
| EGFR
exon19 | c.2235_2249delG
GAATTAAGAG
AAGC
E746_A750del | 1.01-1.19 | 0.009-0.043 | 0.023 | 0.019 | 0.013 | 56.5% | 2/5 | 40.0% (5.3%, 85.3%) |
| FLT3
exon20 | c.2503G>T
D835Y | 0.97-1.02 | 0.037-0.059 | 0.045 | 0.043 | 0.008 | 17.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| GNA11
exon5 | c.626A>T Q209L | 1.41-1.48 | 0.036-0.054 | 0.046 | 0.044 | 0.008 | 17.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| IDH1
exon4 | c.395G>A R132H | 0.5-0.53 | 0.038-0.049 | 0.035 | 0.044 | 0.020 | 57.1% | 4/5 | 80.0% (28.4%, 99.5%) |
| KRAS
exon2 | c.34G>A G12S | 0.9-1.03 | 0.026-0.057 | 0.041 | 0.039 | 0.011 | 26.8% | 5/5 | 100.0% (47.8%, 100.0%) |
| KRAS
exon2 | c.38G>A G13D | 0.91-1.06 | 0.217-0.249 | 0.231 | 0.229 | 0.012 | 5.2% | 5/5 | 100.0% (47.8%, 100.0%) |
| KRAS
exon4 | c.436G>A A146T | 0.82-0.88 | 0.031-0.055 | 0.042 | 0.044 | 0.009 | 21.4% | 5/5 | 100.0% (47.8%, 100.0%) |
| NRAS
exon3 | c.183A>T Q61H | 1.01-1.14 | 0.039-0.065 | 0.051 | 0.051 | 0.01 | 19.6% | 5/5 | 100.0% (47.8%, 100.0%) |
| PIK3CA
exon10 | c.1624G>A
E542K | 0.67-0.87 | 0.038-0.047 | 0.042 | 0.042 | 0.004 | 9.5% | 5/5 | 100.0% (47.8%, 100.0%) |
| PIK3CA
exon21 | c.3140A>G
H1047R | 0.62-0.72 | 0.222-0.331 | 0.276 | 0.258 | 0.05 | 18.1% | 5/5 | 100.0% (47.8%, 100.0%) |

16

17

18

19

20

iii. 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. Results are summarized in Table 8.

| Specimen | Total No
unique
mutations
detected
across
all 5
replicates* | Positive call
rate
per mutation (two-sided 95% (two-sided 95% CI) |
|--------------------- | M15-22924 100.0% (86.3%, 100.0%) | M15-3038 100.0% (78.2%, 100.0%) | M16-19000 4/5 for 1 98.0% (89.4%, 99.9%) | M1688-5C 1/5 for 1 95.6% (89.0%, 98.8%) 80.0% (28.4%,
99.5%) |
| M-1698-A9 100.0% (86.3%, 100.0%) | M-1654-CA 100.0% (88.4%, 100.0%) | M-1612-28 100.0% (83.2%, 100.0%) | M1648-D5 100.0% (92.9%, 100.0%) | M-1707-12 3/5 for 1;
2/5 for 1 | 20/25
80.0% (59.3%, 93.2%) 60.0% (14.7%,
94.7%) |
| Commercial
sample | 13 4/5 for 1 ;
3/5 for 1;
2/5 for 1 | 59/65
90.8% (81.0%, 96.5%) 60.0% (14.7%,
94.7%) | | Positive call rate
CI) | Negative call rate
-|-------------------------------------------------------------------------------|-------------------------------------------------------|-------------------------------------------|------------------------------------------|
| 5 | 5/5 for all | 25/25
| - |
| 3 | 5/5 for all | 15/15
| - |
| 10 | 5/5 for 9
| 49/50
| - |
| 18 | 5/5 for 17
| 86/90
| 4/5
| 5 | 5/5 for all | 25/25
| - |
| 6 | 5/5 for all | 30/30
| - |
| 4 | 5/5 for all | 20/20
| - |
| 10 | 5/5 for all | 50/50
| - |
| 5 | 5/5 for 3
| 3/5
| 5/5 for 10;
| 3/5

Table 8. Precision per specimen across all reportable mutations (N - 5 replicates)

*Positive call rate is calculated based on variants with majority call detected as positive #Negative call rate is calculated based on variants detected at least once, but with majority call as negative. For all other locations, the negative call rates are 100%.

The precision study was also evaluated for the intra-assay repeatability (withinrun). All results were concordant except for ARID1B exon 2 insertion from clinical specimen M-1688, and BRAF V600M point mutation in the commercial control sample as described previously. Additionally, performance with respect to quality metrics (i.e., total depth of coverage and mutant allele coverage) in all replicates was also summarized and shown to meet the pre-specified acceptance criteria (data not shown).

21

iv. Precision Well-characterized reference material: The precision of MSK-IMPACT was assessed through repeated measurements of a well characterized reference standard (HapMap cell line NA20810). To determine sequencing error rates for the reference sample, DNA extracted from the HapMap cell line was included in each run tested in the accuracy study. The study investigated whether the SNPs in the targeted exons were detected at their expected frequencies. Reference genotypes for 11,767 SNPs in the targeted exons using a whole genome sequencing BAM file for NA20810, were obtained from the 1000 Genomes database. A total of 11.443 SNPs (97.2%) were homozygous for the major allele (relative to the hg19 reference genome), 212 SNPs (1.8%) were heterozygous and 112 SNPs (0.95%) were homozygous for the minor allele. The strong bias towards alleles matching the reference genome was expected, given that these SNPs occur in coding exons and there is likely strong selective pressure against deviations from the reference sequence. NA20810 was profiled with the assay multiple times across different runs, for a total of 23 replicates. Zygosity results were 100% concordant and high levels of concordance specifically, the difference between the expected and mean observed mutation frequencies was very small (absolute difference = 0.09%±0.45%). The data provide additional supplemental evidence of the reproducibility of the assay.
v. Precision for Microsatellite Instability (MSI): Precision of the MSI calling by MSIsensor was demonstrated with a total of 12 specimens: 6 MSI-H specimens (at three MSI-score levels, 3 replicates per sample) and 6 MSS specimens. Each DNA extracted sample was tested with 3 inter- and 3 intra-run replicates. Multiple barcodes were included. All samples had 100% agreement between calls. The total number of unstable loci relative to the total number of sites surveyed along with the mean, median and standard deviation (SD) and coefficient of variance (%CV) was also presented for each specimen and score. The results supported the precision of the MSIsensor scores greater than 0.5 Results are shown in Table 9.

| N | Total
Sites_
range | Unstable
Loci_range | Mean | Median | SD | %CV | Positive Call
Rate (two-
sided 95% CI) |
|---|--------------------------|------------------------|-------|--------|------|-------|----------------------------------------------|
| 5 | 1227-1458 | 518-650 | 43.00 | 43.00 | 1.22 | 2.8% | 100%(47.8%,
100.0%) |
| 5 | 1158-1477 | 483-646 | 43.00 | 43.00 | 0.71 | 1.7% | 100%(47.8%,
100.0%) |
| 5 | 1187-1429 | 500-613 | 42.00 | 42.00 | 0.71 | 1.7% | 100%(47.8%,
100.0%) |
| 5 | 1287-1400 | 303-359 | 24.80 | 25.00 | 0.84 | 3.4% | 100%(47.8%,
100.0%) |
| 5 | 1251-1303 | 240-318 | 23.40 | 24.00 | 2.51 | 10.7% | 100%(47.8%,
100.0%) |
| 5 | 1154-1379 | 153-175 | 12.60 | 12.00 | 0.89 | 7.1% | 100%(47.8%,
100.0%) |

Table 9. Precision of the MSIsensor Score Using 12 Specimens

22

| N | Total
Sites_
range | Unstable
Loci_range | Mean | Median | SD | %CV | Positive Call
Rate (two-
sided 95% CI) |
|---|--------------------------|------------------------|------|--------|------|--------|----------------------------------------------|
| 5 | 1321-1545 | 46-58 | 3.60 | 4.00 | 0.55 | 15.3% | 100%(47.8%,
100.0%) |
| 5 | 1535-1604 | 44-64 | 3.40 | 3.00 | 0.55 | 16.2% | 100%(47.8%,
100.0%) |
| 5 | 1411-1612 | 28-38 | 2.20 | 2.00 | 0.45 | 20.5% | 100%(47.8%,
100.0%) |
| 5 | 1438-1528 | 6-9 | 0.48 | 0.50 | 0.08 | 16.7% | 100%(47.8%,
100.0%) |
| 5 | 1315-1487 | 0-2 | 0.02 | 0.00 | 0.04 | 223.6% | 100%(47.8%,
100.0%) |
| 5 | 1312-1532 | 0-1 | 0.01 | 0.00 | 0.03 | 223.6% | 100%(47.8%,
100.0%) |

b) Analytical Sensitivity Limit of Detection (LoD): The LoD of the IMPACT 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. In the first part, a dilution series was conducted to identify the lowest reliable mutant fraction. In part 2, the putative LoD was confirmed with multiple replicates.
- Part 1: Dilution Series: The mean normalized coverage for all exons was i. determined for 10 normal FFPE specimens and the LoD was assessed with samples containing mutations in 5 validation exons (defined as representative exons harboring cancer mutations with evidence of clinical significance assessed in the accuracy study) with the lowest and highest coverage.
  - . The 5 validation exons with lowest coverage correspond to 3 exons harboring SNVs, (ERBB2 exon 20 (V777L), PDGFRA exon 18 (D842V), PIK3CA exon 10 (E545K), and 2 exons harboring indels (EGFR exon 19 and KIT exon 9).
  - The 5 validation exons with highest coverage correspond to 3 exons harboring SNVs (BRAF exon 15 (V600E), KRAS exon 2 (G12D) and PIK3CA exon 2 (R88Q) and 2 exons harboring indels (KIT exon 11 and EGFR exon 20).

