| Part ⅠEfficacy of Combined Icotinib and Pemetrexed in EGFR Mutation-Positive Lung Adenocarcinoma XenograftsBackgroundAt present,the prevalence and mortality of lung cancer rank first in all malignant tumors worldwide.Approximately 80-85%of lung cancers are non-small-cell lung cancer(NSCLC),and more than half of the patients diagnosed with NSCLC are in advanced stage of diseases.Adenocarcinoma is the most frequent histological subtype,and accounts for more than 50%of all NSCLC cases.Historically,Platinum doublet chemotherapy was main treatment method for all patients with advanced NSCLC.However,its efficacy seems to have reached a platform,with a median survival of approximately 8-10 months.With the continuous development of tumor molecular biology,molecular targeted drugs based on the development of key carcinogenic driving genes have been successively introduced,which has completely changed the clinical practice of advanced NSCLC,especially in lung adenocarcinoma.Epidermal growth factor receptor(EGFR)gene mutations represent the most prominent molecular subsets of NSCLC and occur mainly in patients with lung adenocarcinoma,which are also important biomarkers for predicting therapeutic response.Several large clinical studies have demonstrated epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)targeting EGFR are significantly superior to chemotherapy as first-line treatments,showing improved overall response rate and progression-free survival(PFS)in patients with advanced NSCLC harboring EGFR mutations.Therefore,EGFR-TKIs have been widely recommended as the initial therapy for patients with EGFR mutation-positive.Nevertheless,acquired EGFR-TKIs resistance and eventually occurs in most of these patients.How to make the patients further benefited from EGFR-TKIs treatment and delay drug resistance has become an urgent problem in clinical practice.In addition,due to the heterogeneity of malignant tumors,there is a limitation in the single treatment.Therefore,the combination of EGFR-TKIs and chemotherapy with different machanisms of action proposed by many scholars might play a role in further improving the efficacy and delaying drug resistance.However,the conclusions of these related studies are inconsistent,and there are still some controversies such as whether this strategy really brings survival benefits to patients with NSCLC harboring EGFR mutations,what kind of chemotherapeutic drugs should be combined with EGFR-TKIs and what combination mode should be used.In recent years,the antitumor activity and mild toxicity of pemetrexed make it a research hotspot for combination with targeted therapies in patients with nonsquamous NSCLC.A number of in vitro and clinical studies have shown the synergistic effects on EGFR-mutant NSCLC using EGFR-TKIs combined with pemetrexed.However,It is still need to further explore the optimal administration schedule and the mechanism of interaction between the two classes of drugs in vivo.Icotinib is the first generation of small molecule EGFR-TKI,which has a shorter half-life and better fat solubility and safety compared with gefitinib and erlotinib.There are no research reports on the anti-tumor effect of icotinib combined with pemetrexed in EGFR mutation-positive lung adenocarcinoma xenograft models.ObjectiveTo investigate the anti-tumor activity and potential mechanisms of different combined modes of icotinib plus pemetrexed in EGFR mutation-positive lung adenocarcinoma nude mice xenograft models.Methods1.Establishment of xenograft models:36 female BALB/c-nu nude mice aged 4-6 weeks were selected and fed freely in SPF environment.Human lung cancer adenocarcinoma HCC827 cells suspension bearing EGFR mutation was subcutaneously inoculated into the right axillary region of nude mice.The modles were successfully established when tumors reached a mean volume of 150mm3.2.Experimental grouping and dosage regimensThe successfully established nude mice models were randomly divided into six groups with six mice per group.The treatment plans for each of the groups were as follows and administered for a total of three weeks.Pemetrexed and icotinib in all the combined groups were used at the same dosage as in the single drug groups.Before each administration,icotinib was prepared by dissolving in 0.5%sodium carboxymethyl cellulose,and pemetrexed was first diluted in normal saline.(1)The Control group(Control):0.5%sodium carboxymethyl cellulose was administered at a dose of 0.01 ml/g daily,five days a week.(2)Icotinib(Ico)group:icotinib was orally administered to the mice at a dose of 60 mg/kg daily from days 1 to 5,five times per week.(3)Pemetrexed(Pem)group:pemetrexed was intraperitoneally administered the mice at a dose of 250 mg/kg once a week.