MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay

Q: Who is the manufacturer of MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay?

Memorial Sloan-Kettering Cancer Center filed the De Novo classification request for MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay (DEN170058).

Q: What is the FDA product code for MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay?

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

Q: What is the regulatory pathway for MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay?

De Novo classification (submission type: Direct).

Q: Who can help prepare an FDA De Novo submission like MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA De Novo submissions. See https://innolitics.com/services/regulatory/ or contact https://innolitics.com/contact.

DEN170058 · Memorial Sloan-Kettering Cancer Center · PZM · Nov 15, 2017 · Pathology

Device Facts

Record ID	DEN170058
Device Name	MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets):a Hybridization-Capture Based Next Generation Sequencing Assay
Applicant	Memorial Sloan-Kettering Cancer Center
Product Code	PZM · Pathology
Decision Date	Nov 15, 2017
Decision	DENG
Submission Type	Direct
Regulation	21 CFR 866.6080
Device Class	Class 2

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 Story

MSK-IMPACT is a targeted NGS assay for tumor profiling. It takes FFPE tumor tissue and matched normal DNA as input. The workflow includes DNA extraction, library preparation (shearing, end repair, A-tailing, adapter ligation, PCR), and hybridization capture using biotinylated probes targeting 468 cancer-associated genes (~1.5Mb). Enriched libraries are sequenced on an Illumina HiSeq 2500. The analysis pipeline performs alignment to hg19, removes PCR duplicates, and calls somatic SNVs, indels, and microsatellite instability (MSI). MSI status is determined using the MSIsensor algorithm. Results are reported by qualified healthcare professionals to aid in cancer management. The device is used at Memorial Sloan Kettering Cancer Center. Clinical interpretation is facilitated by the OncoKB knowledge base, categorizing mutations by clinical significance.

Clinical Evidence

No clinical data provided in the document. The document outlines requirements for analytical validity (precision, accuracy, limit of detection) and clinical validation via method comparison studies to medically established tests for specific mutations.

Technological Characteristics

Targeted next-generation sequencing (NGS) assay. Analyzes FFPE tumor tissue matched with normal specimens. Detects single nucleotide variants, insertions, deletions, copy number variations, and gene rearrangements. Requires specific DNA extraction, quality/quantity metrics, and bioinformatics pipeline for mutation calling. Single-site implementation.

Indications for Use

Indicated for previously diagnosed cancer patients with solid malignant neoplasms. Requires formalin-fixed, paraffin-embedded tumor tissue matched with normal specimens.

Regulatory Classification

Identification

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

Special Controls

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

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Submission Summary (Full Text)

