| Part 1 The clinical and biological features of 176 adult patients with mixed-phenotype acute leukemia:a single-center studyObjectivesThis study retrospectively analyzed the clinical and laboratory characteristics of adult patients with mixed-phenotype acute leukemia(MPAL),and compared the clinical,laboratory features and clinical outcomes between different subtypes of MPAL.Methods1.We collected the clinical and laboratory data of 247 potential MPAL patients admitted to our center from January 2006 to November 2019,and analyzed the clinical and laboratory characteristics of these patients diagnosed by different criteria.2.Based on the World Health Organization(WHO)2016 criteria,MPAL can be classified as five subtypes:t(9;22)(q34.1;q11.2)/BCR-ABL1(Ph+),t(v;11q23.3)/KMT2A-rearrangement(KMT2A-r),B/myeloid NOS(B/My),T/myeloid NOS(T/My),MPAL NOS rare types(B/T or B/T/My).This study focused on the clinical,genetic and survival characteristics of Ph+,T/My,B/My and B/T subtypes.Results1.Between 2006 and 2019,247 potential MPAL patients were identified in our institute,including 12 children and 235 adults.Among the adult cases,176 cases fulfilled the 2016 WHO classification with a median age of 48(range,16-87)years,50 cases fulfilled the EGIL criteria with a median age of 44(range,16-82)years,9 cases fulfilled neither of the criteria with a median age of 59(range,19-73)years.In the WHO group,t(9;22)(q34.1;q11.2)had a higher incidence(25.9%vs 10.9%,P=0.031)than that in the EGIL group.2.Of the 176 cases,T/My subtype was the most common(48.9%),followed by Ph+(23.9%),B/My(20.5%),B/T(4.0%),KMT2A-r(2.3%)and B/T/My(0.6%).The majority of T/My cases were male while female was more common in B/My.Ph+MPAL was significantly associated with higher white blood cell count and higher lactate dehydrogenase serum levels.According to FAB classification,the T/My MPAL mainly showed myeloid features including M1 and M2,while B/T MPAL showed lymphoid features including ALL and M0.The incidence of chromosomal abnormalities in the entire cohort was 63.6%,and complex karyotypes were more frequently observed in T/My MPAL than other subtypes.The Ph chromosome was the most frequently observed cytogenetic aberration.In 35.7%of Ph+cases,additional chromosomal abnormalities were detected.Of all cases,the incidence of T/M immunophenotype was similar to that of B/M but higher than that of B/T and B/T/M.3.Of the 98 patients received conventional induction chemotherapy,68 patients(69.4%)achieved CR after one course of combinational chemotherapy.In the studied cohort,there was no significant difference in CR rates between ALL-type(85.7%),combined regimens(69.5%),CAG regimens(75.0%)or AML-type(46.2%).The outcome of T/My and B/My groups showed no difference in overall survival(OS)or event-free survival(EFS).The older patients(age>60 years)had a much poorer outcome(2-year OS:22.7%± 12.2%versus 58.7%± 5.8%,P<0.001;2-year EFS:20.1%±10.9%versus 47.3%±5.7%,P=0.010).In total,48 patients underwent allogeneic hematopoietic stem cell transplantation(allo-HSCT).Donors were mismatched related(n=29),matched sibling(n=10),matched unrelated(n=7),and double unit cord blood(n=2).Patients underwent allo-HSCT had longer OS and EFS than those without receiving allo-HSCT(2-year OS:71.1%± 7.1%versus 36.5%±7.2%,P<0.0001;2-year EFS:61.3%±7.2%versus 26.2%± 6.3%,P<0.0001).However,there was no significant difference in OS between different stem cell sources.In our multivariable analysis,post-induction CR and receiving allo-HSCT were associated with encouraging long-term survival.Conclusions1.There was no significant difference in clinical and laboratory characteristics between MPAL patients diagnosed by 2016 WHO or EGIL criteria.2.MPAL subtypes were heterogeneously different in the clinical,genetic and immunophenotypic features.3.Allo-HSCT is an effective treatment for adult patients with MPAL and can improve the clinical outcome of patients of MPAL.Part 2 Genomic and transcriptomic features in adult patients with mixed-phenotype acute leukemiaObjectives1.To identify the mutation spectrum,copy