Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development - PubMed (original) (raw)

. 2011 Jul 20;3(92):92ra66.

doi: 10.1126/scitranslmed.3002543.

Hanno Matthaei, Anirban Maitra, Marco Dal Molin, Laura D Wood, James R Eshleman, Michael Goggins, Marcia I Canto, Richard D Schulick, Barish H Edil, Christopher L Wolfgang, Alison P Klein, Luis A Diaz Jr, Peter J Allen, C Max Schmidt, Kenneth W Kinzler, Nickolas Papadopoulos, Ralph H Hruban, Bert Vogelstein

Affiliations

Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development

Jian Wu et al. Sci Transl Med. 2011.

Abstract

More than 2% of the adult U.S. population harbors a pancreatic cyst. These often pose a difficult management problem because conventional criteria cannot always distinguish cysts with malignant potential from those that are innocuous. One of the most common cystic neoplasms of the pancreas, and a bona fide precursor to invasive adenocarcinoma, is called intraductal papillary mucinous neoplasm (IPMN). To help reveal the pathogenesis of these lesions, we purified the DNA from IPMN cyst fluids from 19 patients and searched for mutations in 169 genes commonly altered in human cancers. In addition to the expected KRAS mutations, we identified recurrent mutations at codon 201 of GNAS. A larger number (113) of additional IPMNs were then analyzed to determine the prevalence of KRAS and GNAS mutations. In total, 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. In addition to defining a new pathway for pancreatic neoplasia, these data suggest that GNAS mutations can inform the diagnosis and management of patients with cystic pancreatic lesions.

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Conflict of interest statement

Competing interests: Johns Hopkins University has filed patent applications on inventions described in this manuscript, with inventors including J.W., H.M., A.M., L.A.D., K.W.K., N.P., R.H.H., and B.V.

Figures

Fig. 1

Fig. 1

Schematic of capture strategy. Overlapping oligonucleotides flanked by universal sequences complimentary to the 169 genes listed in table S1 were synthesized on an array. The oligonucleotides were cleaved off the array, amplified by PCR with universal primers, ligated into concatamers, and amplified in an isothermal reaction. They were then bound to nitrocellulose filters and used as bait for capturing the desired fragments. An Illumina library was constructed from the sample DNA. The library was denatured and hybridized to the probes immobilized on nitrocellulose. The captured fragments were eluted, PCR-amplified, and sequenced on an Illumina GAIIX instrument.

Fig. 2

Fig. 2

Ligation assays used to assess KRAS and GNAS mutations. (A) Schematic of the ligation assay. Oligonucleotide probes complementary to either the wild-type or the mutant sequences were incubated with a PCR product containing the sequence of interest. The wild-type– and mutant-specific probes were labeled with the fluorescent dyes 6-FAM and HEX, respectively, and the wild-type–specific probe was 11 bases longer than the mutant-specific probe. After ligation to a common anchoring primer, the ligation products were separated on a denaturing polyacrylamide slab gel. Further details of the assay are provided in Materials and Methods. (B) Examples of the results obtained with the ligation assay in the indicated patients. Templates were derived from DNA of normal duodenum or IPMN tissue. Each lane represents the results of ligation of one of four independent PCR products, each containing 200 template molecules. The probe in the left panel was specific to the GNAS R201H mutation, and the probe on the right panel was specific for the GNAS R201C mutation.

Fig. 3

Fig. 3

BEAMing assays used to quantify mutant representation. PCR was used to amplify KRAS or GNAS sequences containing the region of interest (KRAS codon 12 and GNAS codon 201). The PCR products were then used as templates for BEAMing, in which each template was converted to a bead containing thousands of identical copies of the templates (34). After hybridization to Cy3- or Cy5-labeled oligonucleotide probes specific for the indicated wild-type or mutant sequences, respectively, the beads were analyzed by flow cytometry. Scatter plots are shown for templates derived from the DNA of IPMN 130 or from normal spleen. Beads containing the wild-type or mutant sequences are widely separated in the scatter plots, and the fraction of mutant-containing beads are indicated. Beads whose fluorescence spectra lie between the wild-type– and the mutant-containing beads result from inclusion of both wild-type and mutant templates in the aqueous nanocompartments of the emulsion PCR. See Materials and Methods for details.

Fig. 4

Fig. 4

IPMN morphologies. (A and B) Typical IPMN before (A) and after (B) microdissection of the epithelium lining the cyst wall. (C and D) Examples of multiloculated IPMNs from two different patients. (E and F) IPMN (E) and associated invasive adenocarcinoma (F) from the same patient. (G and H) Another patient with an IPMN (G) and associated invasive adenocarcinoma (H). All photographs were of formalin-fixed, paraffin-embedded samples stained with hematoxylin and eosin. Scale bars, 500 μm [(A) to (C)], 200 μm [(D) and (E)], and 100 μm [(F) to (H)].

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