
==== Front
NPJ Precis Oncol
NPJ Precis Oncol
NPJ Precision Oncology
2397-768X
Nature Publishing Group UK London

679
10.1038/s41698-024-00679-7
Article
Pan-tumor validation of a NGS fraction-based MSI analysis as a predictor of response to Pembrolizumab
http://orcid.org/0000-0003-0105-9760
Lin Douglas I. dlin@foundationmedicine.com

1
Quintanilha Julia C. F. 1
http://orcid.org/0000-0003-2642-7160
Danziger Natalie 1
Lang Lixin 2
Levitan Diane 2
Hayne Cynthia 3
Hiemenz Matthew C. 1
Smith David L. 1
http://orcid.org/0000-0002-5070-1783
Albacker Lee A. 1
Leibowitz Jeffrey 1
http://orcid.org/0000-0002-8059-8668
Mata Douglas A. 1
Decker Brennan 1
Lakis Sotirios 4
http://orcid.org/0000-0001-6013-5562
Patel Nimesh R. 1
Graf Ryon P. 1
Elvin Julia A. 1
Ross Jeffrey S. 1
Pattani Varun 1
http://orcid.org/0000-0001-8395-5168
Huang Richard S. P. 1
Wehn Amy K. 2
1 https://ror.org/02ackr434 0000 0004 0599 7276 Foundation Medicine, Inc., Boston, MA USA
2 grid.417993.1 0000 0001 2260 0793 Merck & Co., Inc., Rahway, NJ USA
3 https://ror.org/04drvxt59 grid.239395.7 0000 0000 9011 8547 Beth Israel Deaconess Medical Center, Boston, MA USA
4 Foundation Medicine GmbH, Penzberg, Germany
14 9 2024
14 9 2024
2024
8 20413 2 2024
26 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) tumor status have been demonstrated to predict patient response to immunotherapies. We developed and validated a next-generation sequencing (NGS)-based companion diagnostic (CDx) to detect MSI-H solid tumors via a comprehensive genomic profiling (CGP) assay, FoundationOne®CDx (F1CDx). To determine MSI status, F1CDx calculates the fraction of unstable microsatellite loci across >2000 loci using a fraction-based (FB) analysis. Across solid tumor types, F1CDx demonstrated a high analytical concordance with both PCR (n = 264) and IHC (n = 279) with an overall percent agreement (OPA) of 97.7% and 97.8%, respectively. As part of a retrospective bridging clinical study from KEYNOTE-158 Cohort K and KEYNOTE-164, patients with MSI-H tumors as determined by F1CDx demonstrated an objective response rate (ORR) of 43.0% to pembrolizumab. In real-world cancer patients from a deidentified clinicogenomic database, F1CDx was at least equivalent in assessing clinical outcome following immunotherapy compared with MMR IHC. Demonstrated analytical and clinical performance of F1CDx led to the pan-tumor FDA approval in 2022 of F1CDx to identify MSI-H solid tumor patients for treatment with pembrolizumab. F1CDx is an accurate, reliable, and FDA-approved method for the identification of MSI-H tumors for treatment with pembrolizumab.

Subject terms

Predictive markers
Cancer genomics
Molecular medicine
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Microsatellites are error-prone segments of repetitive DNA composed of short tandem repeats that are abundantly interspersed throughout the genome1–3. Microsatellite instability (MSI), or variation in microsatellite length, is particularly high in cancers from patients with defects in DNA mismatch repair (dMMR), which is a mechanism to correct errors during DNA replication. Tumors with high levels of MSI (MSI-H) have a significant number of mutations leading to neo-antigen production, which can make them recognizable by the immune system2,3. As such, MSI-H has emerged as a biomarker for advanced cancer patients who may benefit from immune checkpoint inhibitors such as pembroluzimab4.

MSI-H status is most common in colorectal (CRC) and endometrial cancers (EMCA) and less prevalent in other cancer types5. MSI-H tumors most often arise sporadically due to somatic inactivation of one of the MMR genes (MSH2, MSH6, MLH1, or PMS2) via mutation, copy-number loss, or MLH1 promoter hypermethylation6. In addition, MSI-H tumors may arise in the context of Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), an inherited cancer susceptibility syndrome due to germline mutations in one of the MMR genes, which predisposes patients to a variety of cancers, including CRC, EMCA, ovarian, upper gastrointestinal, pancreatobiliary, skin, and upper urological tract cancers, among others7. Regardless of the pathogenic mechanism underlying the MSI-H phenotype, immune checkpoint inhibitors are approved by the US Food and Drug Administration (FDA) for patients with tumors that are MSI-H as determined by an approved assay. Pembrolizumab received full approval in 2023 by the FDA for the treatment of MSI-H or dMMR advanced solid tumors as determined by an FDA-approved test8.

Several laboratory assays and techniques are available to determine MSI or MMR status in formalin-fixed, paraffin-embedded (FFPE) solid tumor tissues, such as direct assessment of microsatellite instability via PCR of five select loci, or, alternatively, loss of MMR expression (dMMR) by immunohistochemistry (IHC), which is relatively inexpensive, and widely available9–11. However, these methods do not provide additional tumor genomic characterization, including other NCCN-recommended NGS-based biomarkers in relevant cancer types. More recently, NGS-based comprehensive genomic profiling (CGP) assays examining a greater number of microsatellite loci have also been developed to directly assess MSI status.