{0}------------------------------------------------ # EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) # DECISION SUMMARY ### A. DEN Number: DEN170058 ### B. Purpose for Submission: De novo request for evaluation of automatic class III designation for the MSK-IMPACT # C. Measurand: Somatic single nucleotide variants, insertions, deletions, and microsatellite instability in genes in human genomic DNA obtained from formalin-fixed, paraffin-embedded tumor tissue. Refer to Appendix 1a for complete list of hotspot mutations and Appendix 1b for complete list of genes included in this assay. # D. Type of Test: Next generation sequencing tumor profiling test # E. Applicant: Memorial Sloan Kettering (MSK) # F. Proprietary and Established Names: MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) # G. Regulatory Information: # 1. Regulation section: 21 CFR 866.6080 # 2. Classification: Class II # 3. Product code: PZM {1}------------------------------------------------ # 4. Panel: Pathology # H. Indications for Use: # 1. Indications for Use: The MSK-IMPACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and microsatellite instability for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. MSK-IMPACT is a single-site assay performed at Memorial Sloan Kettering Cancer Center. # 2. Special conditions for use statement(s): For prescription use. For in vitro diagnostic use. # 3. Special instrument requirements: Illumina HiSeq™ 2500 Sequencer (qualified by MSK) # I. Device Description: A description of required equipment, software, reagents, vendors, and storage conditions were provided, and are described in the product labeling (MSK-IMPACT manual). MSK assumes responsibility for the device. # 1. Sample Preparation: The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve the necessary quality performance are shown in the Table 1 below: | Tissue Type | Volume | Minimum Tumor Proportion | Macrodissection requirements (Based on tumor proportion) | Limitations | Storage | |------------------|----------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------| | FFPE sections | 5-20 unstained sections, 10 microns thick | More than 10% of tumor cells; sections containing >20% viable tumor are preferred. For MSI testing, >25% tumor cells. | Yes, macrodissection to obtain non- neoplastic tissue for analysis | Archival paraffin- embedded material subjected to acid decalcification is unsuitable for analysis because acid decalcification severely damage nucleic acids. | Room temp | # Table 1. Specimen Handling and Processing for Validated Specimen Types {2}------------------------------------------------ Genomic DNA is extracted from tissue specimens per protocol. DNA is quantified and concentrated if necessary. The amount of DNA required to perform the test is 100-250ng. DNA is run in singlicate. DNA shearing is conducted per protocol and a quality control check is performed. Average fragment size should be ~200bp. Sheared DNA is stored at -20°C if not proceeding directly to Library Preparation. The DNA can be stored at 37°C for 10-20 minutes, stored at 2-8°C for 24 hours, or at -20°C for longer periods. ## 2. Library Preparation: Sequence libraries are prepared using KAPA Biosystems Library Preparation Reagents by first producing blunt-ended, 5'-phosphorylated fragments. To the 3' ends of the dsDNA library fragments, dAMP is added (A-tailing). Next, dsDNA adapters with 3'dTMP is ligated to the A-tailed library fragments. Library fragments with appropriate adapter sequences are amplified via ligation-mediated pre-capture PCR. A quality control check on the amplified DNA libraries is performed: Samples should be a smear; average fragment size with the peak at ~200bp; and concentration between 5-300ng/uL to ensure adequate hybridization for capture. # 3. Hybrid Capture NGS: Library capture is conducted using NimbleGen Capture reagents. Pooled sequencing libraries are hybridized to the vendor oligo pool. Capture beads are used to pull down the complex of capture oligos and genomic DNA fragments. Unbound fragments are washed away. The enriched fragment pool is amplified by ligation mediated-PCR. The success of the enrichment is measured as a quality control step: Samples should be a smear, average fragment size with the peak at ~300bp; the concentration of the amplified DNA library should be 5-45ng/uL; the LM-PCR yield should be ≥ 250ng. Reactions can be stored at 4°C until ready for purification, up to 72 hours. ### 4. Sequencing and Data Analysis: Sequencing is conducted with the Illumina HiSeq2500 Sequencing Instruments and reagents and PhiX Control v3. The sequencing process uses multiple quality checks. - a) Data Management System (DMS): Automated sample tracking and archival of runassociated metadata (barcode, run name, samples accession number, patient medical record number, source (class), specimen type, and panel version) is conducted with the following key functions: Tracking sample status through various stages of data analysis; tracking iterations of analysis applied to a given sample; recording versions of databases and algorithms used in analysis; archival of selected pipeline output files (FASTO, BAM, VCF) and sequencing run statistics (e.g., cluster density, %clusters passing filter, unassigned read indices). - b) Demultiplexing and FASTQ generation: The analysis pipeline uses software provided by Illumina. Two FASTQ files are generated per samples corresponding to full length forward and reverse reads. Demultiplexing quality control includes quality metrics for per-base sequence quality, sequence content. GC content and sequence length distribution, relative percentages of unmatched indices. {3}------------------------------------------------ - c) Indexing OC check: The potential for index contamination is managed by demultiplexing all sequencing reads for all possible barcodes. If the number of reads > 15,000 for any