Microsatellite instability detection in cell-free dna

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.

1 . A system comprising:
 a hardware processing unit;   a computer-readable storage media storing computer-executable instructions that, when executed by the hardware processing unit, cause the system to perform operations comprising:
 (a) accessing at least one dataset comprising a plurality of microsatellite loci, each comprising a plurality of repeat lengths from a biological sample from a patient; 
 (b) quantifying the plurality of repeat lengths to generate a site score (SS) for each of a plurality of microsatellite loci; 
 (c) generating a microsatellite instability (MI) score for each of the plurality of microsatellite loci by comparing their site score (SS) to a site-specific threshold, wherein SS exceeding their site-specific threshold indicate unstable microsatellite loci; and 
 (d) determining the microsatellite instability status (MSI-S) of the sample as being microsatellite unstable when the MI score exceeds a population threshold. 
   
     
     
         2 . The system of  claim 1 , wherein the dataset further comprises sequencing reads obtained by sequencing the sample. 
     
     
         3 . The system of  claim 2 , wherein the sample comprises cell free DNA(cfDNA) molecules. 
     
     
         4 . The system of  claim 3 , wherein the cfDNA molecules are enriched prior to sequencing. 
     
     
         5 . The system of  claim 4 , wherein the enrichment is performed using capture probes selected for at least one panel. 
     
     
         6 . The system of  claim 5 , wherein the enrichment is performed using probe sets of different relative concentrations to differentially tile across genomic regions. 
     
     
         7 . The system of  claim 1 , wherein the patient is a cancer patient. 
     
     
         8 . The system of  claim 1 , wherein the site score of a given microsatellite locus includes an AIC-based site score that tests for a presence of somatic indels at that microsatellite locus. 
     
     
         9 . The system of  claim 8 , wherein the AIC-based site score is given by AIC=k−log-likelihood. 
     
     
         10 . The system of  claim 1 , wherein the microsatellite instability score comprises at least one unstable microsatellite loci from the plurality of the microsatellite loci. 
     
     
         11 . The system of  claim 1 , wherein the MSI-S further comprises MSI-H or MSI-low. 
     
     
         12 . The system of  claim 1 , wherein the population threshold comprises a predetermined value derived from a population of training normal or non-tumor cfDNA samples. 
     
     
         13 . The system of  claim 7 , wherein the cancer is selected from the group consisting of: biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast carcinoma, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal carcinoma, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinomas, gastrointestinal stromal tumors (GISTs), endometrial carcinoma, endometrial stromal sarcomas, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder carcinomas, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinomas, Wilms tumor, leukemia, acute lymphocytic leukemiaF (ALL), acute myeloid leukemia (AML), chronic lymphocytic (CLL), chronic myeloid (CML), chronic myelomonocytic (CMML), liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, Lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphomas, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, Mantle cell lymphoma, T cell lymphomas, non-Hodgkin lymphoma, precursor T-lymphoblastic lymphoma/leukemia, peripheral T cell lymphomas, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral cavity squamous cell carcinomas, osteosarcoma, ovarian carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasms, acinar cell carcinomas. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine carcinomas, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer, and uterine sarcoma. 
     
     
         14 . The system of  claim 1 , wherein the therapy comprises at least one of surgical therapy, radiation therapy, chemotherapy, or immunotherapy. 
     
     
         15 . The system of  claim 14 , wherein the immunotherapy comprises a checkpoint molecule. 
     
     
         16 . The system of  claim 14 , wherein the immune checkpoint molecule comprises an inhibitory molecule that reduces a signal involved in the T cell response to antigen. 
     
     
         17 . The system of any one of  claim 15 , wherein the immune checkpoint molecule comprises at least one of CTLA4, PD-1, PD-L1, PD-L2, CTLA4, CD80, CD86, lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GALS), or adenosine A2a receptor (A2aR). 
     
     
         18 . The system of  claim 15 , wherein the immune checkpoint molecule is a co-stimulatory molecule or a ligand of a co-stimulatory molecule comprising CD28, CD80, CD86, B7RP1, B7-H3, B7-H4, CD137L, OX40L, or CD70. 
     
     
         19 . A system comprising:
 a hardware processing unit;   a computer-readable storage media storing computer-executable instructions that, when executed by the hardware processing unit, cause the system to perform operations comprising:
 (a) accessing at least one dataset comprising a plurality of sequence reads of a plurality of microsatellite loci, each comprising a plurality of repeat lengths from a biological sample comprising cfDNA molecules, wherein the cfDNA molecules are enriched using capture probes comprising at least one probe set of different relative concentrations; 
 (b) quantifying the plurality of repeat lengths to generate a site score (SS) for each of a plurality of microsatellite loci; 
 (c) generating a microsatellite instability (MI) score for each of the plurality of microsatellite loci by comparing their site score (SS) to a site-specific threshold, wherein SS exceeding their site-specific threshold indicate unstable microsatellite loci; and 
 (d) determining the microsatellite instability status (MSI-S) of the sample as being microsatellite unstable when the MI score exceeds a population threshold and not calling the given microsatellite locus as being unstable if the SS of the given microsatellite locus does not exceed the site specific trained threshold for the given microsatellite locus; and 
 (e) determining a therapy based on the determination in (d), wherein the therapy comprises immunotherapy comprising at least one of:
 an immune checkpoint molecule selected from the group consisting of: an inhibitory molecule that reduces a signal involved in the T cell response to antigen, is a co-stimulatory molecule and a ligand of a co-stimulatory molecule, 
 an antibody specific for an antigen selected from the group consisting of: 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 the group consisting of: IL-113, IL-6, and TNF-α and activated T-cells. 
 
   
     
     
         20 . A computer-implemented method comprising:
 receiving at least one dataset in a computer system comprising a hardware processor and a computer-readable storage media,   
       wherein the dataset comprises a plurality of microsatellite loci, each comprising a plurality of repeat lengths from a test sample, 
       wherein the computer-readable media comprises instructions that, when executed by the processor, cause the hardware processor to quantify the plurality of repeat lengths to generate a site score (SS) for each of a plurality of microsatellite loci, wherein the SS is compared to a site-specific threshold to determine a microsatellite instability (MI) score, wherein the MI score determines at least one classification of the test sample.