The FDA has recently approved an NGS-based assay, FoundationOne®CDx (F1CDx; Foundation Medicine, Inc., Boston, MA), as a companion diagnostic (CDx) to identify patients with MSI-H solid tumors for treatment with pembrolizumab12. F1CDx is a CGP assay that tests for pathogenic alterations of 324 genes as well as for complex biomarkers, such as MSI, tumor mutational burden (TMB), and homologous recombination deficiency (HRD). To determine the MSI status of a tumor by NGS, F1CDx employs a novel fraction-based (FB) MSI analysis that analyzes >2,000 microsatellite loci to categorize a tumor specimen as MSI-High (MSI-H), microsatellite stable (MSS), or microsatellite equivocal (MSI-E).

Recently, the College of American Pathologists (CAP) released guidelines favoring use of MMR IHC or PCR over NGS by pathologists for detecting MSI-H tumors from patients being considered for immunotherapy1. These CAP guidelines were further endorsed by the American Society of Clinical Oncology (ASCO)13 since there are limited validation data demonstrating equivalence of NGS-based assays to more traditional PCR and MMR IHC assays. However, ASCO also acknowledged the utility of NGS in capturing other relevant alterations and biomarkers and of conserving tissue when the amount of available tissue limits the ability to perform multiple sequential tests. Therefore, the current recommendation is that if an NGS-based assay is utilized, equivalency should be demonstrated to MMR IHC or PCR in detecting MSI-H tumors1.

Here, the robust pan-tumor analytical and clinical validity of a novel FB MSI analysis via F1CDx for the identification of MSI-H solid tumor patients, and which led to the FDA approval of F1CDx as a CDx for pembrolizumab, is demonstrated. Analytical and clinical validity was further established in a real-world analysis consisting of samples from a deidentified clinicogenomic database, which evaluated concordance with reported MMR IHC status and response to immunotherapy based on F1CDx MSI status.

Methods

F1CDx Assay

F1CDx is an FDA-approved, NGS-based CGP, in vitro diagnostic device that uses hybridization-based capture technology for the detection of substitutions, insertion and deletion alterations, copy-number alterations, and select rearrangements in 324 genes, as well as an evaluation of complex biomarkers, including MSI, TMB and HRD. F1CDx sequencing methods have been described previously14. Sequence data were analyzed using proprietary software developed by Foundation Medicine (FMI), and variant calling was performed as previously described15. Discordances between F1CDx and PCR were further investigated by examining the mutational signature using the decomposition method of Zehir et al.16 with the 96-feature single-base substitution COSMIC reference signatures (version 2, March 2015) generated by Alexandrov et al.17 to yield coefficient weights representing the contributions of the signatures in each sample16–18. At least 10 variants were required for signature analysis, which included all predicted somatic point variants with unknown functional status.

Fraction-based (FB) MSI analysis by F1CDx

This study was conducted in accordance with The Declaration of Helsinki. Institutional Review Board approval was obtained prior to use of samples in the described validation studies and all the data was anonymized prior to performing the described analysis. The following IRBs were utilized: 1) Protocol No. 120160955 for the bridging analytical studies, and 2) WCG IRB, Protocol No. 420180044, for CGDB studies. Only biospecimens from participants that signed an IRB/EC approved trial level consent were used for bridging to the FoundationOne CDx test.

F1CDx employs a fraction-based (FB) MSI analysis to categorize a tumor specimen as MSI-High (MSI-H), MSI-equivocal/intermediate or microsatellite stable (MSS). The F1CDx assay and associated FB-MSI method are performed and FDA approved in conjunction with the pre-analytical F1CDx DNA extraction method. Formalin-fixed paraffin embedded (FFPE) tumor tissue (FFPE blocks or unstained slides) is a pre-requisite. Minimum tumor content and overall DNA extraction yield of a sample for MSI analysis is the same as for the F1CDx assay. Board-certified Foundation Medicine pathologists initially assess tumor content through the estimation of nucleated tumor cells. F1CDx requires at least 20% nucleated tumor cells to enter the DNA extraction procedure, and a minimum of 50 ng of DNA is needed to proceed with sequencing. Non-F1CDx DNA extraction methods were not evaluated relative to the FB-MSI performance. To determine MSI status, the lengths of repetitive loci (minimum of five repeat units of mono-, di-, and trinucleotides) are assessed in comparison to the expected lengths based on an internal database of >3,000 clinical samples. A locus containing an unique repeat length that is not observed in the internal database is considered to be unstable. The FB-MSI analysis calculates the fraction of microsatellite loci determined to be altered or unstable (i.e., the fraction unstable loci score) based on an analysis across >2,000 microsatellite loci. For a given microsatellite locus, non-somatic alleles are discarded, and the qualified microsatellite is categorized as unstable if remaining alleles differ in length from the reference genome. An MSI score is generated by calculating the fraction of unstable loci for each distinct sample, which only considers those loci that achieve adequate coverage for the sample. Post-sequencing, MSI status reportability is determined by assessment of the required downstream sequencing QC metrics that include adequate computational tumor purity, adequate sequencing coverage, and lack of detectable contamination. The final fraction unstable loci score is calculated as the number of unstable microsatellite loci divided by the number of evaluable microsatellite loci. Two FB-MSI score thresholds are applied to classify the MSI status of a tumor specimen: MSI-H tumors have FB-MSI scores ≥ 0.0124; MSS tumors have FB-MSI scores ≤ 0.0041; MSI-Equivocal tumors have FB-MSI intermediate scores >0.0041 and <0.0124. The cutoffs for F1CDx FB-MSI analysis were developed using a set of 380 samples for which results from the Promega MSI PCR Analysis System v1.2 test results were available.

