Silencing of Hint1, a novel tumor suppressor gene,by promoter hypermethylation in hepatocellular carcinoma

The Hint1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. Previous studies in mice provided evidence that Hint1 may be haploinsufficient with respect to its function as a tumor suppressor. In the present study, we investigated the aberrant methylation of Hint1 and explored possible relationships between aberrant methylation and clinicopathological features in hepatocellular carcinoma (HCC). Hypermethylation of Hint1 was evaluated by the methylation specific PCR (MSP) method in 40 patients with HCC (tumor and paired adjacent non-tumor tissues) from Taiwan, 22 cases of normal liver tissue (14 from Taiwan and 8 from the US). HINT1 expression in tissues was detected by immunohistochemistry. The frequencies of hypermethylation of Hint1 in tumor, paired adjacent non-tumor and normal liver tissue were 55.0%, 37.5% and 9.1%, respectively. A statistically significant inverse association was found between Hint1 methylation status and expression of the HINT1 protein in tumor tissues (p = 0.003). The relationship between Hint1 methylation status and clinical features and other, previously measured biomarkers was also analyzed. p16 hypermethylation was statistically significantly associated with Hint1 methylation status (p = 0.035). There were no correlations between Hint1 methylation and hepatitis B (HBV) or hepatitis C (HCV) infection status or aflatoxin B₁ (AFB₁-) and polycyclic aromatic hydrocarbons (PAHs)-DNA adduct levels. These results suggest that promoter hypermethylation of Hint1 may play a role in hepatocarcinogenesis.     

Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia

DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of −1189 and −1176 was inversely correlated with mRNA expression (Spearman correlation: −0.333, P = 0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P = 0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.     

Promoter methylation of BRMS1 correlates with smoking history and poor survival in non-small cell lung cancer patients

Purpose

To investigate whether methylation of BRMS1 is associated with clinical outcomes in patients with NSCLC.

Methods

Methylation status of BRMS1 was examined in 325 NSCLC patients who were treated with surgery. We analyzed associations between the methylation of BRMS1 genes separately and available epidemiologic and clinical information including smoking status, gender, age, and histological type, or the stage of the tumor.

Results

In the cohort of 325 NSCLC cases, 152 samples were identified as methylated (46.77%). Promoter methylation of BRMS1 was present only in 6 specimens (8.42%) in adjacent non-cancerous tissues (P = 2.257 × 10⁻¹⁴). Patient smoking history had a positive correlation with methylation rate of BRMS1 (OR = 2.508, 95%CI(1.516, 4.151)). Compared with unmethylated group, methylated group showed the lower level of BRMS1 mRNA (P = 0.013). And patients with a high level of BRMS1 mRNA expression had significantly better overall survival than those with low expression (P = 0.002). Multivariate Cox proportional hazard regression analysis also showed that promoter methylation of BRMS1 was significantly unfavorable prognostic factors (hazard ratio, 1.912; 95% CI, and 1.341-2.726).

Conclusions

These results provide clinical evidence to support the notion that BRMS1 is a NSCLC metastasis suppressor gene. Measuring methylation status of BRMS1 promotor is a useful marker for identifying NSCLC patients with worse disease-free survival.     

Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP− primary glioblastoma

To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP− primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ?18 months) and 20 short-term survivors (STS; overall survival ?9 months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP− samples. Methylation levels of 11 CpG loci (10 genes) were statistically significantly lower, and 43 CpG loci (40 genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP− samples (P < 0.01). Of the 43 CpG loci that were hypermethylated in LTS G-CIMP− samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP− primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP− primary GBMs.     

Aberrant methylation of the RIZ1 gene in myelodysplastic syndrome and acute myeloid leukemia

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p = 0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p = 0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.     

