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K Number
DEN170058
Device Name
MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay
Manufacturer
Memorial Sloan-Kettering Cancer Center
Date Cleared
2017-11-15

(51 days)

Product Code
PZM
Regulation Number
866.6080
Type
Direct
Panel
Pathology
Reference & Predicate Devices
N/A
Predicate For
N/A
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended 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.

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.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text for the MSK-IMPACT assay.

Device Name: MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets)
Type of Test: Next generation sequencing tumor profiling test
Purpose: Qualitative in vitro diagnostic test for detecting somatic mutations (point mutations, small insertions and deletions) and microsatellite instability (MSI) in formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients with solid malignant neoplasms.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally embedded within the "Performance" section and the "Reporting" section's "Table 3. Sample Level Quality Control Metrics." The reported performance is found throughout the "Performance" section.

Acceptance Criteria (from text)Reported Device Performance (from text)
Specimen Requirements:
Minimum Tumor Proportion: >10% of tumor cells; >20% viable tumor preferred; >25% for MSI testing.The minimum tumor proportion required for the MSI assay was established as 25% (based on CRC specimens, assay and score reproducible to 8% tumor proportion qualitatively, but decreased trend quantitatively) (Table 1). The DNA extraction method was validated with historic data from >10,000 specimens, demonstrating invalid rates of 7.2% to 18.4%, supporting performance across FFPE tumor types (Table 5).
Quality Control Metrics (Table 3):
Average target coverage: > 200XFor normal samples, mean coverage across all targeted exons was 571X (SD = 373X). Analysis of normal samples showed that with mean sample coverage of 571X, 98% of exons are sequenced with coverage greater than 306X (or normalized coverage >0.54), leading to a conservative threshold of 200X mean sample coverage. In silico downsampling to 203X coverage detected 94% of mutations with 10% VAF (Performance L.1.b and Table 3).
Coverage Uniformity: ≥ 98% target exons above 100X coverage99.5% of exons were sequenced to a depth of 100X or greater, and 98.6% to 250X or greater. It’s expected that 98% of exons will be sequenced to >100X coverage when mean sample coverage is 185X. (Performance L.1.b)
Base Quality: > 80% of bases with QS above > Q30Not explicitly detailed in the performance section but stated as a QC metric in Table 3. Implicitly met if overall performance is approved.
% Cluster passing filter (Cluster PF): > 80%Not explicitly detailed in the performance section but stated as a QC metric in Table 3. Implicitly met if overall performance is approved.
% Reads passing filter (Reads PF): > 80%Not explicitly detailed in the performance section but stated as a QC metric in Table 3. Implicitly met if overall performance is approved.
Hotspot Mutation calling threshold: DP ≥ 20, AD ≥ 8, VF ≥ 2%Filtering scheme designed to reject false positives while maintaining detection capability. Example: pre-filter SNVs (hotspot) had 1 false positive, post-filter 0 (Table 4). LoD confirmation: 5 replicates for 6 SNVs at 5% MAF showed 100% positive call rates, except one replicate failing on PTEN exon 6 due to low read depth below 5% (Performance L.2.b.ii and Table 11).
Non-hotspot Mutation threshold: DP > 20, AD ≥ 10, VF ≥ 5%Filtering scheme designed to reject false positives while maintaining detection capability. Example: pre-filter SNVs (non-hotspot) had 342 false positives, post-filter 0 (Table 4). LoD study showed most mutations detected at low VAFs (e.g., 2-9% in Tables 10A-J). Confirmed LoD study (Part 2) for various mutations showed 100% positive call rates for variant types except one discordant case (PTEN exon 8 deletion) at 3.6-7.9% VF (Table 11).
Indels: Fewer than 20% of samples in an established 'standard normal' database (This seems to be a filtering criteria for indels, not a reporting metric.)Indels had 40,793 pre-filter false positives, reduced to 8 post-filter (Rejection Rate 0.999) (Table 4). LoD confirmation: 5 replicates for 3 deletions and 4 insertions at 5% MAF showed 100% positive call rates, except one deletion (PTEN exon 6), which also failed read depth (Performance L.2.b.ii and Table 11).
Positive Run Control: The difference between the observed and expected frequencies for the known mutations should be within 5%.Mixed positive control sample with expected VFs: Results reviewed to confirm known mutations called and observed frequencies match expected values within 5% (Controls, b).
Negative Run Control: The correlation between expected and observed mutation frequencies should be 0.9 or higher.Pooled negative control: Observed mutation frequencies compared against expected for 862 common SNPs; correlation expected to be 0.9 or higher (Controls, c). Figure 2 shows correlation of 0.975 (with slope 0.971 and intercept -0.004) for observed vs. expected variant frequency, establishing consistent correlation >0.9.
Sample-Mix up QC: Flagged if pairs of samples from the same patient with > 5% discordance and from different patients with < 5% discordance.Pipeline computes 'percent discordance'; expected discordance between tumors and matched normal should be low (<5%), between different patients high (~25%). Samples flagged if >5% for same patient ("unexpected mismatches") or <5% for different patients ("unexpected matches") (Device Description, 4.e.i).
Major Contamination QC: % heterozygous sites at fingerprint SNPs < 55%; Average MAF at homozygous fingerprint SNPs < 2%.Samples flagged if average minor allele frequency at homozygous SNP sites exceeds 2% (Device Description, 4.e.ii).
Criteria for calling test failure: If a sample presents with mean coverage across all exons < 50x and no mutations are detected due to the low overall coverage, the test is deemed "failed" for the sample.Not explicitly detailed in the performance section but stated in Table 3. Implicitly met if overall performance is approved.
Analytical Performance (General):
Precision (Within-run, Between-run, Total Variability): Using clinical samples, covering all mutation types (positive/negative), including samples near LoD. Assessed by agreement within replicates and sequencing quality metrics.Panel-Wide Reproducibility: 69 mutations in clinical specimens and 13 in commercial cell line (total 82). All mutations showed 100% concordance except 4 in clinical specimens and 3 in commercial sample. Discordant cases were in repetitive regions, or had low frequencies near 2% (Performance L.2.a.ii and Table 7). Positive call rates varied per mutation and specimen (Table 7 and 8). Per Specimen Precision: (N=5 replicates). Overall positive call rates ranged from 80% to 100% across various specimens (Table 8). Intra-assay repeatability: All results concordant except for ARID1B exon 2 insertion and BRAF V600M point mutation (commercial control). Reference Material (NA20810): 23 replicates. Zygosity results were 100% concordant. Difference between expected and mean observed mutation frequencies was very small (absolute difference = 0.09%±0.45%), providing supplemental evidence of reproducibility (Performance L.2.a.iv). MSI Precision: 12 specimens (6 MSI-H, 6 MSS) tested with 3 inter- and 3 intra-run replicates. All samples had 100% agreement between calls (Performance L.2.a.v and Table 9).
Analytical Sensitivity (LoD): Defined as mutant allele fraction at which 95% of replicates are reliably detected. Confirmed with multiple replicates.Part 1 (Dilution Series): Serial dilutions of patient samples were used to identify lowest reliable mutant fraction. Most mutations were called at lowest dilution (e.g., BRAF V600E at 2% VF, KRAS G12D at 6% VF, EGFR ins at 3% VF), except PIK3CA (PIK3CA Exon 2 (R88Q) was WT at 1:16 dilution) (Tables 10A-J). Part 2 (Confirmation): 5 replicates tested for 3 deletions, 4 insertions, and 6 SNVs at 5% minor allele frequency. All variants had 100% positive call rates except one replicate for a PTEN exon 6 deletion (mutation read depth below estimated LoD of 5%) (Performance L.2.b.ii and Table 11). LoD is stated as 2% for hotspot and 5% for non-hotspot mutations (Assay Cut-off).
Analytical Specificity: Maintained by paired tumor/matched normal sequencing.Established during assay optimization; paired tumor/matched normal sequencing minimizes interference (Performance L.2.g).
Accuracy (Method Comparison): Using clinical specimens representing intended specimen type and range of tumor types. Specific criteria for SNV/MNVs, insertions, deletions, and MSI.Overall Accuracy: 432 out of 433 cases (99.8% with 95% CI (98.7%, 100.0%)) successfully detected known mutations compared to orthogonal methods. One discordant case (EGFR exon 20 duplication) was identified due to filtering algorithm, which was subsequently modified. (Performance L.2.i.i) PPA by Mutation Type/Gene: SNV/MNVs showed 100% PPA for all listed genes (Table 15A). Insertions showed PPA from 93.8% (EGFR) to 100% (Table 15B). Deletions showed 100% PPA for all listed genes (Table 15C). Wildtype Calls (Supplemental Study): 95 specimens with 109 mutations and 3026 wild-type calls across 33 hotspots in 10 genes. Variant-level concordance: PPA was 100% (96.7%, 100.0% CI), NPA was 100% (99.9%, 100.0% CI) (Performance L.2.i.ii). MSI Accuracy (MSIsensor): CRC/EC (Training): Cut-off of 10 established based on concordance with MSI-PCR or MMR IHC using 138 CRC and 40 EC specimens. CRC (Validation): 135 CRC patients, 66 with both MSK-IMPACT MSI and IHC results. PPV = 92.3% (12/13, 95% CI 64.0%-99.8%), NPV = 98.1% (52/53, 95% CI 90.0%-100.0%) (Table 16). Non-CRC/EC: 119 non-CRC/EC samples assessed by MSIsensor and MSI-PCR. Excluding missing data: PPV = 93.9% (46/49, 83.1%-98.7% CI), NPV = 96.7% (58/60, 88.5%-99.6% CI). Including missing data: PPV = 78.0% (46/59, 65.3%-87.7% CI), NPV = 96.7% (58/60, 88.5%-99.6% CI). (Table 17).

2. Sample Size Used for the Test Set and Data Provenance

  • Test Set Sample Sizes:

    • Precision Studies:
      • 10 samples (9 FFPE specimens, 1 commercial cell line) used for panel-wide reproducibility (Table 6).
      • Well-characterized reference standard (HapMap cell line NA20810) in 23 replicates for sequencing error rates and reproducibility.
      • 12 specimens (6 MSI-H, 6 MSS) for MSI precision.
    • Analytical Sensitivity (LoD):
      • Part 1 (Dilution Series): Patient samples (number not specified, but for 5 validation exons, implying at least 5 patients) with 5-8 serial dilutions.
      • Part 2 (Confirmation): Unspecified number of samples, providing variants for 3 deletions, 4 insertions, and 6 SNVs, each tested with 5 replicates.
      • MSI LoD: CRC specimens (number not specified, but 5 replicates run).
    • DNA Input Assessment: Unspecified number of samples (historical data from >10,000 samples mentioned in pre-analytical performance context). Table 13 presents data by DNA input amounts but not sample count for each bin.
    • Accuracy (Method Comparison):
      • 267 unique mutations in 433 FFPE tumor specimens for the main comparison (Table 14).
      • 95 specimens for the supplemental wildtype calls study.
      • 138 colorectal cancer (CRC) and 40 endometrial carcinoma (EC) specimens (training set) for MSI cutoff establishment.
      • 135 CRC patients (66 with both MSK-IMPACT and IHC) for MSI cutoff validation.
      • 119 unique non-CRC and non-EC tumor-normal pair samples for MSI comparison in other cancer types.