Pan-tumor F1CDx analytical validation and concordance with PCR

403 samples from various solid tumor types were tested by Promega PCR MSI Analysis system v1.2 (Promega PCR) according to the manufacturer’s recommendations at Almac laboratory in Craigavon, UK and by F1CDx for determination of concordance between the two assays. Samples were derived from commercially procured tumor banks and 56 samples from KEYNOTE-158 screen-failed subjects. Samples not meeting full F1CDx FB-MSI or Promega PCR quality control (QC) criteria (n = 139) were discarded from the analysis, resulting in a final cohort of 264 cases with evaluable F1CDx and PCR results. Point estimates for OPA, PPA, NPA, and 95% two-sided Wilson Score confidence intervals (Cis) were calculated from the final cohort samples.

Pan-tumor F1CDx concordance with MMR IHC

A retrospective chart review from the FMI genomic database was performed in a randomly, consecutively selected set of 279 patients with various types of cancers, including 179 CRC, 69 EMCA and 31 non-CRC and non-EMCA cancers. The tumors from these patients had evaluable MSI status from previous F1CDx FB MSI testing at FMI and had external MMR IHC testing data available from various institutions during routine clinical care. MMR IHC results were extracted from the accompanying pathology reports. Point estimates for OPA, PPA and NPA and 95% two-sided Wilson Score CIs were calculated for the entire cohort (pan-tumor), CRC only, EMCA only, and non-CRC and non-EMCA tumor types only.

Clinical validation of F1CDx in KEYNOTE-158 (NCT02628067) Cohort K and KEYNOTE-164 (NCT02460198)

To establish the clinical validity of the F1CDx FB MSI analysis as a CDx for pembrolizumab, available samples from KEYNOTE-158 Cohort K and KEYNOTE-164 were retrospectively tested with F1CDx as part of a bridging study. KEYNOTE-158 is an ongoing multicenter, global, open-label trial of pembrolizumab in participants with multiple types of advanced (unresectable and/or metastatic) rare cancers (except CRC) who have failed prior therapy. KEYNOTE-164 is a single-arm, multisite, multicohort, Phase 2 study designed to evaluate the efficacy of pembrolizumab in participants with locally advanced unresectable or metastatic (Stage IV) MSI-H/dMMR CRC. All participants in KEYNOTE-158 Cohort K and KEYNOTE-164 were enrolled using a local MSI-H PCR or dMMR IHC test result as the clinical trial assay (CTA).

Clinical validity of F1CDx MSI-H status in a deidentified clinicogenomic database of real-world samples

Metastatic CRC and advanced EMCA patients underwent tissue genomic testing using FMI comprehensive genomic profiling (CGP) assays and were included in the US-wide Flatiron Health and FMI clinicogenomic database between March 2014 and December 2022. Retrospective de-identified longitudinal clinical data were derived from electronic health records (EHR) from approximately 280 US cancer clinics ( ~ 800 sites of care), comprising patient-level structured and unstructured data, curated via technology-enabled abstraction of clinical notes and radiology/pathology reports. These were linked to genomic data derived from FMI testing by de-identified, deterministic matching19. The cohort included patients treated with immunotherapy in any line of therapy (LOT), the majority receiving pembrolizumab, that had MSI status by the F1CDx FB-MSI analysis and MMR status by IHC as assessed locally via a tissue specimen and the results included in EHR. Validated outcome measurements were used as endpoints for this study, including time to next treatment (TTNT), and time to treatment discontinuation (TTD), and OS for EMCA patients, and TTNT, PFS, and TTD for CRC patients. The analyses performed in this study were pre-specified in a prospectively declared statistical analysis plan (SAP), which followed the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) guidelines20, including the specification of the research question, pre-specified analytical plans such as inclusion and exclusion criteria, potential biases, primary and secondary outcome measures, and handling of missing data. The pre-specified analyses included the examination of the power of the association with immunotherapy outcomes by biomarkers (MSI by FB F1CDx analysis, dMMR by IHC) in mCRC and EMCA patients treated with immunotherapy in any LOT. Chi-square tests and Wilcoxon rank-sum tests were used to assess differences between groups of categorical and continuous variables, respectively. Missing values were imputed with the expected values based on observed covariates using random forests (R package ‘missforest’). Differences in TTNT, PFS, OS, and TTD were evaluated with the log-rank test and Cox proportional hazard models.

For outcomes measurement, TTNT was calculated from the immunotherapy treatment start date until the start of the next treatment line (due to any cause), or death, and patients not yet reaching the next treatment line or death were right-censored at the date of the last clinical visit, laboratory results, or medication order. PFS was calculated from the treatment start date until the time of disease progression or death, and patients not yet reaching progression or death were right-censored at the date of the last clinical visit, laboratory result, or medication order. TTNT and PFS are time-to-event proxies for drug clinical effectiveness21. OS was calculated from the start of treatment to death from any cause, and patients with no record of mortality were right-censored at the date of the last clinic visit or structured activity. OS risk intervals were left truncated to the date of the CGP report to account for immortal time, as patients cannot enter the database until a CGP report is provided22,23. TTD was calculated from the treatment start date until the immunotherapy treatment discontinuation for any reason or death, and patients not yet reaching treatment discontinuation or death were right-censored at the date of last clinical visit, laboratory result, or medication use. The mortality information in the Flatiron Health database is a composite derived from documents within the EHR, Social Security Death Index, and a commercial death dataset mining data from obituaries and funeral homes. This mortality information has been externally validated in comparison to the National Death Index with > 90% accuracy24.

MSI prevalence by F1CDx FB-MSI analysis across advanced cancers of various sites of origin and in tumors derived from Lynch syndrome patients

MSI-H prevalence as determined by the F1CDx FB-MSI analysis was evaluated in 174,166 solid tumor samples across various advanced cancer types from the FMI research database of clinical samples from routine clinical care. To determine the frequency of MSI by the FB-MSI analysis in tumors derived from Lynch syndrome patients, we identified 29 patients with clinically confirmed (n = 25) or suspected (n = 4) Lynch syndrome based on the accompanying submitted clinical documents, such as pathology reports, oncologist clinical note or test requisition sheet. The F1CDx FB-MSI analysis was used to analyze NGS data from these 29 tumors, and the presence of MMR gene alterations was evaluated by F1CDx. For the four patients with suspected Lynch syndrome, a somatic‐germline‐zygosity method was utilized to predict whether the MMR gene alteration was of germline origin25.

