Method for Detecting Tumor DNA in a cfDNA Sample Collected from a Patient that has Previously Undergone Cancer Therapy

Abstract

The present invention provides a method for detecting a genetic variant in a region of interest in a DNA sample comprising (i) determining, for a given sequencing platform, sequencing process and sequencing depth, the distribution of the number of reads supporting a genetic variant or plurality of genetic variants expected to be observed in the sequencing results of amplification reactions due to amplification and sequencing error (read count distribution); (ii) based on the read count distribution determined in step (i), establishing a threshold frequency at or above which the genetic variant must be observed in sequencing results of amplification reactions to assign a positive determination for the presence of the genetic variant in a given amplification reaction; (iii) partitioning the DNA sample into a plurality of replicate amplification reactions, so that the mean number of amplifiable template molecules of the region of interest in a replicate amplification reaction is fewer than the reciprocal of the threshold frequency determined in step (ii); (iv) performing the amplification reactions of step (iii) and sequencing the products of amplification reactions, (v) based on step (ii) and the results of step (iv), determining the presence/absence of the genetic variant in each replicate amplification reaction; and (vi) integrating the results of (v) to determine the presence/absence of the genetic variant in the region of interest in the DNA sample.

1 - 30 . (canceled) 
     
     
         31 . A method for detecting circulating tumour DNA in a post-therapy sample, comprising:
 (a) obtaining a first cell-free DNA (cfDNA) sample from a patient, wherein the cfDNA sample is obtained from the patient after the patient has received a cancer therapy;   (b) partitioning the first cfDNA sample into at least two replicate reactions, wherein each replicate reaction has, on average, at least two amplifiable template molecules for each of a plurality of regions of interest;   (c) amplifying the plurality of regions of interest from each of the replicate reactions of step (b), using multiplex PCR, to produce amplification products;   (d) sequencing the amplification products of (c) to produce sequencing results;   (e) analysing the sequencing results of (d) for each of the regions of interest in each replicate amplification reaction by comparing the allelic fraction for each of the sequence variations to a corresponding read count distribution, wherein the read count distribution indicates the frequency at which a sequence variation generated by an amplification and/or sequencing error is expected to be observed at each sequencing depth; and   (f) integrating the results of (e) to determine the presence/absence of circulating tumour DNA in the cfDNA sample.   
     
     
         32 . The method of  claim 31  wherein step (e) comprises determining the presence/absence of a sequence variation of each of the regions of interest in each replicate amplification reaction. 
     
     
         33 . The method of  claim 31  wherein step (e) comprises determining the probability of the presence of a sequence variation of each of the regions of interest in each replicate amplification reaction. 
     
     
         34 . The method of  claim 31 , further comprising performing the method on a second cfDNA sample, wherein the second cfDNA is obtained from the patient at a different time to the first cfDNA sample. 
     
     
         35 . The method of  claim 34 , wherein the second cfDNA sample is obtained from the patient prior to treatment with the cancer therapy. 
     
     
         36 . The method of  claim 31 , further comprising determining if the patient is responding to the cancer therapy based on the presence and/or quantity of the circulating tumour DNA in the first cfDNA sample. 
     
     
         37 . The method of  claim 31 , further comprising determining if the patient is developing drug resistance to the cancer therapy based on the presence and/or quantity of the circulating tumour DNA in the first cfDNA sample. 
     
     
         38 . The method of  claim 31 , further comprising analysing tumour evolution in response to the therapy based on the presence and/or quantity of the circulating tumour DNA in the first cfDNA sample. 
     
     
         39 . The method of  claim 31 , further comprising analysing tumour load based on the presence and/or quantity of the circulating tumour DNA in the first cfDNA sample. 
     
     
         40 . The method of  claim 31 , wherein a change in the amount of circulating tumour DNA of the first sample relative to a second, earlier, sample indicates that the tumour is getting larger or smaller after treatment. 
     
     
         41 . The method of  claim 31 , further comprising quantifying the circulating tumour DNA in the cfDNA sample. 
     
     
         42 . The method of  claim 31 , wherein step (b) comprises partitioning the first cfDNA sample into at least three replicate reactions, wherein each replicate reaction has, on average, at least two amplifiable template molecule for each of a plurality of regions of interest; 
     
     
         43 . The method of  claim 31 , wherein each replicate reaction of step (b) has, on average, 5 to 1000 amplifiable template molecules for each of the plurality of regions of interest. 
     
     
         44 . The method of  claim 31 , wherein step (e) comprises comparing the allelic fraction of a sequence variation to a threshold frequency established using the read count distribution. 
     
     
         45 . The method of  claim 44 , wherein the threshold frequency is determined using a binomial, over-dispersed binomial, Beta, Normal, Exponential or Gamma probability distribution model based on the read count distribution. 
     
     
         46 . The method of  claim 31 , further comprising performing the method on a second sample, wherein the second sample is a tissue sample obtained from the patient.