  • Data Provenance:

    • General: The device is performed at Memorial Sloan Kettering Cancer Center (MSK), indicating the data likely originates from their patient population.
    • Retrospective/Prospective:
      • The pre-analytical performance (specimen invalid rates) used historical data from >10,000 specimens, implying a retrospective chart review.
      • The MSI validation study (CRC patients) was a retrospective-prospective chart review.
      • The clinical performance section mentions a large-scale, prospective clinical sequencing initiative using MSK-IMPACT involving >10,000 patients, whose data are publicly accessible. This cohort likely informed the broader context and understanding of the device but was not explicitly stated as the test set for the analytical validation.
      • The analytical performance studies (precision, LoD, accuracy) used clinical samples/specimens, which could be retrospective or prospectively collected for the purpose of the study. The text doesn't explicitly state for each study.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts

The text does not specify the number of experts used to establish the ground truth for the test set, nor their specific qualifications (e.g., "radiologist with 10 years of experience").

However, it does indicate:

  • For the accuracy studies, results were compared to "original results obtained with the validated orthogonal methods." This implies that the ground truth was established by these validated orthogonal methods, which are presumably performed and interpreted by qualified personnel using established clinical diagnostics.
  • For MSI, the MSIsensor results were compared to "a validated MSI-PCR or MMR IHC test," a "commercially available PCR assay," or a "validated IHC panel (MLH1, MSH2, MSH6 and PMS2)." Again, this suggests ground truth from established, clinical laboratory methods.
  • The "Clinical Evidence Curation" section mentions that "OncoKB undergoes periodic updates through the review of new information by a panel of experts," which informs the clinical interpretation of detected mutations. This expert panel contributes to the broader clinical context of the mutations, but not directly the ground truth for the analytical test set itself.

4. Adjudication Method (e.g., 2+1, 3+1, none) for the Test Set

The text does not describe a formal adjudication method (like 2+1 or 3+1 consensus with experts) for establishing the ground truth of the test set cases. Instead, the ground truth was derived from "validated orthogonal methods."

For example, in the accuracy study, the MSK-IMPACT results were "compared to the original results obtained with the validated orthogonal methods." This indicates that the results from the comparison methods served as the reference standard, rather than requiring an additional expert adjudication process on top of those existing validated methods.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done

No, an MRMC comparative effectiveness study was not described. This document pertains to the analytical validation of a genetic sequencing assay, which inherently does not involve human readers interpreting images in a multi-reader, multi-case setup. Therefore, a comparative effectiveness study measuring human reader improvement with AI assistance (which is typical for imaging AI) is not applicable here.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, the analytical performance studies (precision, analytical sensitivity, and analytical accuracy) described are all measures of the standalone performance of the MSK-IMPACT assay, which relies on its sequencing and bioinformatics pipeline without direct human-in-the-loop diagnostic interpretation to produce the raw mutation calls.

  • The "Mutation calling SNVs and Indels" section and "Summary of mutation filtering scheme" (Figure 1) describe the automated pipeline for identifying mutations.
  • The "Performance" section details how characteristics like precision, LoD, and accuracy were determined for the assay itself by comparing its outputs to known or established results from other validated methods. These do not involve a human interpreting the device's output to make a diagnosis within the performance evaluation but rather assess the accuracy of the device's genomic calls directly.

7. The Type of Ground Truth Used

The primary type of ground truth used was:

  • Orthogonal Methods / Comparator Assays: For the accuracy studies, the MSK-IMPACT results were compared against "original results obtained with the validated orthogonal methods." This included comparison to:
    • Validated orthogonal methods for SNVs and indels.
    • Established MSI-PCR or MMR IHC tests for Microsatellite Instability status.
  • Known Reference Material: For precision, a "well characterized reference standard (HapMap cell line NA20810)" was used, with reference genotypes obtained from the 1000 Genomes database.
  • Expected Values/Dilution Series: For Limit of Detection studies, serial dilutions of patient samples with "known mutations" and "expected frequencies" were used.

Therefore, the ground truth is a combination of established methods, known reference materials, and empirically derived expected values.

8. The Sample Size for the Training Set

The document explicitly mentions training data primarily in the context of the MSI cutoff:

  • MSI Cutoff Training: 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."

For the mutation calling pipeline (SNVs and indels), the text refers to:

  • Optimization of thresholds: "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." The size of this "reference dataset" or "replicates of normal FFPE samples" used for training/optimization of filtering thresholds is not explicitly stated as a defined "training set sample size" for the SNV/indel calling. It implies an internal dataset used during development.

9. How the Ground Truth for the Training Set Was Established

For the MSI cutoff training set:

  • The ground truth was established by "validated MSI-PCR or MMR IHC test" results. These are existing, established clinical diagnostic methods for determining MSI status.

For the SNV/indel pipeline optimization/threshold establishment:

  • The ground truth for optimizing filtering thresholds was based on "paired-sample mutation analysis on replicates of normal FFPE samples" and "reference dataset." This suggests that the "true" status of these calls (i.e., whether they were true positives, false positives, etc.) would have been known or definitively determined through external means (e.g., highly confident calls from a different (perhaps more laborious or deeply sequenced) method, or a known characteristic of the "normal FFPE samples"). However, the specific method for establishing this ground truth for the filtering optimization is not explicitly detailed beyond being from a "reference dataset."

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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

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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:

TissueTypeVolumeMinimum TumorProportionMacrodissectionrequirements(Based on tumorproportion)LimitationsStorage
FFPEsections5-20unstainedsections,10micronsthickMore than 10% of tumorcells;sections containing>20% viable tumor arepreferred.For MSI testing, >25%tumor cells.Yes,macrodissectionto obtain non-neoplastic tissuefor analysisArchival paraffin-embedded materialsubjected to aciddecalcification isunsuitable for analysisbecause aciddecalcification severelydamage nucleic acids.Roomtemp

Table 1. Specimen Handling and Processing for Validated Specimen Types

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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.

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  • 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%). Conversely, the expected discordance between samples from different patients should be high (~ 25%). Pairs of samples from the same patient with > 5% discordance ("unexpected mismatches") and from different patients with <5% discordance ("unexpected matches") are flagged.
    • ii. Sample contamination checks: Alternate alleles (percent heterozygous) at homozygous SNP sites (fingerprint SNPs) are assessed. A sample is flagged for review if the average minor allele frequency at these SNPs exceeds 2%.
    • iii. Check for presence of tumor in normal: Normal samples are expected to be free of known SNVs and insertions and deletions (indels) that are commonly (somatically) recurrent in tumor samples. As a first pass check, the pipeline genotypes normal samples at several known 'hotspot' locations derived from somatic mutation catalogs. If a known tumor-specific mutation (i.e. BRAF V600E) is detected with mutation frequency > 1% in a normal sample, the normal sample is flagged for review and possible exclusion from analysis. Tumor

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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 (< 50X), tumor samples will be considered as unmatched samples, and will be compared against a standard, in-batch pooled FFPE normal control for mutation calling. Filtering is performed to remove low quality sequence data, sources of sequencing artifacts, and germline results.
    • Analysis of pooled FFPE positive and negative controls: data from controls is i. used to confirm lack of contamination as well as analytical sensitivity.
    • ii. Filters on sample coverage: A sequence coverage > 100X is required to achieve 95% power to detect mutations with underlying variant frequency of 10% or greater. To ensure that at least 98% of targeted exons meet this coverage, 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.

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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 (<50X), tumor samples will be compared against a standard, in-batch pooled FFPE normal control for mutation calling; mutations called under these circumstances may include rare germline mutations and cannot be guaranteed to be somatic.

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  • b) Positive control: The positive control sample is a mixture of 3 tumor samples, each sample with a different confirmed SNV and at least one insertion or deletion, representing a range of mutation allele frequencies. Results are compared against a pooled FFPE negative control as an unmatched normal. Data generated from the mixed positive control sample are analyzed using the pipeline, and frequencies of the detected mutations are reviewed to determine if (1) the known mutations are among those called, and (2) the observed frequencies for the known mutations match their expected values within 5% of their values. The mixed FFPE positive control sample pools with expected variant frequency (VF) prior to pooling are shown in Table 2.
Mixed Positive IDSample IDVFKnown Mutation
M-1682-C3-T17%KRAS Q61H
M-1791-8C-T66%EGFR L858R
M-1913-BFM-1754-DB-T61%KITexon9ins
M-1671-CE-T25%KITexon11del
M-1693-5E-T24%PIK3CA H1047R
M-1914-A2M-1646-FC-T41%BRAF V600E
M-1612-28-3-T32%EGFR exon19 del
M-1627-D9-T52%NRAS Q61H
M-1915-CAM-1625-1A-2A-T28%KRAS G12D

Table 2. Positive Controls and Expected Mutation Frequencies

  • c) Negative control: The negative control sample is a mixture of FFPE normal samples verified in previous reruns to be free of turnor contamination and germline copy number mutations in target genes. Polymorphisms unique to each constituent normal sample in the pool have been identified in prior analyses and the expected frequencies for each polymorphism in the pooled negative control are confirmed. The observed mutation frequencies are compared against the expected mutation frequencies for the 862 common SNPs, and the degree of concordance is measured using Pearson's correlation. The correlation between expected and observed mutation frequencies is expected to be 0.9 or higher.
  • d) PCR reagent control [No Template Control (NTC)]: The NTC control should have a Qubit measurement of < 1.0ng/uL. Sequencing data from the NTC control sample will also be subjected to analysis using the pipeline, to verify that no known hotspot mutations are detected. Similar to the pooled FFPE negative control, if a hotspot mutation is detected, any samples containing that mutation in the pool will be reviewed to determine if a re-run is necessary.

6. Result Reporting:

  • Oncopanel results are reported out under one of the two categories: "Cancer . Mutations with Evidence of Clinical Significance" or "Cancer Mutations with

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Potential Clinical Significance". The two categories are based on the supporting level of clinical evidence. Refer to the Clinical Performance Section for more information.

  • Results are reported for point mutations and small insertions and deletions in protein-● coding exons of the 468 gene panel. Refer to Appendix 1b for a list of genes.
  • The MSK-IMPACT does not report mutations in 73 exons due to consistently low coverage in those exons. Refer to Appendix 1c for a list of excluded exons.
  • Reporting takes in account the following quality metrics in the Table 3 below.
QC MetricsAcceptance Criteria
CoverageAverage target coverage > 200X
Coverage Uniformity≥ 98% target exons above 100X coverage
Base Quality> 80% of bases with QS above > Q30
% Cluster passingThe percent cluster passing filter (Cluster PF) > 80%
% Reads passingfilterThe percent reads passing filter (Reads PF) > 80%
Mutation Coverage (DP) ≥ 20,
Hotspot Mutation*calling thresholdNumber of Mutant Reads (AD) ≥ 8,
Mutation Frequency (VF) ≥ 2%
Non-hotspotMutation**thresholdDP > 20, AD ≥ 10, VF ≥ 5%
IndelsFewer than 20% of samples in an established 'standardnormal'database
Positive Run ControlThe difference between the observed and expected frequencies for theknown mutations should be within 5%.
Negative RunControlThe correlation between expected and observed mutation frequenciesshould be 0.9 or higher
Sample-Mix up QCCheck over 1000 custom intergenic/intronic "fingerprint" SNPs.Flagged if pairs of samples from the same patient with > 5%discordance and from different patients with < 5% discordance
MajorContamination QC% heterozygous sites at fingerprint SNPs < 55%; Average MAF athomozygous fingerprint SNPs < 2%
Criteria for callingtest failureIf a sample presents with mean coverage across all exons < 50x and nomutations are detected due to the low overall coverage, the test isdeemed "failed" for the sample.
Table 3. Sample Level Quality Control Metrics

*Defined as Hotspot SNVs in COSMICv68, mutation hotspots reported in TCGA, reported in Cheng at. al.(Nature Biotech, 2016) and indels in selected exons of established oncogenes.