Results

Analytical Accuracy as Compared to a PCR-based MSI Detection Method

To demonstrate the analytical accuracy of our novel MSI method by the F1CDx FB-MSI analysis, we assessed concordance of F1CDx MSI status with Promega PCR in 264 solid tumor samples (see distribution of tumor types in Supplemental Table 1). Results from the Promega PCR assay were dichotomized into MSI-H ( ≥ 2 unstable MSI loci) and non-MSI-H. The non-MSI-H group consisted of tumor samples that were either microsatellite stable (MSS, no unstable loci) or MSI low (MSI-L, 1 unstable loci) for concordance with F1CDx MSI status. The PPA and NPA were 98.9% (95% CI [94.1–99.8%]) and 97.1% (95% CI [93.4–98.8%]), respectively (Table 1), with a total of six discordant cases, and an overall percent agreement of 97.7% (95% CI [94.8–99.1%].Table 1 Pan-tumor concordance results for FB-MSI detection via F1CDx and PCR

		PCR	
		MSI-H	Not MSI-H	
F1CDx	MSI-H	91	5	
	Not MSI-H	1	167	
		PPA [95% CI] = 98.9% [94.1–99.8%]	NPA [95% CI] = 97.1% [93.4–98.8%]	

We further analyzed the six discordant cases. One kidney chromophobe carcinoma sample was not-MSI-H by F1CDx and MSI-H by PCR (F1CDx-/PCR + ) and had a FB-MSI score of 0.0105. Five patients had F1CDx + /PCR- results consisting of three ECMA, one thyroid anaplastic carcinoma, and one brain glioblastoma. These five patients had a median FB-MSI score of 0.0163 (range 0.0135-0.0216). All six discordant cases were near the MSI-H threshold for MSI-H status (0.0124) by F1CDx score (Table 2). Additional genomic analysis from F1CDx platform data integrating MMR gene mutation, COSMIC mutational signature and TMB scores was performed (Table 2). The F1CDx non-MSI-H/PCR MSI-H kidney chromophobe carcinoma had a MSH2 Q344_P349del alteration and a dominant MMR COSMIC mutational signature. This sample had an equivocal FB-MSI score and thus was classified as non-MSI-H. All remaining five F1CDx MSI-H/PCR non-MSI-H discordant cases harbored either deleterious MMR gene alterations and/or an MMR COSMIC mutational signature as well as elevated TMB ( > 10 mut/Mb), lending credibility to the F1CDx-determined MSI-H status (Table 2).Table 2 Analysis of F1CDx FB-MSI analysis and PCR discordances

n	Tumor type	PCR status	F1CDx MSI status	FB-MSI score	MMR gene mutation	COSMIC mutational signature	TMB score (mut/Mb)	
1	Chromophobe RCC	MSI-H	Non-MSI-H	0.0105	MSH2 Q344_P349del (39.9% VAF)	dominant MMR signature	9	
2	Endometrial adenocarcinoma	Not-MSI-H	MSI-H	0.0135	MSH6 Y850* and R1035* (46.6% & 22.5% VAF)	dominant MMR signature	29	
3	Thyroid anaplastic carcinoma	Not-MSI-H	MSI-H	0.0163	None detected	nominally dominant MMR	13	
4	Glioblastoma	Not-MSI-H	MSI-H	0.0174	MSH2 V342fs*15 (81.1% VAF)	dominant MMR	34	
5	Endometrial adenocarcinoma	Not-MSI-H	MSI-H	0.0216	MSH2 D379Y & MSH6 D390N (6% & 38% VAF)	dominant POLE	474	
6	Endometrial adenocarcinoma	Not-MSI-H	MSI-H	0.0191	None detected	dominant MMR signature	11	

There were 12 samples with F1CDx MSI-equivocal status with scores >0.0041 and <0.0124. Of these 12 samples, one was MSI-H by PCR (discordant from F1CDx MSI status), and eleven were non-MSI-H by PCR (concordant with F1CDx). Out of the latter eleven, nine were MSS, and two were MSI-L. All MSI-equivocal samples were from non-CRC tumors.

We further sub-stratified our concordance results based on tumor type. For CRC samples (n = 40), there was 100% concordance between the FB-MSI analysis and PCR. For EMCA samples (n = 33), the observed PPA and NPA for F1CDx and PCR were 100% and 84.2%, respectively. All three (3) discordant EMCA cases were MSI-H by F1CDx and not MSI-H by PCR (Table 2). For other tumor types (i.e., non-CRC and non-EMCA, n = 191), the observed PPA and NPA were 98.3% and 98.4%, respectively. Two (2) non-CRC/non-EMCA discordant cases were MSI-H by F1CDx but not MSI-H by PCR, while one (1) case was not MSI-H by F1CDx but MSI-H by PCR (Table 2).

Analytical Accuracy as Compared to MMR IHC Extracted from Clinical Pathology Reports

To further interrogate the performance of the F1CDx FB-MSI analysis as compared to orthogonal MMR IHC, we randomly and consecutively selected a set of 279 cases from the FMI research database, including 179 CRC, 69 EMCA, and 31 other non-CRC/non-EMCA patient data. Selected cases had evaluable F1CDx FB MSI status and existing external MMR IHC testing results provided on pathology reports that were submitted to FMI at the time of genomic testing. The greater number of CRC and EMCA cases may reflect the fact that MMR IHC testing is standard of care in these two tumor types to screen for Lynch syndrome.

