Ping Chang

Oxidative DNA damage and 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase in human breast cancer.

To test the hypothesis that oxidative stress is involved in breast cancer, we compared the levels of 8‐hydroxy‐2‐deoxyguanosine (8‐oxo‐dG), an oxidized DNA base common in cells undergoing oxidative stress, in normal breast tissues from women with or without breast cancer. We found that breast cancer patients (N = 76) had a significantly higher level of 8‐oxo‐dG than control subjects (N = 49). The mean ( ± SD) values of 8‐oxo‐dG/105 dG, as measured by high‐performance liquid chromatography electrochemical detection, were 10.7 ± 15.5 and 6.3 ± 6.8 for cases and controls, respectively (P = 0.035). This difference also was found by immunohistochemistry with double‐fluorescence labeling and laser‐scanning cytometry. The average ratios (×106) of the signal intensity of antibody staining to that of DNA content were 3.9 ± 7.2 and 1.1 ± 1.4 for cases (N = 57) and controls (N = 34), respectively (P =  0.008). There was no correlation between the ages of the study subjects and the levels of 8‐oxo‐dG. Cases also had a significantly higher level of 8‐hydroxy‐2‐deoxyguanosine DNA glycosylase/apurinic lyase (hOGG1) protein expression in normal breast tissues than controls (P = 0.008). There was no significant correlation between hOGG1 expression and 8‐oxo‐dG. Polymorphism of the hOGG1 gene was very rare in this study population. The previously reported exon 1 polymorphism and two novel mutations of the hOGG1 gene were found in three of 168 cases and two of 55 controls. In conclusion, normal breast tissues from cancer patients had a significantly higher level of oxidative DNA damage. The elevated level of 8‐oxo‐dG in cancer patients was not related to age or to deficiency of the hOGG1 repair gene.

DNA Repair Gene Polymorphisms and Risk of Pancreatic Cancer

Purpose: The current research was undertaken to examine the association between genetic variations in DNA repair and pancreatic cancer risk. Experimental Design: We analyzed 9 single nucleotide polymorphisms of 7 DNA repair genes (LIG3, LIG4, OGG1, ATM, POLB, RAD54L, and RECQL) in 734 patients with pancreatic adenocarcinoma and 780 healthy controls using the Taqman method. Information on cigarette smoking, alcohol consumption, medical history, and other risk factors was collected by personal interview. Results: The homozygous mutant genotype of LIG3 G-39A [odds ratio (OR), 0.23; 95% confidence interval (CI), 0.06-0.82; P = 0.027] and ATM D1853N (OR, 2.55; 95% CI, 1.08-6.00; P = 0.032) was significantly associated with altered risk for pancreatic cancer. A statistically significant interaction of ATM D1853N and LIG4 C54T genotype with diabetes on the risk of pancreatic cancer was also detected. Compared with nondiabetics with the ATM D1853N GG genotype, nondiabetics with the GA/AA, diabetics with the GG, and diabetics with the GA/AA genotypes, respectively, had ORs (95% CI) of 0.96 (0.74-1.24), 1.32 (0.89-1.95), and 3.23 (1.47-7.12; Pinteraction = 0.032, likelihood ratio test). The OR (95% CI) was 0.91 (0.71-1.17), 1.11 (0.73-1.69), and 2.44 (1.34-4.46) for nondiabetics carrying the LIG4 CT/TT genotype, diabetics with the CC genotype, and diabetics carrying the CT/TT genotype, respectively, compared with nondiabetics carrying the CC genotype (Pinteraction = 0.02). Conclusions: These observations suggest that genetic variations in DNA repair may act alone or in concert with other risk factors on modifying a patients risk for pancreatic cancer.