**SNVs and Indels other than the ones defined as hotspot mutations above.

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J. Standard/Guidance Document Referenced (if applicable):

Not applicable

K. Test Principle:

The MSK-IMPACT assay is a custom targeted sequencing platform, utilizing solution-phase exon capture and sequencing, to detect somatic alterations (point mutations, small insertions and deletions, and microsatellite instability) in tumor specimens. The MSK-IMPACT assay involves hybridization capture and deep sequencing of all protein-coding exons of 468 cancer-associated genes. The assay uses custom DNA probes corresponding to all exons and selected introns of oncogenes and tumor suppressor genes. Probes are synthesized by a secondary manufacturer and are biotinylated to enable sequence enrichment through capture by streptavidin-conjugated beads. Probes were designed to tile the entire length of each target sequence in an overlapping fashion, typically extending 20-50 base pairs beyond the boundaries of the target. In total, the probes target approximately 1.5Mb of the human genome.

Genomic DNA is extracted from tumor and patient-matched blood/normal tissue as a normal control when available. Sequence libraries are prepared through a series of enzymatic steps including shearing of double-stranded DNA, end repair, A-base addition, ligation of barcoded sequence adaptors, and low cycle PCR amplification. Multiple barcoded sequence libraries are pooled and captured using the custom-designed biotinylated probes. Captured DNA fragments are then sequenced on an Illumina HiSeq2500 as paired-end reads. Sequence reads are then aligned to the reference human genome. By comparing the identity of bases from the tumor DNA to the matched normal DNA and the reference human genome, somatic alterations are identified in the tumor.

L. Performance:

1. Determination of pipeline thresholds:

  • a) Requirements on exon coverage were established: A power analysis to compute the coverage or total number of reads needed to detect a mutation with true underlying mutation frequency 2% or greater, for varying levels of power (0.8 to 0.99), assuming a fixed alpha (Type I error rate) of 0.05 was conducted. Additionally, the 95% confidence interval ranges of observed mutation frequency as a function of coverage was also calculated. When the mutation is present at 10%, the 95% confidence interval with a coverage of 500X is expected to fall between 7.5% and 13%. When the overall coverage is 100X, the 95% CI for a mutation at 10% is estimated to fall between 5.0% and 17.6%.
    To confirm these estimates, empirical data was obtained to measure the range of observed VF to expected VF using DNA from 10 normal FFPE samples from unrelated individuals which was mixed in equimolar parts so as to create a range of SNPs with expected frequencies as low as 5%. A total of 862 common SNPs were considered for this experiment.

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A boxplot showing the observed mutation frequencies for the 862 common SNPs genotyped in the pooled normal sample binned by their true underlying mutation frequency is shown below. The results demonstrated that an observed VF range from 5.0% to 13.9% for a SNP with true underlying mutation frequency of 10% when the mean coverage of the sample was 480X. This range in values is roughly in line with what the theoretical statistical assessment for a coverage depth of 500X (7.5% to 13.0%). This data provided support for using a 5% as the lower limit for reporting mutations detected with true underlying frequency of 10%.

The boxplot in Figure 2 shows the correlation is 0.975, with a slope of 0.971 and intercept of -0.004. Consistent correlation is established as >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 databaseMutations
Filter criteriaDP ≥ 20X, AD ≥ 8,VF ≥ 2%DP ≥ 20X, AD ≥ 10,VF ≥ 5%
SNVsIndelsSNVsIndels
Pre-filter12434240,793
Post-filter0008
Rejection Rate1.001.001.000.999
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

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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-RunInvalidsPre-RunInvalidsPost-RunInvalids
Tumor TypeSpecimenTypeNumberof TestsTumorInsufficient(Tumor %<20%)DNAInsufficient(DNA yield<50ng)SequencingFailure(Coverage<50X)PercentInvalids
Non-Small CellLung CancerFFPE1995532087516.8
BreastCarcinomaFFPE1588411269716.6
ColorectalCancerFFPE11052939319.0
Prostate CancerFFPE87928637118.4
GliomaFFPE601133168.3
PancreaticCancerFFPE58415382914.0
Soft TissueSarcomaFFPE479321137.7
Bladder CancerFFPE4801220259.8
MelanomaFFPE4117221711.2
Renal CellCarcinomaFFPE40312151610.7
HepatobiliaryCancerFFPE39811171510.8
EsophagogastricCarcinomaFFPE374512168.8
Germ CellTumorFFPE332913308.1
Thyroid CancerFFPE2582121310.5
Ovarian CancerFFPE2444888.2
EndometrialCancerFFPE2352877.2
Head and NeckCarcinomaFFPE20888610.5
Cancer ofUnknownPrimaryFFPE22415151017.8

Table 5. Specimen Invalid Rates for 17 FFPE Tumor Types

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3. Analytical performance:

The hybridization-capture-based targeted re-sequencing assay is designed to detect point mutations [also referred to as single nucleotide variants (SNVs)] as well as small insertions/deletions (indels) < 30bp in length in the coding exons of 468 genes (Appendix 1b). A total of 6,357 exons are sequenced, 73 exons were excluded during assay development due to low sequence coverage and high GC content (Appendix 1c). A paired-sample analysis pipeline (tumor vs. matched normal) is used to identify somatic mutations in the targeted exons. MSK took a representative approach to validation of the SNVs and indels targeted in this panel, which is appropriate for variants of this type.1