Point estimates and 95% two-sided CI for PPA and NPA for the overall sample set were 90.5% (95% CI [79.7% - 95.9%]) and 99.5% (95% CI [97.5–99.9%]), respectively (Table 3) with an overall percent agreement of 97.8% (95% CI [95.1–99.1%]) between F1CDx and locally performed MMR IHC. We further sub-stratified our results according to tumor type. For the CRC subset, the observed PPA and NPA were 88% (95% CI [70.0% - 95.8%]) and 99.4% (95% CI [96.4% - 99.9%]), respectively. For the EMCA subset, the observed PPA and NPA using were 95.5% (95% CI [78.2% - 99.2%]) and 100% (95% CI [92.4% - 100%]), respectively; whereas for tumor types other than CRC or EMCA, PPA was 83.3% (95% CI [43.6% - 97%]) and NPA was 100% (95% CI [86.7% - 100%]).Table 3 Concordance results for F1CDx FB-MSI and locally performed MMR IHC

MSI by F1CDx vs MMR IHC status	F1CDx + dMMR	F1CDx-dMMR	F1CDx + pMMR	F1CDx-pMMR	PPA [95% CI]	NPA [95% CI]	
Pan-tumor	48	5	1	225	90.5% [79.7%, 95.9%]	99.5% [97.5%, 99.9%]	
CRC only	22	3	1	153	88% [70.0%, 95.8%]	99.4% [96.4%, 99.9%]	
EMCA only	21	1	0	47	95.5% [78.2%, 99.2%]	100.0% [92.4%, 100.0%]	
Non-CRC, non-EMCA tumor types	5	1	0	25	83.3% [43.6%, 97%]	100% [86.7%, 100%]	

A representative concordant MSS EMCA by F1CDx and pMMR by IHC is shown in Fig. 1A-E, while a representative concordant MSI-H EMCA by F1CDx and dMMR by IHC is shown in Fig. 1F-J. Representative MMR images for the remaining cases are not available; however, the specific pattern of MMR staining for discordant cases was extracted from pathology reports.Fig. 1 Representative MMR IHC stains for MSS and MSI-H tumors as determined by F1CDx FB-MSI.

A–E High-grade EMCA (A) in a 73-year-old woman showing intact MSH2 (B), MSH6 (C), MLH1 (D), PMS2 (E) expression by IHC. This tumor was MSS by F1CDx. F–J Endometrioid EMCA (F) in an 80-year-old woman showing intact MSH2 (G), MSH6 (H), and loss of MLH1 (I) and PMS2 (J) expression by IHC with internal positive controls (lymphocytes and stromal cells). This tumor was MSI-H by F1CDx. Scale bar = 0.1 mm.

Overall, six samples with discordance between F1CDx FB-MSI and locally performed MMR IHC results were identified in this study, four within the CRC subset, one EMCA sample, and one sample within the non-CRC/non-EMCA subset. Five discordant samples were F1CDx non-MSI-H/dMMR, and one discordant sample was MSI-H by F1CDx and pMMR (Table 4). For three F1CDx non-MSI-H/dMMR cases (1 EMCA, 2 CRC), integrated analysis revealed the presence of an MSH6 alteration in one case and the presence of dominant COSMIC MMR mutational signature in the other two cases favoring the dMMR result over the FB-MSI F1CDx status. In contrast, for the other three discordant cases, further genomic analysis favored the validity of the F1CDx MSI result over IHC. For instance, one F1CDx MSI-H/pMMR CRC exhibited a deleterious MLH1 mutation, dominant DNA MMR COSMIC signature, and high TMB, consistent with further evidence of the validity of the F1CDx MSI-H status (Table 4). Furthermore, for two F1CDx non-MSI-H/dMMR cases (1 CRC, 1 unknown primary adenocarcinoma), the pattern of IHC staining, such as loss of MLH1 only by IHC by report, lack of MMR gene alteration, lack of dominant DNA mismatch mutational signature and low TMB (Table 4) favored the validity of the F1CDx MSI status over IHC.Table 4 Analysis of F1CDx FB-MSI and MMR IHC discordances

n	Tumor type	MMR IHC status	F1CDx MSI status	FB-MSI score	MMR gene mutation	COSMIC mutational signature	TMB score (mut/Mb)	
1	Colorectal adenocarcinoma (CRC)	dMMR (PMS2)	Not-MSI-H	0.0025	None detected	nominally co-dominant MMR	7.6	
2	Colorectal adenocarcinoma (CRC)	dMMR (PMS2)	Not-MSI-H	0.0014	None detected	nominally no dominant signature	5	
3	Colorectal adenocarcinoma (CRC)	dMMR (MSH6)	Not-MSI-H	0.0026	MSH6 frameshift (46% VAF)	cannot be assessed (only 2 assessable alterations)	2.5	
4	Colorectal adenocarcinoma (CRC)	pMMR	MSI-H	0.0613	MLH1 frameshift (43% VAF)	dominant MMR	50	
5	Endometrial adenocarcinoma	dMMR (MLH1/PMS2)	Not-MSI-H	0.0062	None detected	nominally dominant MMR	7.6	
6	Unknown primary adenocarcinoma	dMMR (MLH1 only)	Not-MSI-H	0.0022	None detected	cannot be assessed ( < 5 alterations)	1	

Pan solid tumor clinical validation of F1CDx in KEYNOTE-158 Cohort K and KEYTNOTE-164

