Methods of monitoring immunosuppressive therapies in a transplant recipient

Abstract

The present disclosure relates to methods of monitoring the status of an allograft in a transplant recipient, as well as to methods of monitoring and adjusting immunosuppressive therapies being administered to the transplant recipient.

What is claimed is: 
     
         1 . A method of quantifying an amount of non-self DNA in a transplant recipient of stem cells or a bone marrow transplant from a transplant donor without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing DNA from a sample obtained from the recipient post-transplantation;   (b) amplifying, in a targeted manner from the DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs,   (c) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs (SNP allele distribution patterns) in the panel; and   (d) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of non-self DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns.   
     
     
         2 . The method of  claim 1 , wherein the DNA is cell-free DNA. 
     
     
         3 . The method of  claim 1 , wherein the DNA is cell-bound DNA. 
     
     
         4 . The method of  claim 2 , wherein an amount of non-self cell-free DNA above a predetermined threshold indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease, and an amount of non-self cell-free DNA below a predetermined threshold indicates a status of engraftment, expansion and/or persistence of the transplanted cells. 
     
     
         5 . The method of  claim 3 , wherein an amount of non-self cell-bound DNA above a predetermined threshold and/or increasing or stable over a time interval indicates a status of engraftment, expansion, and/or persistence of the transplanted cells, and an amount of non-self cell-bound DNA below a predetermined threshold and/or decreasing over a time interval indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease. 
     
     
         6 . The method of  claim 1 , wherein the cells are stem cells. 
     
     
         7 . The method of  claim 1 , wherein the cells are a bone marrow transplant. 
     
     
         8 . The method of  claim 1 , wherein each SNP in the panel is selected to have greater than 0.4 minor allele frequency and low linkage with each other. 
     
     
         9 . The method of  claim 1 , further comprising testing for the presence of an infectious agent. 
     
     
         10 . A kit for carrying out the method of  claim 1 , comprising: primers, reagents, controls for targeted single nucleotide polymorphism (SNP) amplification and sequencing, instructions for use, and instructions for accessing and using software for quantifying an amount of non-self DNA in a transplant recipient of stem cells or a bone marrow transplant from a transplant donor. 
     
     
         11 . A method of quantifying an amount of DNA molecules from non-self DNA in a transplant recipient of stem cells or bone marrow transplant from a transplant donor without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing DNA from a sample obtained from the recipient post-transplantation;   (b) amplifying, in a targeted manner from the DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs,   (c) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs (SNP allele distribution patterns) in the panel; and   (d) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of DNA molecules from non-self DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns.   
     
     
         12 . The method of  claim 11 , wherein the DNA is cell-free DNA. 
     
     
         13 . The method of  claim 11 , wherein the DNA is cell-bound DNA. 
     
     
         14 . The method of  claim 12 , wherein an amount of DNA molecules from non-self cell-free DNA above a predetermined threshold indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease, and an amount of DNA molecules from non-self cell-free DNA below a predetermined threshold indicates a status of engraftment, expansion and/or persistence of the transplanted cells. 
     
     
         15 . The method of  claim 13 , wherein an amount of DNA molecules from non-self cell-bound DNA above a predetermined threshold and/or increasing or stable over a time interval indicates a status of engraftment, expansion, and/or persistence of the transplanted cells, and an amount of DNA molecules from non-self cell-bound DNA below a predetermined threshold and/or decreasing over a time interval indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease. 
     
     
         16 . The method of  claim 11 , wherein the cells are stem cells. 
     
     
         17 . The method of  claim 11 , wherein the cells are a bone marrow transplant. 
     
     
         18 . The method of  claim 11 , wherein each SNP in the panel is selected to have greater than 0.4 minor allele frequency and low linkage with each other. 
     
     
         19 . The method of  claim 11 , further comprising testing for the presence of an infectious agent. 
     
     
         20 . The method of  claim 11 , further comprising adding at least one reference DNA molecule to the sample before the amplifying step and quantifying an amount of DNA molecules from the non-self DNA in comparison to the at least one reference DNA molecule. 
     
     
         21 . The method of  claim 20 , wherein the amount of DNA molecules from the non-self DNA is quantified as copies of DNA molecules per volume. 
     
     
         22 . A kit for carrying out the method of  claim 11 , comprising: primers, reagents, controls for targeted single nucleotide polymorphism (SNP) amplification and sequencing, instructions for use, and instructions for accessing and using software for quantifying an amount of DNA molecules from non-self DNA in a transplant recipient of stem cells or a bone marrow transplant from a donor. 
     
     
         23 . A method of preparing amplified SNPs useful for quantifying an amount of non-self DNA in a transplant recipient of stem cells, or a bone marrow transplant from a transplant donor without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing DNA from a sample obtained from the recipient post-transplantation;   (b) extracting DNA from the sample, wherein the DNA comprises non-self DNA and recipient-derived DNA;   (c) amplifying, in a targeted manner from the DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs;   (d) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs (SNP allele distribution patterns) in the panel; and   (e) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of non-self DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns.   
     
     
         24 . The method of  claim 23 , wherein the DNA is cell-free DNA. 
     
     
         25 . The method of  claim 23 , wherein the DNA is cell-bound DNA. 
     
     
         26 . The method of  claim 24 , wherein an amount of non-self cell-free DNA above a predetermined threshold indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease, and an amount of non-self cell-free DNA below a predetermined threshold indicates a status of engraftment, expansion and/or persistence of the transplanted cells. 
     
     
         27 . The method of  claim 25 , wherein an amount of non-self cell-bound DNA above a predetermined threshold and/or increasing or stable over a time interval indicates a status of engraftment, expansion, and/or persistence of the transplanted cells, and an amount of non-self cell-bound DNA below a predetermined threshold and/or decreasing over a time interval indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease. 
     
     
         28 . The method of  claim 23 , wherein the cells are stem cells. 
     
     
         29 . The method of  claim 23 , wherein the cells are a bone marrow transplant. 
     
     
         30 . The method of  claim 23 , further comprising testing for the presence of an infectious agent.