Five to eight serial dilutions were prepared using patient samples positive for the mutations listed above, where tumor samples were either diluted with their respective matched FFPE normal sample (when available) or a previously sequenced, unmatched normal FFPE sample. One replicate at each dilution was tested and the ability to detect the mutation of interest was measured. All results were called at the lowest dilution except for PIK3CA which was called wild-type at the lowest dilution. Results are shown in Tables 10A-J.

23

		Table 10A. Limit of Detection -Part 1
--	--	---------------------------------------

SNV BRAF Exon 15 (Sample M-1648-D5-T)
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat	c.1799T>A	V600E	1018	410	0.4	Called
1:2			1044	319	0.31	Called
1:4			888	173	0.19	Called
1:8			999	91	0.09	Called
1:16			783	26	0.03	Called
1:32			845	20	0.02	Called

Table 10B

SNV KRAS Exon 2 (sample M-1807-ED-T)
Dilution	cDNA change	AA Change	DP	AD	VF	Result
Neat		G12D	907	405	0.45	Called
1:2			820	298	0.36	Called
1:4	c.35G>A		400	97	0.24	Called
1:8			660	121	0.18	Called
1:16			ર્ભર્ટ	ਦੇ ਰੇ	0.09	Called
1:32			632	41	0.06	Called

Table 10C

SNV PIK3CA Exon 2 (Sample M-1729-E1-T)
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat		R88Q	2029	629	0.31	Called
1:2	c.263G>A		1008	211	0.21	Called
1:4			1140	145	0.13	Called
1:8			997	62	0.06	Called
1:16						WT

Table 10D

Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat			2503	922	0.37	Called
1:2			1986	688	0.35	Called
1:4			1513	430	0.28	Called
1:8	c.1667_1681delAGT	556_561del	1049	250	0.24	Called
1:16	GGAAGGTTGTTG		792	138	0.17	Called
1:32			761	66	0.09	Called
1:64			618	37	0.06	Called
1:125			736	18	0.02	Called

Table 10E

EGFR exon 20 Insertion (sample M-1674-10-T)
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat			1484	400	0.27	Called
1:2			777	166	0.21	Called
1:4	c.2308_2309insACT	D770_N771insY	566	105	0.19	Called
1:8			595	55	0.09	Called
1:16			581	33	0.06	Called
1:32			608	21	0.03	Called

24

Table 10F

SNV ERBB2 exon 20 (sample M-1801-98-T)
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat			1471	408	0.28	Called
1:2			1482	240	0.16	Called
1:4	c.2525A>T	D842V	864	73	0.08	Called
1:8			903	38	0.04	Called
1:16			873	24	0.03	Called

Table 10G

SNV PDGFR α Exon 18 (sample M-1670-A6-T
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat			448	236	0.53	Called
1:2			636	142	0.22	Called
1:4	c.1633G>A	E545K	962	95	0.1	Called
1:8			647	45	0.07	Called
1:16			707	16	0.02	Called

Table 10H

SNV PK3CA exon 10 (sample M-1434-A5-T)
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat			448	236	0.53	Called
1:2			636	142	0.22	Called
1:4	c.1633G>A	E545K	962	95	0.1	Called
1:8			647	45	0.07	Called
1:16			707	16	0.02	Called

Table 10I

EGFR exon 19 deletion (sample M-1809-C4-T)
Dilution	cDNAchange	AA Change	DP	AD	VF	Result
Neat	c.2236_2250delG
AATTAAGAGA
AGCA	746_750del	1278	790	0.62	Called
1:2			1137	484	0.43	Called
1:4			792	207	0.26	Called
1:8			666	94	0.14	Called
1:16			622	49	0.08	Called
1:32		746_750del	499	17	0.03	Called

Table 10J

Kit Exon 9 insertion (sample M-1754-DB-T)
Dilution	cDNA change	AA Change	DP	AD	VF	Result
Neat			517	314	0.61	Called
1:2			512	187	0.37	Called
1:4	c.1502_1503insTGCCTA	S501_A502insAY	641	89	0.14	Called
1:8			486	27	0.06	Called
1:16			447	17	0.04	Called
1:32			521	14	0.03	Called

25

ii. Part 2: Confirmation of the LoD. A total of 5 replicates were tested for each of the 3 deletions, 4 insertions and 6 SNVs at 5% minor allele frequency. All variants have 100% positive call rates except for one replicate for a deletion on PTEN exon 6. This replicate also failed the mutation read depth and was below the estimated LoD of 5%. The results are shown in Table 11.

| Type | Mutation | GeneExon | Range
DP | Range
AD | Range
MAF | Range
NormDP | Positive
Call
Rate |
|------|---------------------------------------------------------------------------|------------------|---------------|-------------|--------------|-----------------|--------------------------|
| DEL | In_Frame_Del
c.1735_1737delGAT
p.D579del | KIT exon11 | 509-693 | 26-38 | 0.051-0.056 | 1.08-1.22 | 100.0% |
| DEL | Frame_Shift_Del
c.956_959delCTTT
p.T319Kfs24 | PTEN exon8 | 197-242 | 7-19 | 0.036-0.079 | 0.31-0.48 | 80.0% |
| DEL | In_Frame_Del
c.1672_1683delGAAATT
GACAAA
p.E558_K561del | PIK3R1
exon13 | 216-313 | 18-36 | 0.067-0.116 | 0.43-0.52 | 100.0% |
| INS | In_Frame_Ins
c.2317_2319dupCAC
p.H773dup | EGFR exon20 | 587-749 | 46-65 | 0.067-0.093 | 1.15-1.19 | 100.0% |
| INS | Frame_Shift_Ins
c.1023dupA p.E342Rfs4 | PIK3R1 exon9 | 236-345 | 15-32 | 0.056-0.102 | 0.41-0.58 | 100.0% |
| INS | Frame_Shift_Ins
c.610dupA p.T204Nfs26 | CTCF exon3 | 344-540 | 14-36 | 0.041-0.072 | 0.68-0.86 | 100.0% |
| INS | In_Frame_Ins
c.2290_2310dupTACGTG
ATGGCCAGCGTGGAC
p.Y764_D770dup | EGFR exon20 | 8601-
9836 | 441-572 | 0.05-0.06 | 14.36-
15.46 | 100.0% |
| SNV | Missense_Mutation
c.134T>G p.V45G | AKT3 exon2 | 535-813 | 28-63 | 0.05-0.078 | 1.14-1.36 | 100.0% |
| SNV | Missense_Mutation
c.1798_1799delinsAA
p.V600K | BRAF exon15 | 489-747 | 33-71 | 0.065-0.095 | 1.04-1.32 | 100.0% |
| SNV | Missense Mutation
c.287C>G p.A96G | SOX17 exon1 | 672-805 | 45-59 | 0.061-0.074 | 1.16-1.51 | 100.0% |
| SNV | Missense_Mutation
c.35G>C p.G12A | KRAS exon2 | 445-571 | 20-55 | 0.044-0.106 | 0.78-0.94 | 100.0% |
| SNV | Missense_Mutation
c.3755G>A p.R1252H | KDM5C
exon23 | 475-733 | 40-68 | 0.064-0.13 | 0.88-1.17 | 100.0% |
| SNV | Nonsense_Mutation
c.1024G>T p.E342 | PIK3R1 exon9 | 242-355 | 18-37 | 0.064-0.108 | 0.42-0.59 | 100.0% |

Table 11. Limit of Detection– Part 2

26

iii. Microsatellite instability (MSI): The minimum tumor proportion required to support the MSIsensor score robustness was assessed using CRC specimens. Five (5) replicates were run using multiple barcodes and runs. The data showed that qualitatively, the assay and score are reproducible to 8% tumor proportion, though a decreasing trend in the quantitative score was observed. Therefore, the minimum tumor proportion required for the assay was established as 25% with an average coverage of 200X. Separately, regardless of the tumor proportion, data showed that the score is robust across the MSIsensor score range (refer to Table 9 above and Table 12).

Tumor Purity	Coverage	# Total site	# Unstable loci	MSIsensor Score (%)
Diluted to 8%	517	1420	182	13
Diluted to 8%	562	1389	175	13
Diluted to 8%	555	1352	185	14
Diluted to 8%	502	1361	135	10
Diluted to 8%	378	1273	152	12

Sample ID	Replicate	MSI Sensor Score
CRC-01	1	0.00000
	2	0.00000
	3	0.00000
CRC-04	1	0.00000
	2	0.00000
	3	0.00000
CRC-08	1	0.00000
	2	0.00000
	3	0.00000

iv. 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 (e.g., ≥20 reads per base), and have 100% positive mutation call rate. The optimized and recommended DNA concentration for the assay is 250ng. The DNA input range 50-250ng. was assessed for accuracy and sequencing failures as a function of the input DNA concentration. The results show that assay performance in terms of sequencing failures is a function of genomic DNA input values as shown in Table 13.