To establish the clinical validity of the F1CDx FB-MSI analysis as a CDx to aid in the identification of patients that may respond to pembrolizumab, available samples from KEYNOTE-158 Cohort K and KEYNOTE-164 were retrospectively tested with the F1CDx as part of a bridging study. Patients (n = 444) were enrolled into both studies using local clinical trial assays (CTA) with dMMR status by IHC or MSI-H status by PCR. The observed objective response rate (ORR) to pembrolizumab per RECIST 1.1 in the 444 CTA-positive patients was 31.8% (95% CI [27.4%-36.3%]) (Table 5).Table 5 Clinical validation and efficacy results in KEYNOTE-164 and KEYNOTE-158 Cohort K Combined

Clinical outcome	CTA positive (N = 444)	F1CDx positive and CTA positive (N = 107)	F1CDx negative and CTA positive (N = 58)	F1CDx result missing and CTA positive (N = 279)	
ORR% [95% CI]	31.8% [27.4, 36.3]	43.0% [33.5, 52.9]	12.1% [5.0, 23.3]	31.5% [26.1, 37.3]	
Complete response	38 (8.6%)	13 (12.1%)	2 (3.4%)	23 (8.2%)	
Partial response	103 (23.2%)	33 (30.8%)	5 (8.6%)	65 (23.3%)	
Database cutoff date: KEYNOTE 164: September 09, 2019. KEYNOTE 158: October 05, 2020

Patients with sufficient available FFPE tumor tissue and confirmed appropriate consent were submitted for analysis by the F1CDx test. Observed objective response rate (per RECIST 1.1) was calculated for patients with evaluable MSI status by F1CDx (n = 165). Patients with tumors that were MSI-H by F1CDx and CTA-positive (n = 107) had an ORR of 43.0% (95% CI [33.5%, 52.9%]) (Table 5). In contrast, patients with tumors that were non-MSI-H by F1CDx and CTA-positive (n = 58) had a lower ORR of 12.1% (95% CI [5.0%, 23.3%)] (Table 5). The ORR for patients whose results was not available from F1CDx was similar to the overall ORR of the cohort at 31.5% (95% CI [26.1%-37.3%]), indicating missing data likely did not influence the observed efficacy results from F1CDx (Table 5).

Clinical validity of F1CDx FB-MSI analysis in real-world samples

We further established the clinical validity of F1CDx MSI-H status and response to immunotherapy in real-world metastatic CRC and advanced EMCA patients with MSI-H tumors from a de-identified clinicogenomic database. In a cohort of 246 CRC patients, agreement between F1CDx FB-MSI status and MMR by IHC was strong among patients that received immunotherapy (majority received pembrolizumab) in any line of therapy, with a Cohen’s kappa coefficient of 0.86. However, the likelihood ratio test indicated that the combination of F1CDx and IHC is better in explaining clinical outcomes than IHC alone for TTNT (p = 0.0004), TTD (p = 0.0101), PFS (p = 0.001) and OS (p = 0.014). TTNT and PFS are time-to-event proxies for drug clinical effectiveness21. The combination of F1CDx + IHC is similar to F1CDx alone in explaining clinical outcomes (Fig. 2A).Fig. 2 Clinical validity of F1CDx FB-MSI in real world samples.

A mCRC patients (n = 246): Time To Next Treatment (TTNT) and Time To Treatment Discontinuation (TTD) of patients treated with immunotherapy. B EMCA patients (n = 105): Time to Next Treatment and Time to Treatment Discontinuation of patients treated with immunotherapy.

Similarly, in a cohort of 105 EMCA patients, agreement between F1CDx FB-MSI status and MMR by IHC was also strong, with a Cohen’s kappa statistic of 0.826. Furthermore, the likelihood ratio test indicated that, in EMCA, the combination of F1CDx and IHC is better in explaining clinical outcomes than IHC alone for TTNT (p = 0.002) and TTD (p = 0.009) (Fig. 2B), but not in OS (p = 0.22). The combination of F1CDx and IHC is also similar to F1CDx alone in explaining clinical outcomes in EMCA patients. F1CDx and MMR by IHC can also be evaluated in the swimmer plots for CRC and EMCA patients (Fig. 2), where patients with MSI-H tumors determined by the F1CDx FB-MSI analysis but pMMR by IHC had favorable outcomes on immunotherapy.

MSI-H prevalence by the FB-MSI analysis across 174,166 advanced cancers of various sites of origin

The F1CDx FB-MSI analysis was deployed across 174,166 diverse real-world, solid tumor samples submitted to FMI for profiling during routine clinical care to determine the distribution of MSI-H across various advanced cancer types. Overall, 2.4% (n = 4224) of all solid tumors were MSI-H (Supplemental Fig. 1 and Supplemental Table 2). The highest MSI-H prevalence was detected in gynecological ( ~ 32%) and gastro-intestinal ( ~ 10%) carcinomas, and lower rates ( < 5%) in other malignancies. Specific tumor types with highest MSI-H rate included endometrioid EMCA (32%), vaginal adenocarcinoma (11%), small intestine adenocarcinoma (10%), CRC (7%), ovarian endometrioid adenocarcinoma (6%), gastric adenocarcinoma (6%), cutaneous squamous cell carcinoma (4%), vaginal squamous cell carcinoma (4%), cervical adenocarcinoma (4%), cutaneous adnexal carcinoma (4%), prostatic adenocarcinoma (3%), and kidney urothelial carcinoma (2%). MSI-H status was very rare ( < 1%) or absent in some tumor types, such as renal cell carcinoma (0.5%), lung adenocarcinoma (0.4%), pleural mesothelioma (0.3%), ovarian serous carcinoma (0.2%), cutaneous melanoma (0.1%), GIST (0%), and sarcomas (0%). MSI-H frequencies by cancer types are included in Supplemental Table 2. In this dataset, there may have been a bias towards advanced or metastatic cancer cases since these comprise the majority of FMI’s database.