unused barcodes, then those reads are analyzed with the pipeline and the fingerprint SNPs are used to identify which of the barcodes used in the pool could be causing the appearance of extra reads. - d) Read alignment and BAM generation: Spurious adapter sequences are trimmed prior to read alignment. Reads are aligned in paired-end mode to the hg19 b37 version of the human genome. Aligned reads are written to a Sequence Alignment Map (SAM) file, which is then converted into Binary Alignment Map (BAM) format. PCR duplicates are removed. Each base within a read is assigned a base quality score by the sequencing software, which reflects the probability an error was made with the base call. To account for systemic biases that may not accurately reflect the actual error probabilities observed empirically, the analysis pipeline uses another tool to adjust the reported quality scores based on the selected covariates. Reassigned quality scores are subject to a threshold of 20, corresponding to a 1/100 chance of error. - e) Sample QC checks: The baits used for hybridization capture include custom intergenic and intronic probes targeting >1000 regions throughout the genome containing common single nucleotide polymorphisms (SNPs). The unique combination of SNPs specific to a given sample serves as a 'fingerprint' for the identity of the corresponding patient, and serves to identify potential sample mix-ups and contamination between samples and barcodes. OC checks involving the use of these 'fingerprint' SNPs are detailed below: - i. Sample mix-up check: The analysis pipeline computes the 'percent discordance' between a reference and query sample, defined as the percent of homozygous sites in the reference sample that are homozygous for the alternate allele in the query sample. The expected discordance between tumors and their respective matched normal should be low (<5%). 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 {4}------------------------------------------------ samples with matched normal controls excluded due to possible tumor contamination will be considered as unmatched tumor samples for subsequent analyses. - f) Mutation calling SNVs and Indels: The analysis pipeline identifies two classes of mutations: (1) single nucleotide variants (SNVs) and (2) indels. Paired sample mutation calling is performed on tumor samples and their respective matched normal controls. In instances where a matched normal sample is unavailable, or where the matched normal sample was sequenced with low coverage (< 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. {5}------------------------------------------------ Image /page/5/Figure/0 description: This image shows a flowchart of the filtering process for SNVs and Indels. The raw pipeline output for SNVs is MuTect, and for Indels, it is SomaticIndelDetector. The standard filter for somatic variants is VFtumor/VFnormal ≥ 5X, AD ≥ 5, and VF ≥ 1%. After annotation with AnnoVar, a two-tiered filtering scheme is applied based on whether the variant is in a hotspot, with different DP, AD, and VF thresholds for each case, and the process ends with manual review. ### Figure 1. Summary of mutation filtering scheme - g) Mutation annotation: Predicted functional effect and clinical interpretation for each mutation is curated by automated software using information from several databases. - h) Microsatellite Instability (MSI) status calling: The somatic MSI status is inferred by interrogating all available genomic microsatellites covered by MSK-IMPACT within tumor samples against the matched normal DNA using the program MSIsensor (Nui B et al. 2014). Essentially, the sequencing results are analyzed via MSIsensor to assess the number and length of homo-polymers / microsatellites within the targeted regions of tumor-normal sample pair. This results in a continuous rather than categorical MSI score assignment for the tumor sample. Loci are considered unstable (somatic) if k-mer distributions are significantly different between the tumor and matched normal using a standard multiple testing correction of x2 p-values. The percentage fraction of unstable sites is reported as the MSIsensor score. The assay uses a MSIsensor score threshold of 10 or greater to define MSI-H by MSIsensor. # 5. Controls - a) Matched normal control: Genomic DNA is extracted from patient-matched normal tissue (when available) or peripheral blood, for use as a matched normal control. In the event a matched normal is unavailable, or where the matched normal sample was sequenced with low coverage (<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. {6}------------------------------------------------ - 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 ID | Sample ID | VF | Known Mutation | |-------------------|----------------|-----|-----------------| | | M-1682-C3-T | 17% | KRAS Q61H | | | M-1791-8C-T | 66% | EGFR L858R | | M-1913-BF | M-1754-DB-T | 61% | KITexon9ins | | | M-1671-CE-T | 25% | KITexon11del | | | M-1693-5E-T | 24% | PIK3CA H1047R | | M-1914-A2 | M-1646-FC-T | 41% | BRAF V600E | | | M-1612-28-3-T | 32% | EGFR exon19 del | | | M-1627-D9-T | 52% | NRAS Q61H | | M-1915-CA | M-1625-1A-2A-T | 28% | 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 {7}------------------------------------------------ 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 Metrics | Acceptance Criteria | |----------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Coverage | Average target coverage > 200X | | Coverage Uniformity | ≥ 98% target exons above 100X coverage | | Base Quality | > 80% of bases with QS above > Q30 | | % Cluster passing | The percent cluster passing filter (Cluster PF) > 80% | | % Reads passing filter | The percent reads passing filter (Reads PF) > 80% | | | Mutation Coverage (DP) ≥ 20, | | Hotspot Mutation* calling threshold | Number of Mutant Reads (AD) ≥ 8, | | | Mutation Frequency (VF) ≥ 2% | | Non-hotspot