Johns Hopkins University

Jian Wu

Bert Vogelstein

Kenneth W Kinzler

Nickolas Papadopoulos

Ralph Hruban

Anirban Maitra

Luis Diaz

Hanno Matthaei

Marco Dal Molin

Assays can be used to detect mutations found in neoplasms of the pancreas, as well as for other neoplasms and other uses. Nucleic acids can be captured from body fluids such as cyst fluids. Thousands of oligonucleotides can be synthesized in parallel, amplified and ligated together. The ligated products can be further amplified. The amplified, ligated products are used to capture complementary DNA sequences, which can be analyzed, for example by massively parallel sequencing.

Enrichment of nucleic acids by complementary capture

To help reveal the pathogenesis of these lesions, we purified the DNA from Intraductal Papillary Mucinous Neoplasm (IPMN) cyst fluids from 19 patients and searched for mutations in 169 genes commonly altered in human cancers. We identified recurrent mutations at codon 201 of GNAS. We found that GNAS mutations were present in 66% of IPMNs and that either KRAS or GNAS mutations could be identified in 96%. In eight cases, we could investigate invasive adenocarcinomas that developed in association with IPMNs containing GNAS mutations. In seven of these eight cases, the GNAS mutations present in the IPMNs were also found in the invasive lesion. GNAS mutations were not found in other types of cystic neoplasms of the pancreas or in invasive adenocarcinomas not associated with IPMNs. These data suggest that GNAS mutations can inform the diagnosis and management of patients with cystic pancreatic lesions.

Mutations in pancreatic neoplasms

Enrichment of nucleic acids by complimentary capture

More than 2% of adults harbor a pancreatic cyst, a subset of which progress to invasive lesions with lethal consequences. To assess the genomic landscapes of neoplastic cysts of the pancreas, we determined the exomic sequences of DNA from the neoplastic epithelium of eight surgically resected cysts of each of the major neoplastic cyst types: serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs) and solid pseudo-papillary neoplasms (SPNs). SPNs are low-grade malignancies, and IPMNs and MCNs, but not SCAs, have the capacity to progress to cancer. We found that SCAs, IPMNs, MCNs, and SPNs contained 10=4.6, 27=12, 16=7.6, and 2.9=2.1 somatic mutations per tumor, respectively. Among the mutations identified, E3 ubiquitin ligase components were of particular note. Four of the eight SCAs contained mutations of VHL, a key component of the VHL ubiquitin ligase complex that has previously been associated both with renal cell carcinomas, SCAs, and other neoplasms. Six of the eight IPMNs and three of the eight MCNs harbored mutations of RNF43, a gene coding for a protein with intrinsic E3 ubiquitin ligase activity that has not previously been found to be genetically altered in any human cancer. The preponderance of inactivating mutations in RNF43 unequivocally establish it as a suppressor of both IPMNs and MCNs. SPNs contained remarkably few genetic alterations, but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (&#x3b2;-catenin) by E3 ubiquitin ligases. These results highlight the essential role of ubiquitin ligases in these neoplasms and have important implications for the diagnosis and treatment of patients with cystic tumors.

Differential identification of pancreatic cysts

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Baltimore, MD, United States of America

Jian Wu