Topographic genotyping for determining the diagnosis, malignant potential, and biologic behavior of pancreatic cysts and related conditions
The application relates to a method of a predicting the presence of invasive pancreatic cancer or high grade dysplasia, pre-cancerous pancreatic states and non-neoplastic conditions comprising detailed molecular analysis incorporating DNA quality and quantity, K-ras mutational analysis and a broad spectrum of tumor suppressor gene linked microsatellite LOH. Methods of diagnosing, determining prognosis of and determining a course of treatment for pancreatic cancer or high grade dysplasia, pre-cancerous pancreatic states and non-neoplastic conditions are also provided.
1. A method for predicting the presence of a pancreatic anomaly in a patient suffering from pancreatic cysts comprising:
a) performing molecular analysis of DNA from an aspirate from a patient pancreatic cyst comprising
(i) performing an optical density analysis of the aspirate to determine DNA quantity;
(ii) performing a quantitative PCR analysis of the aspirate to determine DNA quality;
(iii) performing competitive template PCR to determine DNA quality; and
b) performing mutation analysis of the DNA in the aspirate comprising:
(i) determining the presence of mutations in D 1 S 407 , D 1 S 1193 , LMYC, D1S1172, D3S1539, D3S2303, or D5S592;
(ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein the tumor suppressor genes are selected from the group consisting of: VHL, APC, P53, PTEN, and P16;
(iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;
(iv) determining other structural alterations in the DNA;
(v) determining the percentage of mutated DNA from steps b)(ii) and b)(iii); and
(vi) determining the specific temporal sequence of mutation accumulation based on step (v), wherein the temporal sequence of mutation accumulation is predictive of the presence of a pancreatic anomaly.
2. The method of claim 1 , further comprising confirming the results of steps (a) and (b) by comparing said results with a pathologic analysis of a cystic lesion surgically obtained from the patient.
3. The method of claim 1 , further comprising analyzing the cyst carcinoembryonic antigen (CEA) level of the aspirate.
4. The method of claim 1 , wherein the DNA in the aspirate is free floating, or free and adherent to the surface of cells or tissue constituents of the cyst.
5. The method of claim 1 , wherein the cycles of quantitative PCR performed in step a)(ii) is greater than a threshold unique for that specific type of DNA.
6. The method of claim 1 , wherein the patient is a mammal.
7. The method of claim 6 , wherein the patient is a human.
8. The method of claim 1 , wherein the pancreatic anomaly is a pancreatic cancer or dysplasia, pre-cancerous pancreatic state, or non-neoplastic condition.
9. The method of claim 8 , wherein the pancreatic cancer or dysplasia is selected from the group consisting of pancreatic ductal adenocarcinoma, pancreatic acinar cell carcinoma, neuroendocrine cell carcinoma, sarcoma of the pancreas, metastatic cancer involving the pancreas, pancreaticoblastoma, and bile duct carcinoma (cholangiocarcinoma).
10. The method of claim 8 , wherein the pre-cancerous pancreatic state is selected from the group consisting of mucinous cystadenoma, serous cystadenoma, islet cell tumor, mucinous duct ectasia, intraductal papillary mucinous Neoplasm, pancreatic intraepithelial neoplasia (PaniN grades 1-3), and solid and cystic papillary tumor of the pancreas.
11. The method of claim 8 , wherein the non-neoplastic condition is selected from the group consisting of pancreatitis, pancreatic pseudocyst, mesothelial cyst, lymphoepithelial cyst of the pancreas, and ischemic necrosis of the pancreas.
12. The method of claim 1 , wherein the other structural alterations in DNA are selected from the group consisting of gene amplification, gene translocation or rearrangement, and epigenetic modification of DNA by DNA methylation.