DNA Input	Success	Sequencing Failure
250ng	97%	3%
201-249ng	87%	13%
151-200ng	87%	13%
101-150ng	81%	19%
50-100ng	78%	22%

Table 13. Sequencing Failures Relative to DNA Input

c) Linearity/assay reportable range:

Not applicable

d) Traceability (controls, calibrators, or methods):

The MSK-IMPACT is not traceable to any known standard. Controls and quality metrics are described in the device description section.

27

e) Stability:

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

f) Expected values:

The laboratory follows protocols for the use of controls consistent with CLIA regulation. The MSK-IMPACT does not use calibrators; however, the verification of mutant allele frequency is maintained by analysis of a pooled control with expected allele frequencies.

g) Analytical specificity:

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

Interference:

The MSK-IMPACT assay pre-analytic steps are designed to minimize interference. The invalid rates in the historical testing from >10,000 samples support that any interference from any challenging tissues is minimized.

h) Assay cut-off:

The MSK-IMPACT does not report mutations below 2% for known hotspot mutations and 5% for non-hotspot mutations.

Comparison studies: i)

i. Method comparison:
The MSK-IMPACT assay is designed to detect SNVs and small indels in 6284 exons from 468 genes. The accuracy of the MSK-IMPACT was assessed by comparison of the MSK-IMPACT result to the original results obtained with the validated orthogonal methods. Testing was conducted per protocol. A total of 267 unique mutations in 433 FFPE tumor specimens representing 48 exons in 20 genes were tested and are listed in Table 14 below.

| Gene
(n=20) | #Samples
(n=433) | Exon
(n=48) | Type | Mutations Assessed |
|----------------|---------------------|----------------|----------|---------------------------------------------------------------------------------------------|
| AKT | 10 | exon3 | SNV | E17K |
| ALK | 3 | exon23 | SNV | F1174V/L;S1205F |
| ALK | 4 | exon25 | SNV | R1275Q;R1260T |
| BRAF | 11 | exon11 | SNV | G466V/R;S467L;G469* |
| BRAF | 19 | exon15 | SNV | D594G;V600*;K601I |
| EGFR | 10 | exon18 | SNV | G719A/S; G724S |
| EGFR | 12 | exon19 | DEL | 745_750del; 746_748del; 746_750del;
747_753del; K754fs |
| Gene
(n=20) | #Samples
(n=433) | Exon
(n=48) | Type | Mutations Assessed |
| | 10 | exon20 | SNV | T790M |
| | 16 | exon20 | INS | M766_A767insASV; V769_D770insDNP;
D770_N771ins*;P772_H773ins*;
H773_V774insY/H |
| | 9 | exon21 | SNV | L858R |
| ERBB2 | 7 | exon19 | SNV | L755S;I767M;D769Y |
| | 16 | exon20 | INS | E770_A771insAYVM;
A771_Y772insYVMA;G776_G778ins* |
| | 3 | exon20 | SNV | V777L;G776V |
| | 7 | exon8 | SNV | S310F/Y; S305C |
| FGFR2 | 1 | exon12 | SNV | L528H |
| | 1 | exon7 | SNV | S252W |
| | 1 | exon9 | SNV | Y375C |
| FGFR3 | 2 | exon18 | SNV | P797L |
| | 1 | exon7 | SNV | A261V;A265V |
| | 5 | exon9 | SNV | F384L |
| | 1 | exon9 | INS | G370_S371insH |
| GNA11 | 7 | exon5 | SNV | Q209L |
| GNAQ | 5 | exon5 | SNV | Q209P/L |
| GNAS | 5 | exon8 | SNV | R201C/H |
| HRAS | 3 | exon2 | SNV | G10A; G13D/V |
| | 5 | exon3 | SNV | A59V; Q61R/L/K |
| IDH1 | 8 | exon4 | SNV | R132G/C/H |
| IDH2 | 5 | exon4 | SNV | R172*;R140Q |
| | 1 | exon4 | DEL | T146Lfs15 |
| | 9 | exon11 | INS; DEL | K550fs; 552_557del; 556_558del;
556_561del; 558_565del; 559_566del;
P573_T574insTQLPS |
| KIT | 9 | exon11 | SNV | V555L; W557G; V559D; D572G;L576P. |
| | 6 | exon13 | SNV | V654A; K642E |
| | 5 | exon17 | SNV | D816H; D820E; N822K |
| | 10 | exon9 | INS | S501_A502insAY; A502_Y503dup |
| KRAS | 16 | exon2 | SNV | G12; G13D |
| | 13 | exon3 | SNV | Q61* |
| | 10 | exon4 | SNV | K117N;G138E;A146* |
| MET | 13 | exon14 | SNV | D1010*; Exon14 skipping |
| | 19 | exon14 | DEL | Exon14 skipping; Other splicing defects |
| NRAS | 4 | exon2 | SNV | G13* |
| | 12 | exon3 | SNV | Q61* |
| PDGFRA | 12 | exon18 | SNV | D842V/I |
| | 1 | exon12 | SNV | V561D |
| | 4 | exon10 | SNV | E545A/K; E542K |
| PIK3CA | 2 | exon21 | SNV | H1047R/Y |
| | 1 | exon21 | INS | X1069delinsFL |
| | 8 | exon2 | SNV/MNV | F83L;R88Q;R93Q;K111E/N |
| | 2 | exon2 | DEL | E110del; 112_113del |
| | 9 | exon5 | SNV | V344M;N345I/K |
| | 9 | exon8 | SNV | E418K;C420R;P449R;E453K/Q |
| | 1 | exon8 | DEL | E453_D454del |
| TP53 | 9 | exon4 | SNV/MNV | W53X;W91X;Q100X;G105V/C; S106R;
F113C |
| | 6 | exon4 | DEL | L355fs;P67fs;A84fs;109_109del;G108fs; |
| Gene | #Samples | Exon | | |
| (n=20) | (n=433) | (n=48) | Type | Mutations Assessed |
| | | | | R110fs |
| | 3 | exon4 | INS | V73fs;L114fs;C124fs |
| | б | exon5 | SNV | K132Q;W146X; Y163C; R175H; R158H |
| | 3 | | INS | P153fs; M160 A161insRA; |
| | | exon5 | | Q167_M170dup |
| | | | | K132fs;A138fs;P152fs; R156fs; |
| | 9 | exon5 | DEL | V157_R158del; K164fs; H178fs;D184fs |
| | 2 | exon6 | SNV | R213L/X |
| | | | | G187fs; L188fs; P191_Q192del; |
| | 8 | exon6 | DEL | R196_L201del; D207fs; R209fs; F212fs |
| | | | | Y234C; Y236C; M237I; R248G/Q; |
| | 10 | exon7 | SNV/MNV | R249S; T256P |
| | 3 | exon7 | INS | S241dup; R249fs; T253dup |
| | | | | S241fs; M243X; G244fs; M246X; |
| | 6 | exon7 | DEL | I255del; L257fs |
| | イ | exon8 | SNV/MNV | V272K; C275X; R282W; T284K |
| | 4 | exon8 | INS | C275fs; N288fs; G302fs |
| | | | | N263 N268del; N263fs; R267fs; P278fs; |
| | 5 | exon8 | DEL | P301fs |
| | б | exon10 | SNV | R337L; R342X; R337C |
| | l | exon10 | INS | L344fs |

Table 14. Mutations Represented in the Accuracy Summary Per Gene

28

29

Of the 433 specimens, 418 met the criteria of ≥200X coverage, 15 samples (3.5%) failed to achieve average coverage above 200X. The known mutation associated with each sample was successfully detected in 432 out of 433 cases (99.8% with two-sided 95% CI of (98.7%, 100.0%)). One discordant case was observed in sample M-1994-BC-T, which was used for the validation of insertions in EGFR exon 20. The known mutation for this sample was a 12bp duplication which began in the intron 5' of EGFR exon 20, potentially creating an alternative splice site acceptor for the exon. This duplication event was detected by the indel calling pipeline but was incorrectly filtered out because of the calling algorithm. (The filtering algorithm was modified to improve the detection accuracy for such mutations.)

The MSK-IMPACT accuracy study included 159 unique SNV/MNVs from 20 genes (45 exons), 49 unique deletions from 6 genes (11 exons), and 39 unique insertions from 6 genes (10 exons). Performance was stratified by mutation type and gene for percent positive agreement (PPA) with 95% confidence interval (CI). Results are shown in Table 15A-C.2

2 Performance may be overestimated because specimens were selected based on the availability of results by the orthogonal methods (i.e., the specimen set may lack challenging specimens).