number variations,gene expression profiles as well as fusion transcripts between Ph+MPAL,T/My MPAL,B/My MPAL and B/T MPAL.2.To clarify the difference of mutation profiles and gene expression profiles between MPAL and other acute leukemias.3.To propose an update to 2016 WHO MPAL subtypes by incorporating critical genomic information.Methods1.In order to characterize the molecular background of MPAL,we performed whole exome sequencing on 3 cases with paired diagnosis/remission samples and targeted deep sequencing on 104 cases to illustrate the mutational landscape of this entity.Meanwhile,we compared mutation profile with that from documented acute myeloid leukemia(AML),B cell acute lymphoblastic leukemia(B-ALL),and T cell acute lymphoblastic leukemia(T-ALL).2.To determine the genomic copy number variations(CNVs),array-based comparative genomic hybridization(array-CGH)analysis was performed in 11 MPAL patients,including 7 cases of Ph+,3 cases of T/My and 1 case of B/T.3.To further compare the gene expression features of MPAL,we analyzed gene expression profile in 25 MPAL cases.Meanwhile,we compared the gene expression features of MPAL with those of 105 BALL,75 AML,and 35 T-ALL cases.We further increased the number of cases for transcriptome sequencing,including 89 MPAL,133 AML,53 B-ALL and 41 T-ALL.Meanwhile,fusion transcripts were analyzed in MPAL cases.4.To re-group MPAL according to the genomic and transcriptome features and analyze the molecular subtypes of each new group.Results1.T/My,Ph+and B/My MPAL showed distinct mutation signatures,in which B/T was similar to T/My MPAL.T/My MPAL had more mutations than Ph+ MPAL and B/My MPAL.Mutations in CEBPA,NOTCH 1 and DNMT3A gene were enriched in T/My MPAL,whereas RUNX1 mutations were more commonly seen in Ph+MPAL.T/My MPAL up-regulated T cell receptor signaling pathway and Ph+MPAL up-regulated classic downstream pathways of BCR-ABL1,such as MAPK signaling pathway,PI3K-Akt signaling pathway and Ras signaling pathway.B/T MPAL was characterized by NOTCH 1 and PHF6 mutations.In the co-occurring mutation analysis,significantly overlapped mutations were observed among CEBPA and GATA2 mutations,NOTCH1 and JAK3,KRAS,IDH2 mutations,DNMT3A and ETV6,IDH2 mutations.In addition,NOTCH1 mutations were mutually exclusive with CEBPA and RUNX1 mutations.There was no significant difference on CR or OS between patients with and without gene mutations.It’s worth noting that JAK3 mutations had poorer survival in patients without receiving allo-HSCT(2-year OS:0%versus 42.7%±9.6%,P<0.0001;2-year EFS:0%vs 30.8%±8.9%,P<0.001).In the multivariable analysis,receiving allo-HSCT was associated with encouraging long-term survival,while JAK3 mutation was associated with poor outcome.2.We then compared the T/My and B/My MPAL mutational spectrum with that of AML,B-ALL and T-ALL.MPAL showed a distinct pattern of mutation.In comparison to AML,T/My MPAL had an increased alteration in transcriptional regulation genes,CEBPA,WT1,PHF6,GATA2,ETV6 as well as NOTCH 1,and lower incidence of FLT3 mutations.The incidence of NOTCH 1 mutation was higher in B/My MPAL,while the incidence of DNMT3A mutation was lower in B/My MPAL than AML.In comparison to T-ALL,WT1,NRAS,FLT3 and KRAS mutations were more common in T/My MPAL,nevertheless NOTCH1 gene was frequently mutated in T-ALL.There was no significant difference in mutation spectrum between B/My MPAL and B-ALL.3.A total of 436 genomic DNA CNVs were detected including 293 losses and 143 gains changes,with a mean of 39.6 genomic alterations per sample.Notably,there was significantly more losses in Ph+MPAL than in T/My&B/T MPAL patients(79.0%versus 43.5%,P<0.0001).In our study,39.5%(64/162)of MPAL patients carried at least one fusion gene.As previously reported,BCR-ABL1 fusion was most common and presented in 25.9%(42/162)adult patients with MPAL.Approximately 81.0%(34/42)of Ph+cases expressed P210 transcripts,with the other 19.0%(8/42)expressing P190.Additionally,SETNUP214 and ZNF3 84-rearrangement occurred in 4 cases,while KMT2A-rearrangement and PICALMMLLT10 were detected in 3 