Influence Of Comorbidities Including Anemia On The Efficacy Of Anti-PD1/PD-L1 Immunotherapy In Patients With Advanced Solid Tumor

Part 1.Effect of comorbidities on outcomes of patients with advanced non-small cell lung cancerBackground:Lung cancer is one of the most common cancers with about 781,000 new cases each year in China.The prognosis for patients with lung cancer is poor with 5-year survival proportions of 10-12%.Approximately 90%of lung cancers,especially non-small cell lung cancer(NSCLC),have been attributed to cigarette smoking,with age as an additional risk factor Furthermore,age and smoking are strongly associated with comorbidity,which also coexisted with lung cancer.Along with the social aging intensifies,clinicians will encounter older patients more frequently and with increasing probability that patients with lung cancer will have comorbidities.It is well established that comorbidity has effect on survivalComorbidity may influence survival in different ways.First,patients with lung cancer frequently present with other diseases,including chronic obstructive lung disease,cerebrovascular diseases,heart failure and myocardial infarction.Such types of comorbidity may by itself have a negative effect on survival.Second,comorbidity may significantly mask symptoms and delay the diagnosis of lung cancer or even prevent a full diagnostic evaluation with proper staging of lung cancer.Third,surgical intervention has a positive effect on the survival of lung cancer,but comorbidity may contradict surgical intervention in patients who need timely operation.Although,comorbidities will have a negative impact on survival,it also can increase the personal contact with the medical practitioners.And therefore,it may indirectly have a positive impact on survival by increasing the likelihood of earlier diagnosisSimultaneous estimation of models describing a patient's comorbid status has established The Simplified Comorbidity Score(SCS)and Charlson Comorbidity Index(CCI)are the most two extensively validated scoring systems for assessing comorbidities and predicting prognosis.These two comorbidity indices have previously been used as prognostic factors in patients with various carcinomas,including NSCLC.Of interest,the SCS was designed specifically for lung cancer.In the present study,we intended to explore the association of comorbidities evaluated by CCI and SCS with clinical outcomes,including survival and immune-related adverse events(irAEs),in a cohort of patients with advanced NSCLC undergoing immunotherapy with anti-PD1 agents in China.Additionally,we also analyzed the influence of blood glucose control level on survival of patients with advanced NSCLC complicated with diabetes mellitusPatients and Methods:A total of sixty-six patients with NSCLC who received PD1 inhibitors(pembrolizumab,nivolumab and toripalimab)in our institution in the past two years were enrolled in the retrospective study.Data on comorbid index(CCI and SCS)and clinical outcomes,including immunotherapy responses,progression-free survival(PFS),and irAEs,were collected Patients were divided into two groups:serious group(CCI? 1 or SCS? 8)and mild group(CCI<1 or SCS<8).In addition,we also collected clinical data of 191 patients with advanced NSCLC complicated with diabetes mellitus.Differences between groups were compared using by a ?2 test or Student's t-test.PFS was plotted using the Kaplan-Meier method and compared by the Wilcoxon test.A p-value of less than 0.05 was referred to denote a significant difference.Results:There were 66 patients with NSCLC who received immunotherapy with PD1 inhibitors from February 2017 to November 2019 in our study.The mean CCI and SCS scores for all subjects were 0.74(range:0-3)and 5.56(range:0-18),respectively.There was a weak correlation between these two SCS scoring systems(r=0.334;P=0.006).The disease control rate was obviously higher among the group of CCI<1 than the CCI? 1 group(P<0.001),but similar between groups with SCS<8 and SCS? 8(P=0.585).The median PFS in the CCI<1 group was 271.0(95%CI:214.3-327.7)days compared with 232.0(95%CI:66.2-397.8)days for the CCI? 1 group(P=0.0084).However,the median PFS showed no difference between the groups with SCS<8 and SCS? 8[271.0(95%CI:138.7-403.3)days vs.222.0(95%CI:196.2-247.8)days;P=0.2106].The incidence of adverse events was similar among patients with high versus low comorbidity indexes(CCI:35.8%vs.23.6%,P=0.286;SCS:28.0%vs.29.3%,P=0.912).At the same time,we also analyzed the survival of 191 patients with advanced NSCLC complicated with diabetes.Compared with patients with advanced NSCLC,PFS of advanced NSCLC patients complicated with diabetes was significantly shorter[157.0(95%CI:134.0-180.0)vs.252.0(95%CI:198.6-305.4),P<0.0001].PFS of patients with good blood glucose control during chemotherapy was significantly higher than that of patients with poor blood glucose