Circulating Methylated MINT2 Promoter DNA Is a Potential Poor Prognostic Factor in Gastric Cancer

Background and Aim

Aberrant DNA methylation has been shown to be associated with the growth, development, metastasis, and prognosis of tumors. Methylated DNAs may be suitable biomarkers for cancer patients. Here, we investigated whether circulating methylated MINT2 DNAs represent a potential poor prognostic factor in gastric cancer (GC).

Methods

MINT2 methylation was detected by real-time methylation-specific PCR in tumor tissues, pairing preoperative peritoneal lavage fluid (PPLF) and blood from 92 GC patients. The theory meaning and clinical practicality value of MINT2 methylation in different specimens were analyzed.

Results

The methylation status of the MINT2 gene was found to be significantly higher in tumor tissues (44.6 %, 41/92) than in adjacent normal tissues (3.3 %, 3/92). No MINT2 methylation was found in healthy controls, and partial MINT2 methylation was observed in three (6.25 %, 3/48) patients with chronic atrophic gastritis. The frequency of MINT2 methylation in pairing PPLF and blood samples from 92 GC patients was 40.2 % (37/92) and 39.1 % (36/92), respectively. Methylated MINT2 in tumor tissues, pairing PPLF, and blood samples were very approximate. Aberrant MINT2 methylation in tumor tissues and pairing PPLF or blood samples were closely related to peritoneal dissemination, tumor progression, and poor prognosis (all P < 0.0001).

Conclusions

Aberrant MINT2 methylation in PPLF/blood may predict peritoneal micrometastasis for GC patients, which is a potential poor prognostic factor in GC.     

Morphological Abnormalities of the Recto-Anal Inhibitory Reflex Reflects Symptom Pattern in Neurogenic Bowel

Background

Bowel dysfunction amongst multiple sclerosis (MS) and spinal cord injury (SCI) patients often manifests as fecal incontinence (FI) or constipation, but the pathophysiology is poorly understood. Anorectal physiology provides an objective assessment of lower bowel functions and is increasingly being used in clinical practice.

Aim

The purpose of this study was to correlate symptoms of bowel dysfunction in patients with spinal cord disease with findings in anorectal physiology. We hypothesized that specific abnormalities will correlate with symptoms: prolonged recto-anal inhibitory reflex in patients with incontinence and decreased rectal mucosal blood flow in patients with constipation.

Methods

Forty-nine patients with MS (35 with predominant FI and 14 constipation), 46 supraconal SCI (mixed symptom load), and 21 healthy volunteers matched for age and sex were studied. Subjects completed validated constipation and FI symptom questionnaires. Patients underwent standard anorectal physiology, including assessment of rectal mucosal blood flow and recto-anal inhibitory reflex (RAIR).

Results

Severity of constipation correlates significantly with distension sensitivity (urge volume [r = 0.68, p = 0.002] and maximal volume [r = 0.39, p = 0.03]). Severity of constipation also correlated with diminished rectal mucosal blood flow in both patient groups (r = ?0.51, p = 0.006). In both groups, constipation correlated with diminished relaxation of the sphincters in the RAIR whilst fecal incontinence correlated with a prolonged duration of RAIR (r = 0.33, p = 0.009) and recovery phase (r = 0.37, p = 0.05).

Conclusion

Bowel symptoms in patients with MS and SCI correlate with specific alterations of anorectal physiology. This provides objective assessment of bowel symptoms and may allow tailored treatment to individual patients.     

Association Between Cyclin D1 Polymorphism with CpG Island Promoter Methylation Status of Tumor Suppressor Genes in Gastric Cancer

Background

CpG island hypermethylation of tumor suppressor genes is highly involved in gastric carcinogenesis, and enhanced cell proliferation could accelerate this process. Cyclin D1 regulates cell cycle function and may play a role in methylation-related carcinogenesis.

Aims

We investigated the association between Cyclin D1 gene G870A polymorphism and the methylation status of tumor suppressor genes in gastric cancer.

