Reverse translational research in colorectal cancer

2010/2011Background. A major clinical problem in oncology is that patients with tumors of the same stage and histological type often do not have the same prognosis and they do not show the same response to the therapeutic treatments. This is a major concern in colorectal cancer (CRC), because of its high incidence and impact on public health. Aim. The main goal of the present work is to identify a set of markers of prognosis and therapy efficacy in CRC patients using a reverse translational research approach. In the first part of the work, we focused on the selection criteria to submit stage II colon cancer patients to 5-FU based adjuvant therapy, while in the second part we searched for new molecular targets to predict the response to cetuximab or panitumumab in the metastatic setting. Methods. To the first purpose, we evaluated the mRNA expression levels of thymidylate synthase (TS) by qRT-PCR and IHC, the MMR (mismatch repair system) status by immunohistochemistry (IHC), the CIMP (CpG island methylator phenotype) status by methylation specific PCR (MSP) and the expression of p53, β-catenin, GSK3β and CD8 by IHC on a retrospective case study comprising 60 stage II colon cancer patients submitted to 5-FU adjuvant therapy and 60 colon cancer patients who did not receive therapy. Results of molecular analyses were compared with clinical-pathological factors and histological features of the cancers. To the second purpose, we evaluated KRAS mutational status by direct sequencing and an alteration at the DNA level in one of the genes belonging to the EGFR pathway in a retrospective case study comprising 98 recurrent CRC patients treated with cetuximab or panitumumab and 65 recurrent patients who did not receive treatment. mRNA expression levels of the EGFR-pathway gene were evaluated by qRT-PCR. Information about the gene and the method for alteration assessment cannot however be disclosed because of patent pending. Results. In the first part of the study, we found a better survival in patients with a high mRNA expression level of TS and treated with 5-FU (87% survival for TS high versus 60% for TS low at 5 years of follow up) while an opposite behaviour was displayed by patients who did not received any therapy (83% survival for low TS versus 60% for high TS expressors at five years of follow up). These results were independent from clinical-pathological and the other examined molecular factors. Molecular parameters and clinical-pathological variables did not show any correlation with patients’ survival, with the exception of the presence of tumoral CD8+ infiltrating lymphocytes, which was a good prognostic factor (84% survival for patients with tumor infiltrated versus 63% for those without infiltration at 5 years of follow up), independently of 5-FU treatment. In the second part of the study we found, as expected, an advantage in progression free survival in patients having a wild type K-RAS, but a greater advantage in patients showing one of the types related to the alteration we evaluated (81% survival for patients with the type 1 alteration versus 51% for KRAS wild type patients at 6 months of follow up). The effect of such alteration was specifically related to the treatment with monoclonal antibodies because it was lost in the group of 65 recurrent patients without cetuximab/panitumumab treatment. We also found that higher mRNA levels of the EGFR pathway gene were predictive of better benefit from the therapy with monoclonal antibodies and this effect was shown to be cumulative with a wild type KRAS status. Conclusions. As far it concerns the first part of the study, we therefore identified mRNA expression level of thymidylate synthase (TS), the 5-FU main cellular target, as the most important discriminator in the decision of which stage II colon cancer patients should be submitted to adjuvant 5-FU therapy. This evidence confirms the results obtained previously with a different training set of patients. With respect to the second part of the study, we identified an alteration at the DNA level in one of the genes belonging to the EGFR pathway as a new biomarker of cetuximab/panitumumab treatment efficacy in recurrent CRC patients.XXIV Cicl

Management of stage II colon cancer - the use of molecular biomarkers for adjuvant therapy decision