Mutation** threshold | DP > 20, AD ≥ 10, VF ≥ 5% | | Indels | Fewer than 20% of samples in an established 'standard normal'database | | Positive Run Control | The difference between the observed and expected frequencies for the known mutations should be within 5%. | | Negative Run Control | The correlation between expected and observed mutation frequencies should be 0.9 or higher | | Sample-Mix up QC | Check over 1000 custom intergenic/intronic "fingerprint" SNPs. Flagged if pairs of samples from the same patient with > 5% discordance and from different patients with < 5% discordance | | Major Contamination QC | % heterozygous sites at fingerprint SNPs < 55%; Average MAF at homozygous fingerprint SNPs < 2% | | 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. | | 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. {8}------------------------------------------------ # 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. {9}------------------------------------------------ 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 {10}------------------------------------------------ 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 {11}------------------------------------------------ calculations, which showed 100X coverage was necessary to call mutations with true underlying mutation frequency 10% or greater, with 95% power at an alpha level of 0.05). To be conservative, a threshold of 200X on mean sample coverage is used to determine if a sample is sequenced to sufficient depth for subsequent analysis. A sample is flagged as being at increased risk of false negatives if its mean coverage is below 200X. To provide empirical data for these requirements, MSK utilizes the pool normal sample with known expected single nucleotide mutations (n = 2436) and the underlying mutation allele fractions (MAF). In silico downsampling analysis was conducted with a pool normal mix down to 45% where the sample coverage decreased from 452X to 203X. At this coverage level, 94% of the mutations with expected underlying VAF of 10% were called. - c) Requirements on mutation coverage, allele depth and frequency for positive calls: Permissive standard filters were used to intentionally generate false positives to identify suitable thresholds for parameters such as mutation coverage (DP), alternate allele depth (AD) and mutation frequency (VF) to optimize specificity. The following criteria allows optimal rejection of false positive SNVs (stratified by whether they are hotspots or not) and indel calls, while maintaining ability to detect true positive events with underlying frequency of 10% (5-17.6% observable). Potential strand-bias is also evaluated in the standard somatic mutation calling pipeline. An example of the number of false positive events detected pre and post filtering for coverage depth(DP), number of mutant reads (AD) and variant frequency (VF) is shown in Table 4. | | Mutations -Cosmic database | | Mutations | | |-----------------|----------------------------|--------|---------------------------|--------| | Filter criteria | DP ≥ 20X, AD ≥ 8,VF ≥ 2% | | DP ≥ 20X, AD ≥ 10,VF ≥ 5% | | | | SNVs | Indels | SNVs | Indels | | Pre-filter | 1 | 24 | 342 | 40,793 | | Post-filter | 0 | 0 | 0 | 8 | | Rejection Rate | 1.00 | 1.00 | 1.00 | 0.999 | | Table 4. Sample error correction by DP/AD/VF filter | | | | |-----------------------------------------------------|--|--|--| |-----------------------------------------------------|--|--|--| - 2. Pre-Analytical performance: Minimum DNA requirements were established by measuring assay performance based on different inputs from normal blood and FFPE tumor samples. DNA samples are normalized to yield 50 - 250 ng input and maximized to 55 ul prior to shearing. The normalization and DNA quantification are performed. DNA extraction method was validated based on the invalid rates across multiple tumor types obtained from historical data. The data demonstrated that the DNA extraction has been optimized across tumor types to reasonably conclude that the analytical {12}------------------------------------------------ performance presented is representative across FFPE tumor types. Table 5 shows the historical data for invalid rates from a retrospective chart review of >10,000 specimens tested with MSK-IMPACT. The range of invalid rates was 7.2% to 18.4%. The data shows that interference effects from different specimens are not significant across different tumor types supporting the performance of the pan-cancer specimen handling. | | | | Pre-Run Invalids | Pre-Run Invalids | Post-Run Invalids | | |---------------------------------|------------------|--------------------|--------------------------------------------|---------------------------------------------|---------------------------------------------|---------------------| | Tumor Type | Specimen Type | Number of Tests | Tumor Insufficient (Tumor % <20%) | DNA Insufficient (DNA yield <50ng) | Sequencing Failure (Coverage <50X) | Percent Invalids | | Non-Small Cell Lung Cancer | FFPE | 1995 | 53 | 208 | 75 | 16.8 | | Breast Carcinoma | FFPE | 1588 | 41 | 126 | 97 | 16.6 | | Colorectal Cancer | FFPE | 1105 | 29 | 39 | 31 | 9.0 | | Prostate Cancer | FFPE | 879 | 28 | 63 | 71 | 18.4 | | Glioma | FFPE | 601 | 1 | 33 | 16 | 8.3 | | Pancreatic Cancer | FFPE | 584 | 15 | 38 | 29 | 14.0 | | Soft Tissue Sarcoma | FFPE | 479 | 3 | 21 | 13 | 7.7 | | Bladder Cancer | FFPE | 480 | 12 | 20 | 25 | 9.8 | | Melanoma | FFPE | 411 | 7 | 22 | 17 | 11.2 | | Renal Cell Carcinoma | FFPE | 403 | 12 | 15 | 16 | 10.7 | | Hepatobiliary Cancer | FFPE | 398 | 11 | 17 | 15 | 10.8 | | Esophagogastric Carcinoma | FFPE | 374 | 5 | 12 | 16 | 8.8 | | Germ Cell Tumor | FFPE | 332 | 9 | 13 | 30 | 8.1 | | Thyroid Cancer | FFPE | 258 | 2 | 12 | 13 | 10.5 | | Ovarian Cancer | FFPE | 244 | 4 | 8 | 8 | 8.2 | | Endometrial Cancer | FFPE | 235 | 2 | 8 | 7 | 7.2 | | Head and Neck Carcinoma | FFPE | 208 | 8 | 8 | 6 | 10.5 | | Cancer of Unknown Primary | FFPE | 224 | 15 | 15 | 10 | 17.8 | Table 5. Specimen Invalid Rates for 17 FFPE Tumor Types {13}------------------------------------------------ # 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 type | Mutation type | Gene/ exon | cDNA change | Amino acid change | Mutation frequency | |----------------------------------|---------------|------------------|--------------------------------------------|-------------------|-----------------------| | Glioblastoma | INS | EGFR exon20 | C2290_2310dup TACGTGATGGCCAGC GTGGAC | p.Y764_D770dup | ~5% | | Cutaneous Melanoma | DNV | BRAF exon15 | c.1798_1799delinsAA | V600K | ~6.5% | | Uterine Endometrial Cancer | SNV | KRAS exon2 | C35G>C | G12A | ~7% | | Lung Adenocarcinoma | INS | ERBB2 exon 20 | 2310_2311ins GCATACGTGATG | E770_A771insAYVM | ~15% | | Lung Adenocarcinoma | SNV | EGFR exon 21 | 2573T>G | L858R | ~20% | Table 6: Summary of the Specimens and Allele Frequencies in the Precision Studies 1 For complex structural variations, such as genomic rearrangements (fusions) and copy number variations (CNVs), the expectation is that the representative approach should be demonstrated at the gene level. {14}------------------------------------------------ | Tissue type | Mutation type | Gene/ exon | cDNA change | Amino acid change | Mutation frequency | |---------------------|---------------|---------------------------------------------------------------------------------|------------------------------|-------------------|--------------------| | CRC | SNV | KRAS exon 2 | C34G>T | G12C | ~30% | | Lung Adenocarcinoma | DEL (15bp) | EGFR exon 19 | 2236_2250delGAATTAA GAGAAGCA | E746_A750del | ~30% | | CRC | SNV | BRAF exon 15 | c.1799T>A | V600E | ~40% | | GIST | DEL (6bp) | Kit exon 11 | 1667_1672delAGTGGA | Q556_K558del | ~50% | | FFPE Cell Line | DEL, SNV | Hotspot mutations in BRAF, EGFR, FLT3, GNA11, IDH1, KRAS, NRAS and PIK3CA genes | | | ~2%-15% | - ii. Precision- Panel-Wide Reproducibility: The precision analysis was performed for the known mutations (as listed in Table 6), and also performed for all additional mutations identified in each specimen in any of the test replicates. A total of 69 mutations in the clinical specimens and 13 mutations in the cell line were detected for a total of 82 mutations. In addition to SNV/MNVs, there were 9 deletions and 8 insertions. The results showed that all mutations have 100% concordance in all replicates except for 4 mutations in the clinical specimens and 3 mutations in the commercial sample. In the clinical specimen discordance was observed for an SNV (pQ64K) and a frameshift mutation (pL54fs) in AR exon1, an insertion (pA445_P446insP) ARID1B exon1; and a frameshift mutation (pT319Kfs*24) in PTEN exon 8. The discordance on AR and ARID1B mutations were due to poor mapping quality in the highly repetitive regions. The 3 mutations from the commercial control sample that were discordant were 2 SNVs and one deletion (IDH1 exon4 R132H; BRAF exon15 V600M; EGFR exon19 E746 A750del). These 3 mutations were believed to be discordant because they have low frequencies near 2%. The coefficient of variation (%CV) for the mutation allele frequency was also calculated for all 5 replicates. Thirty-four (45) of the 69 mutations in the clinical specimens had %CV ≤10%, 17/69 were between 10 and 20% and 7/69 were >21%. All results are summarized in Table 7. Each specimen is separated by a dark gray line. Known mutation within each specimen are in bold. Discordant cases are denoted in light grey. All runs passed the quality metrics criteria. Table 7. Panel-wide precision summary for all 5 replicates Abbreviations: NC (normalized coverage); MAF (Mutant allele frequency) {15}------------------------------------------------ | Gene Exon | Mutation (cDNA/Protein Changes) | NC range | MAF range | MAF mean | MAF median | MAF (SD) | MAF (%CV) | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) | |-------------------|---------------------------------------------------------------|-------------|-------------|-------------|---------------|-------------|--------------|-----------------------------|------------------------------------------| | EGFR exon19 | c.2236_2250delG AATTAAGAGA AGCA 746_750del | 0.84-1 | 0.311-0.342 | 0.323 | 0.316 | 0.013 | 4.0% | 5/5 | 100.0% (47.8%, 100.0%) | | PTEN exon2 | c.T83G I28S | 0.62-0.73 | 0.502-0.569 | 0.543 | 0.544 | 0.027 | 5.0% | 5/5 | 100.0% (47.8%, 100.0%) | | TET2 exon3 | c.C311G S104C | 1.04-1.32 | 0.085-0.103 | 0.098 | 0.102 | 0.008 | 8.2% | 5/5 | 100.0% (47.8%, 100.0%) | | TP53 exon7 | c.C742T R248W | 0.97-1.22 | 0.648-0.664 | 0.66 | 0.663 | 0.007 | 1.1% | 5/5 | 100.0% (47.8%, 100.0%) | | BRAF exon15 | c.T1799A V600E | 1.26-1.44 | 0.415-0.454 | 0.431 | 0.425 | 0.015 | 3.5% | 5/5 | 100.0% (47.8%, 100.0%) | | BRCA2 exon14 | c.A7388G N2463S | 0.84-0.96 | 0.19-0.23 | 0.209 | 0.21 | 0.015 | 7.2% | 5/5 | 100.0% (47.8%, 100.0%) | | BRD4 exon19 | c.G3922A A1308T | 0.44-0.56 | 0.5-0.636 | 0.553 | 0.54 | 0.054 | 9.8% | 5/5 | 100.0% (47.8%, 100.0%) | | FBXW7 exon9 | c.G1268T G423V | 0.91-1.05 | 0.369-0.418 | 0.395 | 0.391 | 0.02 | 5.1% | 5/5 | 100.0% (47.8%, 100.0%) | | GRIN2A exon7 | c.C1514A A505E | 0.92-1.1 | 0.194-0.211 | 0.202 | 0.203 | 0.006 | 3.0% | 5/5 | 100.0% (47.8%, 100.0%) | | PTPRD exon12 | c.G10A V4I | 0.5-0.63 | 0.281-0.361 | 0.336 | 0.35 | 0.034 | 10.1% | 5/5 | 100.0% (47.8%, 100.0%) | | RUNX1 exon9 | c.806-1G>A NA | 1.01-1.23 | 0.185-0.21 | 0.202 | 0.207 | 0.01 | 5.0% | 5/5 | 100.0% (47.8%, 100.0%) | | SPEN exon12 | c.C10445T P3482L | 0.94-1.03 | 0.189-0.235 | 0.208 | 0.2 | 0.018 | 8.7% | 5/5 | 100.0% (47.8%, 100.0%) | | SYK exon13 | c.C1768T R590W | 1.13-1.22 | 0.233-0.292 | 0.273 | 0.279 | 0.023 | 8.4% | 5/5 | 100.0% (47.8%, 100.0%) | | TP53 exon6 | c.G610T E204X | 0.9-1.01 | 0.525-0.56 | 0.547 | 0.551 | 0.013 | 2.4% | 5/5 | 100.0% (47.8%, 100.0%) | | APC