Polymorphisms of key chemokine genes and survival of non-small cell lung cancer in Chinese

Chemokines play an important role in the pathogenesis of non-small cell lung cancer (NSCLC). Although the deregulations of chemokines have been reported to be associated with the development and progression of many human cancers including lung cancer, polymorphisms of chemokine genes have not been examined with the survival of NSCLC.We systematically investigated associations of 23 common potentially functional SNPs in the key chemokine genes (CCL2, CCL5, CCL8, CCL20, CCL22, CXCL1, CXCL6, CXCL9 and CXCL12) with the survival of NSCLC in a case cohort of 568 NSCLC patients in a Chinese population. The results showed that variant genotypes of CCL2 rs3760396 and CCL8 rs3138035 were associated with a significantly decreased risk of death for NSCLC (dominant model: adjusted HR = 0.65, 95% CI = 0.48-0.89 for rs3760396; dominant model: adjusted HR = 0.65, 95% CI = 0.49-0.86 for rs3138035), while CXCL12 rs1804429 was associated with an increased risk of death for NSCLC (CC vs AA: adjusted HR = 6.03, 95% CI = 1.44-25.24). Further stepwise regression analysis suggested that only rs3138035, a SNP located at 5′-flanking region of CCL8, was an independently favorable factor for the prognosis of NSCLC and the protective effect was more evident in smokers (adjusted HR = 0.61, 95% CI = 0.42-0.87), patients with squamous cell cancer (adjusted HR = 0.58, 95% CI = 0.35-0.96), patients with early stage (adjusted HR = 0.32, 95% CI = 0.15-0.67) and patients treated with surgical operation (adjusted HR = 0.47, 95% CI = 0.31-0.71). In addition, the interaction analysis demonstrated that stage and surgical operation interacted with the genetic effect of rs3138035 in relation to NSCLC survival (adjusted P_interaction = 0.02 and 0.01, respectively). These findings suggest that CCL8 rs3138035 may be one of the candidate biomarkers for NSCLC survival and may modify death risk associated with stage and surgical operation. Larger studies incorporating functional evaluations are warranted to validate our findings.     

Identification of subgroup patients with stage IIIB/IV non-small cell lung cancer at higher risk for brain metastases

Purpose

Brain metastases (BM), a common occurrence in non-small cell lung cancer (NSCLC), usually lead to a poor prognosis. Recently, the selection of treatment modalities for BM has modestly improved patient survival and quality of life. Treatment choice is largely based on the number of BM, the presence of BM-related symptoms, and performance status. Therefore, early BM detection is crucial. In this study, we aimed to elucidate the factors associated with BM and identify subgroups of patients at higher risk for BM.

Methods and patients

The medical records of 596 consecutive patients with stage I–IV NSCLC were reviewed between January 2006 and November 2011. A multivariate logistic regression (MLR) model was used to identify factors associated with BM.

Results

Among 482 eligible stage IIIB/IV NSCLC patients, 173 (36%) experienced BM during their disease course. On MLR analysis, female gender, age <60 years and adenocarcinoma were associated with BM (OR = 1.71, 95% CI = 1.06–2.75, P = 0.028; OR = 2.11, 95% CI = 1.38–3.22, P = 0.001; and OR = 2.39, 95% CI = 1.16–4.92, P = 0.018, respectively). The actuarial incidence of BM varied widely from 14% to 59% in different subgroups; younger patients with adenocarcinoma tended to experience BM more than older patients with squamous cell carcinoma (OR = 6.88, 95% CI = 2.97–15.94, P < 0.001). Furthermore, the incidence of BM correlated closely with survival after NSCLC diagnosis, and it was 42%, 54% and 64% in patients who survived more than 3, 12 and 24 months, respectively. Notably, the number of BM, the size of the largest BM and the proportion of multiple BM, defined as more than 4 metastatic tumors in brain, were significantly different in NSCLC patients with and without BM-related symptoms or signs (4.0 ± 2.1 vs 2.7 ± 1.9, P < 0.001; 2.6 ± 1.5 vs 1.3 ± 1.0 CM, P < 0.001, and 50% vs 21%, P < 0.001, respectively).

Conclusion

We found that subgroups of NSCLC patients characterized by younger age, female gender and adenocarcinoma are at higher risks for BM. These findings might be helpful to detect BM earlier and facilitate the design of clinical trials aiming at their prevention.     

Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients

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 (HR_{multivariable} = 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 (HR_{multivariable} = 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 (HR_{multivariable} = 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.     

Influence of length of time to diagnosis and treatment on the survival of children with acute lymphoblastic leukemia: A population-based study

The objectives were to describe times to diagnosis and initiation of treatment in pediatric ALL in Ontario from 1997 to 2007, and to measure their impact on OS and EFS. In 1000 children, the median times to diagnosis and treatment were both 1day (IQR = 1–2). Those who began treatment >3 days after diagnosis had inferior OS (AHR = 2.49; 95% CI = 1.40–4.43; p = 0.002), and inferior EFS (AHR = 1.73; 95% CI = 1.01–2.96; p = 0.047) compared to those who began treatment ≤3 days after diagnosis. There was no statistically significant relationship between time to diagnosis and survival. Longer time to treatment was associated with worse survival in pediatric ALL; reasons for this relationship may be multi-factorial.     

CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection

Inactivations of DNA repair genes, O(6)-methylguanine-DNA methyltransferase (MGMT) and hMLH1, by promoter hypermethylation have been reported in several types of primary human neoplasia. This epigenetic inactivation mechanism remains elusive in hepatocellular carcinoma (HCC). To investigate the relation between the expression of MGMT and hMLH1 and the CpG methylation within their promoters in HCCs with or without hepatitis viral infection, we performed immunohistochemistry and urea/bisulphite sequencing on 46 HCCs, corresponding noncancerous tissues, and 20 normal liver tissues. MGMT- and hMLH1-negative HCCs were 60.9% (28 out of 46) and 21.8% (10 out of 46), respectively. HCCs lacking both proteins were 10.9% (five out of 46). The frequency and extent of CpG methylation in the MGMT promoter increased along with hepatitis viral infection and pathological progression. MGMT-negative tumours showed very frequent and widespread methylation in the promoter compared with MGMT-positive tumours. Half of the hMLH1-negative HCCs showed promoter hypermethylation. These data suggested that MGMT gene silencing in a subset of HCCs was likely caused by epigenetic alteration, such as promoter hypermethylation, and that the promoter hypermethylation silenced the hMLH1 gene in half of the hMLH1-negative tumours. A correlation between the promoter methylation status and viral infection, although it was weak, intimated that hepatitis viral infections could play a role in the CpG methylation of the MGMT promoter.     

Polymorphisms in DNA repair and apoptosis-related genes and clinical outcomes of patients with non-small cell lung cancer treated with first-line paclitaxel-cisplatin chemotherapy

This study was conducted to analyze a comprehensive panel of single nucleotide polymorphisms (SNPs) in genes in DNA repair and apoptosis pathways and determine the relationship between polymorphisms and treatment outcomes of patients with non-small cell lung cancer (NSCLC) treated with first-line paclitaxel-cisplatin chemotherapy. Three hundred eighty two patients with NSCLC were enrolled. Seventy-four SNPs in 48 genes (42 SNPs in 27 DNA repair pathway genes and 32 SNPs in 21 apoptotic pathway genes) were genotyped and their associations with chemotherapy response and overall survival (OS) were analyzed. Among SNPs in DNA repair genes, BRCA1 rs799917 was significantly associated with both chemotherapy response and OS. XRCC1 rs25487 exhibited a significant association with chemotherapy response and ERCC2 rs1052555 with OS. Four SNPs in apoptotic genes (TNFRSF1B rs1061624, BCL2 rs2279115, BIRC5 rs9904341, and CASP8 rs3769818) were significantly associated with OS, but not with response to chemotherapy. When the six SNPs which were associated with OS in individual analysis were combined, OS decreased as the number of bad genotypes increased (P_trend = 2 × 10⁻⁶). Patients with 3, and 4–6 bad genotypes had significantly worse OS compared with those carrying 0–2 bad genotypes (adjusted hazard ratio [aHR] = 1.54, 95% CI = 1.14–2.08, P = 0.005; aHR = 2.10, 95% CI = 1.55–2.85, P = 2 × 10⁻⁶, respectively). In conclusion, these findings suggest that the six SNPs identified, particularly their combined genotypes, could be used as biomarkers predicting chemotherapy response and survival of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy.     