Methods

Polymorphisms at G870A in the Cyclin D1 gene were genotyped, and methylation status of the p14, p16, DAP-kinase, and CDH1 genes were determined by methylation-specific-polymerase chain reaction in 139 gastric cancer tissues. CIHM high was defined as three or more methylated CpG islands.

Results

Although no association was found between methylation status and different stages and Lauren’s subtypes, patients with CIHM of DAP-kinase showed significantly worse survival than those without (p = 0.017). In addition, the number of methylated sites was also associated with survival curves (p = 0.0397). The 870G carrier a significantly lower prevalence of CIHM high compared to the AA genotype in advanced-stage gastric cancer (adjusted OR = 0.32, p = 0.047). A weak correlation between the same genotypes and CIHM of p14 were found in the same subtype (adjusted OR = 0.32, p = 0.052). The mean methylation number was significantly lower in G carriers than in AA genotypes in advanced-stage gastric cancer (p = 0.017).

Conclusions

Genetic polymorphism of CCND1 is associated with CIHM status in gastric cancer, especially in the advanced stage, but is independent of clinico-pathological features.     

Predicting the pathologic response of locally advanced rectal cancer to neoadjuvant concurrent chemoradiation using enzyme-linked immunosorbent assays (ELISAs) for biomarkers

Purpose

To investigate the role of biomarkers including serum tissue inhibitor of metalloproteinases-1 (TIMP-1), urokinase plasminogen activator receptor, vascular endothelial growth factor, and epidermal growth factor receptor in predicting pathologic response to neoadjuvant chemoradiation (NACRT) for rectal cancer.

Methods

Between 2007 and 2009, 50 clinical TNM stage II or III patients were analyzed in this prospective study. Pre- and post-NACRT serum levels of biomarkers were assessed using ELISAs. The primary and secondary endpoints were pathologic complete response (pCR) and Mandard regression grade (MRG).

Results

The pCR was reported in 5 patients (10.0 %). According to the MRG, fifteen patients (30.0 %) were divided into group A (Grade I–II), the others in group B (Grade III–V). On univariate analysis, post-NACRT TIMP-1 showed notable significance with pCR (P = 0.092) and significant correlation with MRG group A (P = 0.003). Post-NACRT TIMP-1 ≤ 204.5 ng/mL as cut-off value by ROC curve was associated with more pCR and MRG group A patients (P = 0.016 and 0.002). Interval between NACRT and surgery was related to pCR with approached trend levels of significance (P = 0.05) and to MRG group A significantly on univariate analysis of clinical factors (P = 0.031). On multivariate analysis, post-NACRT TIMP-1 was not significantly related to pCR (P = 0.187), while it was significantly associated with MRG (P = 0.009). Among clinical responders, post-NACRT TIMP-1 level ≤ 204.5 ng/mL was significantly associated with pCR (P = 0.021) and MRG group A (P = 0.003).

Conclusions

Post-NACRT serum TIMP-1 could be used as a predictive marker of pathologic response to NACRT in rectal cancer, even in patients with clinical response.     

Aberrant methylation of the MSH3 promoter and distal enhancer in esophageal cancer patients exposed to first-hand tobacco smoke

Purpose

Polymorphisms in MSH3 gene confer risk of esophageal cancer when in combination with tobacco smoke exposure. The purpose of this study was to investigate the methylation status of MSH3 gene in esophageal cancer patients in order to further elucidate possible role of MSH3 in esophageal tumorigenesis.

Methods

We applied nested methylation-specific polymerase chain reaction to investigate the methylation status of the MSH3 promoter in tumors and matching adjacent normal-looking tissues of 84 esophageal cancer patients from a high-risk South African population. The Cancer Genome Atlas data were used to examine DNA methylation profiles at 17 CpG sites located in the MSH3 locus.

