Beyond the Abstract - Analysis of hOGG1 genotype as a prognostic marker for muscle invasive bladder cancer: A novel approach using peptide nucleic acid-mediated, real-time PCR clamping, by Wun-Jae Kim, MD, PhD

BERKELEY, CA (UroToday.com) - DNA lesions that contain the mutagenic base 8-oxoguanine are repaired by base excision repair.

[1] Human 8-oxoguanine DNA glycosylase 1 (hOGG1) encodes a DNA glycosylase that catalyzes the excision of 8-oxoguanine from oxidatively damaged DNA.[2,3] A Ser326Cys polymorphism of hOGG1 has been identified in bladder, lung and esophageal cancers.[4-6] We carried out a long-term follow-up study of patients with bladder cancer (BC) to assess whether hOOG1 genotype influenced the clinical course of disease. Data were drawn from a previous study population as well as new cases, and the genotype was analyzed using a novel PNA-mediated, real-time PCR clamping method.

 

A total of 377 patients with transitional cell carcinoma of the urinary bladder were recruited from Chungbuk National University Hospital. To delineate a more homogenous study population, patients with concomitant carcinoma in situ (CIS), short-term follow-up periods (less than 6 months) and incomplete data were excluded. Within the study population, 264 patients had primary NMIBC and 113 had MIBC.

PNA-mediated real-time PCR clamping method

All PNA oligomers were synthesized and purified using HPLC (Panagene, Daejeon, Korea). Representative melting curves were obtained with clamped assay. Threshold cycle (Ct) values of all amplified products were obtained, and nucleotide (Nt) sequences were determined for samples with △Ct values that were more than 2 as compared to the control (no PNA probe) Ct value. A PNA probe that is complementary to the wild-type sequence will hybridize specifically with the wild-type sequence and block amplification, while amplification of the variant sequence, which is an imperfect match to the PNA probe, will proceed. A single base mismatch is sufficient to cause differential amplification of mutant and wild-type sequences. Thus, the sample was designated as a Cys genotype if its Ct value was higher than the Ser control sample when using the Cys PNA probe, and a Ser genotype if the Ct value was higher than the Cys control sample when using the Ser PNA probe. [Fig. 1. Representative melting curves obtained with clamped assay. (A) Ser326Ser type; (B) Cys326Cys type; (C) Ser325Cys type.]

Relationship between the genetic polymorphism of hOGG1, and clinicopathological parameters

There was a notable correlation between the hOGG1 genotype and tumor grade (P = 0.023), and tumor size was significantly associated with the hOGG1 genotype (P = 0.033). In cases of MIBC, the hOGG1 genotype was not related to tumor stage, but was significantly associated with progression and cancer-specific survival (P = 0.004 and 0.030, respectively)

Genotype of hOGG1 and prognosis in MIBC

Kaplan-Meier estimates revealed a significant difference in time to progression between the hOGG1 genotypes (log-rank test, P = 0.006). When Ser326Ser and Cys326Cys polymorphisms were compared, Cys326Cys was associated with a greater survival benefit than Sys326Sys (log-rank test, P = 0.039). Multivariate Cox regression analysis revealed that the hOGG1 genotype is a strong predictor of progression only in cases of MIBC. Moreover, the Cys326Cys genotype, tumor stage and operation type were independent predictors of cancer-specific survival in MIBC.

Strong points of the this study

The results of the current study are strengthened by several aspects of its design and methodology. First, we analyzed a relatively large-scale population with long-term follow-up and performed definitive subgroup and survival analyses (i.e., Kaplan-Meier and Cox regression models) after stratifying cases as either NMIBC or MIBC. Recurrence for the NMIBC group was not associated with the hOGG1 genotype, which was in contrast to previous data.[4] This discrepancy may be due to limitations of the previous study, which was a cross-sectional study with a small sample size. Second, we used a novel PNA-mediated, real-time PCR clamping method to determine the genotype. Previous studies have used single-stranded conformational polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) analysis.[7,8] There are several advantages to the PNA method. Restriction enzymes used in RFLP are unstable and expensive, and the procedure is time consuming and involves multiple steps, including direct DNA sequencing and agarose gel electrophoresis. By comparison, the results derived by PNA-mediated, real-time PCR clamping are more accurate and the process is easier to carry out.

We have shown that the hOGG1 Cys326Cys polymorphism is associated with progression and cancer-specific survival in patients with MIBC. hOGG1 genetic polymorphisms represent a promising marker for assessing MIBC prognosis.



[Fig. 1. Representative melting curves obtained with clamped assay. (A) Ser326Ser type; (B) Cys326Cys type; (C) Ser325Cys type.]




References:

  1. Cunningham RP. DNA repair: how yeast repairs radical damage. Curr Biol 1996;6:1230-3.
  2. Lee YS, Lee HS, Park MK, et al. Identification of 8-hydroxyguanine glycosylase activity in mammalian tissues using 8-hydroxyguanine specific monoclonal antibody. Biochem Biophys Res Commun 1993;196:1545-51.
  3. Tchou J, Kasai H, Shibutani S, et al. 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. Proc Natl Acad Sci U S A 1991;88:4690-4.
  4. Kim EJ, Jeong P, Quan C, et al. Genotypes of TNF-alpha, VEGF, hOGG1, GSTM1, and GSTT1: useful determinants for clinical outcome of bladder cancer. Urology 2005;65:70-5.
  5. Sugimura H, Kohno T, Wakai K, et al. hOGG1 Ser326Cys polymorphism and lung cancer susceptibility. Cancer Epidemiol Biomarkers Prev 1999;8:669-74.
  6. Xing DY, Tan W, Song N, Lin DX. Ser326Cys polymorphism in hOGG1 gene and risk of esophageal cancer in a Chinese population. Int J Cancer 2001;95:140-3.
  7. Gangwar R, Ahirwar D, Mandhani A, Mittal RD. Do DNA repair genes OGG1, XRCC3 and XRCC7 have an impact on susceptibility to bladder cancer in the North Indian population? Mutat Res 2009;680:56-63.
  8. Arizono K, Osada Y, Kuroda Y. DNA repair gene hOGG1 codon 326 and XRCC1 codon 399 polymorphisms and bladder cancer risk in a Japanese population. Jpn J Clin Oncol 2008;38:186-91.

 




Written by:
Wun-Jae Kim, MD, PhD as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.

Analysis of hOGG1 genotype as a prognostic marker for muscle invasive bladder cancer: A novel approach using peptide nucleic acid-mediated, real-time PCR clamping - Abstract

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