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

PMID: 22158988 · DOI: 10.1073/pnas.1118046108 · Journal: Proceedings of the National Academy of Sciences (2011)

TL;DR

This study performed whole-exome sequencing on 32 surgically resected neoplastic cysts of the pancreas (8 each of SCA, IPMN, MCN, and SPN) and matched normal tissues to define their genomic landscapes. The authors discovered that RNF43, an E3 ubiquitin ligase gene not previously implicated in human cancer, is recurrently inactivated in IPMNs (6/8) and MCNs (3/8), establishing it as a tumor suppressor. VHL was mutated or had LOH in all 8 SCAs, CTNNB1 was mutated in all 8 SPNs, and KRAS and GNAS were frequently mutated in IPMNs and MCNs. The convergence on ubiquitin-dependent pathway defects across all four cyst types suggests diagnostic and therapeutic implications.

Cohort & data

• 32 microdissected neoplastic pancreatic cysts (8 SCA, 8 IPMN, 8 MCN, 8 SPN) with matched normal tissue from the same patients.
• Dataset: pact_jhu_2011.
• Cancer types: IPMN, MCN, SPN; SCAs (serous cystadenomas, no OncoTree code); progression target: PAAD.
• Whole-exome sequencing using Agilent 50-Mb SureSelect capture on Illumina GAIIx/HiSeq; average unique coverage 120-fold +/- 40 per base pair across 64 libraries.
• An additional 18 SCA cyst fluids were analyzed for VHL mutations using a customized VHL capture chip; 28 IPMN and 3 MCN cyst fluids were also screened.

Key findings

• SCAs contained 10 +/- 4.6 nonsynonymous somatic mutations per tumor – significantly fewer than PDAs (48 +/- 23; P < 0.001). 4/8 SCAs harbored inactivating VHL mutations (N78S, W117L, C162W, S80R); all 8 had LOH of chromosome 3p in or adjacent to VHL. VHL mutations were confirmed by independent ligation-based assays. VHL mutations were also found in 9/18 (50%) SCA cyst fluids.
• IPMNs contained 27 +/- 12 nonsynonymous mutations per tumor. RNF43 was the most commonly mutated gene, altered in 6/8 IPMNs (5 nonsense mutations, 1 nonsense); the probability of RNF43 being a passenger was < 10^-12 (binomial test). KRAS (codon 12) was mutated in 5/8 and GNAS (codon 201) in 5/8 IPMNs. LOH of chromosome 17q (containing RNF43) was observed in 4/8 IPMNs. Two APC mutations were found (one nonsense R450X, one missense R99W).
• MCNs contained 16 +/- 7.6 nonsynonymous mutations per tumor. RNF43 was mutated in 3/8 MCNs (two nonsense, one missense). KRAS was mutated in 6/8 MCNs (all at codon 12). TP53 was mutated in 2/8 MCNs.
• SPNs contained remarkably few alterations: only 2.9 +/- 2.1 nonsynonymous mutations per tumor – fewer than any tumor type yet evaluated by genome-wide sequencing. All 8/8 SPNs harbored activating CTNNB1 mutations at codons 32, 33, 34, or 37 (beta-catenin phosphodegron). Only 1/8 SPNs exhibited any LOH.
• All four cyst types converge on ubiquitin ligase pathway defects: VHL (ubiquitin ligase complex component) in SCAs, RNF43 (intrinsic E3 ubiquitin ligase) in IPMNs/MCNs, and CTNNB1 (resistant to ubiquitin-mediated degradation) in SPNs.

Genes & alterations

Gene	Alteration	Cyst type	Frequency	Notes
VHL	Inactivating point mutations + chr3p LOH	SCA	4/8 mutated; 8/8 LOH	All mutations previously seen in RCC; 9/18 SCA fluids also VHL-mutant
RNF43	Truncating/inactivating mutations + chr17q LOH	IPMN	6/8	5 nonsense mutations; E3 ubiquitin ligase; established as tumor suppressor
RNF43	Truncating/inactivating mutations + chr17q LOH	MCN	3/8	2 nonsense, 1 missense (R127P)
KRAS	Activating missense (codon 12)	IPMN, MCN	5/8 IPMN, 6/8 MCN	G12D, G12R, G12V, G13D
GNAS	Activating missense (R201C/H)	IPMN	5/8	Codon 201 hotspot
CTNNB1	Activating missense (codons 32-37)	SPN	8/8	Inhibits phosphorylation and ubiquitin-mediated degradation of beta-catenin
TP53	Inactivating mutations	MCN	2/8	Associated with aggressive behavior
APC	Nonsense (R450X) + missense (R99W)	IPMN	2/8	Known colorectal tumor suppressor

Clinical implications

• Diagnostic biomarker panel: A five-gene panel (VHL, RNF43, CTNNB1, GNAS, KRAS) can distinguish among the four neoplastic cyst types in cyst fluid samples, potentially reducing unnecessary surgical resections. SCAs always showed VHL alterations without mutations in the other four genes; IPMNs showed RNF43/GNAS/KRAS without VHL or CTNNB1; MCNs showed KRAS/RNF43 without GNAS, CTNNB1, or VHL; SPNs showed CTNNB1 exclusively.
• Reduction of unnecessary surgery: Accurate preoperative cyst classification using molecular markers could spare patients with benign SCAs from surgery (currently misdiagnosed as IPMNs in up to one-third of cases).
• Progression risk stratification: The number and type of genetic alterations in GNAS, RNF43, and other genes in IPMNs and MCNs may help gauge the risk of progression to invasive PAAD. TP53 mutations in MCNs may mark those most likely to progress.

Limitations & open questions

• Small sample size (n = 8 per cyst type) limits statistical power for detecting low-frequency driver events.
• Study used surgically resected specimens with high neoplastic cell content (>33%); clinical cyst fluid samples are often acellular or paucicellular, and sensitivity in that context needs validation.
• The targets of RNF43 ubiquitin ligase activity in vivo remain unknown; functional studies are needed to confirm its tumor-suppressive mechanism.
• SPNs contained remarkably few mutations; whether epigenetic events or translocations contribute to their formation is unanswered.
• No prospective clinical validation of the five-gene cyst fluid diagnostic panel was performed.
• The 4 SCAs without detectable VHL point mutations may have inactivation by deletions, translocations, or epigenetic silencing not detectable by exome sequencing.

Citations from this paper used in the wiki

• “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.” PMID:22158988
• “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.” PMID:22158988
• “The preponderance of inactivating mutations in RNF43 unequivocally establishes it as a suppressor of both IPMNs and MCNs.” PMID:22158988
• “SPNs contained remarkably few genetic alterations but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (beta-catenin) by E3 ubiquitin ligases.” PMID:22158988
• “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.” PMID:22158988

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