cases,respectively.In addition to these previously reported leukemic fusions,we identified three new fusion genes in 3 cases,NRIP1-BCL2L1,IKZF1-TRB and MED14-HOXA9.Furthermore,B/My MPAL was accompanied by more fusions compared with T/My(29.0%vs 10.0%,P=0.028).4.We compared the gene expression features of 25 MPAL with those of 105 B-ALL,75 AML,and 35 T-ALL cases.An unsupervised hierarchical cluster analysis revealed that 56.0%,24.0%and 20.0%of MPAL had B-ALL-like,T-ALL-like and AML-like gene expression profile,respectively.Notably,patients who received the matched treatment were more likely to achieve CR than those who received the unmatched treatment,just falling short of statistical significance(78.6%vs 33.3%,P=0.191).However,it was not further confirmed when we increased the number of samples for transcriptome sequencing(P=1.000).5.Notably,t-Distributed Stochastic Neighbor Embedding and hierarchical clustering analyses of transcriptome sequencing data identified two subtypes with a distinct gene expression profile.There were 9 samples in clusterl and 15 samples in cluster2.Of the 9 cases,6 were T/M MPAL and 3 were ETPALL.77.8%(7/9)of cases harbored FLT3 mutations.These cases exhibited a distinct immunophenotype CD7+CD2+cCD3+CD13+CD117+CD34+.Of 15 cases,14 cases were T/M and 1 case was B/M MPAL.73.3%of cases harbored CEBPA mutations.These results suggest that FLT3 mutation and CEBPA mutation may be two new subtypes.6.MPAL was classified into 8 subtypes(G1-G8)according to hierarchical clustering of transcriptome sequencing,which associated with genomic alterations.In G1-G4 groups,97.6%of the immunophenotypes were T/M.and B/T,and only 2.4%were B/M.In G5-G8 groups,93.6%of the immunophenotypes were B/M,and only 6.4%were T/M.Of the 8 groups,G1 clustered with AML,G2,G3,G4 clustered with T-ALL,83.3%of G5 clustered with AML and 16.7%of G5 clustered with B-ALL,80.0%of G6 clustered with AML,10.0%of G6 clustered with B-ALL and 10.0%of G6 clustered with TALL,both G7 and G8 clustered with B-ALL.We further analyzed the correlation of clinical characteristics with different subtypes.G1 was significantly associated with lower platelet count and normal karyotype as compared to other groups.G2 was associated with lower white blood cell count and patients aged ≥60 years.G3 was correlated with higher white blood cell count and complex karyotype.T(9;22)(q34;q11)/BCR-ABL1 was found exclusively in G5 and G8 and the majority were P210 transcript.Based on FAB criteria,69.2%(9/13)of G1 showed M1/M2 morphologies,the majority of G5(100%)and G6(70.0%)showed M4/M5 morphologies and 55.6%(5/9)of G7 showed ALL/M0 morphologies.Mutation spectrum analysis showed that G1 was associated with CEBPA mutations,G2 was associated with NOTCH1 and DNMT3A mutations,G3 was correlated with NOTCH1,JAK3 and PHF6 mutations,G4 was correlated with FLT3 mutations and higher expression of BCL11B and KIT,and RUNX1 mutations were enriched in G8.Interestingly,G1 and G4 were consistent with cluster 1 and cluster 2,respectively.Pathway analysis showed AML with NPM1 mutated,AML with MLL fusions,myeloid cell development and NF-kB signaling pathway were significantly up-regulated in G5 and G6.Meanwhile,B lymphocyte progenitor,BCR-ABL1 fusion and BCR pathway were up-regulated in G7 and G8.7.We proposed an update to the 2016 WHO classification of MPAL based on genomic and transcriptomic characterization that included five new subtypes of Ph-like,KMT2Ar-like,CEBPA-mutant T/My MPAL,NOTCH1-mutant T/My MPAL,FLT3-mutant T/My MPAL.Conclusions1.T/My,Ph+and B/My MPAL showed distinct mutation and gene expression signatures,in which B/T was similar to T/My MPAL.T/My MPAL was associated with CEBPA,NOTCH1,DNMT3A mutations and up-regulated T cell receptor signaling pathway.Ph+MPAL was characterized by high incidence of RUNX1 mutations and up-regulated classic downstream pathways of BCR-ABL1.2.According to genomic and transcriptomic characterization,MPAL was classified into eight subtypes,which had unique morphological and mutant features.Our study recommends an update to the 2016 WHO MPAL subtypes