| Part ⅠEffects of Icotinib Combined With Anti-angiogenic Drugs on Human Non-small Cell Lung Cancer Xenograft ModelsObjective:This study aimed to evaluate the inhibitory effects and potential mechanisms of icotinib combined with two different anti-angiogenic drugs on lung adenocarcinoma in vivo.At the same time,the effects of combined medication on microvessels of important organs and whether the rebound phenomenon of tumor growth and angiogenesis occurs after drug withdrawal were observed.Meanwhile,the adverse reactions of the combination drugs were also observed.Methods:1.To establish a tumor-bearing nude mouse model of human HCC827 lung adenocarcinoma cell line:the cryopreserved cell line was recovered,cultured and passaged,and then the cells were collected and the cell suspensions were prepared.72 BALB/c-nu female nude mice of 4-6 weeks old were acclimated in SPF laminar flow cabinet for 1 week,and then the cells at a concentration of 2.5×10^7/ml were injected into the right axilla of nude mice.After 10 days,the subcutaneous tumors grew to 5-8 mm in diameter,and the model was bulit successfully.2.Experimental scheme:72 nude mice were randomly divided into 6 groups with 12 mice in each group.The treatment was terminated after 16 days and the observation was continued for another 16 days.The specific medication plan is as follows:Control group:0.5%sodium carboxymethyl cellulose 0.1ml/kg,fed once a day;Icotinib group:Icotinib 60 mg/kg,fed once a day;Bevacizumab group:Bevacizumab 5 mg/kg,intraperitoneal injection,twice a week;Recombinant Human Endostatin group:Endostatin 10mg/kg,subcutaneous injection around the tumor,once a day;Icotinib combined with bevacizumab group:the drug dose is the same as that of the single drug group;Icotinib combined with Recombinant Human Endostatin group:the drug dose is the same as that of the single drug group;3.Body weights and diameter of the tumors were measured before and after drug administration.After 16 days of drug administration,6 nude mice in each group were sacrificed.The remaining mice were stopped using drugs and continued to be observed for another 16 days,and then the mice were sacrificed.Half of tumor specimens and heart,liver and brain tissue specimens were stored in a refrigerator at-80℃,and the remaining specimens were embedded in paraffin for use.Determination of Tumor Volume:Diameters of subcutaneous tumors were measured every 4 days during the experiment and then the tumor volume was calculated.Tumor volume=3.142(ab2)/6,where a represents the long diameter of the tumor tissue,and b represents the short diameter.4.Observation of body weights and general condition of nude miceDuring the experiment,body weights of nude mice were measured every 4 days.At the same time,the general conditions such as foraging,spirit and activity of nude mice were observed during the experiment.5.Immunohistochemical methods were used to determine the microvessel density and Ki67 proliferation index of the tumors in nude mice at the end of the medication period and the end of the observation period.The same methods were used to determine the microvessel density of the heart,liver and brain tissues of nude mice.The differences of microvascular density and Ki67 proliferation index in each group before and after medication were compared.Microvessel density was counted using the Weidner method.6.Western Blot(WB)and Enzyme-linked immunosorbent assay(ELISA)were used to determine the expression of VEGFA in the transplanted tumor tissues at the end of the medication period.Results:1.The nude mice in each group were generally in good condition before and after the medication,with normal foraging and drinking.There was no significant difference in the body weight in each group before and after treatment(P>0.05).2.Tumor growth curve during medication:At the beginning of the experiment,the tumor growth rate in all groups was slower than that of the control group.By the 8th day after administration,compared with the control group,the growth rate of tumor in the bevacizumab group had significantly slowed down(P=0.003),while there was no statistical difference in the endostatin group and icotinib group(P>0.05).Among the three single-drug groups,the bevacizumab group also had a statistically significant difference in tumor volume compared with the endostatin group(P=0.005),while there was no statistically significant difference among the other groups.The tumor growth rate of the two combined treatment groups was also significantly lower than that of icotinib group,endostatin group and control group(P<0.05).There was also a trend of slowing down compared with the bevacizumab group,although not reached to statistical difference(P>0.05).There was no significant difference between the two combined