5Background: There is uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colorectal cancers. The aim of this study is to investigate the combined role of clinical, pathological and molecular parameters to identify those stage II patients who better benefit from adjuvant therapy. Methods: We examined 120 stage II colon cancer patients. Of these, 60 patients received adjuvant 5-FU chemotherapy after surgery and the other 60 did not receive therapy. Immunohistochemical (IHC) analyses were performed to evaluate the expressions of Thymidylate synthetase (TYMS), TP53 (p53), beta-catenin (CTNNB1) and CD8. For TYMS, its mRNA expression levels were also investigated by real time qRT-PCR. The entire case study was characterized by the presence of a defect in the MMR (mismatch repair) system, the presence of the CpG island methylator phenotype (CIMP or CIMP-High) and for the V600E mutation in the BRAF gene. At the histo-pathological level, the depth of tumour invasion, lymphovascular invasion, invasion of large veins, host lymphocytic response and tumour border configuration were recorded. Results: The presence of the V600E mutation in the BRAF gene was a poor prognostic factor for disease free and overall survival (DFS; hazard ratio [HR], 2.57; 95% CI: 1.03 -6.37; p = 0.04 and OS; HR, 3.68; 95% CI: 1.43-9.47; p < 0.01 respectively), independently of 5-FU treatment. Adjuvant therapy significantly improved survival in patients with high TYMS levels (p = 0.04), while patients with low TYMS had a better outcome if treated by surgery alone (DFS; HR, 6.07; 95% CI, 0.82 to 44.89; p = 0.04). In patients with a defect in the MMR system (dMMR), 5-FU therapy was associated to reduced survival (DFS; HR, 37.98; 95% CI, 1.04 to 1381.31; p = 0.04), while it was beneficial for CIMP-High associated tumours (DFS; HR, 0.17; 95% CI, 0.02 to 1.13; p = 0.05). Conclusions: Patients' characterization according to MMR status, CIMP phenotype and TYMS mRNA expression may provide a more tailored approach for adjuvant therapy in stage II colon cancer.Background: There is uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colorectal cancers. The aim of this study is to investigate the combined role of clinical, pathological and molecular parameters to identify those stage II patients who better benefit from adjuvant therapy. Methods: We examined 120 stage II colon cancer patients. Of these, 60 patients received adjuvant 5-FU chemotherapy after surgery and the other 60 did not receive therapy. Immunohistochemical (IHC) analyses were performed to evaluate the expressions of Thymidylate synthetase (TYMS), TP53 (p53), beta-catenin (CTNNB1) and CD8. For TYMS, its mRNA expression levels were also investigated by real time qRT-PCR. The entire case study was characterized by the presence of a defect in the MMR (mismatch repair) system, the presence of the CpG island methylator phenotype (CIMP or CIMP-High) and for the V600E mutation in the BRAF gene. At the histo-pathological level, the depth of tumour invasion, lymphovascular invasion, invasion of large veins, host lymphocytic response and tumour border configuration were recorded. Results: The presence of the V600E mutation in the BRAF gene was a poor prognostic factor for disease free and overall survival (DFS; hazard ratio [HR], 2.57; 95% CI: 1.03 -6.37; p = 0.04 and OS; HR, 3.68; 95% CI: 1.43-9.47; p < 0.01 respectively), independently of 5-FU treatment. Adjuvant therapy significantly improved survival in patients with high TYMS levels (p = 0.04), while patients with low TYMS had a better outcome if treated by surgery alone (DFS; HR, 6.07; 95% CI, 0.82 to 44.89; p = 0.04). In patients with a defect in the MMR system (dMMR), 5-FU therapy was associated to reduced survival (DFS; HR, 37.98; 95% CI, 1.04 to 1381.31; p = 0.04), while it was beneficial for CIMP-High associated tumours (DFS; HR, 0.17; 95% CI, 0.02 to 1.13; p = 0.05). Conclusions: Patients' characterization according to MMR status, CIMP phenotype and TYMS mRNA expression may provide a more tailored approach for adjuvant therapy in stage II colon cancer.noneopenM. Donada; S. Bonin; R. Barbazza; D. Pettirosso; G. StantaDonada, Marisa; Bonin, Serena; R., Barbazza; D., Pettirosso; Stanta, Giorgi

A synonymous EGFR polymorphism predicting responsiveness to anti-EGFR therapy in metastatic colorectal cancer patients

Genetic factors are known to affect the efficiency of therapy with monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer (mCRC). At present, the only accepted molecular marker predictive of the response to anti-EGFR mAbs, is the somatic mutation of KRAS and NRAS as a marker of resistance to anti-EGFR. However, only a fraction of KRAS wild type patients benefit from that treatment. In this study we show that the EGFR gene polymorphism rs1050171 defines, independently of RAS mutational status, a sub-population of 11% of patients with a better clinical outcome after anti-EGFR treatment. Median PFS for patients with the GG genotype was 10.17 months compared to 5.37 of those with AG+AA genotypes. Taken together our findings could be used to better define CRC populations responding to anti EGFR therapy. Further studies in larger independent cohorts are necessary to validate the present observation that a synonymous polymorphism in EGFR gene impacts on clinical responsiveness