In this study we find that the pancreatic precursor neoplasms known as IPMNs are characterized by widespread aberrant hypermethylation affecting several hundred CpG islands. Extensive aberrant hypermethylation has been demonstrated in pancreatic and other invasive cancers, but the timing of such aberrant methylation during tumor development has not been well established. In our study we find that even low-grade IPMNs harbor aberrant methylation of hundreds of CpG islands, with evidence of increased levels of methylation with increasing neoplastic grade. Consistent with this trend is the finding that significantly more genes are methylated in pancreatic ductal adenocarcinomas than in IPMNs.
Among the genes tested, both BNIP3
had promising performance characteristics as diagnostic markers of IPMNs with high-grade dysplasia. Aberrant methylation of BNIP3
has been described in pancreatic ductal adenocarcinomas, but not in IPMNs (37
is a trans-membrane protein and shares a PTCH/DISP homologous domain and aberrant methylation of PTCHD2/DISP3
has not been described in any solid cancer.
Our MCAM analysis identified other genes that were selectively methylated in high-grade IPMNs including the gene TFPI-2
which we have previously described as much more commonly methylated in high-grade IPMNs and infiltrating adenocarcinomas than in low- or intermediate-grade of IPMNs (25
). Since most IPMNs are detected incidentally when they are small neoplasms of low malignant potential, they are managed by surveillance and only undergo resection when there is a concern for high-grade neoplasia or malignancy. Since imaging tests cannot differentiate low-grade from high-grade neoplastic lesions, markers that can reliably identify the grade of neoplasia in IPMNs could help determine the optimal timing of resection for patients undergoing surveillance (38
). Genes with methylation profiles highly specific for high-grade dysplasia in IPMNs could be useful as markers for detection in fine needle aspirates of IPMN samples (3
Of the 2244 genes identified as aberrantly methylated in any IPMN, many have been identified as aberrantly methylated in pancreatic cancers, including genes bound by polycomb and those involving pathways and gene families commonly targeted in pancreatic ductal adenocarcinomas. For example, of the 331 aberrantly methylated genes involved in transcription regulation, many were members of the homeobox superfamily. Indeed, 41 of the 245 aberrantly methylated genes in high-grade IPMNs were homeobox genes. Homeobox genes encode transcription factors and play a key role in development by activating or repressing down-stream target genes involved in many cellular processes, including adhesion, apoptosis, body patterning, differentiation, migration, proliferation, polarity and proliferation and are commonly observed in cancers (41
Several members of the Wnt signaling pathway were also found to be hypermethylated in IPMNs by MCAM analysis including multiple genes previously shown to be epigenetically silenced both in invasive pancreatic and other types of cancer such as FZD10, SFRP1, APC2, SOX11
and SOX17. SOX17
is expressed in the developing pancreas and has an important role in endoderm development (42
). Sox17 represses the Wnt/β-catenin signaling pathway (43
) and its silencing has been reported in colorectal and stomach cancers (43
). We and others have previously described aberrant hypermethylation of SOX17
in pancreatic cancers (31
). In the present study, we find that aberrant hypermethylation of SOX17
and silencing of its protein product is evident early during pancreatic neoplastic development and the prevalence of methylation increases with IPMN grade, and this was associated with silencing of SOX17 expression in ~1/4 of IPMNs and pancreatic ductal adenocarcinomas.
Another gene we found to be commonly methylated in IPMNs was the putative tumor suppressor gene EBF3/COE3
which encodes a transcription factor involved in regulating differentiation and neurogenesis (45
). Although aberrant methylation of EBF3
has not been reported in pancreatic neoplasms previously, such methylation has been reported in several cancers, including glioblastoma and head and neck squamous cell carcinomas (45
). Furthermore, EBF3
mutation has been described (47
). Methylation of EBF3
by MSP was detected in only 1 of 40 normal pancreas tissues.
Many genes involved in cell adhesion were also hypermethylated in IPMNs, including members of the cadherin superfamily CDH2, CDH4, PCDH7, PCDH8, PCDH10, PCDH17, and PCDHB7. Loss of the protein product of CDH2
(n-cadherin) expression has been reported in infiltrating pancreatic ductal adenocarcinomas (48
). We and others have previously reported occasional silencing, mutation and focal loss of expression of CDH1
in pancreatic cancers (49
). Interestingly, occasional somatic mutations of cadherins and protocadherins have been identified in pancreatic cancers (47
In summary, genome-wide CpG island methylation profiling of IPMNs reveals that IPMNs harbor aberrant methylation of hundreds of CpG islands relative to normal pancreatic duct and that aberrant CpG island methylation increases with IPMN grade. The specificity of methylation of genes such as BNIP3 for high-grade dysplasia highlights the potential diagnostic applications of methylated genes in the evaluation of IPMNs.