Liver cancer methods and compositions
The present invention provides a novel method for detection of liver cancer. This method detects high-sensitively, high-specifically, simply and accurately liver cancer, especially that in early stage by identifying and/or quantifying methylation on particular genes and/or their DNA fragments in clinical specimens, and by combining said methylated DNA values with existing tumor marker values and/or DNA amounts in blood. This invention also detects a precancerous lesion, detects a risk of recurrence after treatment of liver cancer, detects malignancy of liver cancer and monitors progression of liver cancer with time by the same method. As for particular genes, BASP1 gene, SPINT2 gene, APC gene, CCND2 gene, CFTR gene, RASSF1 gene and SRD5A2 gene are mentioned.
1. A method for detecting the presence or amount of methylated cytosine specific to liver cancer on a region containing CpG sequences in BASP1 gene and SRD5A2 gene comprising the following steps of (a) to (d):
(a) isolating genomic DNA from a patient sample;
(b) contacting the DNA with a reagent for chemical or enzymatic treatment of the DNA in order to discriminate between methylated and unmethylated cytosines;
(c) amplifying methylated cytosine-containing regions of BASP1 gene and SRD5A2 gene present in the genomic DNA using PCR utilizing at least one of SEQ ID NOs: 1, 2, 13, 14, 15, 16, 27, 28, 31 and 37; and
(d) determining the presence or amount of methylated cytosine in BASP1 gene and SRD5A2 gene,
wherein the presence or amount of methylated cytosine is an indication that said patient is afflicted with liver cancer, has an increased risk of occurrence or recurrence after treatment of liver cancer or has a progression of liver cancer with time.
2. The method according to claim 1 , wherein amplifying in step (c) is performed in the presence of at least one PCR primer complementary to at least one of said cytosine-containing regions that specifically hybridizes to said cytosine-containing regions when said cytosines are methylated.
3. The method according to claim 1 , wherein amplifying in step (c) is performed in the presence of blocker oligonucleotides which partially overlap with amplification primers and are complementary to at least one of said cytosine-containing regions and specifically hybridize to said regions when the cytosines are not methylated.
4. The method according to claim 1 , wherein amplifying in step (c) is performed by real-time PCR with a fluorescent probe.
5. The method according to claim 1 , wherein determination of the presence or amount of methylated cytosine in step (d) is performed by sequencing or by mass spectrometry.
6. The method according to claim 1 , wherein chemical treatment in step (b) consists of conversion of unmethylated cytosine to uracil.
7. The method according to claim 1 , wherein the chemical reagent in step (b) is a bisulfite-containing reagent.
8. The method according to claim 7 , wherein the bisulfite-containing reagent comprises sodium bisulfate or sodium sulfite or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
9. The method according to claim 1 , wherein enzymatic treatment in step (b) is carried out with a methylation-sensitive restriction enzyme.
10. The method according to claim 1 , further comprising step (e) wherein protein concentration or free floating DNA amounts of one or more genes selected from SPINT2, APC, CCND2, CFTR and RASSF1 are further determined in the sample.
11. The method according to claim 10 , further comprising determining protein concentration or free floating DNA amounts of one or more genes selected from GSTP1, AFP and PWKA-II.
12. The method according to claim 1 , wherein liver cancer is in early stage.
13. The method according claim 1 , wherein the sample is derived from human sera, plasma, whole blood, tissues, blood cells, excretions or secretions.
14. The method of claim 1 , wherein amplifying methylated cytosine-containing regions of BASP1 gene is performed utilizing SEQ ID NOs: 1, 2 and 31 or 15, 16 and 31, and amplifying methylated cytosine-containing regions of SRD5A2 gene is performed utilizing SEQ ID NOs: 13, 14 and 37 or 27, 28 and 37.