  • a) Precision Studies: The objective of the precision studies was to assess between-run and within-run precision. Extracted DNA was run once per day for 3 days using different barcodes for inter-day assessment (n=3). For one run, a sample was run in triplicates for intra-day assessment, resulting in a total of 3+1+1=5 replicates. For each replicate tested, all observed mutations were reported and assessed for precision. Details of the study are described below.
    • Precision Panel: The precision of the MSK-IMPACT assay was assessed using i. 10 samples (9 FFPE specimens and one commercial cell line) to represent different tumor types, different mutation types, and the range of mutant allele frequencies. The panel included challenging specimens. The specimen panel was selected based on known mutations corresponding to "Cancer Mutations with Evidence of Clinical Significance" as well as the associated target tissue. The representative list of specimens is shown in Table 6.
Tissue typeMutation typeGene/exoncDNA changeAmino acid changeMutationfrequency
GlioblastomaINSEGFRexon20C2290_2310dupTACGTGATGGCCAGCGTGGACp.Y764_D770dup~5%
CutaneousMelanomaDNVBRAFexon15c.1798_1799delinsAAV600K~6.5%
UterineEndometrialCancerSNVKRASexon2C35G>CG12A~7%
LungAdenocarcinomaINSERBB2exon 202310_2311insGCATACGTGATGE770_A771insAYVM~15%
LungAdenocarcinomaSNVEGFRexon 212573T>GL858R~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 typeMutation typeGene/ exoncDNA changeAmino acid changeMutation frequency
CRCSNVKRAS exon 2C34G>TG12C~30%
Lung AdenocarcinomaDEL (15bp)EGFR exon 192236_2250delGAATTAA GAGAAGCAE746_A750del~30%
CRCSNVBRAF exon 15c.1799T>AV600E~40%
GISTDEL (6bp)Kit exon 111667_1672delAGTGGAQ556_K558del~50%
FFPE Cell LineDEL, SNVHotspot 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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GeneExonMutation(cDNA/ProteinChanges)NCrangeMAF rangeMAFmeanMAFmedianMAF(SD)MAF(%CV)Positive/TotalCallsPositive Call Rate(two-sided 95% CI)
EGFRexon19c.2236_2250delGAATTAAGAGAAGCA746_750del0.84-10.311-0.3420.3230.3160.0134.0%5/5100.0% (47.8%, 100.0%)
PTENexon2c.T83G I28S0.62-0.730.502-0.5690.5430.5440.0275.0%5/5100.0% (47.8%, 100.0%)
TET2exon3c.C311G S104C1.04-1.320.085-0.1030.0980.1020.0088.2%5/5100.0% (47.8%, 100.0%)
TP53exon7c.C742T R248W0.97-1.220.648-0.6640.660.6630.0071.1%5/5100.0% (47.8%, 100.0%)
BRAFexon15c.T1799A V600E1.26-1.440.415-0.4540.4310.4250.0153.5%5/5100.0% (47.8%, 100.0%)
BRCA2exon14c.A7388G N2463S0.84-0.960.19-0.230.2090.210.0157.2%5/5100.0% (47.8%, 100.0%)
BRD4exon19c.G3922A A1308T0.44-0.560.5-0.6360.5530.540.0549.8%5/5100.0% (47.8%, 100.0%)
FBXW7exon9c.G1268T G423V0.91-1.050.369-0.4180.3950.3910.025.1%5/5100.0% (47.8%, 100.0%)
GRIN2Aexon7c.C1514A A505E0.92-1.10.194-0.2110.2020.2030.0063.0%5/5100.0% (47.8%, 100.0%)
PTPRDexon12c.G10A V4I0.5-0.630.281-0.3610.3360.350.03410.1%5/5100.0% (47.8%, 100.0%)
RUNX1exon9c.806-1G>A NA1.01-1.230.185-0.210.2020.2070.015.0%5/5100.0% (47.8%, 100.0%)
SPENexon12c.C10445TP3482L0.94-1.030.189-0.2350.2080.20.0188.7%5/5100.0% (47.8%, 100.0%)
SYKexon13c.C1768T R590W1.13-1.220.233-0.2920.2730.2790.0238.4%5/5100.0% (47.8%, 100.0%)
TP53exon6c.G610T E204X0.9-1.010.525-0.560.5470.5510.0132.4%5/5100.0% (47.8%, 100.0%)
APCexon16c.G3856T E1286X0.8-1.050.326-0.390.3510.3490.0267.4%5/5100.0% (47.8%, 100.0%)
APC exon7c.C646T R216X0.87-1.060.148-0.1850.1620.160.0159.3%5/5100.0% (47.8%, 100.0%)
CREBBPexon29c.G4837AV1613M1-1.190.159-0.1960.1780.180.0179.6%5/5100.0% (47.8%, 100.0%)
KRASexon2c.G34T G12C1.13-1.310.289-0.3520.3140.3050.0247.6%5/5100.0% (47.8%, 100.0%)
NOTCH1exon34c.7541dupCP2514fs1.28-1.50.144-0.2110.1840.1890.02513.6%5/5100.0% (47.8%, 100.0%)
SMAD4exon11c.C1333T R445X0.76-0.950.206-0.2380.2230.2290.0146.3%5/5100.0% (47.8%, 100.0%)
ALOX12Bexon11c.G1406A R469Q1.03-1.310.333-0.3770.3550.3560.0164.5%5/5100.0% (47.8%, 100.0%)
ARID1Bexon1c.1333_1334insCGC A445_P446insP0.2-0.20.2-0.20.20.2NANA1/520.0% (0.5%, 71.6%)
CDK8exon10c.C1014A D338E0.59-0.70.256-0.3360.3030.3150.03210.6%5/5100.0% (47.8%, 100.0%)
DNMT1exon36c.T4380G H1460Q1.18-1.510.51-0.5580.5340.530.0173.2%5/5100.0% (47.8%, 100.0%)
ERBB2exon2c.G140A R47H1.16-1.590.596-0.7120.6560.6660.0456.9%5/5100.0% (47.8%, 100.0%)
ERBB2exon20c.2310_2311insGCATACGTGATGE770_A771insAYVM1.02-1.380.142-0.1990.1730.1710.02313.3%5/5100.0% (47.8%, 100.0%)
ERCC2exon21c.C1904T A635V1.19-1.470.363-0.4660.4090.4230.04511.0%5/5100.0% (47.8%, 100.0%)
IRS1 exon1c.C3639A S1213R0.42-0.490.384-0.4940.4490.4550.048.9%5/5100.0% (47.8%, 100.0%)
MED12exon37c.5258_5282delCTCCTACCCTGCTAGAGCCTGAGAA A1753fs1.08-1.360.141-0.1870.1640.170.01911.6%5/5100.0% (47.8%, 100.0%)
MED12exon43c.6339_6340insCAGCAACACCAGQ2113_Q2114insQQHQ0.96-1.430.37-0.4220.40.3990.0215.3%5/5100.0% (47.8%, 100.0%)
NF1exon51c.C7595T A2532V0.92-1.040.627-0.680.6640.6760.0223.3%5/5100.0% (47.8%, 100.0%)
NTRK1exon1c.G53A G18E0.28-0.550.6-0.6680.6310.630.0274.3%5/5100.0% (47.8%, 100.0%)
PDGFRBexon7c.G946A V316M0.73-1.140.615-0.6810.6460.6420.0264.0%5/5100.0% (47.8%, 100.0%)
PIK3CBexon15c.A2150G N717S0.67-0.850.273-0.3170.2990.3080.0186.0%5/5100.0% (47.8%, 100.0%)
PTPRSexon32c.C4822TR1608W0.79-1.060.526-0.5620.5430.5420.0132.4%5/5100.0% (47.8%, 100.0%)
RB1 exon2c.138-2A>Gsplicing mutation0.51-0.750.231-0.3450.2910.2840.04716.2%5/5100.0% (47.8%, 100.0%)
TET1exon4c.G3476A R1159Q0.86-1.340.499-0.6060.5330.5220.0448.3%5/5100.0% (47.8%, 100.0%)
TP53exon5c.G524A R175H0.75-1.110.247-0.3440.3140.3370.0412.7%5/5100.0% (47.8%, 100.0%)
EGFRexon21c.T2573G L858R1.4-1.440.172-0.2250.1990.2030.0210.1%5/5100.0% (47.8%, 100.0%)
HNF1Aexon4c.C934T L312F0.35-0.540.033-0.0770.0570.0590.01628.1%5/5100.0% (47.8%, 100.0%)
MLL3exon42c.G9671A R3224H1.27-1.40.089-0.1180.1040.1050.01110.6%5/5100.0% (47.8%, 100.0%)
NTRK3exon14c.1401delC P467fs0.49-0.540.062-0.0860.0740.0770.0113.5%5/5100.0% (47.8%, 100.0%)
TP53exon10c.A1051T K351X0.74-0.840.075-0.1160.1030.1080.01615.5%5/5100.0% (47.8%, 100.0%)
AR exonlc.161_171delTGCTGCTGCTGL54fs0.34-0.390.079-0.0970.0880.0870.00910.2%3/560.0% (14.7%, 94.7.0%)
AR exon1c.C190A Q64K0.25-0.290.134-0.1350.1340.1340.0010.7%2/540.0% (5.3%, 85.3%)
KITexon11c.1667_1672delAGTGGA556_558del1.65-1.860.554-0.5950.5690.5660.0162.8%5/5100.0% (47.8%, 100.0%)
KITexon17c.T2467G Y823D1.28-1.490.619-0.6580.6460.6550.0162.5%5/5100.0%(47.8%, 100.0%)
RPS6KB2exon10c.G840T K280N0.93-1.190.435-0.4730.4620.4680.0153.2%5/5100.0%(47.8%, 100.0%)
CARD11exon25c.3382T>Ap.V1128I1.34-1.580.276-0.2930.2840.2780.0093.2%5/5100.0% (47.8%, 100.0%)
EGFRexon20c.2290_2310dupTACGTGATGGCCAGCGTGGACp.Y764_D770dup14.36-15.460.05-0.060.0550.0550.0047.3%5/5100.0% (47.8%, 100.0%)
EGFRexon7c.874G>Tp.V292L21.51-21.820.934-0.9390.9370.9390.0020.2%5/5100.0% (47.8%, 100.0%)
NOTCH3exon22c.3646G>Ap.A1216T1.35-1.520.247-0.3180.2810.2810.0269.3%5/5100.0% (47.8%, 100.0%)
PTENexon5c.395G>Cp.G132A0.6-0.720.605-0.6670.6350.6310.0294.6%5/5100.0% (47.8%, 100.0%)
RUNX1exon8c.899C>Tp.T300M0.81-0.920.244-0.2740.260.2660.0155.8%5/5100.0% (47.8%, 100.0%)
STAG2exon17c.1544_1547delATAG p.D515Gfs*60.19-0.270.677-0.8420.7530.7410.0678.9%5/5100.0% (47.8%, 100.0%)
TERTPromoterg.1295228C>Tnon-coding0.55-0.670.388-0.4670.4210.4170.0337.8%5/5100.0% (47.8%, 100.0%)
AKT3exon2c.134T>G p.V45G1.14-1.360.05-0.0780.0660.0670.01218.2%5/5100.0% (47.8%, 100.0%)
BRAFexon15c.1798_1799delinsAA p.V600K1.04-1.320.065-0.0950.0720.0670.01318.1%5/5100.0% (47.8%, 100.0%)
KITexon11c.1735_1737delGAT p.D579del1.08-1.220.051-0.0560.0530.0540.0023.8%5/5100.0% (47.8%, 100.0%)
CTCFexon3c.610dupAp.T204Nfs*260.68-0.860.041-0.0720.0570.0610.01424.6%5/5100.0% (47.8%, 100.0%)
EGFRexon20c.2317_2319dupCAC p.H773dup1.15-1.190.067-0.0930.0780.0790.01114.1%5/5100.0% (47.8%, 100.0%)
KDM5Cexon23c.3755G>Ap.R1252H0.88-1.170.064-0.130.0880.0840.02629.5%5/5100.0% (47.8%, 100.0%)
KRASexon2c.35G>C p.G12A0.78-0.940.044-0.1060.0760.0740.02330.3%5/5100.0% (47.8%, 100.0%)
PIK3R1exon13c.1672_1683delGAAATTGACAAAp.E558_K561del0.43-0.520.067-0.1160.0850.0810.01922.4%5/5100.0% (47.8%, 100.0%)
PIK3R1exon9c.1023dupAp.E342Rfs*40.41-0.580.056-0.1020.0830.0860.01720.5%5/5100.0% (47.8%, 100.0%)
PIK3R1exon9c.1024G>Tp.E342*0.42-0.590.064-0.1080.0930.0950.01718.3%5/5100.0% (47.8%, 100.0%)
PTENexon6c.493-1G>Ap.X165_splice0.53-0.640.173-0.2080.1920.1870.0157.8%5/5100.0% (47.8%, 100.0%)
PTENexon8c.956_959delCTTTT p.T319Kfs*240.28-0.480.006-0.0790.0490.0520.02959.2%3/560.0% (14.7%, 94.7.0%)
SOX17exon1c.287C>G p.A96G1.16-1.510.061-0.0740.0690.0690.0057.2%5/5100.0% (47.8%, 100.0%)
BRAFexon15c.1798G>AV600M0.97-1.060.016-0.0410.0270.0270.0137.0%3/560.0% (14.7%, 94.7.0%)
BRAFexon15c.1799T>A V600E0.97-1.060.051-0.080.0640.0670.01218.8%5/5100.0% (47.8%, 100.0%)
EGFRexon18c.2155G>AG719S1.23-1.330.125-0.1790.1580.1640.02213.9%5/5100.0% (47.8%, 100.0%)
EGFRexon19c.2235_2249delGGAATTAAGAGAAGCE746_A750del1.01-1.190.009-0.0430.0230.0190.01356.5%2/540.0% (5.3%, 85.3%)
FLT3exon20c.2503G>TD835Y0.97-1.020.037-0.0590.0450.0430.00817.8%5/5100.0% (47.8%, 100.0%)
GNA11exon5c.626A>T Q209L1.41-1.480.036-0.0540.0460.0440.00817.4%5/5100.0% (47.8%, 100.0%)
IDH1exon4c.395G>A R132H0.5-0.530.038-0.0490.0350.0440.02057.1%4/580.0% (28.4%, 99.5%)
KRASexon2c.34G>A G12S0.9-1.030.026-0.0570.0410.0390.01126.8%5/5100.0% (47.8%, 100.0%)
KRASexon2c.38G>A G13D0.91-1.060.217-0.2490.2310.2290.0125.2%5/5100.0% (47.8%, 100.0%)
KRASexon4c.436G>A A146T0.82-0.880.031-0.0550.0420.0440.00921.4%5/5100.0% (47.8%, 100.0%)
NRASexon3c.183A>T Q61H1.01-1.140.039-0.0650.0510.0510.0119.6%5/5100.0% (47.8%, 100.0%)
PIK3CAexon10c.1624G>AE542K0.67-0.870.038-0.0470.0420.0420.0049.5%5/5100.0% (47.8%, 100.0%)
PIK3CAexon21c.3140A>GH1047R0.62-0.720.222-0.3310.2760.2580.0518.1%5/5100.0% (47.8%, 100.0%)

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{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.
SpecimenTotal Nouniquemutationsdetectedacrossall 5replicates**Positive callrateper mutationPositive call rate*(two-sided 95% CI)Negative call rate(two-sided 95% CI)
M15-2292455/5 for all25/25100.0% (86.3%, 100.0%)-
M15-303835/5 for all15/15100.0% (78.2%, 100.0%)-
M16-19000105/5 for 94/5 for 149/5098.0% (89.4%, 99.9%)-
M1688-5C185/5 for 171/5 for 186/9095.6% (89.0%, 98.8%)4/580.0% (28.4%,99.5%)
M-1698-A955/5 for all25/25100.0% (86.3%, 100.0%)-
M-1654-CA65/5 for all30/30100.0% (88.4%, 100.0%)-
M-1612-2845/5 for all20/20100.0% (83.2%, 100.0%)-
M1648-D5105/5 for all50/50100.0% (92.9%, 100.0%)-
M-1707-1255/5 for 33/5 for 1;2/5 for 120/2580.0% (59.3%, 93.2%)3/560.0% (14.7%,94.7%)
Commercialsample135/5 for 10;4/5 for 1 ;3/5 for 1;2/5 for 159/6590.8% (81.0%, 96.5%)3/560.0% (14.7%,94.7%)

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).

