An Exploratory Study Of Chidamide Combined With VDDT In The Treatment Of Relapsed Or Refractory Diffuse Large B-cell Lymphoma

BackgroundDiffuse large B-Cell Lymphoma(DLBCL)is the most common subtype of non-Hodgkin lymphoma(NHL)with strong heterogeneity in clincopathological features,treatment response and prognosis,accounting for 30%to 40%of adult NHL.Before the advent of rituximab,anthracycline-based regimen CHOP was used as the first-line therapy for DLBCL patients and one-third of patients achieve long-term survival.Following the emerging of rituximab,its combination with CHOP significantly improved the long-term survival rate.However,approximately one-third of patients remained to develop relapsed or refractory disease and succumbed to it.It becomes a great challenge to improve the outcome of relapsed or refractory patients.The deep understanding to the pathogenesis and advance in the technology develop many potential anti-tumor drugs.Some of them have shown efficacy in relapsed or refractory DLBCL.Chidamide is a new generation of histone deacetylase inhibitors(HDACI),originally designed and developed in China.Chidamide increases histones and non-histones acetylation levels,promoting the expression of tumor suppressor genes,cell cycle arrest,apoptosis,and anti-tumor effect of tumor microenvironment.At present,it has shown a favorable efficacy and has been approved for the treatment of peripheral T cell lymphoma.However,the efficacy of HDACI monotherapy in DLBCL is limited.Mocetinostat alone,a kind of HDACI,induced the objective response rate of 17%,the stable disease rate of 32%,and the total disease control rate of 49%in patients with relapsed or refractory DLBCL.In the exploratory study of single-drug chidamide from China,3 patients were enrolled with DLBCL with 2 stable diseases and 1 progression disease.in a similar study from Japan,2 patients were enrolled with 1 stable disease and 1 progression disease.In order to raise the efficacy of montherapy,this prospective,one-arm,exploratory clinical trial evaluated the safety and efficacy of chidamide combined with vinorelbine,liposomal doxorubicin,dexamethasone,and thalidomide(VDDT)regimen in relapsed or refractory patients with DLBCL.The relationship between efficacy and driver gene mutations was explored,either.Concurrently,DLBCL cell lines were used to confirm the relation of chidamide effect with driver gene mutations.Part 1:A Prospective,Single Arm Clinical Study on Chidamide Combined with VDDT in the Treatment of Relapsed or Refractory Diffuse Large B-cell LymphomaObjectiveThis prospective,one-arm,exploratory clinical study on chidamide combined with VDDT regimen evaluated the efficacy and safety in the treatment of relapsed or refractory DLBCL,as well as relationship between the efficacy and driver gene mutations.Methods1.This study was a prospective,single arm,exploratory study(clinicaltrials.gov,clinicaltrial no.NCT02733380).All enrolled patients met the inclusion and exclusion criteria.They were diagnosed based on the 2008 WHO classification of tumours of haematopoietic and lymphoid tissues,and all patients had measurable lesions.The dosage and treatment cycle of VDDT regiment were adjusted according to the protocol design.We summarized and analyzed clinicopathological characteristics,molecular biomarkers,and their association with clinical efficacy.2.The treatment efficacy was evaluated according to the International Working Group(IWG)criteria.3.Safety was evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events(NCI CTCAE V4.03).4.93 hot-spot driver genes were detected with tumor tissues using next generation sequencing.5.SPSS software was used for statistical analysis.Results1.8 patients were included in this study,including 3 males and 5 females,with median age of 53(44-73)years and median treatment lines of 2.5(2-5).2.All of patients did not complete treatment as planned,with a median of 2(1-6)cycles.2 patients terminated the treatment due to disease progression,and 6 patients due to adverse events,and none died from treatment.Efficacy analysis showed that disease control rate was 75%,including the partial remission rate of 25%,and the stable disease rate of 50%;the progression disease rate was 25%.Median follow-up time was 178(30-427)days,with 7 cases disease progression and related death.The median PFS and OS was 36(26-150)and 178(30-427)days,respectively.3.The safety analysis showed that