Characteristics Of Peripheral Blood Lymphocytes And Gene Mutations In Patients With MSI-H Colorectal Cancer

Objectives: By comparing with patients with microsatellite stable(MSS)colorectal cancer(CRC),we analyzed the status of 16 common cancer driver genes and the level of peripheral blood T lymphocyte subsets in patients with microsatellite instability-high(MSI-H)CRC,to further enrich the molecular and immunological information of CRC patients with different microsatellite status.Methods: A total of 378 patients with CRC who had been treated in X Hospital from January 2020 to June 2021 were chosen.All of them were required to have a clear status of microsatellite and 16 genes needed in our study.Among the 378 cases,78 patients with MSI-H and 300 patients with MSS.We retrospectively collected clinicopathological and molecular immunology information from all patients,including 16 genes status,initial(without any prior treatment)tumor size,clinical stage and the level of T lymphocyte subset in peripheral blood,etc.And then performed statistical analysis to compare the differences between the two groups.T lymphocyte subsets in peripheral blood,including the CD45+CD3+T cell ratio,CD4+Th cell ratio,CD8+Ts cell ratio,and Th/Ts value,were determined by flow cytometry.16 genes included TP53,APC,KRAS,NRAS,HRAS,BRAF,PIK3 CA,SMAD4,PTEN,ERBB2(HER2),KIT,PDGFRA,EGFR,MET,VEGFA and KDR(VEGFR2),were determined by the Next-generation sequencing(NGS).Results: 1.Among the 378 CRC patients included,229 were males and 149 were females,aged 23-85 years.The median age of the patients in MSI-H group was lower than that of the MSS group(53 years vs 58 years,P=0.01).For the location of tumor,CRCs with MSI-H were more frequently located in the right colon(53.8% vs17.3%,P<0.001),whereas MSS cases were more frequently located in the rectum(28.2% vs 58.0%,P<0.001).In addition to the common adenocarcinoma component,MSI-H CRCs frequently had a combined mucinous adenocarcinoma or signet ring cell carcinoma component(41.0% vs 13.0%,P<0.001).Regarding imaging findings at initial diagnosis,CRCs in the MSI-H group exhibited larger volumes of the primary tumor(median: 6.1cm vs 4.5cm,P<0.001)and regional lymph nodes(median: 0.9CM vs 0.8cm,P=0.015).And the primary tumors of MSI-H CRCs were often more than or equal to 5cm(70.5% vs 40.3%,P<0.001).Meanwhile,the local invasion of CRCs with MSI-H was more serious,manifested as later T stage,higher proportion of T4 b stage(24.4% vs 17.7%,P=0.021),and more patients in stage III(59.0% vs 44.7%,P=0.024).Furthermore,intestinal obstruction was also more likely to occur in patients with MSI-H CRC(46.2% vs 30.3%,P=0.008).No significant statistical differences between the two groups were observed concerning gender,ethnicity,CEA level,c N stage,and distant metastatic organs at initial diagnosis.2.Of the 16 genes studied,the TP53 gene had the highest probability of mutation in all CRC cases(67.99 %),and mutation was more common in patients with MSS(75.00% vs 41.03%,P<0.001).Additionally,significant differences also were found in the status of the other 8 genes between the two groups,each of them with a higher probability of mutation occurring in MSI-H CRCs,including BRAF(19.23% vs6.33%,P<0.001),PTEN(23.36% vs 2.00%,P<0.001),ERBB2(HER2)(14.10% vs4.00%,P=0.002),KIT(11.54% vs 1.33%,P<0.001),PDGFRA(10.26% vs 0.63%,P<0.001),EGFR(8.97% vs 0.33%,P<0.001),MET(6.41% vs 0.33%,P=0.001)and HRAS(3.85% vs 0%,P=0.009).However,there was no significant correlation between the status of the remaining 7 genes and microsatellite status(all P >0.05).3.Among all 378 patients with CRC,302 had complete data on initial peripheral T lymphocyte subsets,including 64 in the MSI-H group and 238 in the MSS group.The levels of CD45+CD3+T cell ratio(69.25% vs 65.65%,P=0.012),CD4+Th cell ratio(38.70% vs 34.30%,P=0.007)and CD8+Ts cell ratio(24.85% vs 21.50%,P=0.031)were higher in the initial peripheral blood samples of patients with MSI-H CRC compared with patients with MSS.No significant statistical difference was observed in the Th/Ts value between the two groups(1.49 vs 1.61,P=0.644).Conclusions: 1.Our study confirmed some clinicopathological features that could identify MSI-H CRC patients at an early stage,including a younger age of onset;the tumors were mainly located in the right colon and rich in mucinous adenocarcinoma or signet ring cell carcinoma component;on imaging,the primary tumor and regional lymph nodes were larger,T stage was later,T4 b accounted for a higher proportion,more patients in stage III,and more prone to intestinal obstruction.2.Patients with MSI-H CRC have a unique mutation spectrum,with a higher probability of mutation in BRAF,PTEN,ERBB2(HER2),KIT,PDGFRA,EGFR,MET and HRAS genes and a lower probability of mutation in TP53 gene relative to patients with MSS.These results suggest that CRCs with different microsatellite status may have different pathogenic mechanisms or molecular pathways,and provide further exploration space for more particular molecular typing,diagnosis and treatment of CRC.3.The “hot” anti-tumor immune response in MSI-H CRC is not only limited to the local tumor,but also manifested in the systemic immune system,as evidenced by higher levels of CD45+CD3+T cell ratio,CD4+Th cell ratio,and CD8+Th cell ratio in the peripheral blood.Peripheral blood-based assessment of the tumor immune environment may be clinically valuable.