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  • 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.
NTotalSites_rangeUnstableLoci_rangeMeanMedianSD%CVPositive CallRate (two-sided 95% CI)
51227-1458518-65043.0043.001.222.8%100%(47.8%,100.0%)
51158-1477483-64643.0043.000.711.7%100%(47.8%,100.0%)
51187-1429500-61342.0042.000.711.7%100%(47.8%,100.0%)
51287-1400303-35924.8025.000.843.4%100%(47.8%,100.0%)
51251-1303240-31823.4024.002.5110.7%100%(47.8%,100.0%)
51154-1379153-17512.6012.000.897.1%100%(47.8%,100.0%)

Table 9. Precision of the MSIsensor Score Using 12 Specimens

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NTotalSites_rangeUnstableLoci_rangeMeanMedianSD%CVPositive CallRate (two-sided 95% CI)
51321-154546-583.604.000.5515.3%100%(47.8%,100.0%)
51535-160444-643.403.000.5516.2%100%(47.8%,100.0%)
51411-161228-382.202.000.4520.5%100%(47.8%,100.0%)
51438-15286-90.480.500.0816.7%100%(47.8%,100.0%)
51315-14870-20.020.000.04223.6%100%(47.8%,100.0%)
51312-15320-10.010.000.03223.6%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.

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Table 10A. Limit of Detection -Part 1
-------------------------------------------
SNV BRAF Exon 15 (Sample M-1648-D5-T)
DilutioncDNAchangeAA ChangeDPADVFResult
Neatc.1799T>AV600E10184100.4Called
1:210443190.31Called
1:48881730.19Called
1:8999910.09Called
1:16783260.03Called
1:32845200.02Called

Table 10B

SNV KRAS Exon 2 (sample M-1807-ED-T)
DilutioncDNA changeAA ChangeDPADVFResult
NeatG12D9074050.45Called
1:28202980.36Called
1:4c.35G>A400970.24Called
1:86601210.18Called
1:16ર્ભર્ટਦੇ ਰੇ0.09Called
1:32632410.06Called

Table 10C

SNV PIK3CA Exon 2 (Sample M-1729-E1-T)
DilutioncDNAchangeAA ChangeDPADVFResult
NeatR88Q20296290.31Called
1:2c.263G>A10082110.21Called
1:411401450.13Called
1:8997620.06Called
1:16WT

Table 10D

DilutioncDNAchangeAA ChangeDPADVFResult
Neat25039220.37Called
1:219866880.35Called
1:415134300.28Called
1:8c.1667_1681delAGT556_561del10492500.24Called
1:16GGAAGGTTGTTG7921380.17Called
1:32761660.09Called
1:64618370.06Called
1:125736180.02Called

Table 10E

EGFR exon 20 Insertion (sample M-1674-10-T)
DilutioncDNAchangeAA ChangeDPADVFResult
Neat14844000.27Called
1:27771660.21Called
1:4c.2308_2309insACTD770_N771insY5661050.19Called
1:8595550.09Called
1:16581330.06Called
1:32608210.03Called

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Table 10F

SNV ERBB2 exon 20 (sample M-1801-98-T)
DilutioncDNAchangeAA ChangeDPADVFResult
Neat14714080.28Called
1:214822400.16Called
1:4c.2525A>TD842V864730.08Called
1:8903380.04Called
1:16873240.03Called

Table 10G

SNV PDGFR α Exon 18 (sample M-1670-A6-T
DilutioncDNAchangeAA ChangeDPADVFResult
Neat4482360.53Called
1:26361420.22Called
1:4c.1633G>AE545K962950.1Called
1:8647450.07Called
1:16707160.02Called

Table 10H

SNV PK3CA exon 10 (sample M-1434-A5-T)
DilutioncDNAchangeAA ChangeDPADVFResult
Neat4482360.53Called
1:26361420.22Called
1:4c.1633G>AE545K962950.1Called
1:8647450.07Called
1:16707160.02Called

Table 10I

EGFR exon 19 deletion (sample M-1809-C4-T)
DilutioncDNAchangeAA ChangeDPADVFResult
Neatc.2236_2250delGAATTAAGAGAAGCA746_750del12787900.62Called
1:211374840.43Called
1:47922070.26Called
1:8666940.14Called
1:16622490.08Called
1:32746_750del499170.03Called

Table 10J

Kit Exon 9 insertion (sample M-1754-DB-T)
DilutioncDNA changeAA ChangeDPADVFResult
Neat5173140.61Called
1:25121870.37Called
1:4c.1502_1503insTGCCTAS501_A502insAY641890.14Called
1:8486270.06Called
1:16447170.04Called
1:32521140.03Called

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  • 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.
TypeMutationGeneExonRangeDPRangeADRangeMAFRangeNormDPPositiveCallRate
DELIn_Frame_Delc.1735_1737delGATp.D579delKIT exon11509-69326-380.051-0.0561.08-1.22100.0%
DELFrame_Shift_Delc.956_959delCTTTp.T319Kfs*24PTEN exon8197-2427-190.036-0.0790.31-0.4880.0%
DELIn_Frame_Delc.1672_1683delGAAATTGACAAAp.E558_K561delPIK3R1exon13216-31318-360.067-0.1160.43-0.52100.0%
INSIn_Frame_Insc.2317_2319dupCACp.H773dupEGFR exon20587-74946-650.067-0.0931.15-1.19100.0%
INSFrame_Shift_Insc.1023dupA p.E342Rfs*4PIK3R1 exon9236-34515-320.056-0.1020.41-0.58100.0%
INSFrame_Shift_Insc.610dupA p.T204Nfs*26CTCF exon3344-54014-360.041-0.0720.68-0.86100.0%
INSIn_Frame_Insc.2290_2310dupTACGTGATGGCCAGCGTGGACp.Y764_D770dupEGFR exon208601-9836441-5720.05-0.0614.36-15.46100.0%
SNVMissense_Mutationc.134T>G p.V45GAKT3 exon2535-81328-630.05-0.0781.14-1.36100.0%
SNVMissense_Mutationc.1798_1799delinsAAp.V600KBRAF exon15489-74733-710.065-0.0951.04-1.32100.0%
SNVMissense Mutationc.287C>G p.A96GSOX17 exon1672-80545-590.061-0.0741.16-1.51100.0%
SNVMissense_Mutationc.35G>C p.G12AKRAS exon2445-57120-550.044-0.1060.78-0.94100.0%
SNVMissense_Mutationc.3755G>A p.R1252HKDM5Cexon23475-73340-680.064-0.130.88-1.17100.0%
SNVNonsense_Mutationc.1024G>T p.E342*PIK3R1 exon9242-35518-370.064-0.1080.42-0.59100.0%

Table 11. Limit of Detection– Part 2

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  • 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 PurityCoverage# Total site# Unstable lociMSIsensor Score (%)
Diluted to 8%517142018213
Diluted to 8%562138917513
Diluted to 8%555135218514
Diluted to 8%502136113510
Diluted to 8%378127315212
Sample IDReplicateMSI Sensor Score
CRC-0110.00000
20.00000
30.00000
CRC-0410.00000
20.00000
30.00000
CRC-0810.00000
20.00000
30.00000
  • 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 InputSuccessSequencing Failure
250ng97%3%
201-249ng87%13%
151-200ng87%13%
101-150ng81%19%
50-100ng78%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.

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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)TypeMutations Assessed
AKT10exon3SNVE17K
ALK3exon23SNVF1174V/L;S1205F
ALK4exon25SNVR1275Q;R1260T
BRAF11exon11SNVG466V/R;S467L;G469*
BRAF19exon15SNVD594G;V600*;K601I
EGFR10exon18SNVG719A/S; G724S
EGFR12exon19DEL745_750del; 746_748del; 746_750del;747_753del; K754fs
Gene(n=20)#Samples(n=433)Exon(n=48)TypeMutations Assessed
10exon20SNVT790M
16exon20INSM766_A767insASV; V769_D770insDNP;D770_N771ins*;P772_H773ins*;H773_V774insY/H
9exon21SNVL858R
ERBB27exon19SNVL755S;I767M;D769Y
16exon20INSE770_A771insAYVM;A771_Y772insYVMA;G776_G778ins*
3exon20SNVV777L;G776V
7exon8SNVS310F/Y; S305C
FGFR21exon12SNVL528H
1exon7SNVS252W
1exon9SNVY375C
FGFR32exon18SNVP797L
1exon7SNVA261V;A265V
5exon9SNVF384L
1exon9INSG370_S371insH
GNA117exon5SNVQ209L
GNAQ5exon5SNVQ209P/L
GNAS5exon8SNVR201C/H
HRAS3exon2SNVG10A; G13D/V
5exon3SNVA59V; Q61R/L/K
IDH18exon4SNVR132G/C/H
IDH25exon4SNVR172*;R140Q
1exon4DELT146Lfs*15
9exon11INS; DELK550fs; 552_557del; 556_558del;556_561del; 558_565del; 559_566del;P573_T574insTQLPS
KIT9exon11SNVV555L; W557G; V559D; D572G;L576P.
6exon13SNVV654A; K642E
5exon17SNVD816H; D820E; N822K
10exon9INSS501_A502insAY; A502_Y503dup
KRAS16exon2SNVG12*; G13D
13exon3SNVQ61*
10exon4SNVK117N;G138E;A146*
MET13exon14SNVD1010*; Exon14 skipping
19exon14DELExon14 skipping; Other splicing defects
NRAS4exon2SNVG13*
12exon3SNVQ61*
PDGFRA12exon18SNVD842V/I
1exon12SNVV561D
4exon10SNVE545A/K; E542K
PIK3CA2exon21SNVH1047R/Y
1exon21INSX1069delinsFL
8exon2SNV/MNVF83L;R88Q;R93Q;K111E/N
2exon2DELE110del; 112_113del
9exon5SNVV344M;N345I/K
9exon8SNVE418K;C420R;P449R;E453K/Q
1exon8DELE453_D454del
TP539exon4SNV/MNVW53X;W91X;Q100X;G105V/C; S106R;F113C
6exon4DELL355fs;P67fs;A84fs;109_109del;G108fs;
Gene#SamplesExon
(n=20)(n=433)(n=48)TypeMutations Assessed
R110fs
3exon4INSV73fs;L114fs;C124fs
бexon5SNVK132Q;W146X; Y163C; R175H; R158H
3INSP153fs; M160 A161insRA;
exon5Q167_M170dup
K132fs;A138fs;P152fs; R156fs;
9exon5DELV157_R158del; K164fs; H178fs;D184fs
2exon6SNVR213L/X
G187fs; L188fs; P191_Q192del;
8exon6DELR196_L201del; D207fs; R209fs; F212fs
Y234C; Y236C; M237I; R248G/Q;
10exon7SNV/MNVR249S; T256P
3exon7INSS241dup; R249fs; T253dup
S241fs; M243X; G244fs; M246X;
6exon7DELI255del; L257fs
イexon8SNV/MNVV272K; C275X; R282W; T284K
4exon8INSC275fs; N288fs; G302fs
N263 N268del; N263fs; R267fs; P278fs;
5exon8DELP301fs
бexon10SNVR337L; R342X; R337C
lexon10INSL344fs

Table 14. Mutations Represented in the Accuracy Summary Per Gene

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{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}------------------------------------------------