(4)Sequential pemetrexed followed by icotinib(Pem-Ico)group:pemetrexed was intraperitoneally administered on day 1 and then icotinib was orally administered from days 2 to 6 per week.(5)Sequential icotinib followed by pemetrexed(Ico-Pem)group:icotinib was orally administered from days 1 to 5 and then pemetrexed was intraperitoneally administered on day 6 per week.(6)Concurrent icotinib and pemetrexed(lco+Pem)group:icotinib was orally administered from days 1 to 5 and concurrent pemetrexed was intraperitoneally administered on day 1 per week.3.The growth curves of the tumor xenografts,the tumor growth inhibition rate and the coefficient of drug interactionThe body weights and tumor volumes of the mice were monitored every three days,and the growth curves of the tumor xenografts were then drawn according to time in all groups.The mice were sacrificed by cervical dislocation at the end of the 21st day.The tumor xenografts were collected by dissection and subsequently weighed.Half of the tumors were refrigerated at-80℃,while the other half were embedded in paraffin for subsequent experiments.The tumor growth inhibition rate(TGIR)was assessed using the formula:[1-(mean tumor weight of the treatment group/mean tumor weight of the control group)]×100%.The coefficient of drug interaction was determined by the formula:CDI=AB/(A×B),where AB is the ratio of average tumor weight between the combined group and the control group,and A or B is the ratio of average tumor weight between the single drug group and the control group.The synergistic effect produced by the combination of the two drugs is judged by CDI<1.4.HE staining was used to observe the histological changes in each group of tumor xenografts.5.Immunohistochemistry was used to detect the expression of CD34,PCNA,Ki-67 and caspase-3 proteins in each group of tumor xenograft tissues.Microvessel density(MVD)of each group were evaluated with CD34 positive cell counting.The proliferation and apoptosis index of tumor cells were assessed by PCNA,Ki-67 and caspase-3 scores respectively and expressed as the percentage of positive cells among all tumor cells.6.Western blotting was used to detect the expression of TS,EGFR,phospho-EGFR,AKT,phospho-AKT,MAPK and phospho-MAPK in each group of tumor xenograft tissues.7.Adverse reactions were evaluated by monitoring the weight and behavior changes of nude mouse in each group.Results1.Transplanted tumor growth and tumor inhibition rateWith the extension of time,The tumor xenograft volumes of the control group gradually increased,while tumor xenograft growth in the Pem,Ico,and Ico-Pem groups slowed.In contrast,decreased tumor volumes were observed in the Pem-Ico and Ico+Pem groups.At the end of the 21st day,the tumor volumes in the Pem-Ico(62.38±30.23 mm3)and Ico+Pem(73.69±30.73 mm3)groups were significantly smaller than those of the control(689.12±253.43 mm3),Pem(393.64±154.73 mm3),Ico(236.56±121.95 mm3),and Ico-Pem(149.33±65.11 mm3)groups(P<0.05).The tumor volumes of the Ico and the Ico-Pem groups were smaller than those of the Pem group(P<0.05),while the tumor volumes of the Pem group were smaller than those of the control(P<0.05).However,there were no significant difference in the xenograft volumes between the Pem-Ico group and the Ico Pem group or the Ico group and the Ico-Pem group(P>0.05).The tumor inhibition rate among the five treatment groups in ascending order were:Pem(41.5%%),Ico(68.3%%),Ico-Pem(73.2%),Ico+Pem(85.4%),and Pem-Ico(87.8%).The CDI of the pem-ico group and Ico+Pem group was 0.66 and 0.79,respectively.The results suggest that modes of sequential pemetrexed followed by icotinib and concurrent combinations of icotinib and pemetrexed can significantly inhibit the growth of xenografts compared with icotinib or pemetrexed alone and produce the synergistic anti-tumor effects,while sequential icotinib followed by pemetrexed has no obvious additive effect on inhibiting the growth of xenografts compared with icotinib alone.2.Histological morphology of xenograftsIn the Control group,tumor cells obviously proliferated and closely arranged,and the mitotic figures were more common with less tumor stroma.After treatment,the contents of the tumor cells were reduced and the fibrous tissue increased among the five drug treatment groups,particularly in the Pem-Ico and Ico+Pem groups.3.Immunohistochemical results of microvascular density in xenograftsThe MVD among the six groups was significantly different(F=86.49;P<0.05).First,the MVDs in the Ico(25.62±2.35),Pem-Ico(18.02±2.94),Ico+Pem(1 9.93±2.14),and the Ico-Pem(26.72±2.32)groups were significantly lower than the control(39.38±1.49)and Pem groups(36.85±2.29)(P<0.01).Furthermore,the Pem-Ico and Ico+Pem group values were significantly lower than those of the Ico and Ico-Pem groups(P<0.01).However,the MVD between the three paired groups(Pem-Ico