Expression of Thymidylate Synthase in Primary Colorectal Adenocarcinoma

Abstract

Thymidylate synthase (TS) is a key regulatory enzyme in the cellular pathway of de novo pyrimidine synthesis and a target enzyme for 5-fluorouracil (5-FU). Most clinical studies have shown that high levels of TS in tumors are associated with decreased sensitivity to 5-FU treatment. In this study TS expression was assessed at DNA, RNA, and protein levels. The study included 69 tumors from patients with primary colorectal adenocarcinoma. At the DNA level TS enhancer polymorphism was measured on whole blood by PCR. At the RNA level TS mRNA expression was measured on formalin-fixed paraffin-embedded tumor tissue and on fresh-frozen tumor tissue by real-time RT-PCR. Protein expression was assessed by IHC. Correlation was found between TS mRNA expression in fresh-frozen tumor tissue and formalin-fixed paraffin-embedded tissue (R=0.71). TS enhancer 3/3 had significantly higher protein levels as assessed by IHC than the TS enhancer 2/2 (P=0.02), although there was no statistically significant correlation between TS enhancer polymorphism and TS mRNA expression. An interesting observation not previously reported is that the predominant IHC reaction pattern in tumors from patients with the TS enhancer genotype 3/3 is different in tumors from patients with genotypes 2/2 and 2/3. The results indicate that clinical studies of the significance of TS with regard to 5-FU-based chemotherapy should be based on assessment of TS activity at DNA, RNA, and protein levels.

Correlation between Thymidylate Synthase Gene Variants, RNA and Protein Levels in Primary Colorectal Adenocarcinomas

“…In accordance with previously published results, 25,32,47 significantly lower protein levels were observed in tumours from patients with TS 2R/2R compared with tumours from patients with TS 3R/3R. No statistically significant difference was observed between tumours from patients with TS 2R/2R and 2R/3R.…”

“…Reactive lymphocytes served as internal controls. Staining intensity and the percentage of stained tumour cells were considered in the staining assessment as described by Lindebjerg et al 47 Intensity was graded on a semiquantitative scale from 0 to 3 (0, no staining; 1, weak staining intensity; 2, strong staining intensity; and 3, very strong staining intensity). The extent of staining was graded on a scale from 0 to 4 (0, no staining; 1, staining of 0% -10% of carcinoma cells; 2, staining of 10% -25% of carcinoma cells; 3, staining of 25% -50% of carcinoma cells; and 4, staining of > 50% of carcinoma cells).…”

Thymidylate synthase expression and p21WAF1/p53 phenotype of colon cancers identify patients who may benefit from 5-fluorouracil based therapy

“…With our cut-off level (histoscore p53 > 7), high p53 (p53+) nuclear expression was seen in 60.3 % of the samples, which is within the range found in the literature. A number of different scoring methods and threshold levels have been reported for the assessment of immunohistochemical TS expression in colorectal cancer: (1) The intensity of TS staining of tumors was arbitrarily graded from 0 to 3 (or 0 to 4) and dichotomized (grades 0 to 1 defined as low level, and 3 to 4 as high level of TS expression) – a review of 13 studies [22] and [24, 26, 40, 41], (2) TS expression was dichotomized using arbitrary thresholds of 10 %, 15 %, 20 % [22] or ≥30 % [42] of stained cells per field, (3) Histoscores were calculated [18, 43]. In these reports the proportion of cases expressing high levels of TS ranged from 14 % to 80 %.…”

Epidermal growth factor (EGF) A61G polymorphism and EGF gene expression in normal colon tissue from patients with colorectal cancer

“…The concentration and purity of the samples was determined by optical density at wavelengths 260 nm and 280 nm (The isolated RNA was quantitated by Spectophotometry) (Eppendorf, Hamburg, Germany). cDNA synthesis was performed using M-MLV RT (Invitrogene) as previously described [17], and subsequently, the mRNA was quantified by Real-time PCR (ABI PRISM 7900 HT FAST Sequence Detection System). The PCR amplification was conducted using the TaqMan † Fast Universal PCR Master mix (2 )) (Applied Biosytems) according to manufactures instructions.…”