Compositions and methods for detecting a neoplasia
The invention provides compositions and methods for detecting a neoplasia (e.g., pancreatic cancer, lung cancer, colon cancer) in a subject sample (e.g., serum, blood, plasma, tissue). In particular embodiments, the invention provides methods for detecting BNC1 and ADAMTS1 promoter methylation in circulating DNA in serum.
1. A method of identifying and treating a subject, the method comprising:
(a) obtaining a biological sample from the subject;
(b) isolating genomic DNA from the biological sample;
(c) bisulfite treating the genomic DNA;
(d) PCR amplifying the bisulfite-treated genomic DNA to obtain one or more PCR products;
(e) hybridizing the one or more PCR products to a fluorescently labeled oligonucleotide comprising SEQ ID NOS: 19 or 20 for detection of methylated nucleotides of the BNC1 gene promoter or exon 1, thereby forming a first PCR product-fluorescently labeled oligonucleotide probe complex;
(f) hybridizing the one or more PCR products to a fluorescently labeled oligonucleotide comprising SEQ ID NOS: 9 or 10 for detection of methylated nucleotides of the ADAMTS1 gene promoter or exon 1, thereby forming a second PCR product-fluorescently labeled oligonucleotide probe complex; and
(g) detecting an elevated level of the first PCR product fluorescently labeled oligonucleotide complex as compared to a first reference level obtained in a sample previously obtained from the subject and detecting an elevated level of the second PCR product-fluorescently labeled oligonucleotide complex relative to a second reference level obtained in a sample previously obtained from the subject, and
(h) administering an epigenetic therapy or resecting the pancreas of the subject having an elevated level of the first and second PCR products detected in step (g), wherein the epigenetic therapy is selected from the group consisting of entinostat, SAHA (suberovlanilide hvdroxamic acid), depsipeptide, azocvtidine, and deazocytidine.
2. The method of claim 1 , further comprising detecting an alteration in the sequence or expression level of a Brca1, Brca2, p16, K-ras, APC, EGFR, and/or EML-ALK4 gene or polypeptide.
3. The method of claim 1 , wherein the biologic sample is a tissue or biologic fluid sample.
4. The method of claim 1 , wherein the biologic sample is selected from the group consisting of blood, serum, plasma, urine, pancreatic juice, pancreatic cyst fluid, or lung lavage.
5. The method of claim 1 , wherein the methylation levels of the BNC1 and ADAMTS1 promoters are quantified.
6. The method of claim 1 , wherein the subject is a smoker, has a Brca1 or Brca2 mutation, pancreatic cyst, chronic pancreatitis, presence of colon polyps or adenomas, or a family history of cancer.
7. A method for diagnosing and treating pancreatic cancer in a subject comprising:
a) detecting methylated nucleotides of the BNC1 gene promoter or exon 1 using an oligonucleotide comprising SEQ ID NO: 19;
b) detecting methylated nucleotides of the ADAMTS1 gene promoter or exon 1 using an oligonucleotide comprising SEQ ID NO: 9;
c) detecting an elevated level of methylated nucleotides in both BNC1 and ADAMTS1 gene promoters or exon 1 nucleotides as compared to a reference level;
d) diagnosing the subject as having pancreatic cancer; and
e) administering an epigenetic therapy or resecting the pancreas of the patient diagnosed as having pancreatic cancer.
8. The method of claim 7 , wherein the epigenetic therapy comprises an therapeutically effective amount of histone deacetylase or methylation inhibitors.