30

| Gene | Number
of exons | Number of
unique
mutations | Number of
samples | PPA (95% CI) |
|--------|--------------------|----------------------------------|----------------------|------------------------|
| AKT1 | 1 | 1 | 10 | 100.0% (69.2%, 100.0%) |
| ALK | 2 | 5 | 7 | 100.0% (59.0%, 100.0%) |
| BRAF | 2 | 13 | 30 | 100.0% (88.4%, 100.0%) |
| EGFR | 3 | 6 | 30 | 100.0% (88.4%, 100.0%) |
| ERBB2 | 3 | 12 | 17 | 100.0% (80.5%, 100.0%) |
| FGFR2 | 3 | 3 | 3 | 100.0% (29.2%, 100.0%) |
| FGFR3 | 3 | 3 | 8 | 100.0% (63.1%, 100.0%) |
| GNA11 | 1 | 1 | 7 | 100.0% (59.0%, 100.0%) |
| GNAQ | 1 | 2 | 5 | 100.0% (47.8%, 100.0%) |
| GNAS | 1 | 2 | 5 | 100.0% (47.8%, 100.0%) |
| HRAS | 2 | 7 | 8 | 100.0% (63.1%, 100.0%) |
| IDH1 | 1 | 3 | 8 | 100.0% (63.1%, 100.0%) |
| IDH2 | 1 | 4 | 6 | 100.0% (54.1%, 100.0%) |
| KIT | 3 | 13 | 20 | 100.0% (83.2%, 100.0%) |
| KRAS | 3 | 15 | 39 | 100.0% (91.0%, 100.0%) |
| MET | 1 | 9 | 13 | 100.0% (75.3%, 100.0%) |
| NRAS | 2 | 6 | 16 | 100.0% (79.4%, 100.0%) |
| PDGFRA | 2 | 3 | 13 | 100.0% (75.3%, 100.0%) |
| PIK3CA | 4 | 19 | 32 | 100.0% (89.1%, 100.0%) |
| TP53 | 6 | 32 | 37 | 100.0% (90.5%, 100.0%) |

Table 15A.Percent Positive Agreement for SNV/MNVs by Gene

Table 15B. Percent Positive Agreement for insertions by gene

| Gene | Number
of exons | Number of
unique
mutations | Number of
samples | PPA (95% CI) |
|--------|--------------------|----------------------------------|----------------------|------------------------|
| EGFR | | 12 | 16 | 93.8% (69.8%, 100.0%) |
| ERBB2 | | oc | 16 | 100.0% (79.4%, 100.0%) |
| FGFR3 | | | 1 | 100.0% (2.5%, 100.0%) |
| KIT | | ಗ | 10 | 100.0% (69.2%, 100.0%) |
| PIK3CA | | | 1 | 100.0% (2.5%, 100.0%) |
| TP53 | ર | 14 | 14 | 100.0% (76.8%, 100.0% |

		Table 15C. Percent Positive Agreement for deletions by gene

| Gene | Number of
exons | No. unique
mutations | Number of
samples | PPA (95% CI) |
|--------|--------------------|-------------------------|----------------------|------------------------|
| EGFR | 1 | 6 | 12 | 100.0% (73.5%, 100.0%) |
| IDH2 | 1 | 1 | 1 | 100.0% (2.5%, 100.0%) |
| KIT | 1 | 7 | 9 | 100.0% (66.4%, 100.0%) |
| MET | 1 | 18 | 19 | 100.0% (82.4%, 100.0%) |
| PIK3CA | 2 | 3 | 3 | 100.0% (29.2%, 100.0%) |
| TP53 | 5 | 14 | 14 | 100.0% (76.8%, 100.0%) |

ii. Supplemental Method Comparison Study for Wildtype Calls: A supplemental study was conducted to assess accuracy for 33 "hotspots" within 10 genes. A total of 95 specimens were tested and the accuracy of

31

MSK-IMPACT results at all 33 positions was compared to results obtained with a single orthogonal method. Within the 95 specimens, there were 109 mutations across samples and 3026 wild-type calls. Variant-level concordance (PPA and NPA) was 100% for all results with two-sided 95% confidence intervals of (96.7%, 100.0%) for mutations (PPA) and (99.9%, 100.0%) for wild-type locations (NPA).

iii. Method Comparison of the MSK-IMPACT MSIsensor:

The somatic MSI status is inferred by interrogating all available genomic microsatellites covered by MSK-IMPACT within tumor samples against the matched normal DNA using the MSIsensor program as described in the Device Description section above. An MSIsensor score assigned to each tumor sample is used to distinguish MSS from MSI-H by MSIsensor.

The cutoff was first established using a training specimen dataset consisting of 138 colorectal cancer (CRC) and 40 endometrial carcinoma (EC) specimens with matched normal and having MSI status results from a validated MSI-PCR or MMR IHC test. MSIsensor scores ranged from 0 to 47.7 for CRC and 0 to 43.7 for EC. Based on concordance to either mismatch repair immunohistochemistry (MMR IHC) for MLH1, MSH2, MSH6 and PMS2 expression, or a commercially available PCR assay that detects 5 mononucleotide microsatellite loci including MR-21, BAT-25, MONO-27, NR-24 and BAT-26, a MSIsensor cut-off of 10 was established to delineate microsatellite stable (MMS) from high microsatellite instability (MSI-H).

A separate data set was obtained to validate this cut-off. A retrospectiveprospective chart review of 135 CRC patients was conducted to identify cases that had both MSK-IMPACT MSI results and results by a validated IHC panel (MLH1, MSH2, MSH6 and PMS2). A total of 66 specimens had both sets of results. Of these, there were two discordant cases. The estimated positive predictive value (PPV) was 92.3% (12/13) with two-sided 95% confidence interval of 64.0%-99.8% and the estimated negative predictive value (NPV) was 98.1%. (52/53) with two-sided 95% confidence interval of 90.0%, 100.0%. The results are shown in Table 16 below.

	CRC/EC Concordance with IHC	MMR-D*	MMR-P*	Total
MSI Sensor	MSI-H ≥ 10	12	1	13
	MSS 200x, ensure that 95% of
exons are covered to 100x
or more. Concordance for
known mutations should
be >95%.
New library preparation protocol, kits,
or reagents	Sequence at least 40
DNA specimens
(tumor / normal
pairs) or three pools
previously
sequenced by MSK-
IMPACT. Measure
sequence coverage
distribution, and call	For cases sequenced to

200x, ensure that 95% of
exons are covered to 100x
or more. Concordance for
calling somatic mutations
with variant allele fraction
10% should be >98%. | |
| Type of change | | Validation Strategy | Pre-specified success
criteria |
| | | somatic mutations in
all samples. | |
| Changes to probes for already
analytically validated genes | | Re-capture existing
sequence libraries
from at least 3 runs
(at least 40 samples)
with new probes,
sequence, and
analyze. | For cases sequenced to
200x, ensure that 95% of
exons in analytically
validated genes are
covered to 100x or more.
Concordance for calling
somatic mutations with
variant allele fraction
10% should be >98%. |
| New sequencing instrument or reagents
using similar chemistry and technology,
and the sequence depth and read length
are not changed from previous
platform. | | Re-sequence
existing captured
libraries from at
least 3 runs, and call
somatic mutations in
all samples. | Sequence coverage
distribution and GC bias
across targeted regions
should be within 5% of
prior sequencing runs.
Concordance for calling
somatic mutations with
variant allele fraction
10% should be >98%. |
| Bioinformatics
pipeline | Update to
underlying
annotation
database or
transcript
isoforms | Reanalyze FASTQ
files (raw
sequencing reads)
from at least 3 runs
(at least 40 samples).
Compare variants
calls between the
clinical analysis
results and the
current modified
results | Confirm the changes do
not change the variant call
results. Confirm the
annotations for the
unaffected transcripts do
not change. Confirm the
annotations for the
affected transcripts are
modified as expected. |
| | Update to data
management
system and
system database | Reanalyze FASTQ
files (raw
sequencing reads)
from at least 3 runs
(at least 40 samples)
in production mode.
Compare variants
calls between the
clinical analysis
results and the
current modified
results | Ensure that all previously
called mutations are
recovered and the variants
in the database of results
are concordant with the
variants in the pipeline
output files |
| Type of change | Validation Strategy | Pre-specified success criteria | |
| Modification to
an existing
component of
the analysis
pipeline (e.g.,
tool or
algorithm)
where the
underlying
algorithm or
main parameter
settings (e.g.
minimal
coverage/VAF
threshold for
SNV/indel
calling;
MSIsensor
score cut-off for
MSI-H calling,
etc.) are not
changed. | Reanalyze FASTQ
files (raw
sequencing reads)
from at least 3 runs
(at least 40 samples).
Compare variants
calls between the
clinical analysis
results and the
current modified
results | Ensure that all previously
called mutations are
recovered and that newly
detected mutations can be
explained by pipeline
modifications. | |

35

36

Q. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable, and the special controls for this device type.

R. Patient Perspectives

This submission did not include specific information on patient perspectives for this device.