GeneNumberof exonsNumber ofuniquemutationsNumber ofsamplesPPA (95% CI)
AKT11110100.0% (69.2%, 100.0%)
ALK257100.0% (59.0%, 100.0%)
BRAF21330100.0% (88.4%, 100.0%)
EGFR3630100.0% (88.4%, 100.0%)
ERBB231217100.0% (80.5%, 100.0%)
FGFR2333100.0% (29.2%, 100.0%)
FGFR3338100.0% (63.1%, 100.0%)
GNA11117100.0% (59.0%, 100.0%)
GNAQ125100.0% (47.8%, 100.0%)
GNAS125100.0% (47.8%, 100.0%)
HRAS278100.0% (63.1%, 100.0%)
IDH1138100.0% (63.1%, 100.0%)
IDH2146100.0% (54.1%, 100.0%)
KIT31320100.0% (83.2%, 100.0%)
KRAS31539100.0% (91.0%, 100.0%)
MET1913100.0% (75.3%, 100.0%)
NRAS2616100.0% (79.4%, 100.0%)
PDGFRA2313100.0% (75.3%, 100.0%)
PIK3CA41932100.0% (89.1%, 100.0%)
TP5363237100.0% (90.5%, 100.0%)

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

Table 15B. Percent Positive Agreement for insertions by gene

GeneNumberof exonsNumber ofuniquemutationsNumber ofsamplesPPA (95% CI)
EGFR121693.8% (69.8%, 100.0%)
ERBB2oc16100.0% (79.4%, 100.0%)
FGFR31100.0% (2.5%, 100.0%)
KITಗ10100.0% (69.2%, 100.0%)
PIK3CA1100.0% (2.5%, 100.0%)
TP53ર1414100.0% (76.8%, 100.0%
Table 15C. Percent Positive Agreement for deletions by gene
GeneNumber ofexonsNo. uniquemutationsNumber ofsamplesPPA (95% CI)
EGFR1612100.0% (73.5%, 100.0%)
IDH2111100.0% (2.5%, 100.0%)
KIT179100.0% (66.4%, 100.0%)
MET11819100.0% (82.4%, 100.0%)
PIK3CA233100.0% (29.2%, 100.0%)
TP5351414100.0% (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

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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 IHCMMR-D*MMR-P*Total
MSI SensorMSI-H ≥ 1012113
MSS < 1015253
Total135366
PPV = 92.3% (12/13) 95% CI 64.0%-99.8%
NPV = 98.1%. (52/53) 95% CI 90.0%, 100.0%

Table 16. MSIsensor Results Compared to IHC MMR for CRC

*MMR-D refers to deficient in mismatch repair proteins and MMR-P indicates not deficient

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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. The results are shown in Table 17. Excluding the specimens without a MSI-PCR result from the total number of specimens analyzed, PPV is 46/49=93.9% (83.1%, 98.7%), and NPV is 58/60=96.7% (88.5%, 99.6%). When including all missing data in the analysis (i.e., consider all PCR unknown data as discordant results), the PPV=46/59=78.0% (65.3%. 87.7%). NPV= 58/60=96.7% (88.5%. 99.6%). (The MSIsensor MSI-H/MSS definition is based on genome wide analysis of over 1000 microsatellite markers and not based on the 5 or 7 MSI loci described in current clinical practice guidelines.)

Non CRC/EC concordance with MSI-PCRPCR Results
MSI-HMSI-L/MSSUnknown*Total
MSIsensorMSI-H(≥10)4631059
MSS(≥2 & <10)258060
Total486110119
Excluding missingspecimens with 95%CIPPV is 46/49=93.9% 95%CI (83.1%, 98.7%)
NPV is 58/60=96.7% (88.5%, 99.6%)
Accounting formissing specimens with95%CIPPV=46/59=78.0% (65.3%, 87.7%)
NPV= 58/60=96.7% (88.5%, 99.6%)

Table 17. MSIsensor Results Compared to PCR 5 Loci MSI Panel for Other Cancer Types

  • In exploratory analysis, the 10 without PCR results were all MMR-D by IHC, consistent with the MSI-H by MSIsensor findings.

MSI Supplemental Information:

The mean, median and range of MSIsensor score was determined in a large cohort of 10,900 patients with 66 different types advanced solid tumor. The MSIsensor scores ranged from 0 to 48.5, mean 1.2, median 0.4. The prevalence of MSI-H by MSIsensor was also determined, and the findings are consistent with the MSI-H prevalence as described in the literature (data not shown).

3. Clinical Performance:

MSK-IMPACT assay is a molecular profiling platform using next generation sequencing to detect somatic alterations (point mutations and small insertions and deletions and microsatellite instability) in tumor specimens using a 468 gene panel. The genes in the panel were selected for their role in cancer pathogenesis and tumor

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suppression, or for clinical or mechanistic information of relevance in the management of cancer patients. The assay reports mutations under two categories: "Cancer mutations with evidence of clinical significance" and "Cancer mutations with potential clinical significance" consistent with the intended use clinical settings. Mutations with evidence of clinical significance are represented in professional guidelines as established by consensus opinion of experts in the health care community.

Clinical Evidence Curation:

MSK-IMPACT uses a clinical evidence curation resource (OncoKB) to facilitate the clinical interpretation of detected mutations. OncoKB is a knowledge base that includes biologic, clinical and therapeutic information curated from multiple information resources including professional guidelines and recommendations, therapeutic labeling, disease specific expert and advocacy group recommendations, and medical literature. OncoKB information is publicly available through an interactive web site. Classification criteria were developed by MSK to communicate the level of clinical evidence available for individual mutations in the test report. The mutations are reported under two categories (i.e., cancer mutations panel with evidence of clinical significance and cancer mutations panel with potential clinical significance) based on the pre-specified classification criteria. OncoKB undergoes periodic updates through the review of new information by a panel of experts.

4. Clinical cut-off:

Not applicable.

5. Expected values:

The prevalence of somatic mutations was explored through a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which tumor and matched normal sequence data from a cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations was compiled. The prevalence of mutations and cancer type via the link to the publicly accessible data on cohort of tested patients and available pathological and clinical annotations was published by Zehir, A. et al., "Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients." 2017. 23(6):703-713. This information is also available at the following website. (http://www.cbioportal.org/study?id=msk impact 2017#summary)

N. Instrument Name

Illumina HiSeq 2500 (qualified by MSK)

O. System Descriptions:

1. Modes of Operation:

The Illumina HiSeq2500 is a high throughput sequencing system using Sequencing-By-Synthesis chemistry.

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2. Software:

FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes X or No

    1. Level of Concern: Moderate
    1. Specimen Handling Refer to Device Description section above.
  • 5 Calibration and Quality Controls: Refer to Device Description section above.

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section Above:

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 changeValidation StrategyPre-specified success criteria
New pre-analytical protocol, kits orreagentsSequence at least 10specimens withknown mutations.Measure sequencecoveragedistribution, and callsomatic mutations inall samples.For cases sequenced to>200x, ensure that 95% ofexons are covered to 100xor more. Concordance forknown mutations shouldbe >95%.
New library preparation protocol, kits,or reagentsSequence at least 40DNA specimens(tumor / normalpairs) or three poolspreviouslysequenced by MSK-IMPACT. Measuresequence coveragedistribution, and callFor cases sequenced to>200x, ensure that 95% ofexons are covered to 100xor more. Concordance forcalling somatic mutationswith variant allele fraction>10% should be >98%.
Type of changeValidation StrategyPre-specified successcriteria
somatic mutations inall samples.
Changes to probes for alreadyanalytically validated genesRe-capture existingsequence librariesfrom at least 3 runs(at least 40 samples)with new probes,sequence, andanalyze.For cases sequenced to>200x, ensure that 95% ofexons in analyticallyvalidated genes arecovered to 100x or more.Concordance for callingsomatic mutations withvariant allele fraction>10% should be >98%.
New sequencing instrument or reagentsusing similar chemistry and technology,and the sequence depth and read lengthare not changed from previousplatform.Re-sequenceexisting capturedlibraries from atleast 3 runs, and callsomatic mutations inall samples.Sequence coveragedistribution and GC biasacross targeted regionsshould be within 5% ofprior sequencing runs.Concordance for callingsomatic mutations withvariant allele fraction>10% should be >98%.
BioinformaticspipelineUpdate tounderlyingannotationdatabase ortranscriptisoformsReanalyze FASTQfiles (rawsequencing reads)from at least 3 runs(at least 40 samples).Compare variantscalls between theclinical analysisresults and thecurrent modifiedresultsConfirm the changes donot change the variant callresults. Confirm theannotations for theunaffected transcripts donot change. Confirm theannotations for theaffected transcripts aremodified as expected.
Update to datamanagementsystem andsystem databaseReanalyze FASTQfiles (rawsequencing reads)from at least 3 runs(at least 40 samples)in production mode.Compare variantscalls between theclinical analysisresults and thecurrent modifiedresultsEnsure that all previouslycalled mutations arerecovered and the variantsin the database of resultsare concordant with thevariants in the pipelineoutput files
Type of changeValidation StrategyPre-specified success criteria
Modification toan existingcomponent ofthe analysispipeline (e.g.,tool oralgorithm)where theunderlyingalgorithm ormain parametersettings (e.g.minimalcoverage/VAFthreshold forSNV/indelcalling;MSIsensorscore cut-off forMSI-H calling,etc.) are notchanged.Reanalyze FASTQfiles (rawsequencing reads)from at least 3 runs(at least 40 samples).Compare variantscalls between theclinical analysisresults and thecurrent modifiedresultsEnsure that all previouslycalled mutations arerecovered and that newlydetected mutations can beexplained by pipelinemodifications.

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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 HealthIdentified Mitigations
Incorrect performance of the testleading to false positives, falsenegativesGeneral controls and special control (b)(1)
Incorrect interpretation of test resultsGeneral controls and special controls(b)(1)(iii)(E) and (b)(2)

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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 FactorsLimitations 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.
ConclusionsDo 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

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(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.

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(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

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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:

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(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.

