IntegratingTYMS,KRASandBRAFtesting in patients with metastatic colorectal cancer

Abstract

AIMTo investigate the impact of thymidylate synthase (TYMS), KRAS and BRAF in the survival of metastatic colorectal cancer (mCRC) patients treated with chemotherapy.METHODSClinical 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.RESULTSThe 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 1st 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).CONCLUSIONThe 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.

TYMS/KRAS/BRAF molecular profiling predicts survival following adjuvant chemotherapy in colorectal cancer

“…Analysis was carried-out as previously described[31,32]. PCR was performed using 1U of Platinum ® Taq DNA Polymerase (Thermo Fisher Scientific, Germany), 1.5 mmol/L of Mg and 200 nmol/L of dNTPs and primers.…”

“…Althoug the same primers were used, 5’-UTR amplification was performed using a GC rich amplification kit (PCRX Enhancer System, Thermo Fisher Scientific, Germany) adding 1× of PRCx Enhancer. Genotyping for the 2R/3R polymorphism was performed by running 10 μL of the PCR product on a 1.5% agarose gel and staining with Ethidium Bromide as previously described (Ntavatzikos et al[31]). Similarly, for the 12G>C substitution, 10 μL of PCR product was digested with 1U of HaeIII restriction enzyme (Takara, Japan) at 37 o C for 1 h and run on an 8% 19:1 polyacrylamide gel.…”

“…Detection of m KRAS in codons 12 and 13 and BRAF activating mutation V600E were performed as previously described with a commercially available Real-Time PCR kit (Therascreen KRAS, DxS Diagnostics, United Kingdom) detecting 6 mutations of codon 12 (G12D, G12A, G12V, G12S, G12R, G12C) and 1 mutation of codon 13 (G13D)[31,33]. A positive reaction mix for all mutations was included.…”

“…Based on the predicted TYMS protein expression, 5’UTR polymorphisms were assigned into low (2RG/2RG, 2RG/3RC, 3RC/3RC), medium (2RG/3RG, 2RG/3RCLOH, 2RG/3RGLOH, 2RGLOH/3RC) and high TYMS protein expression group (3RG/3RG, 3RG/3RC, 2RGLOH/3RG)[31]. The effect of each 3’UTR polymorphism was examined against all the others by applying univariate analysis and it was found that only the ins/LOH polymorphism had a statistically significant effect.…”

Therapeutic Applications of Vegf Inhibitors in Breast Cancer.

“…Bevacizumab is a humanised, function-blocking monoclonal antibody that selectively binds .The anti-VEGF antibody is currently approved in combination with intravenous 5-fluorouracil (5-FU)-based chemotherapy, which typically is a combination of irinotecan, 5-FU, and leucovorin (IFL), for first-or second-line treatment of metastatic carcinoma of the colon or rectum (34)(35) and, in conjunction with paclitaxel and carboplatin, for first-line treatment of unresectable, locally advanced, recurrent or metastatic nonsquamous, non-small cell lung cancer (NSCLC) (36). The Bevacizumab also has activity in breast cancer and kidney cancer.…”