S. Identified Risks to Health and Identified Mitigations:

Identified Risks to Health	Identified Mitigations
Incorrect performance of the test
leading to false positives, false
negatives	General controls and special control (b)(1)
Incorrect interpretation of test results	General controls and special controls
(b)(1)(iii)(E) and (b)(2)

37

1. Benefit/Risk Determination
Summary of the Benefit(s)	The MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) test provides comprehensive genomic profiling of tumor samples (point mutations, small insertions and deletions and microsatellite instability), in previously diagnosed cancer patients, for use by qualified health professionals in accordance with professional guidelines. There is probable clinical benefit of the device based on evidence from peer-reviewed clinical literature and analytical performance of the device in identifying genomic alterations.
Summary of the Risk(s)	Erroneous device results could adversely influence clinical interpretation and consultation for patients. The risk of an erroneous test result is mitigated by the analytical performance of this device. The accuracy of the test was demonstrated using clinical specimens covering a variety of clinically relevant variants across multiple tumor types and variant categories (i.e., point mutations, small insertions and deletions and microsatellite instability). The output of this device demonstrated a high degree of analytical concordance to comparator assays across multiple tumor types. Thus, the probable risk of this device is mitigated by the supportive analytical performance for the device, when clinical limitations and the established special controls, in combination with general controls, are considered.
Summary of Other Factors	Limitations statements in the test report and the established special controls, in combination with general controls, serve to mitigate the risks associated with the use of this device.
Conclusions
Do the probable benefits outweigh the probable risks?	The probable clinical benefits of this device, which allows for detection of somatic mutations and MSI status in patients previously diagnosed with cancer, outweigh the probable risks that are mitigated by the special controls established for this device type, in combination with general controls.

T. Benefit/Risk Determination

U. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.6080. FDA believes that special controls, along with the applicable general controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	PZM
Device Type:	Next Generation Sequencing Based Tumor Profiling Test
Class:	II (special controls)
Regulation:	21 CFR 866.6080

38

(a) Identification. A next generation sequencing (NGS) based tumor profiling test is a qualitative in vitro diagnostic test intended for NGS analysis of tissue specimens from malignant solid neoplasms to detect somatic mutations in a broad panel of targeted genes to aid in the management of previously diagnosed cancer patients by qualified health care professionals.

(b) Classification. Class II (special controls). A next generation sequencing based tumor profiling test must comply with the following special controls:

(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, paraffinembedded 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:

( /) 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.

39

(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., formalinfixed, 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

40

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:

41

(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, paraffinembedded 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)(ii), (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 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.

42

Appendix 1a:

List of hotspot mutations (i.e., commonly somatically mutated in cancers) for all genes in the MSK-IMPACT panel

Gene	Codons
ABL1	G250, Q252, Y253, E255, T315, F317, M351, F359, H396R
AKT1	E17,Q124,G171,E170
AKT2	V140
ALK	K1062,D1091,C1156,M1166,I1171,F1174,L1196,A1234,F1245,I1250,R1275,Y1278
APC	S1234,I1307,E1309,E1317,P1319,G1339,S1341,P1361,P1372,P1373,R1399,S1400,
S1407,S1411,V1414,S1415,S1421,T1438,P1439,P1440,T1445,P1453,N1455,E1464,
S1465,T1487,L1488,F1491,T1493,E1494,T1537,K1555,T1556,I1557,C1578
AR	T878,T8782,Q581
ARAF	S214
ARID1A	D1850,G2087
ARID2	R314,S297,R285,A1773
ASXL1	Y591,E635,G645,G646,E1102D
ASXL2	R591
ATM	D1853,R3008,R3376,E2164
ATRX	K1936,E625
BARD1	P24
BCL6	R594,R618
BCOR	N1425,N14591
BRAF	G464,G466,G469,Y472,N581,D594,F595,G596,L597,A598_T599,V600,V600_K601,
K6010,V60010,K6010,G4694,N5810,G4660
CARD11	R170
CBL	Y371,L380,C384,C404,R420Q
CDH1	T263
CDK4	R24
CDKN2A	S43,P48,A57,A68,D74,L78,P81,H83,D84,L97,D108,P114,H831,D1081,P1140
CEBPA	P23,H24,Q83,K304_Q305,E309_T310,Q312_K313,K313_V314,K313_V314,K313,
E316_L317,E316_L317insQ
CHEK2	K373,K3732
CIC	R215
CREBBP	R1446,S1680,R14460
CRLF2	F232C
CSF1R	Y969C
CTCF	R377
CTNNB1	D32,S33,G34,I35,H36,S37,T40,T41,T42,A43,P44,S45,G48,K49,E53,K335,S376,
S336,D324,T412,G349,S455,C619

DICER1	E1813
DIS3	R382,D488
DNMT1	E432
DNMT3A	G543,R635,S714,F731,R882,R8820
DOT1L	G1386
EGFR	R108,A289,G598,R677,E709,G719,K745_E749,K745_E746,E746_A750,E746_S752,
E746_T751,E746_E749,E746_T751,L747_P753,L747_A750,L747_T751,L747_S752,
L747_T751,L747_E749,L747,T751,S752_I759,D761,S768,V769_D770,D770_N771,
H773_V774,R776,T790,L833,H835,T847,P848,T854,L858,L861,G863,L8587,A2898,
R252,R222
EP300	D1399,D13990,C1164
EPHB1	R170
ERBB2	S310,L755,D769,A775_G776,G776,V777,V842,S3108,L7553,E930,R678
ERBB3	V1043,D297,M91
ERBB4	R711
ERCC2	D312
ESR1	Y537
ETV1	R187
ETV6	R369
EZH2	Y646,R690
FBXW7	G423,R465,R479,R505,S582,R689,R4652,R5054,R4792
FGFR2	S252,P253,C382,N549,N550,K659
FGFR3	R248,S249,G370,S371,Y373,G380,A391,K650,G697,S2492,Y3730
FGFR4	V550
FLT3	D835,I836,D8358
FOXL2	C134W
FUBP1	R430
GATA1	M1,S30,V74I
GATA2	G320,L321,L359,R362Q
GNA11	R183,Q209,R256
GNAQ	R183,Q209
GNAS	R201,Q227,R8448
GRIN2A	R1067
HIST1H3E	E74
HNF1A	W206,P291,G292
HRAS	G12,G13,Q61,E62,Q614,G136,G122
IDH1	G70,V71,R132,V178,R13239,P33
IDH2	R140,R172,V294,R1402,R1721
IL7R	K395
IRS2	G1057
JAK1	R873
JAK2	F537_K539,H538_K539,K539,I540_E543,R541_E543,N542_E543,E543_D544,V617
JAK2	R683
JAK3	A572,A573,R657Q
KDR	S1100,E759
KEAP1	R470
KIT	D52,D419,Y503_F504,K509,M541,K550_K558,P551_V555,P551_E554,P551_M552,
Y553_K558,E554_K558,Q556_V560,W557_K558,W557,W557_V559,W557_E561,
W557_V559,K558_E562,K558,K558_V560,V559,V559_V560,V559_E561,V560,E56
Y570_L576,D572,L576,D579,K642,V654,T670,S715,D816,K818,D820,N822,Y823,V
25, D8160
KMT2C	V656
KRAS	G10_A11,G12,G13,V14,L19,Q22,T58,A59,Q61,K117,A146,G1242,G133,Q619,A146
LATS2	A3243,G3630
MAP2K1	Q56,K57,D67,P124,P1240,F53,E203
MAP2K4	R134
MAP3K1	S1330,S939
MAPK1	E322
MAX	R600
MED12	L36,Q43,G44,L1224,L12240
MEF2B	D83V
MET	T1010,Y1248,Y1253,M1268,K1360
MLL3	K2797
MPL	S505,W515,W515R
MSH6	F1088,T1219I
MTOR	S22152,F1888
MYC	T58
MYCN	P44
MYD88	S219,S243,L265P
NF1	L844
NFE2L2	D29,L30,G31,R34,E79,T80,G81,E82,E794,D294,R342
NOTCH1	L1574,L1575,V1578,L1585,L1586,F1592,L1593,L1594,R1598,R1599,L1600,
L1601,L1678,L1679,Q2460,P2514,A1944
NOTCH2	E385,N463
NPM1	W288,W290
NRAS	G12,G13,A18,G60,Q61,Q6193,G128,G138
NTRK1	T264
PAK7	E144
PARP1	I562
PAX5	P80R
PDGFRA	V561,S566_E571,N659,D842,I843_D846,D1071N
PIK3C2G	S670
PIK3CA	R38,E81,R88,R93,G106,R108,K111,G118,V344,N345,C378,E418,C420,E453,
P539,E542,E545,Q546,E547,S553,K567,H701,E726,C901,G1007,Y1021,T1025,
M1043,N1044,D1045,A1046,H1047,G1049,T1052,A1066,N1068,E54534,H104715,
E54217,Q5467,R887,N3453,C4209,G1187,E7265,E4535, K1113, R932, R382, R1080,
E39
PIK3R1	G376,D560,N564,K567
POLE	P2864,V4111
PPP2R1A	P179,R182,R183,S256,W257,R258,R1832
PREX2	G233C
PTCH1	P1315
PTEN	K6,P38,L42,H61,Y68,Y76,Y88,H93,I101,C105,L112,H123,A126,G129,R130,C136,
A151,Y155,R159,K164,G165,S170,R173,N184,E242,P246,P248,C250,K267,V290,
L318,T319,T321,N323,F347,R1309,R1730,K128
PTPN11	G60,D61,E69,A72,T73,E76,S502,G503,Q510
PTPRD	S431,P666
RAC1	P295
RAF1	S2570
RET	E632_T636,E632_L633,C634,M918T
RHOA	E40,Y42
RICTOR	S1101
RIT1	M90
RUNX1	L56,R107,D198,R201,R204,R162,R205
SDHA	S4560,A466,R465
SF3B1	E622,R625,H662,K666,K700,K7002
SMAD4	A118,D351,R361,G386,R3619,D537,P356
SMARCA	T910,G1232
SMARCB	R377,A382,P383
SMO	W535L
SPOP	F133,F1338,W131,F102
SRSF2	P95,P95_R102,P107H
STAG2	R370
STK11	D194,P281,F354L
TET2	C25,C262,Q764,F868,R1261,H1380,V1718L
TNFAIP3	L324
TP53	E11,D49,P82,T102,G105,Y107,R110,L111,F113,K120,T125,Y126,Y126_K132,S127,
P128,L130,N131,K132,M133,F134,C135,A138,K139,T140,C141,P142,V143,Q144,
I145,V147,S149,P151,R152,P153,G154,T155,R156,V157,R158,A159,M160,A161
I162,Y163,K164,S166,H168,M169,T170,E171,V172,V173,R174,R175,C176,P177,
P177_C182,H178,H179,E180,R181,C182,D184,D186,G187,P190,P191,Q192,H193,
L194,I195,R196,V197,E198,G199,N200,R202,V203,Y205,D208,R209,T211,F212,R21

