Prognostic Relevance of Aberrant DNA Methylation in G1 and G2 Pancreatic Neuroendocrine Tumors

Background/Aims: The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. Methods: In a well-characterized series of 56 sporadic G1 and G2 PanNETs, Methylation-Sensitive Multiple Ligation-dependent Probe Amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNA) of 53 chromosomal regions. Long Interspersed Nucleotide Element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. Results: Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. Twelve tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only five PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). Conclusion: The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progressio

Diagnostic utility of MS-MLPA in DNA methylation profiling of adenocarcinoma and neuroendocrine carcinomas of the colon-rectum

Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) is a fast, new, inexpensive method that has rarely been exploited in DNA methylation profiling of colorectal cancers (CRCs). The aim of this study was to test the diagnostic utility of MS-MLPA to evaluate the methylation status of 34 genes in normal colonic mucosa samples and in a well-characterized series of 83 adenocarcinomas and 21 neuroendocrine carcinomas of colon-rectum. Two commercial MS-MLPA kits (SALSA MS-MLPA ME001-C1 Tumor suppressor-1 Kit and SALSA MS-MLPA ME002-B1 Tumor suppressor-2 Kit) were used to perform promoter methylation analysis on formalin-fixed and paraffin-embedded tissues. MS-MLPA analysis was validated by bisulfite pyrosequencing, bisulfite cycle sequencing, and methylation-specific PCR. MS-MLPA analysis identified a subset of 27 CRCs (26 % of cases) showing high levels of gene methylation involving a mean percentage of 34 % of the promoters examined. These tumors exhibited all the main clinicopathological and genetic features described for CRCs with CpG island Methylator Phenotype-High. High levels of methylation were observed with similar frequency in adenocarcinomas and in neuroendocrine carcinomas (25 % versus 29 %, respectively), but different methylation profiles were observed in the two tumor types. In both groups, tumors with microsatellite instability and widespread methylation represented a homogeneous clinicopathological entity. MS-MLPA assay is an easy and reliable system for epigenetic characterization of tumor tissues and leads to a rapid identification of CRCs with the highest levels of gene methylation. Aberrant gene methylation is a common abnormality in CRC initiation and may be observed in tumors with very different genetic and clinicopathological profile

Supplementary Material for: Prognostic Relevance of Aberrant DNA Methylation in G1 and G2 Pancreatic Neuroendocrine Tumors

<strong><em>Background/Aims:</em></strong> The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. <b><i>Methods:</i></b> In a well-characterized series of 56 sporadic G1 and G2 PanNETs, methylation-sensitive multiple ligation-dependent probe amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNAs) of 53 chromosomal regions. Long interspersed nucleotide element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. <b><i>Results:</i></b> Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene-specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. 12 tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only 5 PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene-specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis, low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). <b><i>Conclusion:</i></b> The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progression

Epigenetic and Genetic Changes of APC Gene in Acinar Cell Carcinoma of the Pancreas

Background: Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinoma (ACC) are largely unknown. ACCs generally lack mutations of KRAS, p53, DPC4 and p16, while mutations of the APC gene have been previously described in 17.6% of ACCs. However, it is not known whether loss of function of APC gene in ACC can occur through multiple alternative genetic mechanisms. Design: We investigated promoter methylation and copy number of APC gene using MS-MLPA and FISH analysis in 14 ACCs. Droplet Digital PCR (ddPCR, Biorad Instrument) and immunohistochemistry were employed to evaluate APC mRNA and protein expression in the same tumors. Results: APC methylation was found in 10/14 (71.4%) cases and FISH analysis revealed loss of APC in 7 ACCs. Interestingly 4 ACCs (4/14 28.6% %) revealed both methylation and loss of APC. Only one case did not show any loss and methylation. Absolute quantification of APC mRNA levels demonstrated a significant reduction of the transcript in all investigated ACCs compared with normal control pancreases (10.5 \ub1 3.2 RNA copies/\u3bcl in ACCs versus 56.77 \ub1 8.1 RNA copies/\u3bcl in normal controls; p<0.0001). APC protein expression was not found in any case investigated. APC gene methylation and loss did not correlate with stage, grade and prognosis. Conclusions: APC gene inactivation is a frequent event in ACCs. Gene methylation and loss might be considered as a mechanism of APC haploinsufficiency. Moreover, our data suggest that APC gene alterations are an early event in ACC tumorigenesis

Epigenetic and Genetic Changes of APC Gene in Acinar Cell Carcinoma of the Pancreas

Background: Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinoma (ACC) are largely unknown. ACCs generally lack mutations of KRAS, p53, DPC4 and p16, while mutations of the APC gene have been previously described in 17.6% of ACCs. However, it is not known whether loss of function of APC gene in ACC can occur through multiple alternative genetic mechanisms. Design: We investigated promoter methylation and copy number of APC gene using MS-MLPA and FISH analysis in 14 ACCs. Droplet Digital PCR (ddPCR, Biorad Instrument) and immunohistochemistry were employed to evaluate APC mRNA and protein expression in the same tumors. Results: APC methylation was found in 10/14 (71.4%) cases and FISH analysis revealed loss of APC in 7 ACCs. Interestingly 4 ACCs (4/14 28.6% %) revealed both methylation and loss of APC. Only one case did not show any loss and methylation. Absolute quantification of APC mRNA levels demonstrated a significant reduction of the transcript in all investigated ACCs compared with normal control pancreases (10.5 \ub1 3.2 RNA copies/\u3bcl in ACCs versus 56.77 \ub1 8.1 RNA copies/\u3bcl in normal controls; p<0.0001). APC protein expression was not found in any case investigated. APC gene methylation and loss did not correlate with stage, grade and prognosis. Conclusions: APC gene inactivation is a frequent event in ACCs. Gene methylation and loss might be considered as a mechanism of APC haploinsufficiency. Moreover, our data suggest that APC gene alterations are an early event in ACC tumorigenesis

Oxidative DNA damage induces hypomethylation in a compromised base excision repair colorectal tumourigenesis

Background:A compromised base excision repair (BER) promotes carcinogenesis by accumulating oxidative DNA-damaged products as observed in MUTYH-associated polyposis, a hereditary colorectal cancer syndrome marked by adenomas and cancers with an accumulation of 8-oxoguanine. Remarkably, DNA global demethylation has been shown to be mediated by BER, suggesting a relevant interplay with early colorectal tumourigenesis. To check this hypothesis, we investigated a cohort of 49 adenomas and 10 carcinomas, derived from 17 MUTYH-associated polyposis patients; as adenoma controls, we used a set of 36 familial adenomatous polyposis and 24 sporadic polyps.Methods:Samples were analysed for their mutational and epigenetic status, measured as global LINE-1 (long interspersed nuclear element) and gene-specific LINE-1 MET methylation by mass spectrometry and pyrosequencing.Results:MUTYH-associated polyposis adenomas were strikingly more hypomethylated than familial adenomatous and sporadic polyps for both DNA demethylation markers (P=0.032 and P=0.007 for LINE-1; P=0.004 and P<0.0001 for LINE-1 MET, respectively) with levels comparable to those of the carcinomas derived from the same patients. They also had mutations due mainly to KRAS/NRAS p.G12C, which was absent in the controls (P<0.0001 for both sets).Conclusions:Our results show that DNA demethylation, together with specific KRAS/NRAS mutations, drives the early steps of oxidative damage colorectal tumourigenesis.British Journal of Cancer advance online publication, 31 January 2017; doi:10.1038/bjc.2017.9 www.bjcancer.com