treatment groups(P>0.05).By the end of the 16th day of medication,the tumor volume of all treatment groups was significantly slower than that of the control group(P<0.05).Among the three single-drug groups,tumor growth was significantly slowed in the bevacizumab group and the icotinib group compared with the endostatin group(P=0.014,0.019,respectively),while there was no statistical difference between the icotinib group and the bevacizumab group(P>0.05).The tumor growth rate of the two combined treatment groups was significantly lower than that of icotinib,endostatin and bevacizumab groups(P<0.05).There was no significant difference between the two combined treatment groups(P>0.05).3.Tumor growth curve during drug withdrawal:After 4 days of drug withdrawal,the tumor growth rate of all groups increased faster than before.By the 8th day of drug withdrawal,the endostatin group had the highest growth curve slope,which was not only higher than that of all treatment groups,but also higher than that of the control group.This suggested that the tumor growth rate in the endostatin group was the fastest.The slope of the growth curve in the other groups was lower than that of the control group.Among them,icotinib and bevacizumab groups were the second,and the two combined treatment groups had the slowest tumor growth rate.However,after 8 days of drug withdrawal,the growth curve slope of all groups tended to be consistent,but it was still lower than that of the control group,suggesting that the growth rate of all groups gradually tended to be consistent after 8 days of drug withdrawal.At the end of the 32-day observation period,the tumor volume in all drug groups was lower than that of the control group(P<0.05).4.MVD of tumor tissue:Compared with the control group,the MVD of endostatin group and bevacizumab group was significantly lower(P<0.001)after 16 days of treatment,while there was no significant difference between the icotinib group and the control group(P>0.05).At the same time,the MVD of tumor tissues in the two combined treatment groups was not only lower than that of the control group(P<0.001),but also significantly lower than that of the corresponding anti-vascular drug group(P<0.05).There was no significant difference in MVD of tumor tissues between the two combined treatment groups(P>0.05).After 16 days of drug withdrawal(day 32),there were no significant differences in the MVD of tumor tissues among all groups(P>0.05).5.MVD of liver,heart and brain tissues:At the end of treatment(day 16),the MVD of liver tissues in the combined treatment groups and the anti-vascular drug group was significantly lower than that of the icotinib group and the control group(P<0.05).In the pair comparison between the combined treatment groups and the single anti-vascular drug groups,the MVD of liver tissues in the icotinib combined with endostatin group was significantly lower than that of the endostatin group(P=0.011).However,the MVD of liver tissues in the icotinib combined with bevacizumab group also tended to be lower than that of the bevacizumab group,although there was no statistical difference(P>0.05).There was no significant difference between the two combined treatment groups(P>0.05).At the end of observation period,the statistical difference between groups disappeared(P>0.05).There were no significant differences in MVD of heart and brain tissues among all groups at the end of treatment or the end of drug withdrawal observation(P>0.05).6.Expression level of Ki67 in tumor tissues:The expression level of Ki67 in tumor tissues of the combined treatment groups and the single drug groups was significantly lower than that of the control group at the end of the treatment period(P<0.05).In the comparison of the three groups,the expression level of Ki67 in the icotinib group and bevacizumab group was significantly lower than that of the endostatin group(P<0.05).The expression level of Ki67 in the two combined treatment groups was also significantly lower than that of the corresponding single anti-vascular drug groups(P<0.05).There was no statistical difference between the two combined treatment groups.At the end of drug withdrawal observation,there was no significant difference in Ki67 expression level between all groups(P>0.05).7.ELISA results:At the end of the treatment period,there was no significant difference in expression level of VEGFA between icotinib group and control group(P>0.05).The expression level of VEGFA of endostatin and bevacizumab groups was significantly lower than that of control group(P<0.001).The expression level of VEGFA of icotinib combined with bevacizumab group was significantly lower than that of the control group,icotinib group and bevacizumab monotherapy group(P=0.002).The expression level of VEGFA