Analysis of cell-free dna in urine and other samples

Abstract

Diseases (e.g., cancer) of a particular organ can be detected by analyzing cell-free DNA. Some embodiments may use an organ-associated sample that is from a particular organ or passes through the particular organ, as may occur, for example, in urine, saliva, blood, and stool samples. In some embodiments, methylation levels of cell-free DNA can be measured in a sample. Tissue-specific methylation patterns can be used to determine fractional contributions from different tissue types. In other embodiments, sizes of organ-associated cell-free DNA can be measured. A statistical measure of the size profile may indicate that cell-free DNA fragments are collectively longer than expected for subjects with healthy tissue compared to non-healthy tissue. In other embodiments, two different samples can be analyzed to determine whether a particular organ has cancer. Cell-free DNA in a blood sample and organ-associated sample can both be analyzed to identify chromosomal regions exhibiting a copy number aberration.

What is claimed is: 
     
         1 . A method of analyzing a urine sample of an organism, the urine sample including cell-free DNA fragments, the method comprising:
 for each size of a plurality of sizes:
 measuring an amount of DNA fragments from the urine sample corresponding to the size; 
   calculating a first value of a first parameter based on the amounts of DNA fragments at multiple sizes, the first parameter providing a statistical measure of a size profile of DNA fragments in the urine sample, wherein the first parameter increases with increasing size of the DNA fragments;   comparing the first value to a reference value; and   determining a classification of a level of bladder cancer based on the comparing.   
     
     
         2 . The method of  claim 1 , wherein the reference value is determined from samples that are respectively known to have bladder cancer, known to not have bladder cancer, or from some samples known to have bladder cancer and from some samples known to not have bladder cancer. 
     
     
         3 . The method of  claim 1 , wherein:
 the first parameter comprises a periodicity index, the periodicity index calculated using a difference in amounts of DNA fragments at a plurality of peaks in the size profile and amounts of DNA fragments at a plurality of troughs in the size profile,   the plurality of peaks are present at regular size intervals, and   the plurality of troughs are present at regular size intervals offset from the plurality of peaks.   
     
     
         4 . The method of  claim 1 , wherein determining the classification of the level of bladder cancer comprises determining that the organism has bladder cancer when the first value is greater than the reference value. 
     
     
         5 . The method of  claim 1 , wherein the level of bladder cancer comprises a stage of bladder cancer. 
     
     
         6 . The method of  claim 1 , wherein the reference value is determined from the organism before an operation to remove a tumor from a bladder of the organism. 
     
     
         7 . The method of  claim 1 , wherein the classification of the level of bladder cancer is whether the bladder cancer is muscle invasive. 
     
     
         8 . The method of  claim 1 , wherein measuring the amount of DNA fragments from the urine sample corresponding to the size comprises:
 for each DNA fragment of a plurality of DNA fragments from the urine sample:
 sequencing the DNA fragment to obtain sequence reads, 
 aligning the sequence reads to a reference genome, and 
 determining the size of the DNA fragment using the aligned reads. 
   
     
     
         9 . The method of  claim 8 , wherein the plurality of DNA fragments is chosen at random. 
     
     
         10 . The method of  claim 1 , wherein the classification of the level of bladder cancer comprises that the organism has bladder cancer. 
     
     
         11 . The method of  claim 10 , further comprising:
 determining a treatment plan for the bladder cancer based on the classification.   
     
     
         12 . The method of  claim 10 , further comprising:
 treating the bladder cancer based on the classification with chemotherapy, drugs, diet, therapy, or surgery.   
     
     
         13 . The method of  claim 1 , wherein the first parameter comprises a median or a proportion of DNA fragments having a threshold size. 
     
     
         14 . The method of  claim 1 , wherein the total amount of DNA fragments at the multiple sizes is greater than 100,000 DNA fragments. 
     
     
         15 . A computer product comprising a computer readable medium storing a plurality of instructions for controlling a computer system to perform the method of  claim 1 . 
     
