Colorectal cancer molecular classification using BRAF, KRAS, microsatellite instability and CIMP status: Prognostic implications and response to chemotherapy.

OBJECTIVE: The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy. DESIGN: This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS). RESULTS: Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P<0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P<0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005). CONCLUSION: We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy.

Efficacy of Adjuvant 5-Fluorouracil Therapy for Patients with EMAST-Positive Stage II/III Colorectal Cancer.

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5-FU) within DNA and subsequent cytotoxicity was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSß (hMSH2-hMSH3 heterodimer) MMR complexes were present, compared to hMutSα > hMutSß alone. We tested if patients with EMAST CRCs (hMutSß defective) had diminished response to adjuvant 5-FU chemotherapy, paralleling in vitro findings. We analyzed 230 patients with stage II/III sporadic colorectal cancers for which we had 5-FU treatment and survival data. Archival DNA was analyzed for EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). Kaplan-Meier survival curves were generated and multivariate analysis was used to determine contribution to risk. We identified 102 (44%) EMAST cancers. Ninety-four patients (41%) received adjuvant 5-FU chemotherapy, and median follow-up for all patients was 51 months. Patients with EMAST CRCs demonstrated improved survival with adjuvant 5FU to the same extent as patients with non-EMAST CRCs (P<0.05). We observed no difference in survival between patients with stage II/III EMAST and non-EMAST cancers (P = 0.36). There is improved survival for stage II/III CRC patients after adjuvant 5-FU-based chemotherapy regardless of EMAST status. The loss of contribution of hMSH3 for 5-FU cytotoxicity may not adversely affect patient outcome, contrasting patients whose tumors completely lack DNA MMR function (MSI-H).

5-Fluorouracil adjuvant chemotherapy does not increase survival in patients with CpG island methylator phenotype colorectal cancer.

BACKGROUND & AIMS: 5-Fluorouracil (5-FU)-based adjuvant chemotherapy does not increase survival times of patients with colorectal tumors with microsatellite instability. We determined the response of patients with colorectal tumors with the CpG island methylator phenotype (CIMP) to 5-FU-based therapy. METHODS: We analyzed a population-based cohort of 302 patients with colorectal cancer (CRC) for a median follow-up time of 50.7 months. CIMP status was determined by analysis of the CACNAG1, SOCS1, RUNX3, NEUROG1, and MLH1 promoters; tumors were considered to be CIMP positive if at least 3 promoters were methylated. RESULTS: Tumors from 29.5% of patients (89/302) were CIMP positive; CIMP status did not influence disease-free survival (DFS; log-rank = 0.3). Of tumors of TNM stages II-III (n = 196), 32.7% were CIMP positive. Among patients with stages II-III CRC who did not receive adjuvant 5-FU chemotherapy, those with CIMP-positive tumors had longest times of DFS (log-rank = 0.04); In patients who received chemotherapy, those with CIMP-positive tumors had shorter times of DFS (log-rank = 0.02). In patients with CIMP-negative tumors, adjuvant 5-FU chemotherapy significantly increased time of DFS (log-rank = 0.00001). However, in patients with CIMP-positive tumors, adjuvant 5-FU chemotherapy did not affect time of DFS (log-rank = 0.7). Multivariate analysis showed a significant, independent interaction between 5-FU treatment and CIMP status (hazard ratio [HR], 0.6; 95% confidence interval [CI], 0.5-0.8). Among patients with CIMP-positive tumors, adjuvant chemotherapy was not an independent predictor of outcome (HR, 0.8; 95% CI, 0.3-2.0). In patients who did not receive adjuvant 5-FU chemotherapy, CIMP status was the only independent predictor of survival (HR, 2.0; 95% CI, 1.1-3.8). CONCLUSIONS: Patients with CIMP-positive colorectal tumors do not benefit from 5-FU-based adjuvant chemotherapy.

The efficacy of adjuvant chemotherapy with 5-fluorouracil in colorectal cancer depends on the mismatch repair status.

AIMS: The aim of this study is to evaluate if mismatch repair (MMR) defective colorectal cancer has a different response to adjuvant 5-fluorouracil (5-FU) chemotherapy in a cohort of patients prospectively followed during 5 years. METHODS: The cohort included 754 surgically treated patients with colorectal cancer. MMR status was diagnosed by MLH1 and MSH2 immunohistochemistry and microsatellite instability analysis. Median follow-up was 49.2 months (range 1-73). At inclusion, 505 patients were diagnosed as TNM II or III stage, analysis of the efficacy of adjuvant chemotherapy was made on this population. Adjuvant chemotherapy was applied to 248 patients (98.2% 5-FU based). RESULTS: MMR deficiency was found in 76 patients (10.1%). No differences were found in overall survival (log-rank p=0.3) or disease-free survival (log-rank p=0.3) regarding MMR status. Adjuvant chemotherapy improves survival in patients in the II or III stage, but this improvement is only evident in patients with MMR-competent tumours (log-rank p=0.00001). Survival of patients with MMR-defective tumours does not improve with adjuvant chemotherapy (log-rank p=0.7). A multivariate analysis showed an independent effect of the interaction between MMR status and adjuvant chemotherapy (Hazard ratio 2.04; 95% confidence interval: 1.42-2.93). CONCLUSION: In a cohort of colorectal cancer patients, those with MMR-deficient tumours seem not to benefit from 5-FU-based chemotherapy.

Brain cholinergic impairment in liver failure.

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (~30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (~20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (~50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.