Methods and systems for tumor detection

Abstract

Methods are provided to improve the positive predictive value for cancer detection using cell-free nucleic acid samples. Various embodiments are directed to applications (e.g., diagnostic applications) of the analysis of the fragmentation patterns and size of cell-free DNA, e.g., plasma DNA and serum DNA, including nucleic acids from pathogens, including viruses. Embodiments of one application can determine if a subject has a particular condition. For example, a method of present disclosure can determine if a subject has cancer or a tumor, or other pathology. Embodiments of another application can be used to assess the stage of a condition, or the progression of a condition over time. For example, a method of the present disclosure may be used to determine a stage of cancer in a subject, or the progression of cancer in a subject over time (e.g., using samples obtained from a subject at different times).

What is claimed is: 
     
         1 . A method of determining a level of cancer in a subject, the method comprising:
 (a) determining a first amount of cell-free nucleic acid molecules from a pathogen in a first biological sample from the subject;   (b) determining, based on sequence reads from a second biological sample of the subject, a second amount of nucleic acid molecules in the second biological sample that are from the pathogen and have a size within a given range, wherein the pathogen is a virus; and   (c) determining the level of cancer of the subject based on the first amount and the second amount.   
     
     
         2 . The method of  claim 1 , wherein the first amount of cell-free nucleic acid molecules from the pathogen in the first biological sample comprises i) a number of copies of cell-free nucleic acid molecules from the pathogen per a volume of the first biological sample; or ii) a proportion of cell-free nucleic acid molecules from the pathogen in the first biological sample. 
     
     
         3 . The method of  claim 1 , wherein the second amount of nucleic acid molecules in the second biological sample that are from the pathogen and have a size within the given range comprises a proportion of nucleic acid molecules being from the pathogen and having a size within the given range in the second biological sample. 
     
     
         4 . The method of  claim 1 , further comprising normalizing an amount of nucleic acid molecules from the second biological sample that are from the pathogen and have a size within the given range using a third amount of nucleic acid molecules from the second biological sample having a size within the given range and being from a human autosomal genome to render the second amount. 
     
     
         5 . The method of  claim 1 , wherein the determining the second amount comprises massively parallel sequencing of the nucleic acid molecules from the second biological sample to generate the sequence reads. 
     
     
         6 . The method of  claim 1 , further comprising determining a size ratio of:
 a first proportion of sequence reads of nucleic acid molecules from the second biological sample that align to a reference genome of the pathogen with a size within the given range; and   a second proportion of sequence reads of nucleic acid molecules from the second biological sample that align to a human reference genome with a size within the given range.   
     
     
         7 . The method of  claim 6 , wherein the given range is 80 to 110 base pairs in length. 
     
     
         8 . The method of  claim 6 , further comprising comparing the size ratio to a cutoff. 
     
     
         9 . The method of  claim 8 , wherein the cutoff is about 7. 
     
     
         10 . The method of  claim 6 , further comprising determining a size index, wherein the size index is an inverse of the size ratio. 
     
     
         11 . The method of  claim 10 , further comprising comparing the size index to a second cutoff value. 
     
     
         12 . The method of  claim 11 , wherein the second cutoff value is at least 0.1. 
     
     
         13 . The method of  claim 1 , wherein the level of cancer comprises nasopharyngeal cancer. 
     
     
         14 . The method of  claim 1 , wherein the first amount is determined from a first assay; wherein the second amount is determined from a second assay; and wherein a positive predictive value for nasopharyngeal cancer in the subject of the first assay and the second assay is at least 5-fold greater than a positive predictive value of the first assay. 
     
     
         15 . The method of  claim 14 , wherein the positive predictive value for nasopharyngeal cancer in the subject of the first assay and the second assay is at least 7.5-fold greater than the positive predictive value of the first assay. 
     
     
         16 . The method of  claim 14 , wherein the positive predictive value for nasopharyngeal cancer in the subject of the first assay and the second assay is at least 15%. 
     
     
         17 . The method of  claim 14 , wherein the positive predictive value for nasopharyngeal cancer in the subject of the first assay and the second assay is at least 25%. 
     
     
         18 . The method of  claim 14 , wherein:
 a false positive rate for nasopharyngeal cancer in the subject of the first assay and the second assay is at least 5-fold lower than a false positive rate of the first assay.   
     
     
         19 . The method of  claim 18 , wherein the false positive rate for nasopharyngeal cancer in the subject of the first assay and the second assay is at least 10-fold lower than the false positive rate of the first assay. 
     
     
         20 . The method of  claim 18 , wherein the false positive rate for nasopharyngeal cancer in the subject of the first assay and the second assay is less than 1%. 
     
     
         21 . The method of  claim 1 , wherein the pathogen is Epstein-Barr Virus (EBV). 
     
     
         22 . The method of  claim 1 , wherein determining the first amount comprises amplification. 
     
     
         23 . The method of  claim 22 , wherein the amplification comprises polymerase chain reaction (PCR). 
     
     
         24 . The method of  claim 23 , wherein the PCR comprises quantitative PCR (qPCR). 
     
     
         25 . The method of  claim 1 , wherein the first biological sample is plasma. 
     
     
         26 . The method of  claim 1 , wherein the second biological sample is plasma. 
     
     
         27 . The method of  claim 1 , wherein the nucleic acid molecules from the second biological sample comprise cell-free nucleic acid molecules. 
     
     
         28 . The method of  claim 1 , wherein the first amount is determined by massively parallel sequencing. 
     
     
         29 . The method of  claim 1 , wherein the second biological sample is obtained at least 2 weeks after the first biological sample is obtained. 
     
     
         30 . The method of  claim 1 , wherein the second biological sample is obtained at least 4 weeks after the first biological sample is obtained.