Integrating TYMS, KRAS and BRAF testing in patients with metastatic colorectal cancer

Abstract

Aim: To investigate the impact of thymidylate synthase (TYMS), KRAS and BRAF in the survival of metastatic colorectal cancer (mCRC) patients treated with chemotherapy.

Methods: Clinical data were collected retrospectively from records of consecutive patients with mCRC treated with fluoropyrimidine-based chemotherapy from 1/2005 to 1/2007. Formalin-fixed paraffin-embedded tissues were retrieved for analysis. TYMS genotypes were identified with restriction fragment analysis PCR, while KRAS and BRAF mutation status was evaluated using real-time PCR assays. TYMS gene polymorphisms of each of the 3' untranslated region (UTR) and 5'UTR were classified into three groups according to the probability they have for high, medium and low TYMS expression (and similar levels of risk) based on evidence from previous studies. Univariate and multivariate survival analyses were performed.

Results: The analysis recovered 89 patients with mCRC (46.1% de novo metastatic disease and 53.9% relapsed). Of these, 46 patients (51.7%) had colon cancer and 43 (48.3%) rectal cancer as primary. All patients were treated with fluoropyrimidine-based chemotherapy (5FU or capecitabine) as single-agent or in combination with irinotecan or/and oxaliplatin or/and bevacizumab. With a median follow-up time of 14.8 mo (range 0-119.8), 85 patients (95.5%) experienced disease progression, and 63 deaths (70.8%) were recorded. The 3-year and 5-year OS rate was 25.4% and 7.7% while the 3-year progression-free survival rate was 7.1%. Multivariate analysis of TYMS polymorphisms, KRAS and BRAF with clinicopathological parameters indicated that TYMS 3'UTR polymorphisms are associated with risk for disease progression and death (P < 0.05 and P < 0.03 respectively). When compared to tumors without any del allele (genotypes ins/ins and ins/loss of heterozygosity (LOH) linked with high TYMS expression) tumors with del/del genotype (low expression group) and tumors with ins/del or del/LOH (intermediate expression group) have lower risk for disease progression (HR = 0.432, 95%CI: 0.198-0.946, P < 0.04 and HR = 0.513, 95%CI: 0.287-0.919, P < 0.03 respectively) and death (HR = 0.366, 95%CI: 0.162-0.827, P < 0.02 and HR = 0.559, 95%CI: 0.309-1.113, P < 0.06 respectively). Additionally, KRAS mutation was associated independently with the risk of disease progression (HR = 1.600, 95%CI: 1.011-2.531, P < 0.05). The addition of irinotecan in 1^st line chemotherapy was associated independently with lower risk for disease progression and death (HR = 0.600, 95%CI: 0.372-0.969, P < 0.04 and HR = 0.352, 95%CI: 0.164-0.757, P < 0.01 respectively).

Conclusion: The TYMS genotypes ins/ins and ins/LOH associate with worst prognosis in mCRC patients under fluoropyrimidine-based chemotherapy. Large prospective studies are needed for validation of our findings.

Keywords: BRAF; Chemotherapy; KRAS; Loss of heterozygosity; Polymorphisms; Survival; TYMS; Thymidylate synthase; mCRC.

Figure 1

Agarose gel with PCR products for the thymidylate synthase 5’untranslated region 28 bp insertion. A DNA ladder of repeated 50 bp fragments was used (M 50 bp). All potential genotypes (2R 241 bp and 3R 242 bp) are depicted as well, as a sample with LOH for 3R (Lane 3). bp: Base pair; LOH: Loss of heterozygosity; UTR: Untranslated region.

Figure 2

Polyacrylamide gel resolution showing the 12G>C substitution in the 5’untranslated region after digestion. Expected bands are 12, 44, 45 and 47 bp for all genotypes. Digestion of a sample with 2R or 3R12G genotype results in production of two bands of 66 and 28 bp, while in 3R12C genotypes those two fragments are left undigested in a single 94 bp fragment. bp: Base pair; LOH: Loss of heterozygosity; UTR: Untranslated region.

Figure 3

Polyacrylamide gel resolution showing the 3’untranslated region products. Expected bands are 104 and 110 bp. In heterozygotes, a second band of approximately 200 bp was observed due to heteroduplex mismatches. bp: Base pair; UTR: Untranslated region.

Figure 4

Kaplan-Meier survival curve. A: Kaplan-Meier curve for PFS according to TYMS 3’UTR groups; B: Kaplan-Meier curve for OS according to TYMS 3’UTR groups. Comparisons were made using long-rank tests. OS: Overall survival; PFS: Progression-free survival; UTR: Untranslated region.