Prognostic value of SUVmax measurements obtained by FDG-PET in patients with non-small cell lung cancer receiving chemotherapy

[¹⁸F]Fluorodeoxyglucose (FDG) uptake has been shown to correlate well with tumor proliferation rates. In patients with non-small cell lung cancer (NSCLC) receiving chemotherapy, we analyzed the relationships between the maximum standardized uptake value (SUVmax) obtained by FDG positron emission tomography (FDG-PET) and other clinical factors, and examined whether or not SUVmax could predict progression-free survival (PFS) and/or overall survival (OS). This retrospective study involved 62 consecutive NSCLC patients (35 male and 27 female: median age, 65 years). All patients underwent FDG-PET examination before treatment. As the first-line treatment, the patients received chemotherapy with (n = 15) or without (n = 47) radiotherapy. Survival curves were obtained by the Kaplan-Meier method, and differences in survival between subgroups were analyzed by the log-rank test and the Cox proportional hazards model. Significant correlations were observed between SUVmax and gender (P = 0.006), histology (P < 0.001), smoking status (P = 0.049), stage (P = 0.015), and treatment modality (P = 0.008), but not other factors, including age (P = 0.402) and performance status (P = 0.421). The median SUVmax was 5.1 (25-75th percentile: 3.45-7.0) in patients with adenocarcinoma and 8.3 (25-75th percentile: 6.9-9.9) in those with other types of NSCLC. Adenocarcinomas showed significantly lower SUVmax than the other tumor types (P < 0.001). Cox analysis adjusting for possible confounding factors, including gender, smoking status, histology and stage, demonstrated that the hazard ratios increased as the SUVmax increased in terms of both PFS (P = 0.008) and OS (P = 0.045), indicating that SUVmax predicts outcome independently of other clinical factors, such as histology and stage. Our findings indicate that FDG-PET examination can provide information useful for prognostication in NSCLC.     

The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas

Our study was designed to clarify the significance of silencing the E-cadherin gene, which is located on 16q22.1, due to CpG methylation during hepatocarcinogenesis. The CpG methylation status of primary hepatocellular carcinomas (HCCs) and corresponding liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, were assessed by digesting DNA with methylation-sensitive and non-sensitive restriction enzymes. CpG methylation around the promoter region of the E-cadherin gene was detected in 46% of liver tissues showing chronic hepatitis or cirrhosis and 67% of HCCs examined. Immunohistochemical examination revealed reduced E-cadherin expression in 59% of HCCs examined. CpG methylation around the promoter region correlated significantly with reduced E-cadherin expression in HCCs (p < 0.05). CpG methylation around the promoter region, which increases during the progression from a precancerous condition to HCC, may participate in hepatocarcinogenesis through reduction of E-cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue morphology. Int. J. Cancer 71:355-359, 1997. © 1997 Wiley-Liss Inc.     

Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection

Background

Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen and may stimulate the proliferation and invasiveness of human hepatocellular carcinoma (HCC) cells through the c-met receptor. This study evaluates the significance of serum HGF levels in patients undergoing HCC resection.

Study design

The peripheral and portal sera and HCC and non-tumorous tissues of 40 HCC patients, with tumor TNM stage I (n = 12), II (n = 17), and III (n = 11) diseases, who underwent hepatic resection were prospectively collected. Serum HGF levels were determined by enzyme-linked immunosorbent assay. The c-met protein expressions were examined by immunohistochemistry. Median follow-up time was 69 months.

Results

The prehepatectomy portal HGF levels (median, 622 pg/mL) were significantly higher than peripheral HGF levels (564 pg/mL) (P = 0.026). The posthepatectomy portal HGF levels (699 pg/mL) were significantly higher than prehepatectomy portal HGF levels (P < 0.001). C-met expression was detected in 87.5% HCC and in 85.0% non-tumorous liver tissues. By Cox multivariate analysis, posthepatectomy portal HGF level >699 pg/mL (P < 0.001), multiple tumors (P = 0.042), and TNM stages II (P = 0.019) and III (P = 0.009) were independent factors related with survival. Patients with a posthepatectomy portal HCG level >699 pg/mL and with a positive c-met expression in HCC tissue have the worst survival.