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Appendix 1a:

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

GeneCodons
ABL1G250, Q252, Y253, E255, T315, F317, M351, F359, H396R
AKT1E17,Q124,G171,E170
AKT2V140
ALKK1062,D1091,C1156,M1166,I1171,F1174,L1196,A1234,F1245,I1250,R1275,Y1278
APCS1234,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
ART878,T8782,Q581
ARAFS214
ARID1AD1850,G2087
ARID2R314,S297,R285,A1773
ASXL1Y591,E635,G645,G646,E1102D
ASXL2R591
ATMD1853,R3008,R3376,E2164
ATRXK1936,E625
BARD1P24
BCL6R594,R618
BCORN1425,N14591
BRAFG464,G466,G469,Y472,N581,D594,F595,G596,L597,A598_T599,V600,V600_K601,K6010,V60010,K6010,G4694,N5810,G4660
CARD11R170
CBLY371,L380,C384,C404,R420Q
CDH1T263
CDK4R24
CDKN2AS43,P48,A57,A68,D74,L78,P81,H83,D84,L97,D108,P114,H831,D1081,P1140
CEBPAP23,H24,Q83,K304_Q305,E309_T310,Q312_K313,K313_V314,K313_V314,K313,E316_L317,E316_L317insQ
CHEK2K373,K3732
CICR215
CREBBPR1446,S1680,R14460
CRLF2F232C
CSF1RY969C
CTCFR377
CTNNB1D32,S33,G34,I35,H36,S37,T40,T41,T42,A43,P44,S45,G48,K49,E53,K335,S376,S336,D324,T412,G349,S455,C619
DICER1E1813
DIS3R382,D488
DNMT1E432
DNMT3AG543,R635,S714,F731,R882,R8820
DOT1LG1386
EGFRR108,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
EP300D1399,D13990,C1164
EPHB1R170
ERBB2S310,L755,D769,A775_G776,G776,V777,V842,S3108,L7553,E930,R678
ERBB3V1043,D297,M91
ERBB4R711
ERCC2D312
ESR1Y537
ETV1R187
ETV6R369
EZH2Y646,R690
FBXW7G423,R465,R479,R505,S582,R689,R4652,R5054,R4792
FGFR2S252,P253,C382,N549,N550,K659
FGFR3R248,S249,G370,S371,Y373,G380,A391,K650,G697,S2492,Y3730
FGFR4V550
FLT3D835,I836,D8358
FOXL2C134W
FUBP1R430
GATA1M1,S30,V74I
GATA2G320,L321,L359,R362Q
GNA11R183,Q209,R256
GNAQR183,Q209
GNASR201,Q227,R8448
GRIN2AR1067
HIST1H3EE74
HNF1AW206,P291,G292
HRASG12,G13,Q61,E62,Q614,G136,G122
IDH1G70,V71,R132,V178,R13239,P33
IDH2R140,R172,V294,R1402,R1721
IL7RK395
IRS2G1057
JAK1R873
JAK2F537_K539,H538_K539,K539,I540_E543,R541_E543,N542_E543,E543_D544,V617
JAK2R683
JAK3A572,A573,R657Q
KDRS1100,E759
KEAP1R470
KITD52,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,E56Y570_L576,D572,L576,D579,K642,V654,T670,S715,D816,K818,D820,N822,Y823,V25, D8160
KMT2CV656
KRASG10_A11,G12,G13,V14,L19,Q22,T58,A59,Q61,K117,A146,G1242,G133,Q619,A146
LATS2A3243,G3630
MAP2K1Q56,K57,D67,P124,P1240,F53,E203
MAP2K4R134
MAP3K1S1330,S939
MAPK1E322
MAXR600
MED12L36,Q43,G44,L1224,L12240
MEF2BD83V
METT1010,Y1248,Y1253,M1268,K1360
MLL3K2797
MPLS505,W515,W515R
MSH6F1088,T1219I
MTORS22152,F1888
MYCT58
MYCNP44
MYD88S219,S243,L265P
NF1L844
NFE2L2D29,L30,G31,R34,E79,T80,G81,E82,E794,D294,R342
NOTCH1L1574,L1575,V1578,L1585,L1586,F1592,L1593,L1594,R1598,R1599,L1600,L1601,L1678,L1679,Q2460,P2514,A1944
NOTCH2E385,N463
NPM1W288,W290
NRASG12,G13,A18,G60,Q61,Q6193,G128,G138
NTRK1T264
PAK7E144
PARP1I562
PAX5P80R
PDGFRAV561,S566_E571,N659,D842,I843_D846,D1071N
PIK3C2GS670
PIK3CAR38,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
PIK3R1G376,D560,N564,K567
POLEP2864,V4111
PPP2R1AP179,R182,R183,S256,W257,R258,R1832
PREX2G233C
PTCH1P1315
PTENK6,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
PTPN11G60,D61,E69,A72,T73,E76,S502,G503,Q510
PTPRDS431,P666
RAC1P295
RAF1S2570
RETE632_T636,E632_L633,C634,M918T
RHOAE40,Y42
RICTORS1101
RIT1M90
RUNX1L56,R107,D198,R201,R204,R162,R205
SDHAS4560,A466,R465
SF3B1E622,R625,H662,K666,K700,K7002
SMAD4A118,D351,R361,G386,R3619,D537,P356
SMARCAT910,G1232
SMARCBR377,A382,P383
SMOW535L
SPOPF133,F1338,W131,F102
SRSF2P95,P95_R102,P107H
STAG2R370
STK11D194,P281,F354L
TET2C25,C262,Q764,F868,R1261,H1380,V1718L
TNFAIP3L324
TP53E11,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
TP63R379
TSC2N1515
TSHRM453,I486,L512,I568,D619,A623,L629,I630,T632,D633,D633E
U2AF1S34,Q157,S347
VHLV62,S65,S72,V74,F76,N78,S80,P81,L85,P86,L89,N90,S111,G114,H115,L118,D121,
VHLL128,V130,G144,F148,I151,L153,V155,L158,E160,C162,V166,R167,L169,L184
WT1V303,R312,A314,R394,D396,R462
XP01E571,R749

{43}------------------------------------------------

{44}------------------------------------------------

{45}------------------------------------------------

{46}------------------------------------------------

{47}------------------------------------------------

Gene NameTranscript ID
ABL1NM_005157
ACVR1NM_001111067
AGO2NM_012154
AKT1NM_001014431
AKT2NM_001626
AKT3NM_005465
ALKNM_004304
ALOX12BNM_001139
AMER1NM_152424
ANKRD11NM_013275
APCNM_000038
ARNM_000044
ARAFNM_001654
ARID1ANM_006015
ARID1BNM_020732
ARID2NM_152641
ARID5BNM_032199
ASXL1NM_015338
ASXL2NM_018263
ATMNM_000051
ATRNM_001184
ATRXNM_000489
AURKANM_003600
AURKBNM_004217
AXIN1NM_003502
AXIN2NM_004655
AXLNM_021913
B2MNM_004048
BABAM1NM_001033549
BAP1NM_004656
BARD1NM_000465
BBC3NM_001127240
BCL10NM_003921
BCL2NM_000633
BCL2L1NM_138578
BCL2L11NM_138621
BCL6NM_001706
BCORNM_001123385
BIRC3NM_182962
BLMNM_000057
BMPR1ANM_004329
BRAFNM_004333
BRCA1NM_007294
BRCA2NM_000059
BRD4NM_058243
BRIP1NM_032043
BTKNM_000061
CALRNM_004343
CARD11NM_032415
CARM1NM_199141
CASP8NM_001080125
CBFBNM_022845
CBLNM_005188
CCND1NM_053056
CCND2NM_001759
CCND3NM_001760
CCNE1NM_001238
CD274CD276NM_014143NM_001024736
CD79ANM_001783
CD79BNM_001039933
CDC42NM_001791
CDC73NM_024529
Appendix 1b: List of genes/transcripts included on the MSK-IMPACT panel
---------------------------------------------------------------------------

{48}------------------------------------------------

CDH1NM_004360
CDK12NM_016507
CDK4NM_000075
CDK6NM_001145306
CDK8NM_001260
CDKN1ANM_078467
CDKN1BNM_004064
CDKN2Ap14ARFNM_058195
CDKN2Ap16INK4ANM_000077
CDKN2BNM_004936
CDKN2CNM_078626
CEBPANM_004364
CENPANM_001809
CHEK1NM_001274
CHEK2NM_007194
CICNM_015125
CREBBPNM_004380
CRKLNM_005207
CRLF2NM_022148
CSDE1NM_001242891
CSF1RNM_005211
CSF3RNM_000760
CTCFNM_006565
CTLA4NM_005214
CTNNB1NM_001904
CUL3NM_003590
CXCR4NM_003467
CYLDNM_001042355
CYSLTR2NM_020377
DAXXNM_001141970
DCUN1D1NM_020640
DDR2NM_006182
DICER1NM_030621
DIS3NM_014953
DNAJB1NM_006145
DNMT1NM_001379
DNMT3ANM_022552
DNMT3BNM_006892
DOT1LNM_032482
DROSHANM_013235
DUSP4NM_001394
E2F3NM_001949
EEDNM_003797
EGFL7NM_201446
EGFRNM_005228
EIF1AXNM_001412
EIF4A2NM_001967
EIF4ENM_001130678
ELF3NM_004433
EP300NM_001429
EPAS1NM_001430
EPCAMNM_002354
EPHA3NM_005233
EPHA5NM_004439
EPHA7NM_004440
EPHB1NM_004441
ERBB2NM_004448
ERBB3NM_001982
ERBB4NM_005235
ERCC2NM_000400
ERCC3ERCC3NM_000122NM_000122
ERCC4NM_005236
ERCC5NM_000123
ERFNM_006494
ERGNM_182918
ERRFI1NM_018948
ESR1NM_001122740
ETV1NM_001163147
ETV6NM_001987
EZH1NM_001991
EZH2NM_004456
FAM175ANM_139076
FAM46CNM_017709
FAM58ANM_152274
FANCANM_000135
FANCCNM_000136
FAT1NM_005245
FBXW7NM_033632
FGF19NM_005117
FGF3NM_005247
FGF4NM_002007
FGFR1NM_001174067
FGFR2NM_000141
FGFR3NM_000142
FGFR4NM_213647
FHNM_000143
FLCNNM_144997
FLT1NM_002019
FLT3NM_004119
FLT4NM_182925
FOXA1NM_004496
FOXL2NM_023067
FOXO1NM_002015
FOXP1NM_001244814
FUBP1NM_003902
FYNNM_153047
GATA1NM_002049
GATA2NM_032638
GATA3NM_002051
GLI1NM_005269
GNA11NM_002067
GNAQNM_002072
GNASNM_000516
GPS2NM_004489
GREM1NM_013372
GRIN2ANM_001134407
GSK3BNM_002093
H3F3ANM_002107
H3F3BNM_005324
H3F3CNM_001013699
HGFNM_000601
HIST1H1CNM_005319
HIST1H2BDNM_021063
HIST1H3ANM_003529
HIST1H3BNM_003537
HIST1H3CNM_003531
HIST1H3DNM_003530
HIST1H3ENM_003532
HIST1H3FNM_021018
HIST1H3GNM_003534
HIST1H3HNM_003536
HIST1H3INM_003533
HIST1H3JNM_003535
HIST2H3CNM_021059
HIST2H3DNM_001123375
HIST3H3NM_003493
HLA-ANM_001242758
HLA-BHLA-BNM_005514NM_005514
HNF1ANM_000545
HOXB13NM_006361
HRASNM_001130442
ICOSLGNM_015259

{49}------------------------------------------------

{50}------------------------------------------------

ID3NM002167
IDHI005896NM
IDH2002168NM
IFNGR1000416NM
IGF100111285NM
IGFIRNM000875
IGF2NM_001127598
IKBKE014002NM
IKZF1NM006060
IL10NM000572
IL7RNM002185
INHANM002191
INHBANM002192
INPP4A001134224NM
INPP4BINPPL1001101669NM001567NM
INSR000208NM
IRF4NM002460
IRSINM005544
IRS2NM_003749
JAK1002227NM
JAK2004972NM
JAK3000215NM
JUN002228NM
KDM5ANM001042603
KDM5C004187NM
KDM6ANM021140
KDR002253NM_
KEAP1NM203500
KIT000222NM
KLF4NM004235
KMT2ANM001197104
KMT2B_014727NM
KMT2C170606NM
KMT2D003482NM
KNSTRN033286NM
KRASNM033360
LATS 1004690NM
LATS2NM014572
LMO1NM002315
LYN002350NM006785
MALT1MAP2K1NMNM002755
MAP2K2NM030662
MAP2K4NM003010
MAP3K1005921NM
MAP3K13NM_004721
MAP3K14NM003954
MAPK1002745NM
MAPK3NM_002746
MAPKAP1_001006617NM
MAXNM002382
MCL1NM021960
MDC1NM_014641
MDM2002392NM
MDM4002393NM
MED12NM005120
MEF2B001145785NM
MENI000244NM
MET000245NM
MGA001164273NM
MITF198159NM
MLHINM_000249
MPL005373NM_
MRE11A005591NM_
MSH2000251NM