vs.Ico+Pem,Ico vs.Ico-Pem,and Pem vs.the control)were not significantly different(P>0.05).These findings showed the significant enhanced effect of MVD inhibition in the modes of sequential pemetrexed followed by icotinib and concurrent icotinib combined with pemetrexed treatments,while sequential icotinib followed by pemetrexed treatment did not show this effect.4.Immunohistochemical results of proliferation and apoptosis index in xenograftsThere were significant decreases in the proliferation indices by PCNA and Ki-67 scores and an increase in the apoptosis index by caspase-3 score in the five drug treated groups compared to the control.The proliferation indices were lower and the apoptosis index was higher in the Pem-Ico,Ico+Pem,Ico,and Ico-Pem groups than in the Pem group(P<0.01).The proliferation indices of the Pem-Ico and Ico+Pem groups were significantly lower than those of the Ico and Ico-Pem groups;by contrast,the apoptosis index of the Pem-Ico and Ico+Pem groups were significantly higher than those of the Ico and Ico-Pem groups(P<0.01).No significant differences were identified between the Pem-Ico and Ico+Pem groups or the Ico and Ico-Pem groups(P>0.05).These results showed significant enhanced antiproliferative and proapoptotic activity in the administration of sequential pemetrexed followed by icotinib and concurrent icotinib combined with pemetrexed but no in sequential icotinib followed by pemetrexed treatment.5.Western blotting resultsThe expression levels of TS in Ico group and Pem group were lower than those in Control group(P<0.05),whereas the expression levels of TS in Pem-Ico group and Ico Pem group were significantly lower than those in other groups(P<0.05).A decrease in the expression levels of phospho-EGFR,phospho-AKT and phospho-MAPK was detected among icotinib containing treatments,with the most significant reductions in the Pem-Ico and Ico+Pem groups(P<0.05).In addition,there were no significant differences in TS,phosphoEGFR,phospho-AKT,and phospho-MAPK expression levels between the Ico and Ico-Pem groups or the Pem-Ico and Ico+Pem groups(P>0.05).Compared with the Control group,the phospho-EGFR levels were slightly increased and the phospho-AKT levels were slightly decreased in the Pem group with no significant difference(P>0.05),while the phospho-MAPK was not affected(P>0.05).These results suggested that the mode of sequential pemetrexed followed by icotinib and concurrent icotinib combined with pemetrexed treatments dramatically enhanced the inhibition of TS expression and phosphorylation of the EGFR signaling pathways.6.Changes in the body weight of nude mice before and after treatment were not significant(P>0.05).The activity and eating behavior of nude mice were not significantly affected during the experiment.Conclusion1.Both modes of sequential pemetrexed followed by icotinib and concurrent combinations of icotinib and pemetrexed showed synergistic growth inhibition effects in EGFR mutation-positive lung adenocarcinoma HCC727 cell line nude mice xenograft models,with significantly better effect than icotinib or pemetrexed alone.But the mode of sequential icotinib followed by pemetrexed had no obvious advantage over icotinib monotherapy on the tumor growth inhibition.2.Both modes of sequential pemetrexed followed by icotinib and concurrent combinations of icotinib and pemetrexed showed significant enhancement of reducing the microvascular density,inhibiting the proliferation of tumor cells,and promoting the apoptosis of tumor cells in xenografts,thus produced better anti-tumor activity.However,the above enhancement was not demonstrated in the mode of sequential icotinib followed by pemetrexed.3.The enhancement of inhibitory effects on TS expression and EGFR signaling pathway activation in tumor xenograft tissues might explain the synergistic anti-tumor activity which produced by sequential pemetrexed followed by icotinib and concurrent combinations of icotinib and pemetrexed from the molecular mechanism.Part Ⅱ Efficacy of Icotinib Combined with Chemotherapy in Untreated Patients with EGFR Mutation-Positive Advanced Lung AdenocarcinomaBackroundNon-small-cell lung cancer(NSCLC)is one of the leading causes of deaths from cancer worldwide,with lung adenocarcinoma being the most common histological subtype.Due to the absence of symptoms in early stages and the lack of efficient screening,more than 65%of NSCLC patients are diagnosed in advanced stage.Platinum-based chemotherapy has been proven to provide a survival benefit for patients with advanced NSCLC.However,more than half of the patients cannot survive to 1 year,as most tumor cells easily acquire resistance to chemotherapeutic drugs.In the past decade,molecular targeted therapy based on specific driver mutations has significantly improved the therapeutic status of NSCLC,especially lung adenocarcinoma.Among them,the EGFR gene mutation is researched on the most mature and most widely recognized.Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)targeting EGFR mutation have been demonstrated to provided better clinical efficacy than chemotherapy as first-line therapy,and recommended as the initial treatment for advanced lung adenocarcinoma patients with sensitizing EGFR mutation.However,most such patients will usually develop aquired resistance around within 12 months after receiving the first or second generation of EGFR-TKIs treatment.Although the third generation of EGFR-TKIs such as osimertinib has been shown prolonged progression-free survival(PFS)compared with two previous in first-line application,those patients who were treated with osimertinib aslo inevitably develop resistance.Therefore,the main issue in the treatment of EGFR-mutant lung adenocarcinoma using TKIs is how to retard or prevent TKIs resistance,so as to prolong PFS and overall survival(OS)of these patients.Several studies have been conducted to explore whether combination of EGFR-TKIs and chemotherapy as first-line treatment can improve the survival benefit of advanced patients with lung adenocarcinoma and EGFR mutation.Although previous studies suggested that gefitinib or erlotinib combined with platinum-containing chemotherapy could prolong PFS and OS of these patients,the results were not completely consistent because of different design schemes.Whether combination therapy can improve the efficacy of EGFR-TKIs in first-line therapy and become a better initial treatment strategy still needs to be further explored.Icotinib is a first generation of EGFR-TKI and has shown similar efficacy and better safety comparable to gefitinib.Platinum combined with pemetrexed is a first-line standard chemotherapy regimen for lung adenocarcinoma.There were no prospective and multicenter studies to investigate whether combination of icotinib and standard chemotherapy as first-line treatment can also improve the PFS and OS of patients with EGFR mutation-positive advanced lung adenocarcinoma.ObjectiveTo explore the efficacy and safety of combination of icotinib and chemotherapy as first-line therapy versus icotinib alone for patients with EGFR mutation-positive advanced lung adenocarcinoma.Methods1.Study objectsUntreated patients diagnosed as EGFR mutation-positive advanced lung adenocarcinoma in 10 general hospitals of Shandong Province from January 16,2014 to December 31,2016 were included.2.Study design(1)General data of all enrolled patients were recorded at baseline.Eligible patients were randomly assigned to either the combination of icotinib and standard chemotherapy treatment group(referred to as the combination group),and to the icotinib alone group(referred to as the icotinib group).The combination group received the intercalated combination of sequential icotinib following chemotherapy,that was,patients first received pemetrexed plus carboplatin on the first day of each cycle and then oral icotinib on days 2-19 until two days before the start of the next chemotherapy.This treatment was repeated every 21 days for four to six cycles,and patients who did not experience cancer progression were maintained with oral icotinib.The icotinib group received oral icotinib monotherapy daily.All therapies were continued until disease progression,unacceptable toxicity or death.(2)If disease progression was observed,the second-line treatment was selected by the attending physician.Three main strategies were used:1)testing for the T790M mutation of the EGFR gene with peripheral blood specimen was performed,and patients positive for the mutation received oral osimertinib;2)patients without the T790M mutation received chemotherapy,radiotherapy or a topical treatment(such as radiofrequency ablation,microwave ablation,etc.);and 3)in patients without T790M mutation detected,some with slow or local progression continued to be treated with oral icotinib or along with topical treatment until the attending physician thought icotinib should be discontinued,and others received chemotherapy,radiotherapy or a topical treatment.3.Outcome measures(1)The primary endpoint in this trial was PFS.