exon16 | c.G3856T E1286X | 0.8-1.05 | 0.326-0.39 | 0.351 | 0.349 | 0.026 | 7.4% | 5/5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | APC exon7 | c.C646T R216X | 0.87-1.06 | 0.148-0.185 | 0.162 | 0.16 | 0.015 | 9.3% | 5/5 | 100.0% (47.8%, 100.0%) | | CREBBP exon29 | c.G4837A V1613M | 1-1.19 | 0.159-0.196 | 0.178 | 0.18 | 0.017 | 9.6% | 5/5 | 100.0% (47.8%, 100.0%) | | KRAS exon2 | c.G34T G12C | 1.13-1.31 | 0.289-0.352 | 0.314 | 0.305 | 0.024 | 7.6% | 5/5 | 100.0% (47.8%, 100.0%) | | NOTCH1 exon34 | c.7541dupC P2514fs | 1.28-1.5 | 0.144-0.211 | 0.184 | 0.189 | 0.025 | 13.6% | 5/5 | 100.0% (47.8%, 100.0%) | | SMAD4 exon11 | c.C1333T R445X | 0.76-0.95 | 0.206-0.238 | 0.223 | 0.229 | 0.014 | 6.3% | 5/5 | 100.0% (47.8%, 100.0%) | | ALOX12B exon11 | c.G1406A R469Q | 1.03-1.31 | 0.333-0.377 | 0.355 | 0.356 | 0.016 | 4.5% | 5/5 | 100.0% (47.8%, 100.0%) | | ARID1B exon1 | c.1333_1334insCG C A445_P446insP | 0.2-0.2 | 0.2-0.2 | 0.2 | 0.2 | NA | NA | 1/5 | 20.0% (0.5%, 71.6%) | | CDK8 exon10 | c.C1014A D338E | 0.59-0.7 | 0.256-0.336 | 0.303 | 0.315 | 0.032 | 10.6% | 5/5 | 100.0% (47.8%, 100.0%) | | DNMT1 exon36 | c.T4380G H1460Q | 1.18-1.51 | 0.51-0.558 | 0.534 | 0.53 | 0.017 | 3.2% | 5/5 | 100.0% (47.8%, 100.0%) | | ERBB2 exon2 | c.G140A R47H | 1.16-1.59 | 0.596-0.712 | 0.656 | 0.666 | 0.045 | 6.9% | 5/5 | 100.0% (47.8%, 100.0%) | | ERBB2 exon20 | c.2310_2311insG CATACGTGAT G E770_A771insAY VM | 1.02-1.38 | 0.142-0.199 | 0.173 | 0.171 | 0.023 | 13.3% | 5/5 | 100.0% (47.8%, 100.0%) | | ERCC2 exon21 | c.C1904T A635V | 1.19-1.47 | 0.363-0.466 | 0.409 | 0.423 | 0.045 | 11.0% | 5/5 | 100.0% (47.8%, 100.0%) | | IRS1 exon1 | c.C3639A S1213R | 0.42-0.49 | 0.384-0.494 | 0.449 | 0.455 | 0.04 | 8.9% | 5/5 | 100.0% (47.8%, 100.0%) | | MED12 exon37 | c.5258_5282delCT CCTACCCTGCT AGAGCCTGAGA A A1753fs | 1.08-1.36 | 0.141-0.187 | 0.164 | 0.17 | 0.019 | 11.6% | 5/5 | 100.0% (47.8%, 100.0%) | | MED12 exon43 | c.6339_6340insCA GCAACACCAG Q2113_Q2114ins QQHQ | 0.96-1.43 | 0.37-0.422 | 0.4 | 0.399 | 0.021 | 5.3% | 5/5 | 100.0% (47.8%, 100.0%) | | NF1 exon51 | c.C7595T A2532V | 0.92-1.04 | 0.627-0.68 | 0.664 | 0.676 | 0.022 | 3.3% | 5/5 | 100.0% (47.8%, 100.0%) | | NTRK1 exon1 | c.G53A G18E | 0.28-0.55 | 0.6-0.668 | 0.631 | 0.63 | 0.027 | 4.3% | 5/5 | 100.0% (47.8%, 100.0%) | | PDGFRB exon7 | c.G946A V316M | 0.73-1.14 | 0.615-0.681 | 0.646 | 0.642 | 0.026 | 4.0% | 5/5 | 100.0% (47.8%, 100.0%) | | PIK3CB exon15 | c.A2150G N717S | 0.67-0.85 | 0.273-0.317 | 0.299 | 0.308 | 0.018 | 6.0% | 5/5 | 100.0% (47.8%, 100.0%) | | PTPRS exon32 | c.C4822T R1608W | 0.79-1.06 | 0.526-0.562 | 0.543 | 0.542 | 0.013 | 2.4% | 5/5 | 100.0% (47.8%, 100.0%) | | RB1 exon2 | c.138-2A>G splicing mutation | 0.51-0.75 | 0.231-0.345 | 0.291 | 0.284 | 0.047 | 16.2% | 5/5 | 100.0% (47.8%, 100.0%) | | TET1 exon4 | c.G3476A R1159Q | 0.86-1.34 | 0.499-0.606 | 0.533 | 0.522 | 0.044 | 8.3% | 5/5 | 100.0% (47.8%, 100.0%) | | TP53 exon5 | c.G524A R175H | 0.75-1.11 | 0.247-0.344 | 0.314 | 0.337 | 0.04 | 12.7% | 5/5 | 100.0% (47.8%, 100.0%) | | EGFR exon21 | c.T2573G L858R | 1.4-1.44 | 0.172-0.225 | 0.199 | 0.203 | 0.02 | 10.1% | 5/5 | 100.0% (47.8%, 100.0%) | | HNF1A exon4 | c.C934T L312F | 0.35-0.54 | 0.033-0.077 | 0.057 | 0.059 | 0.016 | 28.1% | 5/5 | 100.0% (47.8%, 100.0%) | | MLL3 exon42 | c.G9671A R3224H | 1.27-1.4 | 0.089-0.118 | 0.104 | 0.105 | 0.011 | 10.6% | 5/5 | 100.0% (47.8%, 100.0%) | | NTRK3 exon14 | c.1401delC P467fs | 0.49-0.54 | 0.062-0.086 | 0.074 | 0.077 | 0.01 | 13.5% | 5/5 | 100.0% (47.8%, 100.0%) | | TP53 exon10 | c.A1051T K351X | 0.74-0.84 | 0.075-0.116 | 0.103 | 0.108 | 0.016 | 15.5% | 5/5 | 100.0% (47.8%, 100.0%) | | AR exonl | c.161_171delTGC TGCTGCTG L54fs | 0.34-0.39 | 0.079-0.097 | 0.088 | 0.087 | 0.009 | 10.2% | 3/5 | 60.0% (14.7%, 94.7.0%) | | AR exon1 | c.C190A Q64K | 0.25-0.29 | 0.134-0.135 | 0.134 | 0.134 | 0.001 | 0.7% | 2/5 | 40.0% (5.3%, 85.3%) | | KIT exon11 | c.1667_1672delA GTGGA 556_558del | 1.65-1.86 | 0.554-0.595 | 0.569 | 0.566 | 0.016 | 2.8% | 5/5 | 100.0% (47.8%, 100.0%) | | KIT exon17 | c.T2467G Y823D | 1.28-1.49 | 0.619-0.658 | 0.646 | 0.655 | 0.016 | 2.5% | 5/5 | 100.0% (47.8%, 100.0%) | | RPS6KB2 exon10 | c.G840T K280N | 0.93-1.19 | 0.435-0.473 | 0.462 | 0.468 | 0.015 | 3.2% | 5/5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | CARD11 exon25 | c.3382T>A p.V1128I | 1.34-1.58 | 0.276-0.293 | 0.284 | 0.278 | 0.009 | 3.2% | 5/5 | 100.0% (47.8%, 100.0%) | | EGFR exon20 | c.2290_2310dupT ACGTGATGGC CAGCGTGGAC p.Y764_D770dup | 14.36-15.46 | 0.05-0.06 | 0.055 | 0.055 | 0.004 | 7.3% | 5/5 | 100.0% (47.8%, 100.0%) | | EGFR exon7 | c.874G>T p.V292L | 21.51-21.82 | 0.934-0.939 | 0.937 | 0.939 | 0.002 | 0.2% | 5/5 | 100.0% (47.8%, 100.0%) | | NOTCH3 exon22 | c.3646G>A p.A1216T | 1.35-1.52 | 0.247-0.318 | 0.281 | 0.281 | 0.026 | 9.3% | 5/5 | 100.0% (47.8%, 100.0%) | | PTEN exon5 | c.395G>C p.G132A | 0.6-0.72 | 0.605-0.667 | 0.635 | 0.631 | 0.029 | 4.6% | 5/5 | 100.0% (47.8%, 100.0%) | | RUNX1 exon8 | c.899C>T p.T300M | 0.81-0.92 | 0.244-0.274 | 0.26 | 0.266 | 0.015 | 5.8% | 5/5 | 100.0% (47.8%, 100.0%) | | STAG2 exon17 | c.1544_1547delAT AG p.D515Gfs*6 | 0.19-0.27 | 0.677-0.842 | 0.753 | 0.741 | 0.067 | 8.9% | 5/5 | 100.0% (47.8%, 100.0%) | | TERT Promoter | g.1295228C>T non-coding | 0.55-0.67 | 0.388-0.467 | 0.421 | 0.417 | 0.033 | 7.8% | 5/5 | 100.0% (47.8%, 100.0%) | | AKT3 exon2 | c.134T>G p.V45G | 1.14-1.36 | 0.05-0.078 | 0.066 | 0.067 | 0.012 | 18.2% | 5/5 | 100.0% (47.8%, 100.0%) | | BRAF exon15 | c.1798_1799delins AA p.V600K | 1.04-1.32 | 0.065-0.095 | 0.072 | 0.067 | 0.013 | 18.1% | 5/5 | 100.0% (47.8%, 100.0%) | | KIT exon11 | c.1735_1737delG AT p.D579del | 1.08-1.22 | 0.051-0.056 | 0.053 | 0.054 | 0.002 | 3.8% | 5/5 | 100.0% (47.8%, 100.0%) | | CTCF exon3 | c.610dupA p.T204Nfs*26 | 0.68-0.86 | 0.041-0.072 | 0.057 | 0.061 | 0.014 | 24.6% | 5/5 | 100.0% (47.8%, 100.0%) | | EGFR exon20 | c.2317_2319dupC AC p.H773dup | 1.15-1.19 | 