Sequence variant analysis of cell-free dna for cancer screening

Abstract

A frequency of somatic mutations in a biological sample (e.g., plasma or serum) of a subject undergoing screening or monitoring for cancer, can be compared with that in the constitutional DNA of the same subject. A parameter can derived from these frequencies and used to determine a classification of a level of cancer. False positives can be filtered out by requiring any variant locus to have at least a specified number of variant sequence reads (tags), thereby providing a more accurate parameter. The relative frequencies for different variant loci can be analyzed to determine a level of heterogeneity of tumors in a patient.

What is claimed is: 
     
         1 . A method for detecting tumor-derived mutations in cell-free DNA molecules, the method comprising:
 obtaining, by a computer system, first sequence reads for cell-free DNA molecules from a biological sample of a subject, the first sequence reads comprising first sequences;   obtaining, by the computer system, second sequence reads for DNA molecules from a plurality of blood cells of the subject, the second sequence reads comprising second sequences; and   detecting, by the computer system, the tumor-derived mutations in the cell-free DNA molecules by filtering out a portion of the first sequences that are also present in the second sequences.   
     
     
         2 . The method of  claim 1 , wherein the plurality of blood cells comprise healthy blood cells. 
     
     
         3 . The method of  claim 1 , wherein the plurality of blood cells are known not to be cancerous. 
     
     
         4 . The method of  claim 1 , wherein the blood cells comprise white blood cells. 
     
     
         5 . The method of  claim 1 , wherein the biological sample of the subject comprises blood plasma. 
     
     
         6 . The method of  claim 1 , wherein the biological sample of the subject comprises one or more of a group consisting of urine, ascetic fluid, peritoneal fluid, saliva, cerebrospinal fluid, and a stool sample. 
     
     
         7 . The method of  claim 1 , further comprising sequencing the cell-free DNA molecules from the biological sample of the subject to obtain the first sequence reads. 
     
     
         8 . The method of  claim 7 , wherein sequencing the cell-free DNA molecules from the biological sample comprises targeted sequencing. 
     
     
         9 . The method of  claim 8 , wherein the targeted sequencing is performed on DNA obtained from use of a solid-phase capture technique, a solution hybridization technique, or amplification via PCR. 
     
     
         10 . The method of  claim 8 , wherein the targeted sequencing comprises target-enriching exons. 
     
     
         11 . The method of  claim 8 , wherein the targeted sequencing targets specific tumor-associated mutations. 
     
     
         12 . The method of  claim 1 , wherein sequencing the cell-free DNA molecules from the biological sample of the subject comprises paired-end sequencing, random sequencing, or massively parallel sequencing. 
     
     
         13 . The method of  claim 1 , further comprising sequencing the DNA molecules from the plurality of blood cells of the subject to obtain the second sequence reads. 
     
     
         14 . The method of  claim 1 , further comprising:
 determining genomic positions for the first sequence reads obtained for the cell-free DNA molecules from the biological sample; and   detecting one or more potential sequence variants in the first sequence reads positioned at one or more loci, wherein the potential sequence variants at the one or more loci are detected in the first sequence reads relative to a reference genome, and   wherein the detecting of the tumor-derived mutations in the cell-free DNA molecules is based on filtering out the one or more potential sequence variants that are also present in the second sequences from the plurality of blood cells of the subject.   
     
     
         15 . The method of  claim 14 , wherein the one or more potential sequence variants comprise single-nucleotide variants. 
     
     
         16 . The method of  claim 14 , wherein the tumor-derived mutations are detected as being associated with a non-synonymous change. 
     
     
         17 . A computer-implemented method for detecting sequence variants in a cancer of a subject, the method comprising:
 obtaining a consensus sequence derived using first sequence reads for DNA molecules from a plurality of blood cells of the subject;   receiving second sequence reads for cell-free DNA fragments in a biological sample of the subject, the biological sample including cell-free DNA;   determining genomic positions for the first sequence reads; and   comparing the second sequence reads to the consensus sequence to detect a plurality of loci, wherein:   at each of the plurality of loci, a number of the second sequence reads having a sequence variant relative to the consensus sequence is above a cutoff value, the cutoff value being greater than one.   
     
     
         18 . The method of  claim 17 , wherein the plurality of blood cells comprise healthy blood cells. 
     