     
         16 . A system comprising:
 the computer product of  claim 15 ; and   one or more processors for executing instructions stored on the computer readable medium.   
     
     
         17 . A method of analyzing a urine sample from a renal pelvis of a kidney of an organism, the urine sample comprising DNA, wherein at least some of the DNA is cell-free, the method comprising:
 for each size of a plurality of sizes:
 measuring an amount of DNA fragments from the urine sample corresponding to the size; 
   calculating a first value of a first parameter based on the amounts of DNA fragments at multiple sizes, the first parameter providing a statistical measure of a size profile of DNA fragments in the urine sample;   comparing the first value to a reference value; and   determining a classification of a level of inflammation in the kidney based on the comparing.   
     
     
         18 . A method of identifying cancer in a first organ of an organism by analyzing a first sample and a blood sample of the organism, the first sample and the blood sample both including DNA originating from normal cells and potentially from cells associated with cancer, wherein the first sample is from the first organ or passes through the first organ as the first sample exits the organism and is different from the blood sample, and wherein at least some of the DNA is cell-free in both the first sample and the blood sample, the method comprising:
 analyzing a plurality of DNA molecules from the first sample and the blood sample, wherein analyzing a DNA molecule includes:
 identifying a location of the DNA molecule in a genome of the organism, and optionally determining whether the DNA molecule is methylated at one or more sites; 
   for each chromosomal region of a plurality of chromosomal regions of the organism:
 determining a classification of whether the chromosomal region exhibits an aberration of at least one of a copy number aberration or a methylation aberration for each of the first sample and the blood sample by:
 identifying a respective group of DNA molecules from the respective sample as being from the chromosomal region based on the identified locations, the respective group including at least one DNA molecule located at each of a plurality of loci of the chromosomal region; 
 calculating, with a computer system, a respective value of the respective group of DNA molecules, the respective value defining a property of the DNA molecules of the respective group, the property being at least one of a copy number or a methylation level; 
 comparing the respective value to a reference value; 
 
 determining a first level of cancer based on whether a first amount of chromosomal regions classified as exhibiting an aberration for the first sample is above a first threshold; 
 determining a second level of cancer based on whether a second amount of chromosomal regions classified as exhibiting an aberration for the blood sample is above a second threshold; and 
 determining that the organism has cancer of the first organ when the first level of cancer indicates that the organism has cancer and the second level of cancer indicates that the organism does not have cancer. 
   
     
     
         19 . A method of analyzing a urine sample of an organism, the urine sample including DNA originating from normal cells and potentially from cells associated with cancer, wherein at least some of the DNA is cell-free in the urine sample, the method comprising:
 analyzing a plurality of DNA molecules from the urine sample, wherein analyzing a DNA molecule includes:
 identifying a location of the DNA molecule in a genome of the organism; 
   for each chromosomal region of a plurality of chromosomal regions of the organism:
 determining a classification of whether the chromosomal region exhibits an aberration of at least one of a copy number aberration or a methylation aberration by:
 identifying a group of DNA molecules from the urine sample as being from the chromosomal region based on the identified locations, the group including at least one DNA molecule located at each of a plurality of loci of the chromosomal region; 
 calculating, with a computer system, a value of the group of DNA molecules, the value defining a property of the DNA molecules of the group, the property being at least one of a copy number or a methylation level; 
 comparing the value to a reference value; 
 
 determining a first level of cancer based on whether a first amount of chromosomal regions classified as exhibiting a copy number aberration for the urine sample is above a first threshold; 
 determining a second level of cancer based on whether a second amount of chromosomal regions classified as exhibiting the methylation aberration for the urine sample is above a second threshold; 
 determining a third level of cancer based on whether a fractional contribution of a tumor tissue is above a third threshold; 
 determining that the organism has cancer when at least one of the first level of cancer, the second level of cancer, or the third level of cancer indicate that the organism has cancer.