0.067-0.093 | 0.078 | 0.079 | 0.011 | 14.1% | 5/5 | 100.0% (47.8%, 100.0%) | | KDM5C exon23 | c.3755G>A p.R1252H | 0.88-1.17 | 0.064-0.13 | 0.088 | 0.084 | 0.026 | 29.5% | 5/5 | 100.0% (47.8%, 100.0%) | | KRAS exon2 | c.35G>C p.G12A | 0.78-0.94 | 0.044-0.106 | 0.076 | 0.074 | 0.023 | 30.3% | 5/5 | 100.0% (47.8%, 100.0%) | | PIK3R1 exon13 | c.1672_1683delG AAATTGACAAA p.E558_K561del | 0.43-0.52 | 0.067-0.116 | 0.085 | 0.081 | 0.019 | 22.4% | 5/5 | 100.0% (47.8%, 100.0%) | | PIK3R1 exon9 | c.1023dupA p.E342Rfs*4 | 0.41-0.58 | 0.056-0.102 | 0.083 | 0.086 | 0.017 | 20.5% | 5/5 | 100.0% (47.8%, 100.0%) | | PIK3R1 exon9 | c.1024G>T p.E342* | 0.42-0.59 | 0.064-0.108 | 0.093 | 0.095 | 0.017 | 18.3% | 5/5 | 100.0% (47.8%, 100.0%) | | PTEN exon6 | c.493-1G>A p.X165_splice | 0.53-0.64 | 0.173-0.208 | 0.192 | 0.187 | 0.015 | 7.8% | 5/5 | 100.0% (47.8%, 100.0%) | | PTEN exon8 | c.956_959delCTTT T p.T319Kfs*24 | 0.28-0.48 | 0.006-0.079 | 0.049 | 0.052 | 0.029 | 59.2% | 3/5 | 60.0% (14.7%, 94.7.0%) | | SOX17 exon1 | c.287C>G p.A96G | 1.16-1.51 | 0.061-0.074 | 0.069 | 0.069 | 0.005 | 7.2% | 5/5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | BRAF exon15 | c.1798G>A V600M | 0.97-1.06 | 0.016-0.041 | 0.027 | 0.027 | 0.01 | 37.0% | 3/5 | 60.0% (14.7%, 94.7.0%) | | BRAF exon15 | c.1799T>A V600E | 0.97-1.06 | 0.051-0.08 | 0.064 | 0.067 | 0.012 | 18.8% | 5/5 | 100.0% (47.8%, 100.0%) | | EGFR exon18 | c.2155G>A G719S | 1.23-1.33 | 0.125-0.179 | 0.158 | 0.164 | 0.022 | 13.9% | 5/5 | 100.0% (47.8%, 100.0%) | | EGFR exon19 | c.2235_2249delG GAATTAAGAG AAGC E746_A750del | 1.01-1.19 | 0.009-0.043 | 0.023 | 0.019 | 0.013 | 56.5% | 2/5 | 40.0% (5.3%, 85.3%) | | FLT3 exon20 | c.2503G>T D835Y | 0.97-1.02 | 0.037-0.059 | 0.045 | 0.043 | 0.008 | 17.8% | 5/5 | 100.0% (47.8%, 100.0%) | | GNA11 exon5 | c.626A>T Q209L | 1.41-1.48 | 0.036-0.054 | 0.046 | 0.044 | 0.008 | 17.4% | 5/5 | 100.0% (47.8%, 100.0%) | | IDH1 exon4 | c.395G>A R132H | 0.5-0.53 | 0.038-0.049 | 0.035 | 0.044 | 0.020 | 57.1% | 4/5 | 80.0% (28.4%, 99.5%) | | KRAS exon2 | c.34G>A G12S | 0.9-1.03 | 0.026-0.057 | 0.041 | 0.039 | 0.011 | 26.8% | 5/5 | 100.0% (47.8%, 100.0%) | | KRAS exon2 | c.38G>A G13D | 0.91-1.06 | 0.217-0.249 | 0.231 | 0.229 | 0.012 | 5.2% | 5/5 | 100.0% (47.8%, 100.0%) | | KRAS exon4 | c.436G>A A146T | 0.82-0.88 | 0.031-0.055 | 0.042 | 0.044 | 0.009 | 21.4% | 5/5 | 100.0% (47.8%, 100.0%) | | NRAS exon3 | c.183A>T Q61H | 1.01-1.14 | 0.039-0.065 | 0.051 | 0.051 | 0.01 | 19.6% | 5/5 | 100.0% (47.8%, 100.0%) | | PIK3CA exon10 | c.1624G>A E542K | 0.67-0.87 | 0.038-0.047 | 0.042 | 0.042 | 0.004 | 9.5% | 5/5 | 100.0% (47.8%, 100.0%) | | PIK3CA exon21 | c.3140A>G H1047R | 0.62-0.72 | 0.222-0.331 | 0.276 | 0.258 | 0.05 | 18.1% | 5/5 | 100.0% (47.8%, 100.0%) | {16}------------------------------------------------ {17}------------------------------------------------ {18}------------------------------------------------ {19}------------------------------------------------ {20}------------------------------------------------ - iii. Per Specimen Precision: Results of the precision studies were combined and precision across all reportable genes was determined for each specimen. The positive call rate based on the total number of mutations along with the 2-sides 95% confidence interval were calculated. Results are summarized in Table 8. | Specimen | Total No unique mutations detected across all 5 replicates* | *Positive call rate per mutation | Positive call rate* (two-sided 95% CI) | Negative call rate (two-sided 95% CI) | |----------------------|-------------------------------------------------------------------------------|-------------------------------------------------------|-------------------------------------------|------------------------------------------| | M15-22924 | 5 | 5/5 for all | 25/25 100.0% (86.3%, 100.0%) | - | | M15-3038 | 3 | 5/5 for all | 15/15 100.0% (78.2%, 100.0%) | - | | M16-19000 | 10 | 5/5 for 9 4/5 for 1 | 49/50 98.0% (89.4%, 99.9%) | - | | M1688-5C | 18 | 5/5 for 17 1/5 for 1 | 86/90 95.6% (89.0%, 98.8%) | 4/5 80.0% (28.4%, 99.5%) | | M-1698-A9 | 5 | 5/5 for all | 25/25 100.0% (86.3%, 100.0%) | - | | M-1654-CA | 6 | 5/5 for all | 30/30 100.0% (88.4%, 100.0%) | - | | M-1612-28 | 4 | 5/5 for all | 20/20 100.0% (83.2%, 100.0%) | - | | M1648-D5 | 10 | 5/5 for all | 50/50 100.0% (92.9%, 100.0%) | - | | M-1707-12 | 5 | 5/5 for 3 3/5 for 1; 2/5 for 1 | 20/25 80.0% (59.3%, 93.2%) | 3/5 60.0% (14.7%, 94.7%) | | Commercial sample | 13 | 5/5 for 10; 4/5 for 1 ; 3/5 for 1; 2/5 for 1 | 59/65 90.8% (81.0%, 96.5%) | 3/5 60.0% (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). {21}------------------------------------------------ - iv. Precision Well-characterized reference material: The precision of MSK-IMPACT was assessed through repeated measurements of a well characterized reference standard (HapMap cell line NA20810). To determine sequencing error rates for the reference sample, DNA extracted from the HapMap cell line was included in each run tested in the accuracy study. The study investigated whether the SNPs in the targeted exons were detected at their expected frequencies. Reference genotypes for 11,767 SNPs in the targeted exons using a whole genome sequencing BAM file for NA20810, were obtained from the 1000 Genomes database. A total of 11.443 SNPs (97.2%) were homozygous for the major allele (relative to the hg19 reference genome), 212 SNPs (1.8%) were heterozygous and 112 SNPs (0.95%) were homozygous for the minor allele. The strong bias towards alleles matching the reference genome was expected, given that these SNPs occur in coding exons and there is likely strong selective pressure against deviations from the reference sequence. NA20810 was profiled with the assay multiple times across different runs, for a total of 23 replicates. Zygosity results were 100% concordant and high levels of concordance specifically, the difference between the expected and