| Introduction Mantle cell lymphoma(MCL)is a phenotypically and genetically heterogeneous malignancy in which the genetic alterations determining clinical behavior are not fully understood.The genetic heterogeneity in MCL motivates us to define different genetic subsets,to delineates clonal evolution patterns and their impacts on clinical outcomes.Methods We performed whole-exome sequencing(WES)on 152 DNA samples derived from 134 MCL patients.This cohort includes 123 untreated and 11 relapsed patients,in which 18 with longitudinal samples.95 patients received high dose cytarabine-based immunochemotherapy from BDH-MCL01 clinical trial(NCT02858804).89 samples with matched normal,115 samples derived from purified cryopreserved peripheral blood or bone marrow cells and 42 samples from FFPE sections.Within all these samples,48 with matched RNA sequencing(RNA-seq)data for which 42 from untreated and 6 from relapsed patients.We used GATK,MutSig2CV,and GISTIC2.0 to identify driver genetic lesions,applied MutationalPatterns to define mutational signatures,and utilized ABSOLUTE and PhylogicNDT to determine pattern of clonal evolution.We applied Non-negative matrix factorization consensus clustering to identify molecular subgroups and evaluated association between genomic alterations with clinical outcomes(PFS and OS).Results The median burden of non-silent mutations was 29 per sample(range 8-72).34 recurrently mutated genes were identified,containing previously reported driver mutations(ATM,TP53,NSD2,KMT2D,CCND1,SMARCA4,ARID1A,NOTCH2,NOTCH1,BIRC3,UBR5,TRAF2)and novel mutations(SP140,PCLO,SVEP1,LRP1B,PCDH10,LRP2,CTNNA2,VCAN,KMT2C,TACC2).7 copy number gain and 13 copy number loss regions were detected as recurrent somatic copy number alterations(SCNAs,frequency>10%,q<0.1).A multivariate Cox model of PFS and OS revealed that TP53 mutation/del(17p13),SP140 mutation/del(2q36),mutations in NOTCH1,PCDH10,and del(9p)remained prognostic value,independent of MIPI risk,IGHV mutation and other genetic alterations.We defined the clonal status of genetic alterations and clonal evolution pattern.Del(11q22)and del(9p)tend to be clonal while mutations in NSD2,LRP1B,CTNNA2 were more likely to be subclonal events(q<0.05).Clonality analysis enabled inference of temporal relationships between pairs of alterations.Then we further determined clonal evolution pattern by measuring the dynamic changes of fractions of cancer cells harboring each genetic alteration.11 of 16 longitudinal samples had extreme clonal evolution(69%,CCF change>0.5),4 with modest evolution(0.2 ? CCF change ? 0.5),and 1 without evolution(CCF change<0.2).Patients with extreme evolution had significantly inferior survival than with those with modest or no evolution(median survival from first sampling was 47.5 months vs not reached,p=0.041;from second sampling was 17.1 months vs not reached,p=0.023).We classified MCL into four subsets based on genetic alterations,each with distinct gene expression profiles and clinical behavior.Cluster 1(C1)was associated with leukemic non-nodal MCL while Clusters 2,3,and 4(C-4)were associated with classical MCL.Consistent with different cellular origins for different types of MCL,C1 was enriched for gene expression signatures of memory B cells and C2,C3,and C4 appeared to have a signature of CCR6 negative light zone B cell.1)C1(16.4%)was featured with mutated IGHV,negative expression of SOX11,CCND1 mutation,amp(11q13)as well as with active BCR signaling gene expression and an indolent clinical course.2)C2(23.1%)was enriched with del(11q22),del(1p21)and ATM mutation and harbored no TP53 mutation and del(17p).Patients in C2 were upregulated for gene expression in NF-?B and DNA repair pathways.3)C3(32.1%)was characterized with mutations in SP140,NOTCH1 and NSD2,del(6p)and amp(13q),and downregulation of gene expression in NF-?B,BCR signaling,MYC and inflammatory pathways.4)C4(28.4%)harbored del(17p),del(13q),del(9p)as well as mutations in TP53 and TRAF2.Interestingly,C4 was associated with a higher incidence of Blastoid or pleomorphic MCL(23.7%,p=0.016)and gene expression signatures of hyperproliferation and MYC pathway activation.Patients with the C1 subtype had a more favorable outcome than those with C2,C3,and C4 subtypes;the median PFS for the four subtypes were not reach,41.2,30.7,and 16.1 respectively.Differences in survival of patients whose diseases reflected each of the 4 subtypes also remained significant among patients who received the REDOCH/RDHAP regimen.Conclusions In summary,the integrative genomics and