In vitro BPDE‐induced DNA adducts in peripheral lymphocytes as a risk factor for squamous cell carcinoma of the head and neck

The level of DNA adducts under the same conditions of carcinogen exposure and cell proliferation reflects an integrated measure of carcinogen metabolism and DNA repair. Therefore, such DNA adduct levels have the potential to be a biomarker for susceptibility to chemical carcinogenesis. In a pilot study of 91 patients with squamous cell carcinomas of the head and neck and 115 controls who were frequency matched by age, sex, ethnicity, and smoking status, we applied a newly developed in vitro assay of benzo[a]pyrene diol epoxide (BPDE)‐induced DNA adducts in short‐term peripheral lymphocytes cultures. Levels of BPDE‐DNA adducts were found to be significantly higher in cases than in controls (mean ± SD, 76.8 ± 77.4/107 and 47.1 ± 48.0/107 nucleotides, respectively; p < 0.001). Using the median level of control values (35/107) as the cut‐off point, about 66% of cases were distributed above this level. Logistic regression analysis revealed that the level of BPDE‐induced DNA adducts was an independent risk factor (odds ratio = 2.22; 95% confidence interval = 1.22–4.04) after adjustment for age, sex and smoking status. Further stratified analyses showed that levels of the induced adducts between cases and controls were significantly higher in both age groups, that is, younger or older than 60, as well as in both men and women. Smoking had a positive effect on the induced adducts. The highest level of induced adducts was seen in current smokers, then former smokers and non‐smokers. There was a statistically significant dose–response relationship between the quartile levels of BPDE‐induced DNA adducts and the risk of head and neck cancer (trend test, p = 0.003). Despite the relatively small sample size, the association of BPDE‐induced DNA adducts and cancer risk suggests that this assay has the potential to complement with other biomarkers in identifying individuals at increased risk of developing tobacco‐related cancers.

Single-Nucleotide Polymorphisms of DNA Damage Response Genes Are Associated with Overall Survival in Patients with Pancreatic Cancer

Purpose: The goals of this study were to determine if single-nucleotide polymorphisms in DNA damage repair genes and cell cycle regulating genes affect clinical response to combined gemcitabine radiation therapy and the overall survival (OS) of patients with pancreatic cancer. Experimental Design: We evaluated six single-nucleotide polymorphisms of the ATM, ATM and Rad3-related (ATR), CHEK1, and CHEK2 genes in 119 patients with potentially resectable pancreatic cancer who were enrolled in clinical trials at The University of Texas M. D. Anderson Cancer Center from February 1999 to January 2006, with follow-up until February 2007. Patients received neoadjuvant concurrent gemcitabine and radiation therapy with or without gemcitabine-cisplatin induction therapy. Genotypes were determined and tested for associations with OS by Kaplan-Meier estimation, the log-rank test, and Cox regression analysis. P values of ≤0.05 were considered significant. Results: The ATM G60A and CHEK1 G35A genotypes were significant (P < 0.05), and the ATR C340T genotype borderline significantly (P = 0.079) associated with OS. The hazard ratio of CHEK1 35AA was 2.01 (95% confidence interval, 1.20-3.37; P = 0.007) compared with CHEK1 35GG/GA with adjustments for race, sex, diabetes status, CA19-9 level, and success of tumor resection. A significant combined genotype effect was observed between ATM 60GA/GG, ATR 340CT/CC, and CHEK1 35AA with median OS times of 31.0, 16.2, and 10.5 months for patients carrying ≤1, 2, and 3 deleterious alleles, respectively (P = 0.004). Conclusions: These observations suggest that polymorphic variations of DNA damage response genes affect clinical response to gemcitabine radiation therapy and OS of patients with resectable pancreatic cancer.

Genetic Variation in the PNPLA3 Gene and Hepatocellular Carcinoma in USA: Risk and Prognosis Prediction