to include critical newer genomic information.Part 3 Analysis of immunophenotypic heterogeneity of patients with mixed-phenotype acute leukemiaObjectivesIn this study,we preliminarily investigated immunophenotypic heterogeneity characteristics of MPAL and the possible factors driving the differentiation into different immunophenotype.Methods1.Flow cytometry analysis and sorting were performed on the cryopreserved bone marrow specimens of 7 cases with bilineage phenotype.Leukemic cells in the CD45 and side scatter-defined blast gate were sorted into subpopulations,including myeloid blasts(My-blast),B lymphoblasts(B-blast)or T lymphoblasts(T-blast).Normal T cells were sorted using side scatter,CD45 lymphocyte gate and secondarily using surface CD3 to collect normal T cells in MPAL cases.WES was performed on sorted cells.2.Single cell RNA sequencing was used to further investigate how differential gene expression correlates intra-sample heterogeneity in MPAL.Results1.Of 7 cases,4 were Ph+MPAL with B/M immunophenotype and 3 were T/My MPAL.We detecetd a total of 156 single nucleotide variants(SNVs)and small insertion and deletions(indels)including 82 SNVs and 74 indels.38 SNVs and 39 indels were detected in My-blast,13 SNVs and 14 indels were detected in B-blast and 31 SNV and 21 indels were detected in T-blast.The most frequently mutated genes were RUNX1(42.9%,3/7)and PHF6(42.9%,3/7),which were present in all the My-blast and B-blast or T-blast.Moreover,RUNX1 and PHF6 mutations had higher variant allele frequencies in three cases,indicating early clonal events.In contrast,NOTCH1,KRAS mutations in patient M6 and NRAS mutations in patient M9 had lower VAFs,suggesting subclonal events.2.Using this latent semantic indexing(LSI)projection framework and healthy hematopoietic landscape,we analyzed the leukemic features of MPAL samples at single cell resolution.First,we clustered MPAL to classify cells as "healthy-like" and "disease-like" cells.Second,we projected MPAL single cells onto normal hematopoietic map and found extensive gene expression diversity.We then divided the different subgroups into HSC-like,CLP-like,CMP-like,GMP-like,Early erythroid-like,monocyte-like,DC-like,Pre-B-like,B-like and T-like.In most cases,these classifications were consistent with the proportion of morphologic or flow cytometric as blasts.It is worth noting that the fraction of monocyte-like and DC-like in Ph+cases was higher than T/My cases(P<0.0001).Meanwhile,the proportion of pDC-like in the 3 cases with RUNX1 mutation was significantly higher than that in the 1 case without RUNX1 mutation(P<0.0001).In addition,RUNX1 mutation was detected in three single cells of a Ph+case,two of which were pDC-like and one of which was GMP-like,suggesting that pDClike might originate from leukemia cells.Further evaluation in a larger cohort is required to confirm the role of RUNX1 mutations in MPAL with increased pDC(pDC-MPAL).3.We examined the differential gene expression between lymphoid subpopulations(CLP-like)and myeloid subpopulations(GMP-like).The result showed that T lymphoid subpopulations had a higher expression level of MSI2 and DNTT,while myeloid subpopulations had upregulated expression of LGALS1 and PLEK in T/My cases.Transcriptional factors TCF4 may play a potential role in B lymphoid differentiation by interacting with TCF12,while PLEK may play a potential role in myeloid differentiation by interacting with ITGB2 in Ph+cases.Conclusions1.Intra-sample immunophenotypic heterogeneity may not be determined by genomic variation.Mutations in transcription factor were consistently present in the major clone,suggesting that transcription factor gene alterations arise early in leukemogenesis.2.Notably,the fraction of monocyte-like and DC-like in Ph+cases was higher than T/My cases.The role of RUNX1 mutation in the pathogenesis of pDC-MPAL is worthy of further investigation.3.The expression of different transcription factors in MPAL leads to differentiation of leukemia cells into myeloid and lymphoid cells. |
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