Results

Overall, promoter methylation was detected in 91.9 % of tumors, which was significantly higher compared to 76.0 % in adjacent normal-looking esophageal tissues (P = 0.008). When samples were grouped according to different demographics (including age, gender and ethnicity) and smoking status of patients, methylation frequencies were found to be significantly higher in tumor tissues of Black subjects (P = 0.024), patients of 55–65 years of age (P = 0.032), males (P = 0.037) and tobacco smokers (P = 0.015). Furthermore, methylation of the MSH3 promoter was significantly more frequent in tumor samples from smokers compared to tumor samples from non-smokers [odds ratio (OR) = 31.9, P = 0.031]. The TCGA data confirmed significantly higher DNA methylation level at the MSH3 promoter region in tumors (P = 0.0024). In addition, we found evidence of an aberrantly methylated putative MSH3-associated distal enhancer element.

Conclusion

Our results suggest that methylation of MSH3 together with exposure to tobacco smoke is involved in esophageal carcinogenesis. Due to the active role of the MSH3 protein in modulating chemosensitivity of cells, methylation of MSH3 should further be examined in association with the outcome of esophageal cancer treatment using anticancer drugs.     

Polymorphisms in DNA repair genes XRCC1, XRCC3 and XPD, and colorectal cancer risk: a case–control study in an Indian population

Purpose

Genetic polymorphisms in DNA repair genes may influence variations in individual DNA repair capacity, which could be associated with the development of cancer. We detected the distributions of three single-nucleotide polymorphisms (XRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln) in DNA repair genes, and assessed the associations of these genetic polymorphisms with colon and rectal cancer susceptibility as well as evaluated the interactions of gene–gene and gene–environment in a case–control study of an Indian population.

Methods

This case–control study was conducted with 302 cases (including 59 colon and 243 rectal cancer patients) and 291 cancer-free healthy controls. Genotypes were determined by PCR–RLFP assays. The effects [odds ratios (ORs) and 95% confidence intervals (95% CIs)] of genetic polymorphisms on colorectal cancer were estimated using unconditional logistic regression.

Results

The XRCC1 399Gln allele was found to be associated with a significantly increased rectal cancer risk among men (OR = 1.65, 95% CI 1.04–2.64). Whereas the XRCC3 241Met allele showed a protective tendency against rectal cancer (OR = 0.68, 95% CI 0.46–1.02) for both men and women. Furthermore, a combination of the XRCC1 399Gln allele with XRCC3 Thr/Thr genotype and the XPD 751Gln allele demonstrated the highest rectal cancer risk (OR = 3.52, 95% CI 1.43–9.44).

Conclusions

The combined effects of putative risk alleles/genotypes for different DNA repair pathways may strengthen the susceptibility to rectal cancer.     

The tissue selecting technique (TST) versus the Milligan–Morgan hemorrhoidectomy for prolapsing hemorrhoids: a retrospective case–control study

Background

Milligan–Morgan hemorrhoidectomy (MMH) is the procedure of choice in the management of hemorrhoidal disease. However, this procedure is associated with significant postoperative pain. Tissue selecting technique (TST) is a segmental stapled hemorrhoidopexy, which aims to reduce the postoperative pain, rectovaginal fistula (RVF) and rectal stenosis. The aim of the present study was to compare the clinical outcomes between TST and MMH.

Methods

A case–control study was undertaken to investigate the difference in clinical characteristics between the patients treated with TST and those treated with MMH. Intraoperative and postoperative parameters in both groups were collected and compared.

Results

One hundred and ninety-five eligible patients underwent either TST (n = 121) or MMH (n = 74). The pain score was significantly less in the TST group than that in the MMH group at the first defecation and at 12 h, day 3 and day 7 postoperatively (P = 0.001). Further analysis revealed that, at the time point of 12 h, day 3, day 7 and during first defecation, the pain score in the TST group and TST + STE group was less than that in the MMH group (P = 0.001). No patient in either group developed postoperative rectal stenosis. Furthermore, no case of RVF was identified in the TST group. The 1-year recurrence rate was 3.3 % (4/121) and 2.7 % (2/74), respectively, in TST and MMH groups (P = 1.0).

Conclusions

The 1-year recurrence rate after TST and MMH for the treatment of patients with grade III–IV hemorrhoids is similar. It is encouraging that TST is associated with less postoperative pain and no RVF or rectal stenosis.     