Nonalcoholic fatty liver disease (NAFLD) is an emerging epidemic with high prevalence in Western countries. Genome‐wide association studies had reported that a variation in the patatin‐like phospholipase domain containing 3 (PNPLA3) gene is associated with high susceptibility to NAFLD. However, the relationship between this variation and hepatocellular carcinoma (HCC) has not been well established. We investigated the impact of PNPLA3 genetic variation (rs738409: C>G) on HCC risk and prognosis in the United States by conducting a case−control study that included 257 newly diagnosed and pathologically confirmed Caucasian patients with HCC (cases) and 494 healthy controls. Multivariate logistics and Cox regression models were used to control for the confounding effects of HCC risk and prognostic factors. We observed higher risk of HCC for subjects with a homozygous GG genotype than for those with CC or CG genotypes, the adjusted odds ratio (OR) was 3.21 (95% confidence interval [CI], 1.68–6.41). We observed risk modification among individuals with diabetes mellitus (OR = 19.11; 95% CI, 5.13–71.20). The PNPLA3 GG genotype was significantly associated with underlying cirrhosis in HCC patients (OR = 2.48; 95% CI, 1.05–5.87). Moreover, GG allele represents an independent risk factor for death. The adjusted hazard ratio of the GG genotype was 2.11 (95% CI, 1.26–3.52) compared with CC and CG genotypes. PNPLA3 genetic variation (rs738409: C>G) may determine individual susceptibility to HCC development and poor prognosis. Further experimental investigations are necessary for thorough assessment of the hepatocarcinogenic role of PNPLA3.

Association of CHFR Promoter Methylation with Disease Recurrence in Locally Advanced Colon Cancer

Purpose: This study was designed to determine whether DNA methylation biomarkers are associated with recurrence and survival in colon cancer patients. Experimental Design: A retrospective analysis of 82 patients who received curative surgical resection for American Joint Committee on Cancer (AJCC) high-risk stage II or III colon cancer (1999–2007) was conducted. DNA methylation status was quantitatively evaluated by the pyrosequencing method. We preselected three tumor suppressor genes and one locus of interest; CHFR, ID4, RECK, and MINT1. Mean methylation levels of multiple CpG sites in the promoter regions were used for analysis; 15% or more was defined as methylation positive. The association of recurrence-free survival (RFS) and overall survival (OS) with methylation status was analyzed by the log-rank test, Kaplan–Meier method, and Cox proportional hazards model. Results: Methylation levels of ID4, MINT1, and RECK did not correlate with RFS or OS. CHFR was methylation positive in 63% patients. When methylation status was dichotomized (negative or low: <30%, high: ≥30%), patients with CHFR methylation-high (44%) had worse RFS (P = 0.006) and reduced OS (P = 0.069). When stratified by stage, CHFR methylation-high was associated with reduced RFS (P = 0.004) and OS (P = 0.010) in stage III patients. CHFR methylation-high was commonly associated with N2 disease (P = 0.04) and proximal tumors (P = 0.002). Multivariate analysis indicated AJCC T4 disease and CHFR methylation-high (P = 0.001 and P = 0.015, respectively) were independent predictors for recurrence. Conclusions: The extent of CHFR promoter methylation correlates with RFS, indicating it is a promising epigenetic marker for recurrence. Clin Cancer Res; 17(13); 4531–40. ©2011 AACR.

In vitro Benzo[a]pyrene Diol Epoxide–Induced DNA Adducts and Risk of Squamous Cell Carcinoma of Head and Neck

In this large confirmatory study of 803 patients with squamous cell carcinoma of head and neck (SCCHN) and 839 controls frequency matched by age, sex, and ethnicity, we further examined potential predictors of benzo[a]pyrene diol epoxide (BPDE)-induced adduct levels and their associations with SCCHN risk. BPDE-DNA adduct levels were determined by the (32)P-postlabeling method in peripheral lymphocytes after in vitro challenged by BPDE. We also genotyped for GSTM1 null, GSTT1 null, GSTP1 Ile(105)Val, and GSTP1 Ala(114)Val. Potential predictors of BPDE-DNA adducts were evaluated by stratification and multivariate linear regression analyses and the association between adduct levels and SCCHN risk by multivariate logistic regression analyses. We found that mean BPDE-DNA adduct levels (the relative adduct labeling x 10(7) +/- SD) were significantly higher in cases (77.6 +/- 111.8) than in controls (57.3 +/- 98.3; P < 0.001). Using the median control value (29.22) as a cutoff, 63% of the cases were distributed above this level (adjusted odds ratio, 1.71; 95% confidence interval, 1.39-2.10). A significant dose-response relationship was observed between adduct quartiles and SCCHN risk (P(trend) < 0.001). Multivariate linear regression analysis revealed that ethnicity and smoking were significant predictors of BPDE-DNA adduct levels in controls. In conclusion, we confirmed the previously reported association between in vitro BPDE-induced DNA adduct levels and SCCHN risk, and the assay may help identify individuals at high risk of developing smoking-related cancers.