13. A method for diagnosing and/or determining the prognosis of a pancreatic anomaly in a patient suffering from pancreatic cysts comprising:
a) performing molecular analysis of DNA from an aspirate from a patient pancreatic cyst comprising
(i) performing an optical density analysis of the aspirate to determine DNA quantity;
(ii) performing a quantitative PCR analysis ofthe aspirate to determine DNA quality;
(iii) performing competitive template PCR to determine DNA quality; and
b) performing mutation analysis of the DNA in the aspirate comprising:
(i) determining the presence of one or more mutations in a tumor suppressor gene and/or the presence of a cancer related genetic marker;
(ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances;
(iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;
(iv) determining other structural alterations in DNA;
(v) determining the percentage of mutated DNA from steps b)(ii) and b)(iii); and
(vi) determining the specific temporal sequence of mutation accumulation based on step v), and diagnosing and/or determining the prognosis of a pancreatic anomaly of the patient in need based on the results of steps a) and b).
14. The method of claim 13 , wherein the pancreatic anomaly is a pancreatic cancer or dysplasia, pre-cancerous pancreatic state or non-neoplastic condition.
15. The method of claim 14 , wherein the pancreatic cancer or dysplasia is selected from the group consisting of pancreatic ductal adenocarcinoma, pancreatic acinar cell carcinoma, neuroendocrine cell carcinoma, sarcoma of the pancreas, metastatic cancer involving the pancreas, pancreaticoblastoma, and bile duct carcinoma (cholangiocarcinoma).
16. The method of claim 14 , wherein the pre-cancerous pancreatic state is selected from the group consisting of mucinous cystadenoma, serous cystadenoma, islet cell tumor, mucinous duct ectasia, intraductal papillary mucinous neoplasm, pancreatic intraepithelial neoplasia (PaniN grades 1-3), and solid and cystic papillary tumor of the pancreas.
17. The method of claim 14 , wherein the non-neoplastic condition is selected from the group consisting of pancreatitis, pancreatic pseudocyst, mesothelial cyst, lymphoepithelial cyst of the pancreas, and ischemic necrosis of the pancreas.
18. A method for determining a course of treatment for a pancreatic anomaly in a patient suffering from pancreatic cysts comprising:
a) performing molecular analysis of DNA from an aspirate from a patient pancreatic cyst comprising:
(i) performing an optical density analysis of the aspirate to determine DNA quantity;
(ii) performing a quantitative PCR analysis of the aspirate to determine DNA quality; and
(iii) performing competitive template PCR to determine DNA quality; and
b) performing mutation analysis of the DNA in the aspirate comprising:
(i) determining the presence of mutations in a tumor suppressor gene and/or the presence of a cancer related genetic marker;
(ii) determining tumor suppressor gene loss of heterozygosity (LOH) by catalyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances;
(iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;
(iv) determining other structural alterations in DNA;
(v) determining the percentage of mutated DNA from steps b)(ii) and b)(iii); and
(vi) determining the specific temporal sequence of mutation accumulation based on step v), wherein the results of steps a) and b) are used to determine a course of therapy for a patient suffering from a pancreatic anomaly.
19. The method of claim 18 , wherein the pancreatic anomaly is a pancreatic cancer or dysplasia, pre-cancerous pancreatic state or non-neoplastic condition.
20. The method of claim 19 , wherein:
the pancreatic cancer or dysplasia is selected from the group consisting of pancreatic ductal adenocarcinoma, pancreatic acinar cell carcinoma, neuroendocrine cell carcinoma, sarcoma of the pancreas, metastatic cancer involving the pancreas, pancreaticoblastoma, and bile duct carcinoma (cholangiocarcinoma);
the pre-cancerous pancreatic state is selected from the group consisting of mucinous cystadenoma, serous cystadenoma, islet cell tumor, mucinous duct ectasia, intraductal papillary mucinous neoplasm, pancreatic intraepithelial neoplasia (PanIN grades 1-3), and solid and cystic papillary tumor of the pancreas; and
the non-neoplastic condition is selected from the group consisting of pancreatitis, pancreatic pseudocyst, mesothelial cyst, lymphoepithelial cyst of the pancreas, and ischemic necrosis of the pancreas.