Conclusions

In HCC patients, high peripheral and portal HGF serum levels related with poor prognosis after hepatic resection. Hepatocyte growth factor and c-met receptor can be targets of future HCC postoperative treatment.     

CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma.

PURPOSE: CpG island methylator phenotype (CIMP) involves hypermethylation targeted toward the promoters of multiple genes. To gain insight into the role of epigenetic aberration of tumor-related genes in hepatocarcinogenesis, we determined a hypermethylation profile in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: We examined the promoter methylation status of nine genes in 50 HCCs, 50 paired nontumor tissues, and 6 normal liver tissues by methylation-specific PCR. CIMP+ was defined as having five genes that are concordantly methylated. RESULTS: The frequency of promoter methylation of nine genes in 50 HCCs varied from 10% in P53 to 94% in c-Myc. The methylation status of P14, P15, P16, ER, RASSF1A, WT1, and c-Myc was significantly correlated with HCC and nontumor tissues (P<0.05). Hypermethylation of one or more genes was found in 96% of HCC. CIMP was more frequent in HCC than in nontumor tissues (70% and 12%, P<0.001). There is a significant association between CIMP and methylation of P14, P15, P16, ER, RSAAF1A, and WT1 (P<0.05) and serum alpha-fetoprotein (AFP) level (P=0.017). CIMP+ was more frequent in HCC with AFP>or=30 microg/L than those with AFP<30 microg/L (P=0.005). In addition, the promoter hypermethylation of P15 and P16 was associated with elevated serum AFP levels in 35 HCC samples with CIMP+ (P<0.05). CONCLUSIONS: Positive correlation of CIMP and AFP levels in HCC suggests that CIMP can serve as a molecular marker of late-stage HCC development.     

Efficacy of epidermal growth factor receptor inhibitors versus chemotherapy as second-line treatment in advanced non-small-cell lung cancer with wild-type EGFR: A meta-analysis of randomized controlled clinical trials

Background

EGFR mutation status is closely related to the efficacy of EGFR-TKIs in advanced non-small cell lung cancer (NSCLC). EGFR-TKIs have become the standard first-line treatment for advanced EGFR-mutation NSCLC, while for EGFR wild-type tumors, the preferred first-line treatment is chemotherapy. However, the efficacy of EGFR-TKIs as second-line treatment in EGFR wild-type NSCLC remains controversial. We sought to evaluate the effectiveness of EGFR-TKI as second-line treatment in EGFR wild-type NSCLC.

Methods

Randomized controlled trials that compared EGFR-TKIs with chemotherapy in previously treated advanced NSCLC with wild-type EGFR were included. We performed a meta-analysis to evaluate the effectiveness of EGFR-TKIs compared with standard chemotherapy. The endpoints were progression-free survival (PFS), overall survival (OS), and objective response rate (ORR).

Results

Six randomized controlled trials with a total of 990 patients with wild-type EGFR were included: 499 in the EGFR-TKIs group and 491 in the chemotherapy group. The results indicated that in the second-line treatment of EGFR wild-type advanced NSCLC, PFS was significantly inferior in the EGFR-TKIs group versus the chemotherapy group (HR = 1.37, 95% CI = 1.20–1.56, P < 0.00001). However, this significant difference did not translate into OS (HR = 1.02, 95% CI = 0.87–1.20, P = 0.81). ORR tended to favor chemotherapy but there was no significant difference compared with EGFR-TKI (RR = 1.77, 95% CI = 0.90–3.50, P = 0.10).

Conclusions

Chemotherapy improves PFS significantly but not OS, compared with EGFR-TKIs as a second-line treatment in advanced NSCLC with wild-type EGFR. Whether EGFR-TKIs should be used in EGFR wild-type patients should be considered carefully.