| Background and Objective:NK/T cell lymphoma(natural killer/T-cell lymphoma,NKTCL)is a rare extranodal non-hodgkin lymphoma formerly known as "vascular center lymphoma"and "fatal midline granuloma ",accounting for about 12-15%of head and neck tumors.Geographical and ethnic preferences are shown in Asia and some Latin American countries.Early stage lymphoma was treated with radiotherapy and chemotherapy,and advanced stage NK/T lymphoma advanced rapidly and had poor prognosis,but there was no definite treatment plan.In recent years,immunotherapy has gradually become one of the main treatment methods for tumor therapy,and PD-1/PD-L1 checkpoint is an important part of it.Hyperthermia,known as "green therapy",has become one of the pillars of adjuvant therapy for cancer,and some studies have proved that hyperthermia combined with chemotherapy and radiotherapy can prolong the survival period of cancer patients and reduce the side effects of treatment..Hyperthermia has not been reported for the treatment of NK/T cell lymphoma.This study is intended to explore:(1)The clinical efficacy and prognosis of radiotherapy and chemotherapy combined with PD-1 inhibitors in the treatment of advanced NK/T lymphoma;(2)The effects of hyperthermia and PD-L1 inhibitors on the proliferation and apoptosis of NK/T lymphoma cells and their mechanisms in vitro.In order to provide a basis for safe and effective immunotherapy,thereby increasing the cure rate,extending the survival period of patients.PART Ⅰ Efficacy and prognosis of radiotherapy and chemotherapy combined with PD-1 inhibitors in the treatment of advanced NK/T lymphomaMethods:From January 2017 to December 2019,53 patients with stage III-IV NK/T cell lymphoma diagnosed by the first affiliated Hospital of Zhengzhou University were retrospectively analyzed.According to the different treatment methods they are divided into observation group and control group.Radiotherapy(50 Gy/25F,radiotherapy dose 2Gy/times,5 times per week)or chemotherapy(GDDP regimen 4-6 cycles:gemcitabine 800 mg/m2dl、8+cisplatin 20 mg/m2d1～4+Dexamethasone 15 mg/m2,d1～5+ intramuscular injection of aspartic enzyme 2500IU/m2,d1,21d as a cycle)were given in the control group.The observation group was given PD-1 antibody(200 mg/times,dl,14d as a cycle,Jiangsu Hengrui)as combined treatment.The short-term efficacy,adverse reactions and overall survival were compared between the two groups.Results:1.Short-term Efficacy CR rate is 52.63%in the observation group as 17.65%in the control group,and the CR rate in the observation group was higher than that in the control group significantly(P=0.008).The ORR of the observation group was higher than that of the control group,but the difference was not statistically significan(P=0.057).2.Adverse Reactions Fever,gastrointestinal reactions,bone marrow suppression and abnormal liver function were the main adverse reactions during treatment.There was no significant difference between the two groups(P<0.05)。3.Median and overall survival The median survival of the observation group was 22.40±5.056 months,and the median survival of the control group was 18.60±3.474 months.The 1-year and 2-year survival rates of the observation group were divided into 84.73%and 37.90%;the 1-year and 2-year survival rates of the control group were 70.02%and 34.46%.There are statistical differences between the two groups in survival rates(P=0.038).Conclusion:Radiotherapy and chemotherapy combined with PD-1 inhibitors in the treatment of advanced NK/T lymphoma can improve the complete response and the overall survival rates at 1 and 2 years,and do not increase the side effects.PART Ⅱ Study on the effect and mechanism of hyperthermia and PD-L1 inhibitors on NK/T lymphoma cellsMethods:1.Three kinds of NK/T lymphoma cells were placed in incubators at different hyperthermia temperatures(39℃,41℃,43℃)for 30min and then returned to normal incubator at 37℃ for culture.After 24h,CCK-8 was used to detect the proliferation inhibition rate of three kinds of NK lymphoma cell lines;2.CCK-8 was used to detect the proliferation inhibition rate of NKL cells after treatment with different concentrations of PD-L1 inhibitor(Fraxinellone,10μM、30μM、50μM、100μM and 200μM)for 24h,48h and 72h;3.NKL cells were treated with hyperthermia(HT)at 43℃ for 30min,100μM PD-L1 inhibitor.After treatment for 24h,48h and 72h,CCK-8 was used to detect the cell proliferation inhibition rate.4.The cells were divided into blank control(NC)group,HT(43℃ 30min)group,Fraxinellone(100 μm)group,HT(43℃ 30min)+Fraxinellone(100 μm)group.After treatment for 48h,the cells in each group were collected:① Flow cytometry were used to detect cell apoptosis;② Flow cytometry were used to detect cell cycle changes;③ Transwell were used to detect cell migration;④ Real-time quantitative PCR were used to detect the expression of related gene HIF-1α、JAK2、STAT3、PD-L1;⑤ Western-Blot were used to detect pathway-related protein expression.Results:1.Both NKL and KHYG-1 cells had the highest proliferation inhibition rate at 43℃,the NKL proliferation inhibition rate was 81.44%and the KHYG-1 was 65.17%.NKYS showed no inhibitory effect on proliferation.2.50μM、100μM inhibition was statistically significant.There was no statistically difference in cell inhibition of 10μM,30μM,200μM.3.HT and Fraxinellone had the most significant inhibitory effects at 24h(82.50%)and 48h(41.88%).Both of them had strong inhibitory effects at 48h(88.10%),and there was an interaction between them.4.① The results showed that the apoptosis rate of NKL lymphoma cells increased gradually in Fraxinellone(100 μm)group,HT(43℃ 30min)group and HT(43℃ 30min)+Fraxinellone(100 μm)group.But there was no statistical difference between Fraxinellon group and NC group(P>0.05).② The results showed that the proportion of S phase in the HT(43℃ 30min)group and the HT(43℃ 30min)+Fraxinellone(100μM)group decreased(P<0.007),but there was no significant difference in others.③ The results showed that the cell migration ability of the Fraxinellone(100μM)group,the HT(43℃ 30min)group,the HT(43℃ 30min)+Fraxinellone(100μM)group gradually decreased,and the difference between the HT group and the HT+Fraxinellon group was significant(P<0.001).Fraxinellone(100μM)group was not significantly weakened(P>0.05).④ The expression trend of each gene was consistent,and there was no significant difference in statistics(P>0.05).⑤ Compared with normal NKL cells,hyperthermia and Fraxinellon can inhibit JAK2/STAT3 protein phosphorylation,inhibit HIF-lα protein synthesis,and down-regulate PD-L1 expression(P<0.001),while JAK2 and STAT3 proteins have no significant changes(P>0.05).Conclusion:(1)Hyperthermia(43 ℃,30 min)and Fraxinellon(100μM)on NKL lymphoma cells inhibited cell proliferation、migration,apoptosis was induced at the same time.(2)Hyperthermia and Fraxinellon may inhibit the proliferation of tumor cells by inhibiting the phosphorylation of JAK2/STAT3 in NKL cells and reducing the HIF-1s protein to reduce the formation of PD-L1 protein. |
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