Thymidylate synthase genotyping is more predictive for therapy response than immunohistochemistry in patients with colon cancer

Abstract

Thymidylate synthase (TS) is a potentially valuable marker for therapy response since it is the molecular target of 5-fluorouracil (5-FU). TS can be analyzed at the DNA (gene polymorphisms and amplification) and protein level (immunohistochemistry). This study investigated the predictive role of TS at the DNA and protein levels in patients with N 1 colon cancer (n 5 38). Tumor and normal tissues were genotyped using PCR for variable number of tandem repeats (VNTR), a single nucleotide polymorphism (SNP) in the 3R allele and a 6 bp deletion (1494del6) in the TS gene. Tumor tissues were additionally analyzed for loss of heterozygosity (VNTR polymorphism). A newly developed real time PCR assay was used to detect the presence of TS gene amplifications in tumor tissues. VNTR analysis in normal tissue was significantly associated with distant tumor recurrence (8% for 2R/2R vs. 52% for patients carrying a 3R allele, p 5 0.038) and cancer-specific survival (p 5 0.021). IHC was not found to be significantly associated with patients' outcome. No correlations between TS gene polymorphisms and IHC were found. However, TS gene amplification was correlated with a strong IHC staining intensity. In conclusion, this study indicates that DNA based analysis is more predictive for patientsÕ outcome than TS IHC. ' 2008 Wiley-Liss, Inc.Key words: thymidylate synthase; polymorphism; 5-fluorouracil; colon cancer; gene amplification A substantial number of patients with colon cancer who received adjuvant 5-fluorouracil (5-FU)-based therapy will not benefit from it. Therefore, predictive markers are needed in order to discriminate between responsive and nonresponsive patients. Thymidylate synthase (TS) is a central enzyme in DNA synthesis and is a potentially valuable marker since it is the molecular target of 5-FU. TS protein expression is affected by 3 different functional polymorphisms in the untranslated regions (UTRs) of the gene. Sensitivity to 5-FU based therapy might be largely influenced by the intracellular levels of the TS protein.TS protein levels can be studied directly by western blotting, enzyme activity assays, 1,2 ELISA 3,4 and immunohistochemistry (IHC). 5,6 The mainstream method is IHC because it is a relatively cheap, widely implemented technique that enables studying protein expression in situ. At the DNA level, TS protein expression is affected by different underlying functional polymorphisms as shown by several functional studies. [7][8][9][10][11][12] These polymorphisms are the following: a variable number of tandem repeats (VNTR) containing 2 (2R) or 3 (3R) repeats of 28 bp, 7 a single nucleotide polymorphism (SNP) of a G to C substitution in the 3R allele 9 in the 5 0 UTR and a 6 bp deletion at nucleotide 1494 in the 3 0 UTR (1494del6). 12 Recently, a SNP of a G to C substitution in the first repeat of the 2R allele has also been found (hereafter referred to as the 2RC allele). 13,14 TS mRNA with 3 repeats has greater translation efficiency than mRNA with 2 repeats. 7,11 Individuals with a 3R/3R genotype will,...

Predictors of survival and toxicity in patients on adjuvant therapy with 5-fluorouracil for colorectal cancer

“…However, our population size was comparable with that of other similar studies and genotype classifications reflected the best available information on gene biological activity. In this respect, literature data are inconclusive in that the same TS polymorphisms have been correlated with both favourable and unfavourable effects, and no correlation has even been reported (Dotor et al, 2006;Gusella and Padrini, 2007;Gosens et al, 2008;Fernandez-Contreras et al, 2009). Besides the methodological ).…”

TS gene polymorphisms are not good markers of response to 5-FU therapy in stage III colon cancer patients

“…3A and B). Although, in a previous publication we reported a predictive value for the VNTR [12], only a small number of patients were studied at that time and the apparently contradictory results could be explained by the difference in patient numbers between studies. In the present larger cohort of patients, we were not able to reproduce our previous results.…”

“…1), TS protein expression quantitation, TS gene amplification and loss of heterozygosity in predicting 5-FU therapy response [12]. From these results, it seemed that genotyping of the 5 -untraslated region polymorphism of the TS gene was more reliable for predicting response to therapy than protein expression, as determined by IHC and than genotyping the rest of polymorphisms in the 3 -UTR.…”

Comparative analysis of thymidylate synthase at the protein, mRNA, and DNA levels as prognostic markers in colorectal adenocarcinoma

“…Finally, TS mRNA transcription and translation could be influenced by the TS gene promoter enhancer region [16,17]. Recently TS genetic polymorphism in 5 0 -promoter enhancer region (5 0 -TSER) and 3 0 -untranslated region (3 0 -TSUTR) in either tumor or normal tissues have been found to be independent prognostic indicators in colorectal cancer patients [17][18][19][20][21][22][23][24].…”