Clinico-epigenetic combination including quantitative methylation value of DKK3 augments survival prediction of the patient with cervical cancer

Purpose

DKK3 is a target of methylation in various cancers and has been studied by a non-quantitative method. We assessed the quantitative methylation levels of DKK3 in cervical carcinoma, determined the potential clinical correlations, and tested whether the combination of clinical and epigenetic factors augmented the prediction power of prognosis.

Methods

Sixty-two patients with cervical squamous cell carcinoma were included in this study. Quantitative methylation levels were evaluated by pyrosequencing. Clinical and pathologic findings were obtained from medical records. Survival data were analyzed using Kaplan–Meier estimates and compared with the log-rank test. The best clinico-epigenetic combinations were found using the Cox proportional hazard model.

Results

Four of five CpG positions of DKK3 were strongly methylated in cervical carcinoma compared to normal controls (p = 0.0048). The methylation in positions 1 and/or 2 were stronger in patients with higher serum levels of the SCC tumor marker and/or larger tumors (p = 0.01). The patients with a methylation level ≥26.3 % at position 1 had a lower survival rate than the patients with methylation levels at position 1 that were <26.3 % (p = 0.03). The combination of methylation level of position 1, position 3, age, parametrial invasion, and lymphovascular space invasion (LVSI) have a significant correlation with survival (p = 0.0006). Recurrence was significantly related to the combination of methylation level of position 2, position 3, age, parametrium, and LVSI (p = 0.0041).

Conclusions

DKK3 methylation is unfavorable to prognosis. This study defined a threshold level of methylation associated with recurrence-free survival and, furthermore, identified novel clinico-epigenetic combinations predicting disease survival or recurrence.     

Treatment for early ultralow rectal cancer: pull-through intersphincteric stapled transection and anastomosis (PISTA) versus low anterior resection

Background

The aim of this study was to compare the functional and oncologic results of pull-through intersphincteric stapled transection and anastomosis (PISTA) with low anterior resection (LAR) in the treatment for early ultralow rectal cancer.

Methods

A total of 278 patients with early ultralow rectal cancer were retrospectively included and analyzed, with 136 in the PISTA group and 142 in the LAR group.

Results

Gender, age, tumor diameter, distance from the dentate line to the inferior margin of the tumor, tumor stage, length of operation and postoperative complications were comparable in the two groups. Compared with the LAR group, the PISTA group had a more accurate distal transection site, a lower daily fecal frequency (6 (5–7) vs. 8 (7–9), p < 0.001) and a lower Wexner incontinence score (13 (10–14) vs. 14 (13–16), p < 0.001) 3 months after ileostomy reversal, and a higher rate of satisfactory fecal continence (97.1 % vs. 90.8 %, p = 0.043). The follow-up period of the PISTA group was similar to that of the LAR group (56 (30–81) months vs. 54 (30–80) months, p = 0.982). The PISTA group was associated with a lower local recurrence rate (2.2 % vs. 11.3 %, p = 0.003). Kaplan–Meier analysis also showed that the PISTA group was associated with longer overall survival (p = 0.018) and longer local recurrence-free survival (p = 0.004) than the LAR group, while distant metastasis-free survival (p = 0.896) was comparable in the two groups. Multivariate analysis identified lymph node metastasis (p < 0.001) and operation (PISTA vs. LAR, p = 0.031) as independent predictive factors for local recurrence-free survival.

Conclusions

PISTA is a technically simple, oncologically safe and functionally favorable procedure for the treatment for early ultralow rectal cancer.     

S100A Expression and Interleukin-10 Polymorphisms Are Associated with Ulcerative Colitis and Diarrhea Predominant Irritable Bowel Syndrome

Background

Both ulcerative colitis (UC) and diarrhea-predominant irritable bowel syndrome (IBS-D) are associated with alterations in enteric serotonin (5-HT) signaling.

Aims

The purpose of this study was to compare the rectal and sigmoid colonic mucosal expression of S100A proteins and functional polymorphisms of the 5-HT transporter (5HTT) and interleukin-10 genes in patients with IBS-D or UC with healthy controls.