of icotinib combined with endostatin group was significantly lower than that of the control group and icotinib group(P<0.001),and showed a downward trend compared with endostatin monotherapy group(P=0.072).8.Western blot(WB):At the end of the treatment period(day16),compared with the control group,the relative expression level of VEGFA in tumor tissues of icotinib group had no significant difference(P>0.05),but the relative expression level of VEGFA in endostatin and bevacizumab groups was significantly decreased(P<0.001).The relative expression level of VEGFA in the two combined treatment groups was significantly lower than that of the control group,icotinib group,bevacizumab group and endostatin group(P<0.001).Conclusion:1.In the EGFR mutation-positive nude mouse subcutaneous xenograft tumor model,the icotinib combined with anti-angiogenic drug group showed stronger tumor inhibitory effect,which was significantly better than the icotinib single-drug group or the anti-angiogenic single-drug group(bevacizumab or recombinant human endostatin).The Ki67 proliferation index in the combined drug group was also significantly lower than that of the single drug group.There was no significant difference between icotinib combined with endostatin and icotinib combined with bevacizumab in tumor inhibition.2.4 days after drug withdrawal,the tumors in all drug groups grew faster than before.Among them,the slowest growth rate was in the combination therapy group,followed by the bevacizumab group and the icotinib group,and the fastest was the recombinant human endostatin group.This may be related to the short half-life of endostatin.However,the difference disappeared on the 16th days after drug withdrawal,and the slope of tumor growth curve in all drug groups tended to be consistent,which was lower than that of the control group.At the end of drug withdrawal observation,the tumor volume of all drug groups was lower than that of the control group,suggesting that there was no obvious rebound after drug withdrawal.3.The icotinib combined with anti-angiogenic drug groups had a stronger inhibitory effects on angiogenesis,and the microvessel density of tumor tissues was significantly lower than that of the anti-angiogenic drug group(bevacizumab or recombinant human endostatin).4.There was no significant difference in the microvessel density of the heart and brain tissues in the combined treatment groups and the anti-angiogenic groups alone compared with other groups.However,the hepatic microvessel density was significantly lower than that of the icotinib group and the control group.In addition,the combined treatment groups had a stronger effect on reducing liver MVD.This difference disappeared soon after drugs withdrawal.5.Icotinib alone has no effect on MVD of tumor tissues,but it has synergistic effect on inhibiting angiogenesis after combined with anti-angiogenic drugs and can significantly down-regulate VEGFA expression,which may be the main mechanism of stronger anti-angiogenic effect in the combined treatment groups.6.Adverse reactions:The side effects of icotinib combined with anti-angiogenic drugs were not significantly increased compared with the single drug group,and the safety was good.Part ⅡThe Clinical Efficacy Observation of Combined low-dose Bevacizumab after Slow Progression of First-line Targeted Drugs in EGFR Mutant NSCLCObjective:Epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)is recommended as the first-line treatment for non-small cell lung cancer with EGFR mutations.However,secondary drug resistance will appear after application.This article aims to explore the efficacy and safety of EGFR-TKI combined with low-dose bevacizumab in the presence of disease progression.Methods:1.Research objects:A total of 81 patients with EGFR(+)advanced non-small cell lung cancer who underwent initial first-line EGFR-TKI therapy were enrolled from January 2017 to December 2021 in Shandong tertiary-grade A class hospital.Specific inclusion criteria are as follows:(1)Non-small cell lung cancer patients with EGFR gene sensitive mutations before first-line EGFR-TKI treatment;(2)All patients were confirmed to have slow disease progression by chest and abdomen CT,cranial MR,bone ECT or PET-CT,and the specific criteria were:①Disease control ≥6 months after application of targeted drugs;②Tumor burden slightly increased compared with before;③Symptom score ≤1 point(5 clinical symptoms related to lung cancer:cough,hemoptysis,chest pain,dyspnea and fever;1 clinical manifestation related to metastasis;0 point for asymptomatic,1 point for stable,2 point for any worsening or new symptoms);(3)All patients had a PS score of ≤2 and at least one measurable lesion according to RECIST 1.1 criteria;(4)≥18 years old;(5)T790M in tissues or