The Effect Of CMTM6 And PD-L1 And Immune Cells In Microenvironment On Immune Checkpoint Blocking Therapy In Colorectal Cancer

Background and PurposeColorectal cancer(CRC)is the third leading cause of cancer death in the world,and it is also on the rise in China.The main causes of death for the patients of colorectal cancer are recurrence and metastasis.Anti PD-1/PD-L1 immunotherapy is one of the major breakthroughs in tumor therapy in recent years,which has been recommended by NCCN(National Comprehensive Cancer Network)guidelines for metastatic or refractory CRC patients with dMMR(Defective Mismatch Repair)or MSI-H(High-frequency Microsatellite Instability).The effective rate of PD-1 inhibitors for advanced CRC patients with dMMR or MSI-H mutations(only 5%)is 40-60%,so finding the appropriate population more accurately and further improving the efficacy of immunotherapy has become an urgent problem to be solved in immunotherapy.CMTM6(CKLF-like MARVEL transmembrane domain containing 6)is a key regulator of PD-L1.It increases the stability of PD-L1 by inhibiting the ubiquitin of PD-L1.A recent study have shown that the co-expression of CMTM6 and PD-L1 in immunotherapy patients with non-small cell lung cancer is significantly associated with longer overall survival,which may enable patients to benefit from blocking the PD-1 axis.The purpose of this study was to investigate the expression of CMTM6,PD-L1 and tumor microenvironment immune cells in CRC dMMR group and pMMR(Proficient Mismatch Repair)group and their clinicopathological significance,and to explore whether CMTM6 can be used as a predictor of immunotherapy decision-making by using PD-1/PD-L1 inhibitors to treat effective or ineffective CRC cohorts.Method1,328 cases of colorectal cancer in the Department of Pathology of Southern Hospital from January 2015 to December 2017 were collected.The expression of mismatch repair proteins(MLH1,MSH2,MSH6 and PMS2)in tumor tissues was detected by immunohistochemistry to determine dMMR CRC.The expression correlation and prognosis of CMTM6,PD-L1 and tumor microenvironment mesenchymal cells(CD4,CD8,CD68,CD 163 and a-SMA)in CRC dMMR group(n=121)and pMMR group(n=127)were analyzed by immunohistochemistry.A total of 32 patients with advanced refractory/metastatic CRC(6 cases of dMMR,26 cases of pMMR)treated with PD-1/PD-L1 inhibitors(Pembrolizumab,Nivolumab,Camrelizumab,Sintilimab and Toripalimab)in three hospitals were collected and divided into effective group(n=6)and ineffective group(n=26)according to the standard response guidelines iRECIST(immunotherapy response evaluation criteriain solid tumor)used in immunotherapy trials.The expression and localization of CMTM6,PD-L1,CD4,CD8,CD68 and CD 163 in 32 cases of CRC tumors were detected by multiple immunofluorescence,and the predictive effect of CMTM6 expression and localization on the treatment of PD-1/PD-L1 inhibitors was analyzed.Results1.The expression level of CMTM6 and PD-L1 in dMMR colorectal cancer was significantly higher than that in pMMR(P<0.001 and P=0.002),and there was a positive correlation between the expression of CMTM6 and PD-L1 in CRC(R=0.456,P<0.001),especially in dMMR(R=0.714,P<0.001).The number of tumor-associated lymphocytes(CD4+and CD8+)and tumor-associated macrophages(CD68+and CD163+)in dMMR group was significantly higher than that in pMMR group(P<0.001,<0.001,=0.001 and<0.001,respectively).There was a positive correlation between the expression of CMTM6 and macrophages(CD68+)in dMMR group(R=0.222,P=0.015),especially in M2 macrophages(CD 163+)(R=0.292,P=0.001),but there was no correlation in pMMR group.2.The expression of CMTM6 and PD-L1 in colorectal cancer is not related to the prognosis of patients,suggesting that CMTM6 or PD-L1 is not a prognostic indicator,and the more CD4+and CD8+lymphocytes in CRC indicate a better prognosis(p<0.001 and p<0.005).3.The total effective rate of immunotherapy in 32 patients with refractory/metastatic colorectal cancer was 18.8%,and the effective rates of dMMR group and pMMR group were 33.3%and 15.4%.There were significant differences in CMTM6/PD-L1,CMTM6/CD8+cell and CMTM6/CD163+cell co-expression between the effective group and the ineffective group(p=0.006,p=0.012 and p=0.001).The effective rates of these 3 groups of co-expression in 32 immunotherapy patients were 50.0%(5/10),57.1%(4/7)and 71.4%(5/7),respectively;and in 6 cases of CRC dMMR patients were 50%(2/4),66.7%(2/3)and 66.7%(2/3),respectively;and in 26 cases of CRC pMMR patients were 50.0%(3/6),50.0%(2/4)and 75.0%(3/4),respectively.The results show that the combined detection of CMTM6 and CD 163 are the best biomarkers for predicting the responsiveness of immunotherapy,with a predictive effective rate of 71.4%in CRC patients,66.7%in the dMMR group and 75.0%in the pMMR group.Conclusion1.The expression level of CMTM6 and PD-L1 in colorectal cancer is not related to the prognosis of patients with colorectal cancer.The detection of CD4+and CD8+T lymphocytes are helpful to judge the prognosis of patients;2.The combined detection of CMTM6 and CD 163 are more accurate than using dMMR alone as a marker of CRC immunotherapy,and can be used as a reference to judge the efficacy of PD-1/PD-L1 in the treatment of CRC.