Methods

mRNA expression of S100 proteins was measured in sigmoid and rectal biopsies and in rectal epithelium isolated by laser-captured microdissection. Leucocyte DNA was analyzed by PCR-based reaction fragment length polymorphisms and direct sequencing. Clinical symptoms were assessed by the self-rating depression scale and by the gastrointestinal symptom rating scale.

Results

Fifty patients with IBS-D, 56 with UC and 50 healthy controls were studied. Colonic mucosal expression of S100A8 and S100A9 in UC was significantly higher than in IBS or controls and correlated with the UC disease activity index (r = 0.65, p < 0.001). S100A10 expression in the rectal epithelium of the IBS patients was significantly higher (0.643 vs. 0.402, p = 0.01) than in controls and correlated with the SDS scores (r = 0.41, p = 0.002). The frequency of IL10-819 CC genotype was significantly higher in IBS-D (10.7 vs. 0 %, p = 0.047) and UC (16 vs. 0 %, p = 0.007) than that in controls.

Conclusion

Overexpression of S100A10 in the rectum may play a role in IBS as it is involved in modulating 5-HT1B receptors. The IL10-819 CC is a candidate genotype for both IBS and UC in Japanese.     

Thymidylate synthase germline polymorphisms in rectal cancer patients treated with neoadjuvant chemoradiotherapy based on 5-fluorouracil

Purpose

Chemoradiotherapy using 5-fluorouracil has shown to be effective treatment for rectal cancer. Thymidylate synthase (TS) is an important target enzyme for the fluoropyrimidines. However, the predictive role of TS levels in early stage rectal cancer is not yet well understood. We analyzed the value of TS gene polymorphisms as a predictive marker in patients with stage II and III rectal cancer treated with preoperative concomitant radiotherapy and fluoropyrimidine-based chemotherapy.

Methods and materials

Between 1998 and 2007, blood samples were obtained from 51 patients with stage II/III rectal cancer. Forty patients were T2–3 (78%), 11 were T4 (22%), and 59% were N+. DNA was extracted from peripheral blood, and the genotypes were analyzed using PCR-restriction fragment length polymorphism and automated sequencing techniques.

Results

The *3/*3 thymidylate synthase genotype was associated with a higher response rate (pathological complete remission and microfoci residual tumor; 61 vs. 22% in *2/*2 and *2/*3; P = 0.013). In the multivariate analysis, the *3/*3 thymidylate synthase genotype was also an independent prognostic factor for better survival (P < 0.05).

Conclusions

The thymidylate synthase genotype might help to identify patients with stage II/III rectal cancer who could benefit from pre- and postoperative fluorouracil-based chemotherapy.     

Add-On Adefovir Is Superior to a Switch to Entecavir as Rescue Therapy for Lamivudine-Resistant Chronic Hepatitis B

Background/Aims

Lamivudine (LAM) has been extensively used to treat hepatitis B, but high incidence of drug resistance has required rescue studies. We validated the optimum treatment strategy for LAM-resistant patients by means of a comparative study of add-on adefovir (ADV) and a switch to entecavir (ETV).

Methods

We assessed the virologic response in consecutive LAM-resistant patients who received add-on ADV or a switch to ETV.

Results

The mean reduction of serum hepatitis B virus (HBV) DNA levels was significantly less in the ETV group than in the add-on ADV group (?3.45 vs. ?4.17; P = 0.047 at week 24 and ?3.81 vs. ?4.68 log₁₀ IU/mL; P = 0.044 at week 48). Achievement of undetectable HBV DNA was significantly lower in the ETV group than in the add-on ADV group (P = 0.043). Multivariate analysis showed that add-on ADV, baseline HBV DNA levels, and initial virologic response were significant predictors of HBV DNA negativity (adjusted OR, 2.582; P = 0.008, 0.304; P = 0.001, and 5.928; P = 0.001). Virologic breakthrough was observed for 12 patients, in the ETV group only.

Conclusions

Add-on ADV was more effective and durable than ETV as rescue therapy. Therefore, add-on ADV might be the preferred strategy for LAM-resistant patients who need long-term antiviral treatment.     

