Cancer detection methods
View Patent ↗The present application provides methods for the detection and diagnosis of cancer. In one aspect, the application provides methods for detecting the presence of cancer in an individual by detecting the methylation state of a region in the promoter of the ZNF154 gene. Methods are provided for detection and diagnosis of cancer from circulating tumor DNA which are minimally invasive and have diagnostic utility across different types and sub-types of cancer. In a further aspect, bioinformatics methods are provided to analyze the methylation state of the ZNF154 promoter and relate the methylation state to the likelihood of cancer in the individual.
1 . A method for detecting the presence of cancer in a human individual, comprising:
treating cell free genomic DNA from a plasma sample from the individual with bisulfite to produce bisulfite-treated genomic DNA; and
PCR amplification of a target nucleic acid molecule comprising nucleotides 58,220,424to 58,220,670 of chromosome 19 according to the human genome version GRCh37/hg19 from the bisulfite-treated genomic DNA to produce amplicons, sequencing the amplicons to produce a plurality of sequence reads; and
detecting the methylation status of 20 CpG sites within the nucleotides 58,220,424 to 58,220,670 of chromosome 19 of the genomic DNA based on the plurality of sequence reads to identify the 20 CpG sites as hypermethylated or not;
wherein detection of the CpG sites as hypermethylated compared to a control identifies the presence of cancer in the individual; and
wherein detection of the CpG sites as not hypermethylated compared to the control identifies the absence of cancer in the individual; and
wherein the cancer is selected from lung cancer, stomach cancer, colon cancer, breast cancer, uterine cancer, bladder cancer, head and neck cancer, kidney cancer, liver cancer, ovarian cancer, pancreas cancer, prostate cancer, and rectum cancer.
2 . The method of claim 1 wherein genomic DNA corresponding to the amplicons comprises or consists of the nucleotide sequence set forth as SEQ ID NO: 5.
3 . The method of claim 1 , wherein detecting the methylation status of the 20 CpG sites comprises:
calculating a ratio X:
X
=
N
2
0
/
(
N
0
+
N
2
0
)
wherein N 0 and N 20 are frequencies of sequence reads in the plurality where 0 or 20 of the 20 CpG sites within the nucleotides 58,220,424 to 58,220,670 of chromosome 19 of the genomic DNA are methylated, respectively; and
wherein an increase in the ratio X as compared with a control is indicative of the presence of cancer in the individual; and
wherein no increase in the ratio X as compared with the control is indicative of the absence of cancer in the individual.
4 . The method of claim 1 wherein detecting the methylation status of the 20 CpG sites comprises:
calculating a ratio Y:
Y
=
N
2
0
/
(
N
0
+
N
1
+
N
2
+
N
3
+
N
4
+
N
5
+
N
2
0
)
wherein N 0 , N 1 , N 2 , N 3 , N 4 , N 5 , and N 20 , are frequencies of sequence reads in the plurality where 0, 1, 2, 3, 4, 5 or 20 of the 20 CpG sites within nucleotides 58,220,424 to 58,220,670 of chromosome 19 of the genomic DNA are methylated, respectively; and
wherein an increase in the ratio Y as compared with a control is indicative of cancer in the individual; and
wherein no increase in the ratio Y as compared with the control is indicative of the absence of cancer in the individual.
5 . The method of claim 1 , wherein the cytosines of the 20 CpG sites are located at nucleotides 58220424, 58220440, 58220443, 58220446, 58220460,58220466, 58220479, 58220482, 58220494, 58220500, 58220513, 58220516, 58220535,58220567, 58220572, 58220595, 58220627, 58220657, 58220662, and 58220669 of chromosome 19.
6 . The method of claim 1 , wherein the control is a threshold value that distinguishes between individuals with and without cancer.