,S215,V216,V217,V218,Y220,E224,G226,S227,D228,C229,T230,I232,Y234,N235,Y2
6, M237,C238,N239,S240,S241,C242,M243,G244,G245,M246,N247,R248,R249,P250
I251,L252,T253,I254,I255,L257,E258,D259,G262,L265,G266,R267,F270,E271,V272,
R273,V274,C275,A276,C277,P278,G279,R280,D281,R282,R283,T284,E285,E286,E28
7, N288,R290,K291,K292,E294,P300,P301,S303,K320,G334,R337,R27328,R24892,
R17538,R2820,G2451,Y2202,H1938,H1797,R1583,C1763,P2783,Y1633,R2800,
G2660,I1950,S2419,R2499,V1577,C2386,E2856,R3375,G2445,V1733,P1512,C2752,
K1321,Y2050,V2720,C1359,D2818,E2718,V2168,M2378,Y2347,E2867,L1946,
A1596,R2675,S1275,C2425,Y2364,C1414,F2704,A1613,V2743,S2153,R2132,H2142,
R1101,N2390,T1550,P1520,P2500,G1050,L1300,Q136,F109
TP63	R379
TSC2	N1515
TSHR	M453,I486,L512,I568,D619,A623,L629,I630,T632,D633,D633E
U2AF1	S34,Q157,S347
VHL	V62,S65,S72,V74,F76,N78,S80,P81,L85,P86,L89,N90,S111,G114,H115,L118,D121,
VHL	L128,V130,G144,F148,I151,L153,V155,L158,E160,C162,V166,R167,L169,L184
WT1	V303,R312,A314,R394,D396,R462
XP01	E571,R749

43

44

45

46

47

Gene Name	Transcript ID
ABL1	NM_005157
ACVR1	NM_001111067
AGO2	NM_012154
AKT1	NM_001014431
AKT2	NM_001626
AKT3	NM_005465
ALK	NM_004304
ALOX12B	NM_001139
AMER1	NM_152424
ANKRD11	NM_013275
APC	NM_000038
AR	NM_000044
ARAF	NM_001654
ARID1A	NM_006015
ARID1B	NM_020732
ARID2	NM_152641
ARID5B	NM_032199
ASXL1	NM_015338
ASXL2	NM_018263
ATM	NM_000051
ATR	NM_001184
ATRX	NM_000489
AURKA	NM_003600
AURKB	NM_004217
AXIN1	NM_003502
AXIN2	NM_004655
AXL	NM_021913
B2M	NM_004048
BABAM1	NM_001033549
BAP1	NM_004656
BARD1	NM_000465
BBC3	NM_001127240
BCL10	NM_003921
BCL2	NM_000633
BCL2L1	NM_138578
BCL2L11	NM_138621
BCL6	NM_001706
BCOR	NM_001123385
BIRC3	NM_182962
BLM	NM_000057
BMPR1A	NM_004329
BRAF	NM_004333
BRCA1	NM_007294
BRCA2	NM_000059
BRD4	NM_058243
BRIP1	NM_032043
BTK	NM_000061
CALR	NM_004343
CARD11	NM_032415
CARM1	NM_199141
CASP8	NM_001080125
CBFB	NM_022845
CBL	NM_005188
CCND1	NM_053056
CCND2	NM_001759
CCND3	NM_001760
CCNE1	NM_001238
CD274	CD276	NM_014143	NM_001024736
CD79A	NM_001783
CD79B	NM_001039933
CDC42	NM_001791
CDC73	NM_024529

Appendix 1b: List of genes/transcripts included on the MSK-IMPACT panel
-------------------------------------------------------------------------	--

48

CDH1	NM_004360
CDK12	NM_016507
CDK4	NM_000075
CDK6	NM_001145306
CDK8	NM_001260
CDKN1A	NM_078467
CDKN1B	NM_004064
CDKN2Ap14ARF	NM_058195
CDKN2Ap16INK4A	NM_000077
CDKN2B	NM_004936
CDKN2C	NM_078626
CEBPA	NM_004364
CENPA	NM_001809
CHEK1	NM_001274
CHEK2	NM_007194
CIC	NM_015125
CREBBP	NM_004380
CRKL	NM_005207
CRLF2	NM_022148
CSDE1	NM_001242891
CSF1R	NM_005211
CSF3R	NM_000760
CTCF	NM_006565
CTLA4	NM_005214
CTNNB1	NM_001904
CUL3	NM_003590
CXCR4	NM_003467
CYLD	NM_001042355
CYSLTR2	NM_020377
DAXX	NM_001141970
DCUN1D1	NM_020640
DDR2	NM_006182
DICER1	NM_030621
DIS3	NM_014953
DNAJB1	NM_006145
DNMT1	NM_001379
DNMT3A	NM_022552
DNMT3B	NM_006892
DOT1L	NM_032482
DROSHA	NM_013235
DUSP4	NM_001394
E2F3	NM_001949
EED	NM_003797
EGFL7	NM_201446
EGFR	NM_005228
EIF1AX	NM_001412
EIF4A2	NM_001967
EIF4E	NM_001130678
ELF3	NM_004433
EP300	NM_001429
EPAS1	NM_001430
EPCAM	NM_002354
EPHA3	NM_005233
EPHA5	NM_004439
EPHA7	NM_004440
EPHB1	NM_004441
ERBB2	NM_004448
ERBB3	NM_001982
ERBB4	NM_005235
ERCC2	NM_000400
ERCC3	ERCC3	NM_000122	NM_000122
ERCC4	NM_005236
ERCC5	NM_000123
ERF	NM_006494
ERG	NM_182918
ERRFI1	NM_018948
ESR1	NM_001122740
ETV1	NM_001163147
ETV6	NM_001987
EZH1	NM_001991
EZH2	NM_004456
FAM175A	NM_139076
FAM46C	NM_017709
FAM58A	NM_152274
FANCA	NM_000135
FANCC	NM_000136
FAT1	NM_005245
FBXW7	NM_033632
FGF19	NM_005117
FGF3	NM_005247
FGF4	NM_002007
FGFR1	NM_001174067
FGFR2	NM_000141
FGFR3	NM_000142
FGFR4	NM_213647
FH	NM_000143
FLCN	NM_144997
FLT1	NM_002019
FLT3	NM_004119
FLT4	NM_182925
FOXA1	NM_004496
FOXL2	NM_023067
FOXO1	NM_002015
FOXP1	NM_001244814
FUBP1	NM_003902
FYN	NM_153047
GATA1	NM_002049
GATA2	NM_032638
GATA3	NM_002051
GLI1	NM_005269
GNA11	NM_002067
GNAQ	NM_002072
GNAS	NM_000516
GPS2	NM_004489
GREM1	NM_013372
GRIN2A	NM_001134407
GSK3B	NM_002093
H3F3A	NM_002107
H3F3B	NM_005324
H3F3C	NM_001013699
HGF	NM_000601
HIST1H1C	NM_005319
HIST1H2BD	NM_021063
HIST1H3A	NM_003529
HIST1H3B	NM_003537
HIST1H3C	NM_003531
HIST1H3D	NM_003530
HIST1H3E	NM_003532
HIST1H3F	NM_021018
HIST1H3G	NM_003534
HIST1H3H	NM_003536
HIST1H3I	NM_003533
HIST1H3J	NM_003535
HIST2H3C	NM_021059
HIST2H3D	NM_001123375
HIST3H3	NM_003493
HLA-A	NM_001242758
HLA-B	HLA-B	NM_005514	NM_005514
HNF1A	NM_000545
HOXB13	NM_006361
HRAS	NM_001130442
ICOSLG	NM_015259