peripheral blood of all enrolled patients was negative or the mutation status was unknown;(6)All patients have a survival period of more than 3 months;(7)There are no contraindication of bevacizumab application;2.Treatment:Eighty-one patients with slow progression continued to receive the original targeted drug combined with bevacizumab 7.5mg/kg every 3 weeks until the targeted drug is terminated.There is no precise standard for when a patient needs to stop using the targeted drug.It is mainly decided by the clinician based on the overall clinical situation and the patient’s will.All patients were informed of their treatment plans and possible adverse reactions and signed the informed consent.During the treatment,blood routine examination,liver and kidney function,urine routine examination,electrocardiogram,etc.,were reviewed every 3 weeks,and tumor changes were evaluated every 6 weeks(chest enhanced CT,abdominal ultrasound,brain MR,bone ECT,etc.).At the same time,adverse reactions such as hypertension,nausea and vomiting,hemoptysis,hematuria,cardiac complications were recorded.3.Clinical indicators:(1)Primary outcomesProgression-free survival 2(PFS2):the time from the beginning of the application of targeted drugs until the targeted drugs are terminated after the addition of bevacizumab(clinicians comprehensively judge whether to discontinue EGFR-TKIs).(2)secondary outcomesProgression-free survival 1(PFS1):the time from first-line application of targeted drugs to the first occurrence of disease progression(PD)defined by RESIST 1.1 or death.Others:Objective remission rate(ORR),Disease control rate(DCR),etc.4.Adverse reactions:The adverse reactions were recorded and graded according to the NCI-CTCAE 4.0.Results:1.The median progression-free survival 1(PFS1)of first-line EGFR-TKI was 12 months.Among the 81 patients,there were 4 CR patients,accounting for 4.94%,63 PR patients,accounting for 77.78%,and 14 SD patients,accounting for 17.28%.ORR 82.72%,DCR 100%.In subgroup analysis,the median PFS1 of gefitinib,erlotinib and icotinib were 10 months,12 months,and 14 months,respectively.2.The median progression-free survival 2(PFS2)was 16 months after EGFR-TKI combined with bevacizumab.In subgroup analysis,the median PFS2 of gefitinib,erlotinib and icotinib were 15 months,17 months,and 20 months,respectively.3.At the data deadline(October 2022),all subjects had an OS of 8-82 months,with a median OS of 30 months.In subgroup analysis,the median OS of gefitinib,erlotinib and icotinib were 30 months,33 months,and 30 months,respectively.4.Adverse reactions:(1)The most common adverse reactions were rash(55.56%),hypertension(38.27%),diarrhea(30.86%),proteinuria(18.52%)and liver dysfunction(16.05%).Among them,most of the adverse reactions were grade 1-2,which could be improved after symptomatic treatment.(2)There were 23 cases(28.40%)of bleeding related events(hematuria,nasal hemorrhage,hemoptysis,gingival hemorrhage),all of which were grade 1-2 and improved after symptomatic treatment.(3)A few patients showed grade 3 adverse reactions,including 5 cases of hypertension(6.17%),3 cases of rash(3.7%),1 case of proteinuria(1.23%),1 case of liver dysfunction(1.23%),1 case of diarrhea(1.23%),and 1 case of leucopenia(1.23%).After symptomatic treatment,they were all improved and did not affect follow-up treatments.(4)No grade 4 adverse reactions were recorded in all patients.Conclusion:After the slow progress of the first-line application of targeted drugs,the combination of anti-angiogenic drugs can further delay tumor growth and the time of salvage chemotherapy,while the side effects are within a controllable range,which is worthy of clinical application.However,large-scale prospective research is still needed for further verificationPart Ⅲ:OverviewProgress of Anti-angiogenic Drugs Combined with EGFR-TKI in Advanced Non-small Cell Lung CancerAntiangiogenic drugs combined with chemotherapy have been proven effective in the treatment of advanced non-small cell lung cancer,and EGFR-TKIs have been the standard first-line treatment for lung cancer patients with EGFR mutations.In recent years,studies have confirmed that anti-angiogenic drugs combined with EGFR-TKIs can further improve the therapeutic effect of these patients.At the same time,a variety of anti-angiogenic drugs are emerging,making it difficult for physicians to choose in clinical practice.Based on the current research status,this paper reviewed a number of large-scale clinical studies,and systematically discussed the clinical efficacy and safety of anti-angiogenic drugs combined with EGFR-TKIs.It is expected to provide help for the selection and precise treatment of patients with advanced non-small cell lung cancer. |
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