Baseline Carcinoembryonic Antigen(CEA) Serum Levels Predict Cetuximab-based Treatment Response In Metastatic Colorectal Cancer And The Mechanism Study

Part One Correlation analysis of original CEA level and the efficacy of cetuximab in mCRC patientsObjective: To investigate the correlation between original CEA level and efficacy of cetuximab in mCRC patients.Method: To collect and collate information of mCRC patients who received cetuximab combined with chemotherapy at the cancer center of Wuhan Union Hospital in 2013-2015. Regarded the original CEA concentration 10ng/ml as the boundary, the patients divided into CEA elevated group and CEA normal group. To retrospective analyze the relationship between clinical pathological characteristics and efficacy of cetuximab and prognosis.Result: According to the inclusion criteria, we get 20 cases in total, which include 13 cases of CEA elevated group and 7 cases of CEA normal group. After cetuximab-based treatment, the DCR of CEA elevated group is higher than CEA normal group(92% VS 28%), the difference is significant(Fisher exact test, P=0.007). However, sex, age, primary tumor site and metastasis extent are irrelevant to the DCR.The mean and median(mos) of OS of CEA elevated group are higher than CEA normal group(18.7 VS 14.2,18 VS 9), the difference is not obvious(Log rank test, P=0.339). The mean and median(mos) of PFS of CEA elevated group are higher than CEA normal group(7.6 VS 5,7 VS 0), the difference is not obvious(Log rank test, P=0.334).Conclusions: the original CEA elevated group has better DCR after cetuximab-based therapy in mCRC patients, compared with CEA normal group. The mean and median of OS and PFS of CEA elevated group are higher than CEA normal group, but the difference has no statistical significance.Part two Correlation analysis of the effect of EGF on the proliferation of LOVO Cells and CEA expressionObjective: To observe the effect of EGF on the proliferation of multiple CRC cells and their correlation with CEA expression.Methods: The effect of EGF on the proliferation of 6 kinds of CRC cells were studied by MTT assay in vitro and confirmed by nude mice transplanted tumor model in vivo. The expression of proteins and m RNA level of CEA were detected by Western blot and RT-PCR. After EGF stimulation, the expression changes of CEA and nuclear transcription factor CEAR of CRC cell were detected by Western blot. The expression of CEA in transplanted tumor tissue was detected by IHC. Using si RNA technique to inhibit the expression of EGFR, the effect of EGF on CEA expression and phosphorylation of AKT and ERK were detected by Western blot.Results: The proliferation effect of LOVO cells stimulated by EGF was significant and dose-dependent, with the tight junction of epithelial cells becoming discrete spindle interstitial cell like morphology, however, the other CRC cells has no proliferation effect and morphologic changes. The proliferation effect of LOVO cells subcutaneous transplantation tumor stimulated by EGF was significant and time and dose-dependent, the maximum proliferation effect was achieved when the concentration of EGF was 500?g/kg/d. Only LOVO cells have high expression of CEA both in protein and m RNA level. EGF can promote the expression of CEA in LOVO cells, and is associated with down-reglation of CEAR expression. The CEA expression of subcutaneous transplantation tumor tissue of EGF group is significantly higher than control group. Inhibiting the expression of EGFR in LOVO cell, the promotion effect of CEA expression and phosphorylation of AKT and ERK stimulated by EGF can be blocked.Conclusions: Only LOVO cells which has high expression of CEA both in m RNA and protein level displays obvious proliferating effect stimulated by EGF, with interstitial cell like morphological change, however, SW620, SW480, HT29, HCT116 and CACO2 have no obvious proliferating effects and morphological change. The leve of CEA in LOVO cells was promoted by EGF stimulation, with down-regulation of CEAR expression. EGF induces AKT and ERK phosphorylation and promotes CEA expression through EGFR in LOVO cells.Part three Correlation analysis of the effect of EGF on the proliferation of LOVO and the status of RAS and EGFRObjective: To observe the effect of EGF on the proliferation of multiple CRC cells and their correlation with RAS mutation and EGFR expression.Methods: RAS and BRAF gene sequencing of 6 kinds of CRC cells. After EGF stimulation, AKT and ERK phosphorylation status of CRC cells were detected by Western blot. The expression level of EGFR of CRC cells was detected by Western blot.Results: LOVO cells and HCT116 cells were KARS G13 D mutation, SW620 and SW480 were KARS G12 V mutation, HT29 and CACO2 has no mutation both in RAS and BRAF. In addition to SW620, LOVO, SW480, HT29, HCT116 and CACO2 are all has high expression of EGFR. Besides, LOVO, SW480, HT29, HCT116 and CACO2 are all has phosphorylation of AKT and ERK after EGF stimulation.Conclusion: There was no significant correlation between the high proliferative activity stimulated by EGF and RAS mutation status in LOVO. EGF sitmulated AKT and ERK phosphorylation in CRC cells was not associated with RAS mutation status, but was associated with EGFR expression. There was no significant correlation between the high proliferative activity stimulated by EGF and EGFR expression.