PPP1R13L variant associated with prognosis for patients with rectal cancer

Background

ERCC1, CD3EAP, and PPP1R13L polymorphisms in the chromosomal region 19q13.2-3 have already been shown to have a synergistic effect on apoptosis and DNA repair pathways. Therefore, the aim of this study was to investigate the association between such genetic variants and the prognosis of colorectal cancer (CRC) following curative surgery.

Methods

DNA was extracted from fresh frozen normal tissue from 349 CRC patients underwent curative surgery and then genotyped for 5 polymorphisms (PPP1R13L rs1970764 and rs1005165; CD3EAP rs967591; ERCC1 rs735482 and rs11615) using PCR-restriction fragment length polymorphism (PCR-RFLP).

Results

Among the 5 polymorphisms, PPP1R13L rs1970764 was significantly associated with relapse-free (RFS) and disease-specific survival (DSS) in a recessive model. In haplotype analysis, three haplotypes (TACC, CGAC, and CGAT) were selected for analysis among 6 haplotypes constructed by the PHASE II program using 4 polymorphisms (rs1005165, rs967591, rs735482, and rs11615) in a moderate to strong linkage disequilibrium (LD) (D′ > 0.4), and a significant difference in RFS was observed among patients with these 3 haplotypes. In the multivariate analysis, the GG genotype of PPP1R13L rs1970764 was identified as an independent prognostic factor for poor RFS and DSS (HR = 1.743 and 1.734; P = 0.003 and 0.010, respectively) when compared with the combine AA/AG genotype adjusted to clinicopathologic variables. In particular, the prognostic impact of PPP1R13L rs1970764 was persistent only in patients with rectal cancer (HR = 3.307 and 3.180; both P < 0.001 for RFS and DSS, respectively).

Conclusions

The present results suggest that the PPP1R13L rs1970764 variant is a possible prognostic marker for patients with rectal cancer.     

Novel Classification and Pathogenetic Analysis of Hypoganglionosis and Adult-Onset Hirschsprung��s Disease

Background and Aims

Researchers have not clearly described the clinical and pathogenetic features of hypoganglionosis and adult-onset Hirschsprung??s disease, which cause pseudo-obstruction or intractable constipation. We conducted this study to explore these features of hypoganglionosis and adult-onset Hirschsprung??s disease in Korean patients.

Methods

We enrolled 24 patients pathologically confirmed as having hypoganglionosis and 11 as having adult-onset Hirschsprung??s disease. We recruited 26 subjects who had undergone operation for nonobstructive colon cancer and 45 healthy volunteers as controls. We described their clinical features, investigated ganglion cells and interstitial cells of Cajal (ICC), and analyzed RET, EDNRB, EDN3, and SOX10 genes.

Results

We classified hypoganglionosis patients into two groups: type I (focal type, n = 13), with focally narrowed transition zone (TZ); and type II (diffuse type, n = 11), without transition zone. Hypoganglionosis patients had significantly fewer ganglion cells than the controls, and those cells were scarcer in the transition zone than in the proximal dilated area (P < 0.05). The ICC numbers in both diseases were significantly lower than in controls; however, they were similar between transition zone and the proximal dilated area in hypoganglionosis. In adult-onset Hirschsprung??s disease, two significant intronic RET polymorphic variants, IVS14-24G>A and IVS19+47T>C, were significantly associated with adult-onset Hirschsprung??s disease (P = 0.0122 and 0.0295, respectively), but not with hypoganglionosis.

Conclusions

Hypoganglionosis and adult-onset Hirschsprung??s disease have different pathophysiologic characteristics, although their clinical presentations are similar. We suggest that there are two subgroups of hypoganglionosis: those with or without a focally narrowed transition zone with a profoundly diminished number of ganglion cells.     

Association of the GNB3 825T-allele with better survival in patients with glioblastoma multiforme

Purpose

Genotypes of the C825T polymorphism of the GNB3 gene encoding the G protein β3 subunit were recently associated with the prognosis of different malignomas. We investigated potential associations of GNB3 genotypes with survival of patients with glioblastoma multiforme (GBM).