transcriptomics analysis described herein has greatly expanded our knowledge regarding the strong associations among genetic clusters,oncogenic pathways,and clinical outcomes.This study provides a framework to assess unappreciated genetic heterogeneity in the clinically defined subtypes of MCLs and forms the basis for designing precision therapies for aggressive MCL with genetic factors and oncogenic pathways as testable targets.Our dataset serves as a valuable genetic and transcriptomic resource for this rare and aggressive type of non-Hodgkin lymphoma.The outcome-associated genetic subsets will guide the choice of therapies in patients with the greatest need.Backgrounds Chronic lymphocytic leukemia(CLL)is one of the most common adult leukemia in the Western population but rare in Chinese descent.Recent reports indicated that Chinese CLL patients are younger and have distinct clinical courses including atypical immunologic features,higher frequency of mutated IGHV,and unique BCR stereotype,indicating there might be different genetic traits leading to CLL for Chinese and Western CLL patients.Genetic factors contributing to the development of CLL have been extensively investigated in the Western CLLs over the last decade,there are limited studies in the Chinese CLLs.Methods To define the mutation pattern in Chinese CLL samples,we started with a discovery cohort which includes 117 untreated and 9 relapsed CLL patients.We assessed 96 CLL driver genes that were assembled from two large scale whole-exome sequencing studies(Landau et al Nature 2015,Puente et al Nature 2015),by deep-targeted sequencing(Illumina MiSeq platform).We further validated the 9 recurrent mutated genes in a validation cohort that includes 212 CLL samples(188 derived from untreated and 24 from relapsed patients).Cytogenetic aberrations and IGHV mutation status were analyzed by FISH and Sanger sequencing,respectively.Results The most frequently mutated genes in untreated patients were MYD88(13%),ATM(11%),TP53(11%),NOTCH1(9%),SF3B1(9%),KMT2D(8%),BIRC3(6%),FBXW7(5%)and BRAF(3%).The most striking difference was the high frequencies of MYD88 and KMT2D mutations in Chinese CLL compared with western CLL.MYD88 mutations were detected in 38 of 303 newly-diagnosed patients(13%)but were not found in 35 patients with relapsed CLL.MYD88 mutant CLL had a significant male predominance with IGHV mutation,a great extent of monoclonal gammopathy and a higher frequency of hepatitis B-virus(HBV)infection.MYD88 mutations were predominantly in patients with IGHV VH3-7 and V3-23 use.Patients with MYD88 mutation had a longer TTT compared with those without mutation.However,MYD88 mutation lost its prognostic significance of TTT if only IGHV-mutated patients are considered.KMT2D mutations are rarely reported in Western CLL with a frequency<1%.We detected 30 KMT2D mutations in 24 untreated and 3 relapse patients with CLL.21/30(70%)were loss-of-function mutations.19 of 27 KMT2D mutations had IGHV mutation with over-representation of variable regions VH4-34 and VH4-39 and enrichment in stereotyped subset#8.We used an immune fluorescence assay to measure H3K4me3 in 3 KMT2D-mutated and 3 KMT2D wild-type CLL cells.KMT2D mutant cells had significantly less fluorescence intensity of H3K4me3 compared with wild-type CLL cells.Next,cells from patients with mutated(N=9)or wild-type KMT2D(N= 6)were treated with decitabine or chidamide alone,or combined with ibrutinib in the Cell Titer-Glo assay.KMT2D-mutated cells were more sensitive to decitabine,reduced sensitivity to ibrutinib and similar sensitivity to chidamide compared with KMT2D wild-type cells.Ibrutinib and decitabine were synergistic suggesting decitabine increases ibrutinib sensitivity in KMT2D-mutated CLL cells.The poor prognostic significance of KMT2D mutation is independent of known prognostic factors.Conclusions we observed high frequency mutations of MYD88 and KMT2D in Chinese CLL compared with European descent.MYD88-mutated CLL had a favorable outcome with male predominance,mutated IGHV status,and higher HBV exposure.KMT2D-mutated CLL showed impaired H3K4 methylation activity and increased sensitivity to decitabine in vitro.The addition of decitabine to ibrutinib may yield synergistic treatment for KMT2D-mutated CLL patients.KMT2D-mutated CLL patients were independently associated with progression adjusting for IGHV mutational status.The data help to explain some of the phenotype and genotype differences between Chinese and Western CLL. |
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