the incidence of grade 1-4 hematological adverse events was 100%,including 100%leukopenia,87.5%neutropenia,75%thrombocytopenia,and 62.5%anemia.The incidence of grade 3-4 hematological adverse events was 75%,including 62.5%leukopenia,75%neutropenia,and 37.5%thrombocytopenia.The incidence of grade 1-4 non-hematological toxicity was 87.5%,including 75%fatigue,75%gastrointestinal toxicity,62.5%liver toxicity,37.5%electrolyte disorder,37.5%pain,and 12.5%infection.The incidence of grade 3 non-hematological adverse events was 12.5%,presenting as fatigue.There were no grade 4 non-hematological adverse events.3.Driver gene mutations were examined in 5 of the 8 patients.The number of the median mutation genes was 6(3-10),and negatively correlated with efficacy(r=-0.913,P=0.03).23 genes mutations were involved.Gene mutations with high frequency included CD79B(60%,3/5),TNFRSF14(40%,2/5),TP53(40%,2/5),and PIMI(40%,2/5).CD79B and TNFRSF14 genes were negatively correlated with efficacy,while TP53 and PIM1 genes did not present correlation.The epigenetic regulatory genes included histone-methylation genes KMT2D/MLL2,SETD2,KMT2A,EZH2,IDH2 and TET2;histone-acetylation genes EP300 and CREBBP;DNA-methylation genes DNMT3A and chromatin-remodeler gene ARID1B.1 patient had EP300 mutation and1 patient had KMT2D/MLL2 mutation.Other epigenetic regulatory genes were not detected.EP300 and KMT2D/MLL2 mutations were not associated with efficacy.Conclusion1.The efficacy of chidamide combined with VDDT is better than chidamide monotherapy in the treatment of relapsed or refractory DLBCL,but the toxicity is not tolerable.The combination regimen should be adjusted.2.The driver gene mutations in patients with relapsed or refractory DLBCL were complicated.Higher gene mutation number,CD79B and TNFRSF14 mutation are related to poor efficacy.The epigenetic regulatory gene mutations have no connection with response to chidamide.Part 2:In-vitro Study on Correlation between Efficacy of Chidamide and Driver Gene MutationsObjectiveTo testify the correlation between the efficacy of chidamide and driver gene mutation in DLBCL cell lines.Methods1.DLBCL cell lines OCI-Ly3,OCI-Ly7,OCI-Ly8,Dohh2,Ros50 and Val were selected to use after recovery,culture and passage.2.Flow cytometry was used to detect the chidamide-induced apoptosis.3.93 hot-spot driver genes was detect using next-generation sequencing in six DLBCL cell lines.Results1.The apoptosis rates among six DLBCL cell lines were significantly different after treatment of 10?mol/L chidamide for 48 hours with OCI-Ly7 of 83.22%,Ros50 of 76.97%,OCI-Ly8 of 68.92%,OCI-Ly3 of 21.78%,Dohh2 of 2.73%and Val of 2.18%.2.The number of gene mutations among six DLBCL cell lines were different,including 8 in OCI-Ly7,13 in Ros50,22 in OCI-Ly8,12 in OCI-Ly3,37 in Dohh2 and 37 in Val.Negative correlation were observed between the gene mutation number and apoptosis(r=-0.812,P=0.05).30 genes were mutated in six DLBCL cell lines.The most common genes were MYC and BCL2,and double mutation of MYC and BCL2 was not correlated with apoptosis.CD79B mutation was found in Dohh2 and Val cells and was negatively correlated with apoptosis(r=-0.828,P=0.042),while TNFRSF14 gene was not found.EP300 mutation was observed in OCI-Ly3.CREBBP mutation was found in OCI-Ly8,Dohh2 and Val cells.KMT2D/MLL2 mutation was found in OCI-Ly3,Dohh2 and Val cells.EZH2 mutation was found in Dohh2,Ros50 and Val cells.ARID1B mutation was found in Ros50 cells.Other epigenetic regulatory gene mutations were not observed.EP300,CREBBP,EZH2 and ARID1B genes mutations were not associated with apoptosis,whereas KMT2D/MLL2 mutation was related to poor response to chidamide(r=-0.878,P=0.021).ConclusionChidamide-induced apoptosis is negatively correlated with the mutation number and CD79B mutation.EP300,CREBBP,EZH2 and ARID1B mutations are not related to chidamide efficacy,while KMT2D/MLL2 mutation indicates negative relavance.General Conclusion1.The efficacy of chidamide combined with VDDT is better than chidamide monotherapy in the treatment of relapsed or refractory DLBCL,but the toxicity is not tolerable.The combination regimen should be adjusted.2.The driver gene mutations in patients with relapsed or refractory DLBCL were complicated.Higher gene mutation number,CD79B and TNFRSF14 mutation are related to poor efficacy.The connection of epigenetic regulatory gene mutations with response to chidamide is not determined.