Methods

One hundred and sixty-one patients suffering from GBM were retrospectively investigated. Inclusion criteria were availability of DNA and a follow-up of at least 24 months. The results were evaluated with respect to the basic clinical data, type of surgical intervention, MGMT promoter methylation, adjuvant therapy, and survival.

Results

After 2 years of first diagnosis, 128 (79.5%) of the 161 patients had died, 33 (20.5%) were alive. Kaplan–Meier curves revealed a significant higher rate of survival for homo- and heterozygous T-allele carriers (P = 0.019) with 38.5 and 25.3%, respectively, but only 11.6% for homozygous C-allele carriers. Multivariable Cox regression identified the heterozygous (hazard ratio 3.3, 95% CI 1.3–8.0, P = 0.010), as well as homozygous GNB3 825 C-allele (hazard ratio 3.7, 95% CI 1.5–9.1, P = 0.004) as an independent negative prognostic factor for 2-year survival according to the GNB3 825 TT genotype reference group.

Conclusions

Our data suggest an association of the GNB3 825TT genotype and better survival in patients with GBM.     

Detection of TFPI2 Methylation in the Serum of Hepatocellular Carcinoma Patients

Background

DNA methylation plays a key role in hepatocellular carcinogenesis and progression. Analysis of aberrant methylation in serum DNA might provide a strategy for noninvasive detection of hepatocellular carcinoma (HCC).

Methods

To explore the feasibility of this approach, we compared TFPI2 methylation status in serum samples of HCC, chronic hepatitis B (CHB) patients and normal control groups using methylation-specific polymerase chain reaction.

Results

Our results showed that the percentage of serum TFPI2 promoter methylation was significantly higher in the HCC group (46.5 %, 20/43) compared with the CHB group (16.7 %, 4/24; p = 0.015) and the normal control group (19.2 %, 5/26; p = 0.022), respectively, indicating that TFPI2 methylation frequently existed in the serum of HCC patients. In our study, the detection rate of HCC using serum TFPI2 methylation was 46.5 % (20/43), which was quite close to the reported detection rate of α-fetoprotein (54 %). In cases where we combined both markers, the detection rate was 61.0 %, suggesting that serum TFPI2 methylation could be used as a potential marker for noninvasive detection of HCC. Then, we evaluated the correlation between the serum TFPI2 methylation status of HCC patients and their clinicopathological parameters. Patients with advanced TNM stage (III–IV) showed a significantly elevated serum methylation percentage of TFPI2 in comparison with those with early TNM stage (I–II) (p = 0.025). Moreover, TFPI2 methylation was observed more frequently according to the progression of TNM stage.

Conclusions

Our present study suggested that TFPI2 methylation in serum tended to be detected more easily in patients with advanced HCC and might be used as a predictor of HCC progression.     

LINE-1 Hypomethylation is Associated with the Risk of Coronary Heart Disease in Chinese Population

Background

Global methylation level in blood leukocyte DNA has been associated with the risk of coronary heart disease (CHD), with inconsistent results in various populations. Similar data are lacking in Chinese population where different genetic, lifestyle and environmental factors may affect DNA methylation and its risk relationship with CHD.

Objectives

To examine whether global methylation is associated with the risk of CHD in Chinese population.

Methods

A total of 334 cases with CHD and 788 healthy controls were included. Global methylation in blood leukocyte DNA was estimated by analyzing LINE-1 repeats using bisulfite pyrosequencing.

Results

In an initial analysis restricted to control subjects, LINE-1 level reduced significantly with aging, elevated total cholesterol, and diagnosis of diabetes. In the case-control analysis, reduced LINE-1 methylation was associated with increased risk of CHD; analysis by quartile revealed odds ratios (95%CI) of 0.9 (0.6-1.4), 1.9 (1.3-2.9) and 2.3 (1.6-3.5) for the third, second and first (lowest) quartile (P_trend < 0.001), respectively, compared to the fourth (highest) quartile. Lower (<median) LINE-1 methylation was associated with a 2.2-fold (95%CI = 1.7-3.0) increased risk of CHD. The lower LINE-1-related CHD risk estimates tended to be stronger among subjects with the highest tertile of homocysteine (P_interaction = 0.042) and those with diagnosis of hypertension (P_interaction = 0.012).

