Reverse translational research in colorectal cancer
Background. 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.