MSI prevalence by F1CDx FB-MSI analysis in a cohort of tumors derived from Lynch syndrome patients

In contrast to the overall 2.4% of all solid tumors that were MSI-H from the FMI database, we sought to determine whether tumors derived from Lynch syndrome patients were enriched for MSI-H as determined by the F1CDx FB-MSI analysis. From the FMI research database, we identified 29 patients with clinically confirmed (n = 25) or clinically suspected (n = 4) Lynch syndrome based on the accompanying medical documents. 65.5% (n = 19) tumors derived from this cohort of patients were MSI-H, while 20.7% (n = 6) were MSI-equivocal (Supplemental Table 3). The remainder of the tumors (13.8%, n = 4) were MSS (Supplemental Table 3). 3 of 4 MSS tumors were of breast or thyroid origin, which are tumor types that are not classically associated with Lynch syndrome. All tumors derived from the patients with clinically suspected Lynch syndrome (n = 4) were MSI-H and harbored deleterious MMR gene mutations as detected by F1CDx, three of which were predicted to be germline via a somatic‐germline‐zygosity classifier method25. Germline status prediction could not be confidently made for the fourth tumor.

Discussion

Immunotherapy has revolutionized cancer care as pan-tumor FDA approved therapies, such as pembrolizumab, have shown clinical benefit across many cancer types with specific biomarkers (i.e., MSI and TMB). Recently, CAP released MMR and MSI testing guidelines for immunotherapy1, in which it was strongly recommended that pathologists use MMR IHC or MSI PCR for MSI/MMR status determination across a variety of tumor types for patients being considered for immunotherapy. In these CAP guidelines, it was stated that MSI-H by an NGS assay must be validated and show equivalence to MMR by IHC or MSI by PCR1. Herein, we present analytical and clinical validation for FB-MSI on F1CDx, a novel targeted hybrid capture NGS-based method of determining MSI-H that indeed shows equivalence to these accepted orthogonal assays. Analytical validation demonstrated that this method has excellent concordance with both dMMR status as assessed using IHC assays and with MSI-H status as determined using Promega PCR. Furthermore, in patients with MSI-H tumors identified using the F1CDx FB-MSI, enrichment in clinical benefit from treatment with pembrolizumab was confirmed. These results further suggest that F1CDx FB-MSI is at least non-inferior to orthogonal PCR or IHC methods for determining tumor MSI/MMR status.

Discordances compared to PCR may be a consequence of PCR assays only analyzing 5 loci, whereas the >2000 loci examined by F1CDx FB-MSI may yield a more comprehensive assessment of microsatellite status. In discordant F1CDx and PCR cases, there were many non-MLH1 mutations, a scenario in which it may be harder to detect MSI in general. In this setting, assaying more microsatellites may be beneficial as evidenced by greater number of discordant cases that were negative by PCR compared with F1CDx FB-MSI. In contrast, discrepancies between MMR IHC and F1CDx may be due to assessing different analytes and biological phenomena. MMR IHC assesses a different analyte (protein) compared to MSI (DNA). It is also worth noting that the concordance assessment of F1CDx FB-MSI to PCR was based on centrally tested PCR results (i.e. – single laboratory), while MMR IHC concordance analysis was based on abstracted IHC results from pathology reports from many different testing laboratories, and likely different antibody clones and assay methods. Also, we did not have fixation time for FFPE tumor samples, but variability in such times could be another source of discrepancy between dMMR IHC and MSI-H, with the possibility that deamination events occurring with longer fixation might lead to discrepant MSI statuses.

Despite discordances between F1CDx FB-MSI and PCR/IHC-based test results, the clinical validation results of F1CDx FB-MSI in KEYNOTE-158 and 164 as well as in real-world settings demonstrated that F1CDx FB-MSI identifies patients with MSI-H tumors that responds to pembrolizumab, as evidenced by a higher ORR for patients that were MSI-H by F1CDx FB-MSI as compared to non-MSI-H. In addition, clinical validity was further demonstrated in real-word samples from a de-identified clinicogenomic database of advanced CRC and EMCA patients, in which MSI-H by F1CDx FB-MSI and dMMR are highly concordant, but the combination of F1CDx FB-MSI and IHC, or FICDx FB-MSI alone, is better in explaining clinical outcomes than IHC alone. These results indicate equivalence of F1CDx FB-MSI in determining outcomes compared to traditional methods.

Some limitations of MSI testing by F1CDx include longer turnaround time compared to MMR IHC and PCR, a requirement of at least 20% tumor content to confidently determine MSI status, less accessibility to F1CDx in surgical pathology practices compared to MMR IHC, and no direct tissue correlation compared with MMR IHC to determine clonal MMR loss26. As a result of the latter, the MSI results may be inconclusive depending on the relative proportion of the dMMR and pMMR tumor cells in the sample being assayed26. Although costs may be decreasing, F1CDx in general is still more expensive and less readily available to pathologists than MMR IHC or PCR. Despite these limitations, comprehensive genomic profiling by an assay such as F1CDx is now becoming not only common but is also now reimbursed by insurance companies for advanced, late-stage cancer patients, who have progressed on traditional chemotherapeutic agents.

Although pediatric patient age was not an exclusion criterion for the PCR or MMR IHC concordance studies, the vast majority of patients in this study were adult patients. In addition, we did not have any known patients with Constitutional Mismatch Repair Deficiency, a scenario in which prior studies have described lack of sensitivity of classic MSI methods to detect MSI-H27. Also, given that patients’ genetic ancestry may influence microsatellite size, an area for future investigation may include how classic MSI methods versus F1CDx FB MSI analysis from patients’ of different genetic ancestries could influence microsatellite size and impact MSI status28.