(2)Secondary endpoints included OS,the objective response rate(ORR),the disease control rate(DCR)and the incidence of adverse reactions.The PFS subgroup analysis was conducted according to gender,age,smoking status,TNM stage,ECOG PS,EGFR mutation type and serum lung cancer tumor markers.Results1.Patients enrollment and follow-up situationA total of 179 patients with advanced lung adenocarcinoma and EGFR mutation positive were enrolled in this study,including 90 cases in the combination group and 80 cases in the icotinib group.There were six patients in the combination group and eight patients in the ecotinib group dropped out of the study.The attrition rates in the combination group and icotinib group did not differ statistically(6.7%vs.9.0%;χ2=0.335,P=0.563).Finally,84 patients in the combination group and 81 patients in the icotinib group were included in the efficacy analysis;85 patients in the combination group and 82 patients in the icotinib group were included in the safety analysis.The follow-up was up to May 23,2018.The shortest follow-up time was 17 months,and the longest follow-up time was 53 months.Sixty-four patients(76.2%)in the combination group experienced disease progression,whereas 20 patients(23.8%)had not by the end of the study.Seventy-two patients(88.9%)in the icotinib alone group experienced disease progression,whereas nine patients(11.1%)had not by the end of the study.There were not statistically significant difference between second-line treatment regimens of the two groups after disease progression(all P>0.05).2.Survival analysisThe median PFS of the combination group was significantly longer than of the icotinib group(16.0 months vs.10.0 months,HR=0.60,95%confidence interval[CI]0.42-0.85;χ2=8.037,P=0.005).Subgroup analyses showed a statistically significant improvement in PFS with combination therapy versus icotinib alone in the following subgroups:male patients aged>65 years,patients who had ever smoked,patients with stage IV disease,patients with an ECOG PS of 1 point,patients with an EGFR exon-21 mutation,patients with normal level of serum lung cancer tumor markers,and patients with elevated levels of other tumor markers but a normal or elevated level of CEA(all P<0.05).In the combination group,41 patients(48.8%)died after disease progression and 43 patients(51.2%)remained alive at the end of the study.By comparison,in the icotinib group,52 patients(64.2%)died after disease progression,and 29 patients(35.8%)were alive.The mortality rate in the combination group was lower than in the exitinib group(48.8%vs.64.2%,χ2=3.970,P=0.046).However,there was no statistically significant difference in median OS between the two groups(36.0 months vs.34.0 months,HR=0.77,95%CI 0.51-1.17;χ2=1.455,P=0.288).3.Reponse rateORR and DCR for the combination group were significantly higher than those for the icotinib group(83.3%vs.70.3%,χ2=3.909,P=0.048;96.4%vs.87.7%,χ2=4.374,P=0.036).4.The incidence of adverse reactionsThe incidence rates of leukopenia in the combination group was significantly higher than the single drug group(40.0%vs.7.3%,χ2= 24.475,P<0.001),and the incidence rates of grade 3-4 leukopenia in the combined group was higher than the icotinib group(10.6%vs.0%,χ2=7.218,P=0.007).The incidence rates of elevated ALT or AST level in the combination group was significantly higher than the icotinib group(49.4%vs.20.7%,χ2=15.026,P<0.001),and the incidence rates of grade 3-4 elevated ALT or AST level in the combined group was higher than the icotinib group(11.8%vs.2.4%,χ2=5.442,P=0.02).The total incidence rates of anemia,anorexia,fatigue,nausea and hair loss in the combination group was significantly higher than that in the icotinib group(all P<0.01),but the difference in the incidence of grade 3-4 of these adverse effects between the two groups were not statistically significant(all P>0.05).There were no statistically significant difference in the total incidence rates of thrombocytopenia,elevated total bilirubin,vomiting,rash,diarrhea and oral mucositis between the two groups(all P>0.05).One patient in each group withdrew from the study due to grade 3 rash,and other patients recovered after symptomatic treatment and drug dose reduction when adverse reactions occurred and received subsequent treatments.No cases of interstitial pneumonia or renal dysfunction occurred in the two groups,and no drug-related deaths were observed in either group.Conclusion1.Compared with icotinib alone treatment,the intercalated combination of icotinib and chemotherapy as first-line therapy significantly improved the PFS,ORR and DCR,but not OS in untreated patients with EGFR mutation-positive advanced lung adenocarcinoma.2.The subgroup analysis of PFS suggested that the patients who were male,aged>65 years,had ever smoked,had stage IV disease,had an ECOG PS of 1point,had an EGFR exon-21 mutation,showed normal levels of serum lung cancer tumor markers,or showed elevated levels of other tumor markers but a normal or elevated level of CEA might benefit more from the combined treatment.3.In terms of safety,the incidence of leukopenia and liver function damage of grades 3 or above was greater with the combination of icotinib and chemotherapy than with icotinib alone,but these adverse effects could be tolerated and managed. |
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