control[197.0(95%CI:136.3-257.7)vs.132.0(95%CI:112.5-151.5)days,P=0.0003]Conclusions:1.Higher CCI values,not SCS,might have been associated with poor survival in patients with NSCLC who received anti-PD1 treatment.2.Poor glycemic control might be one of the risk factors for survival in advanced NSCLC patients with diabetes3.The comorbidity burden had no significant impact on the incidence of irAEs in patients with NSCLCPart 2.Influence of anemia on the efficacy of anti-PD1/PD-L1 immunotherapy in patients with advanced solid tumorsBackground:Anemia is very common in malignant tumor tissues,especially in advanced stage patients.A study on the detection of hemoglobin levels in patients with different malignancies found that the proportion of anemia in patients with lung cancer was much higher than that of other cancer patients,and especially in patients with advanced lung cancer and receiving chemoradiotherapy,the clinical incidence can reach about 90%.Since anemia mainly reduces oxygen supply to tissues of the body,each organ system of the patient will be affected to different degrees.Hypoxia can not only directly affect the quality of life of the patient,but also have an inevitable impact on the treatment effect and survival time of tumor patients.In addition,our previous study found that the erythrocyte differentiation of patients with advanced tumor complicated with anemia and tumor-bearing mouse was blocked,leading to a significant increase of CD45+EPCs(erythrocyte progenitor cells),which were enriched in spleen and played an immunosuppressive role through the expression of reactive oxygen species(ROS).The effect of anemia on the prognosis of patients,especially patients with anti-PD1/PD-L1 antibody immunotherapy has not been reported.In this study,we intended to explore the effect of pre-treatment anemia on the survival of patients with advanced solid tumor who received anti-PD1/PD-L1 antibody immunotherapy,the number and activity of immune cells,and to investigate a new source of MDSC development in cancer patients with anemia.Materials&Methods:(1)Effect of anemia on the prognosis of patients with advanced solid tumor treated with anti-PD1/PD-L1 antibodyThe study was analyzed retrospectively patients with advanced NSCLC(stage ?B or ?)who were admitted to Shanghai pulmonary hospital of Tongji university from January 2016 to January 2019,and 70 patients with malignant solid tumors other than lung cancer who were hospitalized at the second affiliated hospital of the Army Medical University from January 2018 to December 2019.These patients were received monotherapy with anti-PD1/PD-L1 antibody.Kaplan-Meier method was used to calculate the progression-free survival(PFS)and overall survival(OS),and the log-rank test was used to compare the difference between groups.A "P" value less than 0.05 indicates a statistically significant difference.(2)Effect of anemia on the number of peripheral blood immune cells in patients with lung cancerIn this study,peripheral blood cells were collected from lung cancer patients with advanced stage in our hospital in 2017 prior to any anticancer treatment and detected by flow cytometry.The hemoglobin content was divided into the anemia group(<110g/L)and the non-anemia group(? 110g/L).Then,flow cytometry was used to analyze the changes of immune cells in peripheral blood.(3)Effect of anemia on the immune function of tumor-bearing mice before and after treatment anti-PD1/PD-L1 antibodyWe first induced anemia mice model with phenylhydrazine,and inoculated MC38 or B16-F10 tumor cells.Flow cytometry was used to detect the number and function of spleen and tumor tissue immune cells in tumor-bearing mice with anti-PD1/PD-L1 antibody immunotherapy.(4)A novel source of MDSC in advanced tumor with anemiaFirst,splenic CD45+EPCs cells of CD45.1+LLC advanced tumor bearing mice were separated by flow,and the tail vein was transferred back to the irradiated CD45.2+mice.The proportion of MDSC in CD45.1+cells in spleen was detected by flow cytometry 5 days after adoptive transfer.Secondly,splenic CD45+EPCs cells of LLC tumor-bearing mice(day 0,day 14,and day 28)and MMTV-PYMT mice(18 weeks)were separated by flow cytometer,and were cultured in vitro based on 37? cell incubator under MDSC induction conditions.After 72 hours,flow cytometry was used to detect the proportion of induced MDSC.The induced MDSC was mixed with CD8+T cells to detect its immunosuppressive effect on T cell proliferation.Finally,CD45+EPCs cells excluding MDSC in peripheral blood and pleural effusion of patients with lung cancer complicated with anemia and their corresponding control group were selected,and differentiation was induced in vitro.The proportion of MDSC was sorted by flow cytometry 72 hours later.Results:(1)A total of 143 patients with advanced NSCLC who received anti-PD1/PD-L1 antibody monotherapy were included.In terms of the overall response rate,73.3%of the patients