49

50

| ID3 | NM
002167 |
|------------------|---------------------------------|
| IDHI | 005896
NM |
| IDH2 | 002168
NM |
| IFNGR1 | 000416
NM |
| IGF1 | 00111285
NM |
| IGFIR | NM
000875 |
| IGF2 | NM_001127598 |
| IKBKE | 014002
NM |
| IKZF1 | NM
006060 |
| IL10 | NM
000572 |
| IL7R | NM
002185 |
| INHA | NM
002191 |
| INHBA | NM
002192 |
| INPP4A | 001134224
NM |
| INPP4B
INPPL1 | 001101669
NM
001567
NM |
| INSR | 000208
NM |
| IRF4 | NM
002460 |
| IRSI | NM
005544 |
| IRS2 | NM_003749 |
| JAK1 | 002227
NM |
| JAK2 | 004972
NM |
| JAK3 | 000215
NM |
| JUN | 002228
NM |
| KDM5A | NM
001042603 |
| KDM5C | 004187
NM |
| KDM6A | NM
021140 |
| KDR | 002253
NM_ |
| KEAP1 | NM
203500 |
| KIT | 000222
NM |
| KLF4 | NM
004235 |
| KMT2A | NM
001197104 |
| KMT2B | _014727
NM |
| KMT2C | 170606
NM |
| KMT2D | 003482
NM |
| KNSTRN | 033286
NM |
| KRAS | NM
033360 |
| LATS 1 | 004690
NM |
| LATS2 | NM
014572 |
| LMO1 | NM
002315 |
| LYN | 002350
NM
006785 |
| MALT1
MAP2K1 | NM
NM
002755 |
| MAP2K2 | NM
030662 |
| MAP2K4 | NM
003010 |
| MAP3K1 | 005921
NM |
| MAP3K13 | NM_004721 |
| MAP3K14 | NM
003954 |
| MAPK1 | 002745
NM |
| MAPK3 | NM_002746 |
| MAPKAP1 | 001006617
NM |
| MAX | NM
002382 |
| MCL1 | NM
021960 |
| MDC1 | NM_014641 |
| MDM2 | 002392
NM |
| MDM4 | 002393
NM |
| MED12 | NM
005120 |
| MEF2B | 001145785
NM |
| MENI | 000244
NM |
| MET | 000245
NM |
| MGA | 001164273
NM |
| MITF | 198159
NM |
| MLHI | NM
000249 |
| MPL | 005373
NM |
| MRE11A | 005591
NM |
| MSH2 | 000251
NM |

51

MSH3	NM_002439
MSH6	NM_000179
MSI1	NM_002442
MSI2	NM_138962
MST1	NM_020998
MST1R	NM_002447
MTOR	NM_004958
MUTYH	NM_001128425
MYC	NM_002467
MYCL1	NM_001033082
MYCN	NM_005378
MYD88	NM_002468
MYOD1	NM_002478
NBN	NM_002485
NCOA3	NM_181659
NCOR1	NM_006311
NEGR1	NM_173808
NF1	NM_001042492
NF2	NM_000268
NFE2L2	NM_006164
NFKBIA	NM_020529
NKX2-1	NM_001079668
NKX3-1	NM_006167
NOTCH1	NM_017617
NOTCH2	NM_024408
NOTCH3	NM_000435
NOTCH4	NM_004557
NPM1	NM_002520
NRAS	NM_002524
NSD1	NM_022455
NTHL1	NM_002528
NTRK1	NM_002529
NTRK2	NM_006180
NTRK3	NM_001012338
NUF2	NM_031423
NUP93	NM_014669
PAK1	NM_002576
PAK7	NM_177990
PALB2	NM_024675
PARK2	NM_004562
PARP1	NM_001618
PAX5	NM_016734
PBRM1	NM_018313
PDCD1	NM_005018
PDCD1LG2	NM_025239
PDGFRA	NM_006206
PDGFRB	NM_002609
PDPK1	NM_002613
PGR	NM_000926
PHOX2B	NM_003924
PIK3C2G	NM_004570
PIK3C3	NM_002647
PIK3CA	NM_006218
PIK3CB	NM_006219
PIK3CD	NM_005026
PIK3CG	NM_002649
PIK3R1	NM_181523
PIK3R2	NM_005027
PIK3R3	NM_003629
PIM1	PIM1	NM_002648	NM_002648
PLCG2	NM_002661
PLK2	NM_006622
PMAIP1	NM_021127
PMS1	NM_000534
PMS2	NM_000535
PNRC1	NM_006813
POLD1	NM 002691
POLE	NM 006231
PPARG	NM 015869
PPM1D	NM_003620
PPP2R1A	NM 014225
PPP4R2	NM_174907
PPP6C	NM 002721
PRDM1	NM_001198
PRDM14	NM 024504
PREX2	NM 024870
PRKAR1A	NM_212471
PRKCI	NM 002740
PRKD1	NM 002742
PTCH1	NM 000264
PTEN	NM 000314
PTP4A1	NM_003463
PTPN11	NM_002834
PTPRD	NM 002839
PTPRS	NM 002850
PTPRT	NM_133170
RAB35	NM_006861
RAC1	NM 018890
RAC2	NM_002872
RAD21	NM 006265
RAD50	NM_005732
RAD51	NM 002875
RAD51B	NM 133509
RAD51C	NM_058216
RAD51D	NM_133629
RAD52	NM_134424
RAD54L	NM_001142548
RAF1	NM 002880
RARA	NM 000964
RASA1	NM_002890
RB1	NM 000321
RBM10	NM 001204468
RECQL	NM_032941
RECQL4	NM_004260
REL	NM 002908
RET	NM 020975
RFWD2	NM 022457
RHEB	NM_005614
RHOA	NM 001664
RICTOR	NM 152756
RIT1	NM 006912
RNF43	NM_017763
ROS1	NM_002944
RPS6KA4	NM 003942
RPS6KB2	NM 003952
RPTOR	NM 020761
RRAGC	NM_022157
RRAS	NM 006270
RRAS2	NM_012250
RTEL1	NM_032957
RUNX1	NM_001754
RXRA	NM 002957
RYBP	NM 012234
SDHA	NM 004168
SDHAF2	NM_017841
SDHB	NM 003000
SDHC	RHEB	NM 003001	NM_005614
RHOA	NM_001664
RICTOR	NM_152756
RIT1	NM_006912
RNF43	NM_017763
ROS1	NM_002944
RPS6KA4	NM_003942
RPS6KB2	NM_003952
RPTOR	NM_020761
RRAGC	NM_022157
RRAS	NM_006270
RRAS2	NM_012250
RTEL1	NM_032957
RUNX1	NM_001754
RXRA	NM_002957
RYBP	NM_012234
SDHA	NM_004168
SDHAF2	NM_017841
SDHB	NM_003000
SDHC	NM_003001
SDHD	NM_003002
SESN1	NM_014454
SESN2	NM_031459
SESN3	NM_144665
SETD2	NM_014159

52

53

| SETD8 | NM
020382 |
|------------------|------------------------------|
| SF3B1 | 012433
NM |
| SH2B3 | 005475
NM |
| SH2D1A | NM
002351 |
| SHOC2 | NM
007373 |
| SHOI | 018130
NM |
| SLX4 | NM_032444 |
| SMAD2 | 001003652
NM |
| SMAD3 | NM
005902 |
| SMAD4 | 005359
NM |
| SMARCA4 | 003072
NM |
| SMARCB1 | NM
003073 |
| SMARCD1 | 003076
NM |
| SMO | NM
005631 |
| SMYD3 | 001167740
NM |
| SOCS 1
SOSI | NM
003745 |
| SOX17 | 005633
NM
022454
NM |
| SOX2 | NM
003106 |
| SOX9 | NM_000346 |
| SPEN | 015001
NM |
| SPOP | 001007228
NM |
| SPRED1 | NM
152594 |
| SRC | NM
198291 |
| SRSF2 | NM
003016 |
| STAG2 | 001042749
NM |
| STAT3 | 139276
NM |
| STAT5A | 003152
NM |
| STAT5B | NM
012448 |
| STK11 | 000455
NM |
| STK19 | NM
004197 |
| STK40 | NM
032017 |
| SUFU | NM_016169 |
| SUZIZ | 015355
NM |
| SYK | 003177
NM |
| TAP1 | 000593
NM |
| TAP2 | 018833
NM |
| TBX3 | 016569
NM |
| TCEB1 | 005648
NM |
| TCF3 | NM
001136139 |
| TCF7L2 | 001146274
NM |
| TEK | 000459
NM |
| TERT | NM
198253 |
| TETI | 030625
NM |
| TET2 | 001127208
NM |
| TGFBR1
TGFBR2 | NM
004612
NM 001024847 |
| TMEM127 | 001193304
NM |
| TMPRSS2 | 001135099
NM |
| TNFAIP3 | NM_006290 |
| TNFRSF14 | NM_003820 |
| TOP1 | NM
003286 |
| TP53 | 000546
NM |
| TP53BP1 | NM 001141980 |
| TP63 | NM
003722 |
| TRAF2 | NM
021138 |
| TRAF7 | NM
032271 |
| TSC1 | NM
000368 |
| TSC2 | NM
000548 |
| TSHR | 000369
NM |
| U2AF1 | NM
006758 |
| UPF1 | NM_002911 |
| VEGFA | NM_001171623 |
| VHL | 000551
NM_ |
| VTCN1 | NM
024626 |
| WHSCI | _001042424
NM |
| WHSC1L1 | NM_023034 |
| WT1 | NM_024426 |
| WWTR1 | NM_001168280 |
| XIAP | NM_001167 |
| XPO1 | NM_003400 |
| XRCC2 | NM_005431 |
| YAP1 | NM_001130145 |
| YES1 | NM_005433 |
| ZFHX3 | NM_006885 |