{51}------------------------------------------------

MSH3NM_002439
MSH6NM_000179
MSI1NM_002442
MSI2NM_138962
MST1NM_020998
MST1RNM_002447
MTORNM_004958
MUTYHNM_001128425
MYCNM_002467
MYCL1NM_001033082
MYCNNM_005378
MYD88NM_002468
MYOD1NM_002478
NBNNM_002485
NCOA3NM_181659
NCOR1NM_006311
NEGR1NM_173808
NF1NM_001042492
NF2NM_000268
NFE2L2NM_006164
NFKBIANM_020529
NKX2-1NM_001079668
NKX3-1NM_006167
NOTCH1NM_017617
NOTCH2NM_024408
NOTCH3NM_000435
NOTCH4NM_004557
NPM1NM_002520
NRASNM_002524
NSD1NM_022455
NTHL1NM_002528
NTRK1NM_002529
NTRK2NM_006180
NTRK3NM_001012338
NUF2NM_031423
NUP93NM_014669
PAK1NM_002576
PAK7NM_177990
PALB2NM_024675
PARK2NM_004562
PARP1NM_001618
PAX5NM_016734
PBRM1NM_018313
PDCD1NM_005018
PDCD1LG2NM_025239
PDGFRANM_006206
PDGFRBNM_002609
PDPK1NM_002613
PGRNM_000926
PHOX2BNM_003924
PIK3C2GNM_004570
PIK3C3NM_002647
PIK3CANM_006218
PIK3CBNM_006219
PIK3CDNM_005026
PIK3CGNM_002649
PIK3R1NM_181523
PIK3R2NM_005027
PIK3R3NM_003629
PIM1PIM1NM_002648NM_002648
PLCG2NM_002661
PLK2NM_006622
PMAIP1NM_021127
PMS1NM_000534
PMS2NM_000535
PNRC1NM_006813
POLD1NM 002691
POLENM 006231
PPARGNM 015869
PPM1DNM_003620
PPP2R1ANM 014225
PPP4R2NM_174907
PPP6CNM 002721
PRDM1NM_001198
PRDM14NM 024504
PREX2NM 024870
PRKAR1ANM_212471
PRKCINM 002740
PRKD1NM 002742
PTCH1NM 000264
PTENNM 000314
PTP4A1NM_003463
PTPN11NM_002834
PTPRDNM 002839
PTPRSNM 002850
PTPRTNM_133170
RAB35NM_006861
RAC1NM 018890
RAC2NM_002872
RAD21NM 006265
RAD50NM_005732
RAD51NM 002875
RAD51BNM 133509
RAD51CNM_058216
RAD51DNM_133629
RAD52NM_134424
RAD54LNM_001142548
RAF1NM 002880
RARANM 000964
RASA1NM_002890
RB1NM 000321
RBM10NM 001204468
RECQLNM_032941
RECQL4NM_004260
RELNM 002908
RETNM 020975
RFWD2NM 022457
RHEBNM_005614
RHOANM 001664
RICTORNM 152756
RIT1NM 006912
RNF43NM_017763
ROS1NM_002944
RPS6KA4NM 003942
RPS6KB2NM 003952
RPTORNM 020761
RRAGCNM_022157
RRASNM 006270
RRAS2NM_012250
RTEL1NM_032957
RUNX1NM_001754
RXRANM 002957
RYBPNM 012234
SDHANM 004168
SDHAF2NM_017841
SDHBNM 003000
SDHCRHEBNM 003001NM_005614
RHOANM_001664
RICTORNM_152756
RIT1NM_006912
RNF43NM_017763
ROS1NM_002944
RPS6KA4NM_003942
RPS6KB2NM_003952
RPTORNM_020761
RRAGCNM_022157
RRASNM_006270
RRAS2NM_012250
RTEL1NM_032957
RUNX1NM_001754
RXRANM_002957
RYBPNM_012234
SDHANM_004168
SDHAF2NM_017841
SDHBNM_003000
SDHCNM_003001
SDHDNM_003002
SESN1NM_014454
SESN2NM_031459
SESN3NM_144665
SETD2NM_014159

{52}------------------------------------------------

{53}------------------------------------------------

SETD8NM020382
SF3B1012433NM
SH2B3005475NM
SH2D1ANM002351
SHOC2NM007373
SHOI018130NM
SLX4NM_032444
SMAD2001003652NM
SMAD3NM005902
SMAD4005359NM
SMARCA4003072NM
SMARCB1NM003073
SMARCD1003076NM
SMONM005631
SMYD3001167740NM
SOCS 1SOSINM003745
SOX17005633NM022454NM
SOX2NM003106
SOX9NM_000346
SPEN015001NM
SPOP001007228NM
SPRED1NM152594
SRCNM198291
SRSF2NM003016
STAG2001042749NM
STAT3139276NM
STAT5A003152NM
STAT5BNM012448
STK11000455NM
STK19NM004197
STK40NM032017
SUFUNM_016169
SUZIZ015355NM
SYK003177NM
TAP1000593NM
TAP2_018833NM
TBX3016569NM
TCEB1005648NM
TCF3NM001136139
TCF7L2001146274NM
TEK000459NM
TERTNM198253
TETI030625NM
TET2001127208NM
TGFBR1TGFBR2NM004612NM 001024847
TMEM127001193304NM
TMPRSS2001135099NM_
TNFAIP3NM_006290
TNFRSF14NM_003820
TOP1NM003286
TP53000546NM
TP53BP1NM 001141980
TP63NM003722
TRAF2NM021138
TRAF7NM032271
TSC1NM000368
TSC2NM000548
TSHR000369NM
U2AF1NM006758
UPF1NM_002911
VEGFANM_001171623
VHL000551NM_
VTCN1NM024626
WHSCI_001042424NM
WHSC1L1NM_023034
WT1NM_024426
WWTR1NM_001168280
XIAPNM_001167
XPO1NM_003400
XRCC2NM_005431
YAP1NM_001130145
YES1NM_005433
ZFHX3NM_006885

{54}------------------------------------------------

Appendix 1c: List of genes/exons excluded from reporting due to consistently low
coverage.
GeneTranscript IDChromosomeCoordinatesExoncDNAAmino Acid
AGO2NM_0121548:141645584-14164560511_221_8
ANKRD11NM_01327516:89334886-89335071137807_79922603_2664
CD276NM_00102473615:73995113-739954274419_733140_245
CD276NM_00102473615:73996517-7399681361073_1369358_457
CHEK2NM_00719422:29085123-29085203141462_1542488_514
FAM58ANM_152274X:152864420-15286452911176_1392_1
FLT3NM_00411913:28674605-2867464711_431_15
H3F3ANM_0021071:226259052-2262591804283_41195_137
HIST2H3CNM_0210591:149812319-1498127291411_1137_1
HIST2H3DNM_0011233751:149784826-14978523611_4111_137
HLA-ANM_0012427586:29911899-299121744620_895207_299
INSRNM_00020819:7293803-729390211_1001_34
KMT2CNM_1706067:151970790-1519709527850_1012284_338
KMT2CNM_1706067:151962123-15196229481013_1184338_395
KMT2CNM_1706067:151935792-151935911152533_2652845_884
KMT2CNM_1706067:151932902-151933018162653_2769885_923
KMT2CNM_1706067:151927008-151927112182872_2976958_992
KMT2CNM_1706067:151921520-151921701192977_3158993_1053
KMT2CNM_1706067:151921100-151921264203159_33231053_11081053_1108
KMT2CNM_1706067:151919658-151919767213324_34331108_1145
KMT2CNM_1706067:151904385-151904513243713_38411238_1281
MST1NM_0209983:49726031-4972612411_941_32
MST1NM_0209983:49724380-6608_728203_243
MST1NM_020998497245003:49724117-497242357729_847243_283
MST1NM_0209983:49723746-497239148848_1016283_339
MST1NM_0209983:49723495-4972362591017_1147339_383
MST1NM_0209983:49722695-49722815131424_1544475_515
MST1NM_0209983:49722445-49722522141545_1622515_541
MST1NM_0209983:49721983-49722089161770_1876590_626
MST1NM_0209983:49721747-49721886171877_2016626_672
MYCL1NM_0010330821:40367480-4036756011_811_27
NOTCH2NM_0244081:120611948-12061202011_731_25
NOTCH2NM_0244081:120572529-120572610274_15525_52
NOTCH2NM_0244081:120547952-1205482113156_41552_139
NOTCH2NM_0244081:120539620-1205399554416_751139_251
NOTCH3NM_00043519:15311599-1531171611_1181_40
PDPK1NM_00261316:2588114-258813711_241_8
PDPK1NM_00261316:2607704-2607964225_2859_95
PDPK1NM_00261316:2611481-26115233286_32896_110
PDPK1NM_00261316:2611772-26119094329_466110_156
PDPK1NM_00261316:2615554-26156985467_611156_204
PDPK1NM_00261316:2616357-26164546612_709204_237
PDPK1NM_00261316:2627426-26275017710_785237_262
PDPK1NM_00261316:2631296-26313648786_854262_285
PDPK1NM_00261316:2631608-26317049855_951285_317
PDPK1NM_00261316:2633413-263358610952_1125318_375
PIK3CANM_0062183:178937737-178937840131912_2015638_672
PIK3R2NM_00502719:18272089-182723056599_815200_272
PMS2NM_0005357:6022455-6022622122007_2174669_725
PMS2NM_0005357:6018227-6018327132175_2275725_759
PMS2NM_0005357:6017219-6017388142276_2445759_815
PMS2NM_0005357:6013030-6013173152446_2589816_863
PPP4R2NM_1749073:73096337-730965073117_28739_96
PTENNM_00031410:89725044-8972522991027_1212343_404
PTPRTNM_13317020:41818286-4181837311_881_30
RECQLNM_03294112:21623128-21623280161798_1950600_650
RECQL4NM_0042608:145743085-14574316811_841_28
SDHANM_0041685:254508-254621141795_1908599_636
SDHCNM_0030011:161332119-1613322236406_405136_135
SDHDNM_00300211:111965529-1119656944315_480105_160
SETD8NM_02038212:123873980-123874101211_1324_44
SETD8NM_02038212:123892040-1238922508849_1059283_353
STAT5ANM_00315217:40452148-404522998682_833228_278
STAT5ANM_00315217:40452733-404528889834_989278_330
STAT5BNM_01244817:40371330-403714817682_833228_278
STAT5BNM_01244817:40370741-403708968834_989278_330
STK19NM_0041976:31948781-3194882681050_1095350_365
SUZ12NM_01535517:30267305-302673512275_32192_107
SUZ12NM_01535517:30267441-302675053322_386108_129
SUZ12NM_01535517:30274636-302747044387_455129_152
SUZ12NM_01535517:30300165-303002506506_591169_197
SUZ12NM_01535517:30310018-303101239918_1023306_341
TGFBR1NM_0046129:101867488-10186758411_971_33

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§ 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.