with severe anemia(Hb<90g/L)showed progressive disease,which was obviously higher than the 33.6%of the control group(Hb?90g/L).No partial response was observed in patients with moderate to severe anemia.Anemia was an independent risk factor for PFS in patients[hazard ratio=2.92(95%CI:1.25-6.81),P<0.0001].Median PFS was significantly higher in patients with mild or non-anemia(4.40 months vs.2.07 months,P<0.0001),and anemia was an independent risk factor for overall survival[hazard ratio=3.69(95%CI:1.30-10.49),P<0.0001],and the median PFS of patients with mild-moderate or non-anemia was significantly higher than that of patients with moderate or severe anemia(16.83 months vs.4.67 months,P<0.0001).In addition,70 patients with advanced solid tumors other than lung cancer who received anti-PD1/PD-L1 antibody monotherapy were enrolled.With the increase of anemia severity,the disease control rate was 71.7%,64.3%,54.6%and 25%,respectively.Patients with severe anemia had significantly shorter PFS than patients with mild-moderate or non-anemia(1.02 months vs.6.83 months).The hazard ratio of severe anemia is 2.89(95%CI:1.05-7.99)compared with that of mild-moderate or non-anemia(P=0.0019)(2)We detected T lymphocytes and natural killer(NK)cells in peripheral blood of patients with tumor complicated anemia or not,and found that the absolute numbers of CD3+T cells,CD4+T cells,CD8+T cells and NK cells in patients with anemia were significantly lower than those in patients without anemia(P<0.01).The absolute number of MDSC and regulatory T cells in patients with anemia was significantly higher than those without anemia(P<0.01)(3)Proportion of immune cells(CD3+T cells,CD4+T cells,CD8+T cells and NK cells)in the spleen of tumor-bearing mice with anemia was significantly lower than that of tumor-bearing mice without anemia(P<0.01).Meanwhile,the levels of CD107a,IFN-? and TNF-? secreted by CD8+T cells in the spleen of tumor-bearing mice with anemia were significantly lower than those of tumor-bearing mice(P<0.001).Anti-PD-L1 antibody can significantly inhibit tumor growth in non-anemic tumor-bearing mice with B16-F10 and MC38.However,in MC38 and B16-F10 tumor-bearing mice treated with anti-PD-L1 antibody,the proportion of CD107a+,IFN-?+,TNF-?+ in CD4+T and CD8+T cells in tumor tissues of anemia mice was significantly lower than that of non-anemia mice(P<0.001)(4)In the adoptive transfer experiment,CD45+EPCs from wild-type mice failed to differentiate into MDSC,and 12%of CD45+EPCs developed into MDSC in the early and middle stages of tumor-bearing stage.About 24%of CD45+EPCs from later stage of tumor-bearing donors developed into MDSC.In vitro,more than 30%of CD45+EPCs in the spleen of anemic mice with advanced tumor stages differentiated into MDSC.Moreover,the induced MDSC cells in vitro can significantly inhibit the proliferation of CD8+T cells(P<0.0001).Nearly 30%of splenic CD45+EPCs of mice with advanced spontaneous breast cancer complicated with anemia could differentiate into MDSC and significantly inhibited the proliferation of CD8+T cells(P<0.0001),too.CD45+EPCs excluded MDSC were isolated from peripheral blood of patients with chronic renal failure anemia and patients with advanced tumor complicated with anemia.Additionally,CD45+EPCs in patients with chronic renal failure and anemia failed to form MDSC,while about 10%of CD45+EPCs in patients with advanced anemia cancer exhibited a typical MDSC-like cell phenotype in vitro,with a statistically significant difference between the two groups(P<0.0001).Furthermore,under the same induction conditions,CD45+EPCs collected from tuberculous pleural effusion did not differentiate into MDSC-like cells,while more than 60%of CD45+EPCs from malignant pleural effusion could express MDSC surface markersConclusions:(1)Anemia can reduce the immune response rate of patients with advanced malignant solid tumor treated with anti-PD1/PD-L1 antibody,shorten the survival of patients,and is one of the factors associated with poor prognosis.In clinical practice,attention should be paid to the correction and improvement of anemia in patients with advanced solid tumors,in order to improve its immunotherapy effect and prolong the survival(2)Immune function of patients with advanced lung cancer complicated with anemia was significantly reduced compared with those without anemia(3)Anemia can decrease the immunity of tumor-bearing mice(4)Anti-PD1/PD-L1 antibody can significantly inhibit the tumor volume of MC38 and B16-F10 tumor-bearing mice,and so achieved the anticancer effect(5)Anti-PD1/PD-L1 antibody had poor immunotherapeutic effect in tumor-bearing mice with anemia,and the possible mechanism was that anemia might lead to the decline of immune active cells function in the body(6)CD45+EPCs cells with anemia in advanced tumors might be a new source of MDSC and promote immunosuppressive function in patients with advanced tumors.