Biomarkers of TGF-β signaling pathway and prognosis of pancreatic cancer

Background Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer. Method We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models. Results The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001). Conclusion Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.

Insulin-like growth factor axis gene polymorphisms modify risk of pancreatic cancer.

OBJECTIVE Insulin-like growth factor (IGF)-axis genes plays a critical role in cancer development and progression via their impact on the RAS/MAPK/ERK and PI3K/AKT/mTOR signaling pathways. We hypothesized that IGF-axis genetic variants modify individual susceptibility to pancreatic cancer. METHODS We retrospectively genotyped 41 single-nucleotide polymorphisms of 10 IGF-axis genes (IGF1, IGF2, IGF1R, IGF2R, IGFBP1, IGFBP3, IGFBP5, IRS1, IRS2, and IRS4) in 706 pancreatic cancer patients and 706 cancer-free controls using Sequenom and TaqMan technology. The association between genotype and pancreatic cancer risk was evaluated using multivariate logistic regression. A P value ≤.007 at a false discovery rate of 10% was set as the significance level. RESULTS We observed that the IGF1 *10212C>A and Ex4+2776G>A and IGF1R IVS2-70184A>G and IVS2+46329T>C variant genotypes were significantly associated with decreased pancreatic cancer risk (odds ratio [OR] range, 0.60-0.75) and that IGFBP1 Ex4+111A>G (I253M) was significantly associated with increased pancreatic cancer risk (OR=1.46) after adjusted for other risk factors and multiple comparisons (P≤.007). IGF2R and IGFBP3 variant haplotypes were associated with increased and decreased pancreatic cancer risk, respectively (P<.001). We also observed a weak interaction of the IGF1R IVS2+46329T>C and IGF2R Ex45+11C>T (L2222L) genotypes with diabetes (P(interaction)=.05) and interaction of IGF2R and IRS1 genotypes with alcohol consumption (P(interaction)=.03 and .019, respectively) on increased pancreatic cancer risk. CONCLUSION These findings support our hypothesis that polymorphic variants of IGF-axis genes act alone or jointly with other risk factors to affect susceptibility to pancreatic cancer.

Micronuclei levels in peripheral blood lymphocytes as a potential biomarker for pancreatic cancer risk.

To find biomarkers for risk prediction of pancreatic cancer (PC), we evaluated the frequency of micronuclei (MN) in peripheral lymphocytes of 346 patients with PC and 449 healthy controls. The levels of baseline MN (mean ± standard error of micronucleated cells per 1000 binucleated cells) were significantly higher in patients (15.3 ± 0.3) than those in controls [9.7 ± 0.5; adjusted for body mass index (BMI), P < 0.001]. Using the median levels found in controls as the cut point, 78.9% of patients and 43.7% of controls had a higher frequency of MN. Logistic regression analysis with adjustment for known risk factors for PC showed that having a higher level of MN was significantly associated with increased risk of PC [odds ratio (OR): 8.32, 95% confidence interval (CI): 5.06-13.67, P < 0.001]; and the risk was much higher in men than in women [OR (95% CI): 14.19 (7.09-28.40) versus 4.19 (1.90-9.27)]. The level of MN was not associated with disease stage or resection status but was related to smoking status in men and to BMI in women among patients. The level of MN was higher in smokers (14.5 ± 0.6) than in nonsmokers (12.1 ± 0.6; P = 0.023) and in obese (25.3 ± 2.8) versus normal weight individuals (17.7 ± 0.8; P = 0.024). These data showed that elevated level of MN in peripheral lymphocytes was associated with increased risk of PC.