During routine oncologic care, an advanced, recurrent, or metastatic cancer sample that is submitted for molecular profiling may be the starting point of testing for MSI and other biomarkers associated with targeted therapies, which may be different from primary tumors that are routinely tested during Lynch syndrome screening in daily surgical pathology practices. In addition, in advanced cancer cases, by utilizing a CGP approach, such as validated F1CDx FB-MSI, MSI-H may be determined while concurrently testing for other clinically relevant genomic biomarkers. Therefore, we propose an algorithm for detecting MSI-H via a validated NGS-based CGP assay (i.e. F1CDx FB-MSI) to determine eligibility for immunotherapy in advanced, metastatic, or recurrent solid tumors (Fig. 3). In this approach, patients whose tumors are definitively classified as MSI-H by F1CDx FB-MSI are eligible for immunotherapy, such as pembrolizumab, while MSS tumors are not eligible based on MSI testing. For the MSI-inconclusive category, an orthogonal method of MSI testing should be performed, such as via MMR IHC or PCR. Reasons for MSI-inconclusive results by F1CDx FB-MSI include test performance specifications in which MSI cannot be determined due to low tumor purity or contamination. Furthermore, tumors whose MSI scores fell short of MSI-H but were above MSS could potentially represent MSI-L. These tumors were classified as MSI-indeterminate/equivocal, and in the clinical setting, an orthogonal method of MSI testing is recommended to confirm MSI status, if clinically indicated, in order to determine eligibility for immunotherapy (Fig. 3).Fig. 3 Proposed algorithm for determining eligibility for immunotherapy in advanced, metastatic or recurrent solid tumors via the validated NGS-based F1CDx assay to detect MSI-H status.

NGS, next-generation sequencing; CGP, comprehensive genomic profiling; dMMR, mismatch repair-deficient; pMMR, mismatch repair-proficient; MSI-H, microsatellite instability-high; MSS, microsatellite stable; TMB, tumor mutational burden; TMB-H, high TMB.

An additional benefit of CGP testing is that an MMR gene (MSH2, MSH6, MLH1, and PMS2) and/or in BRAF mutation may also be identified in conjunction with MSI-H, a scenario in which may inform work-up for Lynch syndrome in certain patients (Fig. 3). In addition to MSI, F1CDx may identify other complex biomarkers, such as high TMB, which is another FDA-approved tumor-agnostic biomarker for pembrolizumab in the advanced, recurrent or metastatic setting29 (Fig. 3). Notably, among six discordant cases with near-threshold fraction-based MSI scores in the PCR concordance study that had available TMB status, five were TMB-H ( > 10 mut/mb) and one was borderline TMB-H with a score of 9 mut/mb. In this approach, the clinical impact of discrepancies between FB-MSI and orthogonal assays may be mitigated by simultaneous evaluation of other biomarkers for immunotherapy.

The F1CDx FB MSI-H rates across different tumor types are in alignment with the expected MSI-H rates for late-stage or advanced tumors, in contrast to rates of MSI-H that are determined during universal screening of newly diagnosed CRC or EMCA patients for Lynch syndrome30–32. The high prevalence of MSI-H in gynecological and gastrointestinal cancer in our cohort is similar to other studies5 and highlight the number of advanced cancer patients who may benefit from immunotherapy based on a validated and commercially available CGP assay (F1CDx) across various cancer types. In contrast to gynecological and gastrointestinal cancers, MSI-H is very rare in most other tumor types; therefore, routine stand-alone testing for only MSI may not be realistic or cost effective, despite the potential immunotherapy benefit to those rare patients. Determining the MSI status via CGP in combination with other complex biomarkers such as TMB, HRD, and other potentially actionable specific gene alterations from a single tumor specimen may be a reasonable approach that is time- and cost-effective and utilizes less tissue.

In summary, a novel FB-MSI analysis by F1CDx, as defined by the fraction of microsatellite loci determined to be altered or unstable based on an analysis across >2,000 microsatellite loci, can accurately detect MSI-H compared with current IHC- and PCR-based methods. It also demonstrates clinical utility to identify MSI-H patients that may respond to pembrolizumab, and similar FB MSI analysis may also be applied to liquid biopsies, such as FoundationOne®Liquid CDx33. F1CDx FB-MSI may be an efficient method for detecting MSI-H to determine patient eligibility for immunotherapy while simultaneously providing a means of generating additional molecular and complex biomarker results to guide targeted therapeutic decisions.

Supplementary information

Supplementary Information

Supplementary information

The online version contains supplementary material available at 10.1038/s41698-024-00679-7.

Author contributions

Conception, D.L.S., J.L., L.A.A. and V.P., Data analysis and visualization, J.C.F.Q., N.D., C.H., L.A.A., J.L. and D.I.L., Manuscript writing and review, D.I.L., J.C.F.Q., N.D., L.L., D.L., C.H., M.C.H., D.L.S, L.A.A., J.L., D.A.M., B.D., S.L., N.R.P., R.P.G., J.A.E., J.S.R., V.P., R.S.P.H. and A.K.W.

Data availability

All relevant data were provided with the manuscript. If needed, additional details may also be obtained by contacting the corresponding authors.

Competing interests

The authors declare the following competing interests: D.I.L., J.C.F.Q., N.D., M.C.H., D.L.S., L.A.A., J.L., D.A.M., B.D., S.L., N.R.P., R.P.G., J.A.E., J.S.R., V.P., and R.S.P.H. are employed by Foundation Medicine, Inc., a wholly-owned subsidiary of Roche, and are stockholders of Roche. L.L., D.L., and A.K.W. are full-time employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and hold stock and/or restricted stock units in Merck & Co., Inc., Rahway, NJ, USA.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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