BackgroundIn colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing.MethodsCouples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured.ResultsFor the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%) for MGMT displayed significant differences in methylation levels.ConclusionsFrozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

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Purpose: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations.Experimental Methods: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations.Results: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P ¼ <0.01) and was strongly associated with CIMP (P < 0.01). Subgroup analysis for tumor stage, MSI, and BRAF status showed no statistically significant differences in survival between CL1, CL2, and CL3 nor between CIMP and non-CIMP CRCs. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, microsatellite stability (MSS), BRAF wild-type (WT) CRCs: multivariate Cox proportional HR ¼ 3.89 [95% confidence interval (CI), 1.58-9.60, P < 0.01; n ¼ 66] and HR ¼ 2.11 (95% CI, 0.95-4.69, P ¼ 0.068, n ¼ 136) in a second independent population-based study.Conclusions: CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs. Clin Cancer Res; 20(12); 3261-71. Ó2014 AACR.

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20-30% of high-risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients. The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis. In the first series, analyzed by MSP, CRC stage II patients (n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HRmultivariable = 2.51, 95%-CI: 1.42-4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients (n = 231) was not statistically significant (HRmultivariable = 1.16, 95%-CI 0.71-1.92). The third series (n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HRmultivariable = 1.91, 95%-CI: 1.04-3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high-risk stage II CRC patients with a poor prognosis. This merits further investigation.

Abstract-We investigated flow (shear stress)-and agonist-induced cGMP release in mesenteric vascular beds of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The mesenteric vascular bed was perfused in situ with Tyrode's solution. Vascular relaxation and cGMP release in the perfusate were determined on stimulation by flow or by acetylcholine (0.1 mol/L) or sodium nitroprusside (0.1 mmol/L). Flow-induced release of cGMP was significantly greater in SHR than in WKY (PϽ0.01), despite a lower flow-induced dilation in SHR. In both strains, N G -nitro-L-arginine methyl ester (L-NAME) completely inhibited cGMP release in response to flow (PϽ0.001), although flow-induced dilation was not affected by L-NAME in SHR. Moreover, the activity of the constitutive nitric oxide synthase (NOS) was significantly greater in SHR (82Ϯ3.5 fmol/min) than in WKY (66Ϯ3.5 fmol/min; PϽ0.05) and was associated with increased expression of endothelial NOS mRNA in SHR. Sodium nitroprusside induced larger increases in cGMP release in SHR (3593Ϯ304 fmol/min) than in WKY (2467Ϯ302 fmol/min; PϽ0.05). The release of cGMP in response to acetylcholine was significantly lower in SHR (292Ϯ80 fmol/min) than in WKY (798Ϯ218 fmol/min; PϽ0.05) in parallel with smaller acetylcholine-induced relaxation in SHR. Despite increased cGMP production in response to flow and NOS activity, flow-induced dilation was decreased in SHR, suggesting an upregulation of the NO/cGMP pathway to compensate for the increased vascular tone in SHR. (Hypertension. 1998;32:1098-1103.)

BackgroundAlready since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series.MethodsIn the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up.ResultsUsing direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97–3.15; HR 1.85, 95 % CI 0.93–3.86; HR 1.83, 95 % CI 0.92–3.65, respectively).ConclusionsOur results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker’s prognostic outcome is minimal.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0211-8) contains supplementary material, which is available to authorized users.

BackgroundTesticular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients.A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age.ResultsComparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N).ConclusionsThis study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0559-z) contains supplementary material, which is available to authorized users.

Retinoid receptors (RRs) play a key role in cell proliferation and differentiation. We characterized the expression of RA receptors and retinoid X receptors (RARs and RXRs) in a series of 111 thyroid tumors and investigated the mechanisms responsible for their deregulation: hypermethylation of the RARB2 promoter, loss of heterozygosity (LOH) in the regions of RARB and RXRA, and altered expression of CRBP1 and enzymes involved in RA biosynthesis (RDH10 and RALDH2). Expression of RALDH2 and RDH10 was conserved in 100 % of adenomas and in 90 and 98 %, respectively, of carcinomas, whereas staining for CRBP1 was decreased in 9 % of FAs and 28 % of carcinomas, mainly anaplastic carcinomas (55 %). We found an abnormal expression of RARA, RARB, RXRA, and RXRB in 67, 69, 66, and 73 %, respectively, of thyroid carcinomas (n = 78) and in 9, 9, 9, and 33 % of follicular adenomas (n = 33) (p < 0.001). An abnormal staining pattern of at least two of these markers had 90 % sensitivity and 91 % specificity for a diagnosis of malignancy. Promoter hypermethylation of RARB2 was observed in some anaplastic carcinomas (14 %). LOH was found to be common at the RARB locus (3p24-3p25) and the RXRA locus (9q34), respectively, in 44 and 55 % of carcinomas and in 27 and 43 % of adenomas. In conclusion, immunohistochemical staining for RARs and RXRs may help in the differential diagnosis between well-differentiated carcinoma and follicular adenoma. Further investigation should be carried out to determine whether the characterization of RR expression might identify patients who could benefit from therapy with RA derivatives.