     
         19 . The method of  claim 17 , wherein the plurality of blood cells are known not to be cancerous. 
     
     
         20 . The method of  claim 17 , wherein the plurality of blood cells comprise white blood cells. 
     
     
         21 . The method of  claim 17 , wherein the biological sample of the subject comprises blood plasma. 
     
     
         22 . The method of  claim 17 , wherein the biological sample of the subject comprises one or more of a group consisting of: urine, ascetic fluid, peritoneal fluid, saliva, cerebrospinal fluid, and a stool sample. 
     
     
         23 . The method of  claim 17 , further comprising sequencing the cell-free DNA fragments from the biological sample of the subject to obtain the first sequence reads. 
     
     
         24 . The method of  claim 23 , wherein sequencing the cell-free DNA fragments from the biological sample comprises targeted sequencing. 
     
     
         25 . The method of  claim 24 , wherein the targeted sequencing is performed using a solid-phase capture technique, a solution hybridization technique, or amplification via PCR. 
     
     
         26 . The method of  claim 24 , wherein the targeted sequencing comprises target-enriching exons. 
     
     
         27 . The method of  claim 24 , wherein the targeted sequencing targets specific tumor-associated mutations. 
     
     
         28 . The method of  claim 23 , wherein sequencing the cell-free DNA fragments from the biological sample of the subject comprises paired-end sequencing, random sequencing, or massively parallel sequencing. 
     
     
         29 . The method of  claim 17 , further comprising sequencing the DNA molecules from the plurality of blood cells of the subject to obtain the second sequence reads. 
     
     
         30 . A computer-implemented method for detecting sequence variants in a tumor of a subject, the method comprising:
 obtaining first sequence reads for DNA molecules from a plurality of blood cells of the subject;   receiving second sequence reads for cell-free DNA fragments in a biological sample of the subject, the biological sample including cell-free DNA;   determining genomic positions for the first sequence reads; and   detecting sequence variants in the tumor of the subject by comparing the second sequence reads to the first sequence reads to detect a plurality of loci, wherein at each of the plurality of loci, a number of the second sequence reads having a sequence variant relative to the first sequence reads is above a cutoff value, the cutoff value being greater than one.   
     
     
         31 . The method of  claim 30 , wherein the plurality of blood cells comprise healthy blood cells. 
     
     
         32 . The method of  claim 30 , wherein the plurality of blood cells are known not to be cancerous. 
     
     
         33 . The method of  claim 30 , wherein the plurality of blood cells comprise white blood cells. 
     
     
         34 . The method of  claim 30 , wherein the biological sample of the subject comprises blood plasma. 
     
     
         35 . The method of  claim 30 , wherein the biological sample of the subject comprises one or more of a group consisting of: urine, ascetic fluid, peritoneal fluid, saliva, cerebrospinal fluid, and a stool sample. 
     
     
         36 . The method of  claim 30 , further comprising sequencing the cell-free DNA fragments from the biological sample of the subject to obtain the first sequence reads. 
     
     
         37 . The method of  claim 36 , wherein sequencing the cell-free DNA fragments from the biological sample comprises targeted sequencing. 
     
     
         38 . The method of  claim 37 , wherein the targeted sequencing comprises target-enriching exons. 
     
     
         39 . The method of  claim 37 , wherein the targeted sequencing targets specific tumor-associated mutations. 
     
     
         40 . The method of  claim 37 , wherein the targeted sequencing is performed using a solid-phase capture technique, a solution hybridization technique, or amplification via PCR. 
     
     
         41 . The method of  claim 36 , wherein sequencing the cell-free DNA fragments from the biological sample of the subject comprises paired-end sequencing, random sequencing, or massively parallel sequencing. 
     
     
         42 . The method of  claim 30 , further comprising sequencing the DNA molecules from the plurality of blood cells of the subject to obtain the second sequence reads. 
     
     
         43 . The method of  claim 30 , wherein the sequence variants comprise single nucleotide variants. 
     
     
         44 . The method of  claim 30 , further comprising detecting cancer or premalignant change in the subject, wherein detecting the cancer or the premalignant change comprises:
 determining a parameter based on a count of sequence reads having a sequence variant at the plurality of loci; and   comparing the parameter to a threshold value to determine a classification of a level of cancer in the subject.