54

Appendix 1c: List of genes/exons excluded from reporting due to consistently low
coverage.

| Gene | Transcript ID | Chromosome
Coordinates | Exon | cDNA | Amino Acid | |
|----------|---------------|-------------------------------------|------|-----------|------------|-----------|
| AGO2 | NM_012154 | 8:141645584-
141645605 | 1 | 1_22 | 1_8 | |
| ANKRD11 | NM_013275 | 16:89334886-
89335071 | 13 | 7807_7992 | 2603_2664 | |
| CD276 | NM_001024736 | 15:73995113-
73995427 | 4 | 419_733 | 140_245 | |
| CD276 | NM_001024736 | 15:73996517-
73996813 | 6 | 1073_1369 | 358_457 | |
| CHEK2 | NM_007194 | 22:29085123-
29085203 | 14 | 1462_1542 | 488_514 | |
| FAM58A | NM_152274 | X:152864420-
152864529 | 1 | 1176_1 | 392_1 | |
| FLT3 | NM_004119 | 13:28674605-
28674647 | 1 | 1_43 | 1_15 | |
| H3F3A | NM_002107 | 1:226259052-
226259180 | 4 | 283_411 | 95_137 | |
| HIST2H3C | NM_021059 | 1:149812319-
149812729 | 1 | 411_1 | 137_1 | |
| HIST2H3D | NM_001123375 | 1:149784826-
149785236 | 1 | 1_411 | 1_137 | |
| HLA-A | NM_001242758 | 6:29911899-
29912174 | 4 | 620_895 | 207_299 | |
| INSR | NM_000208 | 19:7293803-
7293902 | 1 | 1_100 | 1_34 | |
| KMT2C | NM_170606 | 7:151970790-
151970952 | 7 | 850_1012 | 284_338 | |
| KMT2C | NM_170606 | 7:151962123-
151962294 | 8 | 1013_1184 | 338_395 | |
| KMT2C | NM_170606 | 7:151935792-
151935911 | 15 | 2533_2652 | 845_884 | |
| KMT2C | NM_170606 | 7:151932902-
151933018 | 16 | 2653_2769 | 885_923 | |
| KMT2C | NM_170606 | 7:151927008-
151927112 | 18 | 2872_2976 | 958_992 | |
| KMT2C | NM_170606 | 7:151921520-
151921701 | 19 | 2977_3158 | 993_1053 | |
| KMT2C | NM_170606 | 7:151921100-
151921264 | 20 | 3159_3323 | 1053_1108 | 1053_1108 |
| KMT2C | NM_170606 | 7:151919658-
151919767 | 21 | 3324_3433 | 1108_1145 | |
| KMT2C | NM_170606 | 7:151904385-
151904513 | 24 | 3713_3841 | 1238_1281 | |
| MST1 | NM_020998 | 3:49726031-
49726124 | 1 | 1_94 | 1_32 | |
| MST1 | NM_020998 | 3:49724380- | 6 | 608_728 | 203_243 | |
| | | | | | | |
| MST1 | NM_020998 | 49724500
3:49724117-
49724235 | 7 | 729_847 | 243_283 | |
| MST1 | NM_020998 | 3:49723746-
49723914 | 8 | 848_1016 | 283_339 | |
| MST1 | NM_020998 | 3:49723495-
49723625 | 9 | 1017_1147 | 339_383 | |
| MST1 | NM_020998 | 3:49722695-
49722815 | 13 | 1424_1544 | 475_515 | |
| MST1 | NM_020998 | 3:49722445-
49722522 | 14 | 1545_1622 | 515_541 | |
| MST1 | NM_020998 | 3:49721983-
49722089 | 16 | 1770_1876 | 590_626 | |
| MST1 | NM_020998 | 3:49721747-
49721886 | 17 | 1877_2016 | 626_672 | |
| MYCL1 | NM_001033082 | 1:40367480-
40367560 | 1 | 1_81 | 1_27 | |
| NOTCH2 | NM_024408 | 1:120611948-
120612020 | 1 | 1_73 | 1_25 | |
| NOTCH2 | NM_024408 | 1:120572529-
120572610 | 2 | 74_155 | 25_52 | |
| NOTCH2 | NM_024408 | 1:120547952-
120548211 | 3 | 156_415 | 52_139 | |
| NOTCH2 | NM_024408 | 1:120539620-
120539955 | 4 | 416_751 | 139_251 | |
| NOTCH3 | NM_000435 | 19:15311599-
15311716 | 1 | 1_118 | 1_40 | |
| PDPK1 | NM_002613 | 16:2588114-
2588137 | 1 | 1_24 | 1_8 | |
| PDPK1 | NM_002613 | 16:2607704-
2607964 | 2 | 25_285 | 9_95 | |
| PDPK1 | NM_002613 | 16:2611481-
2611523 | 3 | 286_328 | 96_110 | |
| PDPK1 | NM_002613 | 16:2611772-
2611909 | 4 | 329_466 | 110_156 | |
| PDPK1 | NM_002613 | 16:2615554-
2615698 | 5 | 467_611 | 156_204 | |
| PDPK1 | NM_002613 | 16:2616357-
2616454 | 6 | 612_709 | 204_237 | |
| PDPK1 | NM_002613 | 16:2627426-
2627501 | 7 | 710_785 | 237_262 | |
| PDPK1 | NM_002613 | 16:2631296-
2631364 | 8 | 786_854 | 262_285 | |
| PDPK1 | NM_002613 | 16:2631608-
2631704 | 9 | 855_951 | 285_317 | |
| PDPK1 | NM_002613 | 16:2633413-
2633586 | 10 | 952_1125 | 318_375 | |
| PIK3CA | NM_006218 | 3:178937737-
178937840 | 13 | 1912_2015 | 638_672 | |
| PIK3R2 | NM_005027 | 19:18272089-
18272305 | 6 | 599_815 | 200_272 | |
| PMS2 | NM_000535 | 7:6022455-
6022622 | 12 | 2007_2174 | 669_725 | |
| PMS2 | NM_000535 | 7:6018227-
6018327 | 13 | 2175_2275 | 725_759 | |
| PMS2 | NM_000535 | 7:6017219-
6017388 | 14 | 2276_2445 | 759_815 | |
| PMS2 | NM_000535 | 7:6013030-
6013173 | 15 | 2446_2589 | 816_863 | |
| PPP4R2 | NM_174907 | 3:73096337-
73096507 | 3 | 117_287 | 39_96 | |
| | | | | | | |
| PTEN | NM_000314 | 10:89725044-
89725229 | 9 | 1027_1212 | 343_404 | |
| PTPRT | NM_133170 | 20:41818286-
41818373 | 1 | 1_88 | 1_30 | |
| RECQL | NM_032941 | 12:21623128-
21623280 | 16 | 1798_1950 | 600_650 | |
| RECQL4 | NM_004260 | 8:145743085-
145743168 | 1 | 1_84 | 1_28 | |
| SDHA | NM_004168 | 5:254508-
254621 | 14 | 1795_1908 | 599_636 | |
| SDHC | NM_003001 | 1:161332119-
161332223 | 6 | 406_405 | 136_135 | |
| SDHD | NM_003002 | 11:111965529-
111965694 | 4 | 315_480 | 105_160 | |
| SETD8 | NM_020382 | 12:123873980-
123874101 | 2 | 11_132 | 4_44 | |
| SETD8 | NM_020382 | 12:123892040-
123892250 | 8 | 849_1059 | 283_353 | |
| STAT5A | NM_003152 | 17:40452148-
40452299 | 8 | 682_833 | 228_278 | |
| STAT5A | NM_003152 | 17:40452733-
40452888 | 9 | 834_989 | 278_330 | |
| STAT5B | NM_012448 | 17:40371330-
40371481 | 7 | 682_833 | 228_278 | |
| STAT5B | NM_012448 | 17:40370741-
40370896 | 8 | 834_989 | 278_330 | |
| STK19 | NM_004197 | 6:31948781-
31948826 | 8 | 1050_1095 | 350_365 | |
| SUZ12 | NM_015355 | 17:30267305-
30267351 | 2 | 275_321 | 92_107 | |
| SUZ12 | NM_015355 | 17:30267441-
30267505 | 3 | 322_386 | 108_129 | |
| SUZ12 | NM_015355 | 17:30274636-
30274704 | 4 | 387_455 | 129_152 | |
| SUZ12 | NM_015355 | 17:30300165-
30300250 | 6 | 506_591 | 169_197 | |
| SUZ12 | NM_015355 | 17:30310018-
30310123 | 9 | 918_1023 | 306_341 | |
| TGFBR1 | NM_004612 | 9:101867488-
101867584 | 1 | 1_97 | 1_33 | |

55

56