mean observed mutation frequencies was very small (absolute difference = 0.09%±0.45%). The data provide additional supplemental evidence of the reproducibility of the assay. - v. Precision for Microsatellite Instability (MSI): Precision of the MSI calling by MSIsensor was demonstrated with a total of 12 specimens: 6 MSI-H specimens (at three MSI-score levels, 3 replicates per sample) and 6 MSS specimens. Each DNA extracted sample was tested with 3 inter- and 3 intra-run replicates. Multiple barcodes were included. All samples had 100% agreement between calls. The total number of unstable loci relative to the total number of sites surveyed along with the mean, median and standard deviation (SD) and coefficient of variance (%CV) was also presented for each specimen and score. The results supported the precision of the MSIsensor scores greater than 0.5 Results are shown in Table 9. | N | Total Sites_ range | Unstable Loci_range | Mean | Median | SD | %CV | Positive Call Rate (two- sided 95% CI) | |---|--------------------------|------------------------|-------|--------|------|-------|----------------------------------------------| | 5 | 1227-1458 | 518-650 | 43.00 | 43.00 | 1.22 | 2.8% | 100%(47.8%, 100.0%) | | 5 | 1158-1477 | 483-646 | 43.00 | 43.00 | 0.71 | 1.7% | 100%(47.8%, 100.0%) | | 5 | 1187-1429 | 500-613 | 42.00 | 42.00 | 0.71 | 1.7% | 100%(47.8%, 100.0%) | | 5 | 1287-1400 | 303-359 | 24.80 | 25.00 | 0.84 | 3.4% | 100%(47.8%, 100.0%) | | 5 | 1251-1303 | 240-318 | 23.40 | 24.00 | 2.51 | 10.7% | 100%(47.8%, 100.0%) | | 5 | 1154-1379 | 153-175 | 12.60 | 12.00 | 0.89 | 7.1% | 100%(47.8%, 100.0%) | Table 9. Precision of the MSIsensor Score Using 12 Specimens {22}------------------------------------------------ | N | Total Sites_ range | Unstable Loci_range | Mean | Median | SD | %CV | Positive Call Rate (two- sided 95% CI) | |---|--------------------------|------------------------|------|--------|------|--------|----------------------------------------------| | 5 | 1321-1545 | 46-58 | 3.60 | 4.00 | 0.55 | 15.3% | 100%(47.8%, 100.0%) | | 5 | 1535-1604 | 44-64 | 3.40 | 3.00 | 0.55 | 16.2% | 100%(47.8%, 100.0%) | | 5 | 1411-1612 | 28-38 | 2.20 | 2.00 | 0.45 | 20.5% | 100%(47.8%, 100.0%) | | 5 | 1438-1528 | 6-9 | 0.48 | 0.50 | 0.08 | 16.7% | 100%(47.8%, 100.0%) | | 5 | 1315-1487 | 0-2 | 0.02 | 0.00 | 0.04 | 223.6% | 100%(47.8%, 100.0%) | | 5 | 1312-1532 | 0-1 | 0.01 | 0.00 | 0.03 | 223.6% | 100%(47.8%, 100.0%) | - b) Analytical Sensitivity Limit of Detection (LoD): The LoD of the IMPACT assay is defined as the mutant allele fraction at which 95% of replicates across all replicates for a variant type are reliably detected. Studies were conducted to demonstrate a putative LoD for each variant type. In the first part, a dilution series was conducted to identify the lowest reliable mutant fraction. In part 2, the putative LoD was confirmed with multiple replicates. - Part 1: Dilution Series: The mean normalized coverage for all exons was i. determined for 10 normal FFPE specimens and the LoD was assessed with samples containing mutations in 5 validation exons (defined as representative exons harboring cancer mutations with evidence of clinical significance assessed in the accuracy study) with the lowest and highest coverage. - . The 5 validation exons with lowest coverage correspond to 3 exons harboring SNVs, (ERBB2 exon 20 (V777L), PDGFRA exon 18 (D842V), PIK3CA exon 10 (E545K), and 2 exons harboring indels (EGFR exon 19 and KIT exon 9). - The 5 validation exons with highest coverage correspond to 3 exons harboring SNVs (BRAF exon 15 (V600E), KRAS exon 2 (G12D) and PIK3CA exon 2 (R88Q) and 2 exons harboring indels (KIT exon 11 and EGFR exon 20). Five to eight serial dilutions were prepared using patient samples positive for the mutations listed above, where tumor samples were either diluted with their respective matched FFPE normal sample (when available) or a previously sequenced, unmatched normal FFPE sample. One replicate at each dilution was tested and the ability to detect the mutation of interest was measured. All results were called at the lowest dilution except for PIK3CA which was called wild-type at the lowest dilution. Results are shown in Tables 10A-J. {23}------------------------------------------------ | | | Table 10A. Limit of Detection -Part 1 | |--|--|---------------------------------------| |--|--|---------------------------------------| | SNV BRAF Exon 15 (Sample M-1648-D5-T) | | | | | | | |---------------------------------------|------------|-----------|------|-----|------|--------| | Dilution | cDNAchange | AA Change | DP | AD | VF | Result | | Neat | c.1799T>A | V600E | 1018 | 410 | 0.4 | Called | | 1:2 | | | 1044 | 319 | 0.31 | Called | | 1:4 | | | 888 | 173 | 0.19 | Called | | 1:8 | | | 999 | 91 | 0.09 | Called | | 1:16 | | | 783 | 26 | 0.03 | Called | | 1:32 | | | 845 | 20 | 0.02 | Called | Table 10B | SNV KRAS Exon 2 (sample M-1807-ED-T) | | | | | | | |--------------------------------------|-------------|-----------|--------|-------|------|--------| | Dilution | cDNA change | AA Change | DP | AD | VF | Result | | Neat | | G12D | 907 | 405 | 0.45 | Called | | 1:2 | | | 820 | 298 | 0.36 | Called | | 1:4 | c.35G>A | | 400 | 97 | 0.24 | Called | | 1:8 | | | 660 | 121 | 0.18 | Called | | 1:16 | | | ર્ભર્ટ | ਦੇ ਰੇ | 0.09 | Called | | 1:32 | | | 632 | 41 | 0.06 | Called | Table 10C | SNV PIK3CA Exon 2 (Sample M-1729-E1-T) | | | | | | | |----------------------------------------|------------|-----------|------|-----|------|--------| | Dilution | cDNAchange | AA Change | DP | AD | VF | Result | | Neat | | R88Q | 2029 | 629 | 0.31 | Called | | 1:2 | c.263G>A | | 1008 | 211 | 0.21 | Called | | 1:4 | | | 1140 | 145 | 0.13 | Called | | 1:8 | | | 997 | 62 | 0.06 | Called | | 1:16 | | | | | | WT | Table 10D | Dilution | cDNAchange | AA Change | DP | AD | VF | Result | |----------|-------------------|------------|------|…