Process for Microsatellite Instability Detection

Abstract

The invention provides methods for determining the MSI status of a patient by liquid biopsy with sample preparation using hybrid capture and non-unique barcodes. In certain aspects, the invention provides a method of detecting microsatellite instability (MSI). The method includes obtaining cell-free DNA (cfDNA) from a sample of blood or plasma from a patient and sequencing portions of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA. A report is provided describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution.

1 . A method for determining a prognosis or therapeutic regimen response for a patient having cancer, the method comprising:
 (a) capturing target circulating tumor DNA (ctDNA) from a first liquid sample from the patient at a first timepoint prior to the patient beginning a therapeutic regimen;   (b) determining a baseline ctDNA level at the first timepoint using the ctDNA from (a);   (c) capturing target ctDNA from a second liquid sample from the patient at a second timepoint after the patient begins the therapeutic regimen;   (d) determining an on-treatment ctDNA level at the second timepoint using the ctDNA from (c);   (e) capturing target ctDNA from additional liquid samples from the patient at one or more additional consecutive timepoints while the patient continues the therapeutic regimen;   (f) determining the on-treatment ctDNA level for each additional liquid sample at each of the one or more additional consecutive timepoints using the ctDNA from (e); and   (g) determining the prognosis or therapeutic regimen response for the patient based on comparing the baseline ctDNA level with each of the on-treatment ctDNA levels,
 wherein the prognosis or therapeutic regimen response is positive if a >80% reduction is found in two or more consecutive on-treatment ctDNA levels when compared to the baseline ctDNA level. 
   
     
     
         2 . The method of  claim 1 , wherein the patient has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers. 
     
     
         3 . The method of  claim 1 , wherein the baseline ctDNA level and each of the on-treatment ctDNA levels are determined by measuring one or more serum tumor protein biomarkers, one or more microsatellite instability (MSI) alleles, or a tumor mutation burden (TMB). 
     
     
         4 . The method of  claim 3 , wherein the serum tumor protein biomarkers are CEA and/or CA19-9. 
     
     
         5 . The method of  claim 1 , wherein the prognosis or therapeutic regimen response is positive if a >90% reduction is found in the two or more consecutive on-treatment ctDNA levels when compared to the baseline ctDNA level. 
     
     
         6 . The method of  claim 5 , wherein the baseline ctDNA level and each of the on-treatment ctDNA levels are determined by measuring one or more microsatellite instability (MSI) alleles or a tumor mutation burden (TMB). 
     
     
         7 . The method of  claim 1 , wherein the therapeutic regimen is a checkpoint inhibitor regimen. 
     
     
         8 . A system for determining a prognosis or therapeutic regimen response for a patient having cancer, the system comprising:
 one or more processors; and   a non-transitory memory device containing instructions which, when executed on the one or more processors, cause the one or more processors to perform processes including:
 (a) determining a baseline ctDNA level at a first timepoint using target circulating tumor DNA (ctDNA) captured from a first liquid sample from the patient at the first timepoint prior to the patient beginning a therapeutic regimen; 
 (b) determining an on-treatment ctDNA level at a second timepoint using ctDNA captured from a second liquid sample from the patient at the second timepoint after the patient begins the therapeutic regimen; 
 (c) determining the on-treatment ctDNA level for each additional liquid sample at each of one or more additional consecutive timepoints using ctDNA captured from each of the additional liquid samples from the patient at the one or more additional consecutive timepoints while the patient continues the therapeutic regimen; and 
 (d) determining the prognosis or therapeutic regimen response for the patient based on comparing the baseline ctDNA level with each of the on-treatment ctDNA levels, wherein the prognosis or therapeutic regimen response is positive if a >80% reduction is found in two or more consecutive on-treatment ctDNA levels when compared to the baseline ctDNA level. 
   
     
     
         9 . The system of  claim 8 , wherein the patient has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers. 
     
     
         10 . The system of  claim 8 , wherein the baseline ctDNA level and each of the on-treatment ctDNA levels are determined by measuring one or more serum tumor protein biomarkers, one or more microsatellite instability (MSI) alleles, or a tumor mutation burden (TMB). 
     
     
         11 . The system of  claim 10 , wherein the serum tumor protein biomarkers are CEA and/or CA19-9. 
     
     
         12 . The system of  claim 8 , wherein the prognosis or therapeutic regimen response is positive if a >90% reduction is found in the two or more consecutive on-treatment ctDNA levels when compared to the baseline ctDNA level. 
     
     
         13 . The system of  claim 12 , wherein the baseline ctDNA level and each of the on-treatment ctDNA levels are determined by measuring one or more microsatellite instability (MSI) alleles or a tumor mutation burden (TMB). 
     
     
         14 . The system of  claim 8 , wherein the therapeutic regimen is a checkpoint inhibitor regimen. 
     
     
         15 . A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform actions including:
 (a) determining a baseline ctDNA level at a first timepoint using target circulating tumor DNA (ctDNA) captured from a first liquid sample from a patient at the first timepoint prior to the patient beginning a therapeutic regimen;   (b) determining an on-treatment ctDNA level at a second timepoint using ctDNA captured from a second liquid sample from the patient at the second timepoint after the patient begins the therapeutic regimen;   (c) determining the on-treatment ctDNA level for each additional liquid sample at each of one or more additional consecutive timepoints using ctDNA captured from each of the additional liquid samples from the patient at the one or more additional consecutive timepoints while the patient continues the therapeutic regimen; and   (d) determining a prognosis or therapeutic regimen response for the patient based on comparing the baseline ctDNA level with each of the on-treatment ctDNA levels, wherein the prognosis or therapeutic regimen response is positive if a >80% reduction is found in two or more consecutive on-treatment ctDNA levels when compared to the baseline ctDNA level.   
     
     
         16 . The computer-program product of  claim 15 , wherein the patient has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers. 
     
     
         17 . The computer-program product of  claim 15 , wherein the baseline ctDNA level and each of the on-treatment ctDNA levels are determined by measuring one or more serum tumor protein biomarkers, one or more microsatellite instability (MSI) alleles, or a tumor mutation burden (TMB). 
     
     
         18 . The computer-program product of  claim 17 , wherein the serum tumor protein biomarkers are CEA and/or CA19-9. 
     
     
         19 . The computer-program product of  claim 15 , wherein the prognosis or therapeutic regimen response is positive if a >90% reduction is found in the two or more consecutive on-treatment ctDNA levels when compared to the baseline ctDNA level. 
     
     
         20 . The computer-program product of  claim 19 , wherein the baseline ctDNA level and each of the on-treatment ctDNA levels are determined by measuring one or more microsatellite instability (MSI) alleles or a tumor mutation burden (TMB).