Conclusion

LINE-1 hypomethylation is associated with the risk of CHD in Chinese population. Potential CHD risk factors such as older age, elevated total cholesterol, and diagnosis of diabetes may have impact on global DNA methylation, whereby exerting their effect on CHD risk.     

TFAP2E hypermethylation was associated with survival advantage in patients with colorectal cancer

Purpose

Hypermethylation of TFAP2E (AP-2E) is associated with the chemotherapy-resistant in patients with colorectal cancer (CRC), but its implications on prognosis directly remain unknown. This study was aimed to investigate the role of AP-2E methylation status and other clinicopathologic parameters as predictors of prognosis.

Methods

We detected the methylation status of AP-2E in tumor and adjacent non-tumor tissues from 311 sporadic CRC patients by methylation-sensitive high-resolution melting analysis. Log-rank tests and multivariate Cox analyses were performed to evaluate the role of AP-2E methylation status and other clinicopathologic parameters as predictors of prognosis.

Results

Hypermethylation of AP-2E was detected in 61 % (190/311) tumor tissues. It occurred more frequently in tumors in earlier stages (I/II; P = 0.02), lower levels of tumor invasion (T1–T3; P = 0.04), fewer lymph nodes involved (N0; P < 0.01), and higher histologic grades (G1/G2; P < 0.01). The overall 5-year survival rates in hypermethylation and hypomethylation group were 76.91 and 47.17 % (P < 0.0001), respectively. AP-2E hypermethylation was significantly associated with a favorable clinical outcome with a hazard ratio of 0.486 (95 % CI 0.342–0.692, P < 0.0001) after controlling for age, gender, tumor location, histologic type, TNM staging, and histologic grade.

Conclusions

AP-2E was frequently hypermethylated in tumors from patients with CRC. Aberrant hypermethylation of AP-2E occurred more frequently in tumors with earlier stages, lower levels of tumor invasion, fewer lymph nodes involved, and higher histologic grades. AP-2E hypermethylation might be an independent predictor of survival advantage in patients with CRC.     

Defining Virologic Relapse in Chronic Hepatitis B

Background and Aims

Different definitions of virologic relapse (VR) are being used. One way of defining VR is ??reappearance of HBV DNA in the serum,?? while another definition is an ??increase in HBV DNA level greater than 1 log in two determinations at least 4 weeks apart.?? The aim of this study was to see the effectiveness of these two definitions

Methods

Forty-five HBeAg-positive chronic hepatitis B patients with a virologic response [negative PCR (<12 IU/ml)] who had discontinued therapy were analyzed retrospectively for VR, HBeAg reversion and biochemical flare.

Results

HBV DNA reappeared in the serum (??12 IU/ml) of all 45 patients (100%). An increase in HBV DNA level greater than 1 log in two determinations at least 4 weeks apart was identified in 20 of 25 patients (80%). Biochemical flare and HBeAg reversion were observed in 18 (40%) and 14 (31%) patients, respectively. Peak off-therapy HBV DNA level was significantly associated with biochemical flare (r = 0.758, P < 0.001) and HBeAg reversion (r = 0.645, P < 0.001). Two patients with high initial off-therapy HBV DNA levels (??4.0 log₁₀ IU/ml) were reassessed at 4 weeks, and both experienced a biochemical flare and HBeAg reversion. Two patients had an increase in HBV DNA level greater than 1 log at a very low level (1 log to 2 or 3 log), but did not experience biochemical flare or HBeAg reversion during follow-up.

Conclusions

Reappearance of HBV DNA was universal when sensitive HBV DNA assay was used. Waiting 4 weeks to confirm VR may be harmful for patients with a high HBV DNA level, and was ineffective to indicate re-therapy for patients with increase in HBV DNA at a very low level. There is a need for improved and standardized definitions of VR.