Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways

Abstract

More than 2% of adults harbor a pancreatic cyst, a subset of which progresses 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 pseudopapillary 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 the von Hippel-Lindau gene (VHL), a key component of the VHL ubiquitin ligase complex that has previously been associated 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 (β-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.

Fig. 1.

Neoplastic cyst histopathology. (A) Typical SCA (SCA 38) shows centrally placed round nuclei without atypia. (Scale bar: 100 μ.) (B) Typical IPMN (IPMN 4) is lined by columnar mucin-producing cells that form large papillary projections into the ductal lumen. (Scale bar: 100 μ.) (C) Typical MCN is also lined by columnar mucin-producing cells, but the neoplastic epithelium is associated with a characteristic ovarian-type stroma. (Scale bar: 100 μ.) (D) Same MCN after laser capture microdissection of the columnar mucin-producing cells. (Scale bar: 100 μ.) (E) Gross appearance of a typical cyst-forming SPN (SPN 2). (Scale bar: 10 mm.) (F) Under the microscope, SPN 17 shows poorly cohesive cells supported by a delicate stroma. (Scale bar: 50 μ.)

Fig. 2.

LOH observed in neoplastic cysts of the pancreas. The lines indicate the observed regions of loss, with the different cyst types denoted by the indicated colors.

Fig. 3.

Representative LOH data based on the sequence evaluation of genome-wide SNPs. The positions of each chromosome are indicated on the x axis. The y axis indicates the ratio of the two alleles of each SNP. In normal cells, this fraction is 0.5; in a pure neoplastic cell population in which every cell loses one allele of a locus, the allele ratio is 0.0. At a locus at which at least some neoplastic cells had undergone LOH, allele ratios greater than 0.0 reflect nonneoplastic cells that “contaminated” the microdissected cell population plus the fraction of neoplastic cells that had not undergone LOH at that locus. (A) SCA 40, exhibiting LOH of the region on chromosome 3p containing the VHL gene. (B) IPMN 12, exhibiting LOH of the region on chromosome 17q containing the RNF43 gene. (C) MCN 169, exhibiting LOH of the region on chromosome 17q containing the RNF43 gene.

Fig. 4.

Ligation assays used to confirm mutations in VHL and RNF43. Each lane represents the results of ligation of one of four independent PCR products, with each containing 100 template molecules. The ligation products were then size-separated on a denaturing acrylamide gel. The green bands are 6-carboxyfluorescein–labeled oligonucleotide probes that ligate to an unlabeled oligonucleotide when WT alleles are present. The red bands are hexachlorofluorescein-labeled oligonucleotide probes that ligate to the same unlabeled oligonucleotide only when mutant (MUT) alleles are present. The WT- and MUT-specific oligonucleotide probes were of different lengths (∼32 and ∼12 bases, respectively); thus, they migrated at different positions in the acrylamide gel. The cyst samples and mutations assessed are indicated.