Microsatellite instability detection in cell-free DNA
Inventors
ARTSIOMENKA, Aliaksandr • SIKORA, Marcin Pawel • Barbacioru, Catalin • Chudova, Darya • LEFTEROVA, Martina I.
Assignees
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Publication Number
US-12344902-B2
Publication Date
2025-07-01
Expiration Date
Abstract
Provided herein are methods for determining the microsatellite instability status of samples. In one aspect, the methods include quantifying a number of different repeat lengths present at each of a plurality of microsatellite loci from sequence information to generate a site score for each of the plurality of the microsatellite loci. The methods also include comparing the site score of a given microsatellite locus to a site specific trained threshold for the given microsatellite locus for each of the plurality of the microsatellite loci and calling the given microsatellite locus as being unstable when the site score of the given microsatellite locus exceeds the site specific trained threshold for the given microsatellite locus to generate a microsatellite instability score, which includes a number of unstable microsatellite loci from the plurality of the microsatellite loci.
Core Innovation
The invention relates to determining microsatellite instability (MSI) status for a human subject using cfDNA molecules and using that MSI status to administer immunotherapy to treat cancer. A sample comprising cell-free deoxyribonucleic acids (cfDNA) molecules obtained from the human subject provides sequence information that is used to quantify different repeat lengths at each microsatellite locus.
For each of a plurality of microsatellite loci, the method generates a site score (SS) based on likelihood scores comprising probabilistic log likelihood-based scores derived from observed allele frequencies of the different repeat lengths and noise in the sequence information. The SS of each locus is compared to a site specific trained threshold defined as the maximum value of a site score for a microsatellite locus to be classified as stable, and loci are called unstable when the SS exceeds the threshold, thereby generating a microsatellite instability (MI) score comprising at least one unstable microsatellite locus.
The sample is classified as unstable at the microsatellite instability status (MSI-S) level if the MI score exceeds a population trained threshold for the population of microsatellite loci in the sample to identify an unstable sample. The invention further uses the classified unstable MSI status to administer at least one immunotherapy, including at least one immune checkpoint molecule or an antibody specific for an antigen selected from PD-1, PD-2, PD-L1, PD-L2, CTLA-4, OX40, B7.1, B7He, LAG3, CD137, KIR, CCR5, CD27, CD40, and CD47, as well as proinflammatory cytokines IL-1β, IL-6, and TNF-α, or activated T-cells, thereby treating the cancer in the human subject.
Claims Coverage
The provided material identifies a single independent claim that covers a treatment method combining cfDNA-based MSI detection with thresholded likelihood scoring and downstream immunotherapy selection. The independent claim includes four inventive features.
CfDNA-derived microsatellite site score generation using probabilistic log-likelihood
Generating a site score (SS) by quantifying different repeat lengths at each of a plurality of microsatellite loci from sequence information from a sample comprising cfDNA molecules obtained from the human subject, with likelihood scores comprising probabilistic log likelihood-based scores derived from observed allele frequencies of the different repeat lengths and noise in the sequence information.
Locus-level unstable calling using site specific trained thresholds
Comparing the SS of a given microsatellite locus to a site specific trained threshold for the given microsatellite locus, wherein the site specific trained threshold is the maximum value of a site score for a microsatellite locus to be classified as stable, and calling the locus unstable if the SS exceeds the threshold to generate a microsatellite instability (MI) score comprising at least one unstable microsatellite locus.
Sample-level MSI-S classification using a population trained threshold
Classifying microsatellite instability status (MSI-S) of the sample as unstable if the MI score exceeds a population trained threshold for the population of microsatellite loci in the sample to identify an unstable sample.
Immunotherapy administration based on unstable MSI status
Administering at least one immunotherapy to a human subject whose MSI status is classified as unstable, where the immunotherapy comprises at least one immune checkpoint molecule, an antibody specific for an antigen selected from PD-1, PD-2, PD-L1, PD-L2, CTLA-4, OX40, B7.1, B7He, LAG3, CD137, KIR, CCR5, CD27, CD40, and CD47, a proinflammatory cytokine selected from IL-1β, IL-6, and TNF-α, and activated T-cells, thereby treating the cancer in the human subject.
The core coverage is a cfDNA-based, repeat-length quantification and probabilistic log-likelihood site scoring framework that produces locus-level unstable calls using site specific trained thresholds, aggregates instability into an MI score, and applies a population trained threshold to classify MSI-S as unstable. That unstable MSI-S classification is linked to administration of immunotherapy comprising immune checkpoint molecules, specified antibodies, specified cytokines, and activated T-cells to treat the cancer.
Stated Advantages
Analytical performance targets are claimed, including limit of detection (LoD), analytical specificity, and concordance versus PCR-based MSI testing.
Example validation results are provided using simulation and clinical cohorts.
The approach supports treatment selection for customized immunotherapies based on MSI status classified as unstable.
High overall accuracy for MSI classification in a tissue-referenced clinical validation cohort, with MSI-H detection sensitivity reported as part of evaluable patients.
Analytical sensitivity and analytical specificity are reported as high, with a high precision/concordance result.
Pan-cancer analysis reports a quantified prevalence of MSI-H across tumor types.
Preliminary clinical utility is reported using immunotherapy in advanced gastric cancer patients, including a reported objective response rate and durable responses.
Documented Applications
Using microsatellite instability (MSI) status to select and administer immunotherapy to a human subject having cancer characterized by MSI, including treatment via immune checkpoint molecules, antibodies, proinflammatory cytokines, and activated T-cells.
Computer-readable media and system implementations for MSI classification, including system embodiments for performing MSI classification.
Immunotherapy application in advanced gastric cancer patients treated with PD-1/PD-L1 immunotherapy, with RECIST response reporting and durable responses.
Pan-cancer landscape analysis application using a large cohort of advanced cancer samples to report MSI-H prevalence across multiple tumor types and characterize burdens in MSI-H samples.
Analytical evaluation of an MSI assessment workflow using diluted MSI-H and MSS cell-line cfDNA to report limit of detection and analytical performance.
Clinical validation application using a cohort of tissue-referenced samples (n=1145) to assess performance of the MSI assessment approach.
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