Background Drug-resistant mutations in the ABL1 kinase domain (KD) of BCR::ABL1 are primary mechanisms of resistance to tyrosine kinase inhibitor (TKI) therapy in Ph-positive leukemia. These mutations alter the conformation of ABL1 within BCR::ABL1, impairing TKI and thus diminishing their anti-leukemia efficacy. Our previous studies, presented at the 61st and 62nd ASH Annual Meeting, demonstrated the superiority of next-generation sequencing (NGS) over Sanger sequencing for detecting BCR::ABL1-KD mutations. NGS offers higher sensitivity, accurate mutation frequency determination, and the ability to identify compound or polyclonal mutations within the same amplicon. However, NGS still needs to improve, including its short read length and the disadvantages in identifying in-cis compound mutations, lack of flexibility, and long turnaround time. Leveraging advancements in a novel third-generation sequencing (TGS) technology - characterized by single-molecule sequencing capability, long reads, real-time sequencing, and high accuracy - we have developed an approach for directly full-length in-cis BCR::ABL1-KD mutation screening. Methods The new approach allows for a comprehensive in-cis analysis of resistance mutations in the BCR::ABL1-KD using a novel TGS platform based on single-molecule side synthesis side nanopore sequencing (NSBS), distinct from PacBio or ONT sequencing. We retrospectively analyzed 30 specimens from 30 cases previously tested using NGS, including 15 mutation-positive and 15 mutation-negative specimens under previous investigation. Among them, there were ten single mutations, four double mutations, and one triple mutation, with the variant allele frequency (VAF) ranging from 7.8% to 99.6%. Results The TGS approach can directly analyze in-cis compound BCR::ABL1-KD mutations in a full-length BCR::ABL1-KD sequencing model with high single-base accuracy and superior to the NGS protocol. The novel TGS approach can detect the complete BCR::ABL1 fusion gene sequence (including p190 and p210 types, sequence range 1696~1717bp), which can more accurately obtain the ABL1 sequence in the BCR::ABL1 fusion gene, thus analyzing the veritable resistance mutations in the ABL1 KD of the BCR::ABL1 fusion gene; it has higher detection sensitivity (average sequencing coverage depth of 13086x, range 8226~24152x; Q30 can reach 99.39%; VAF as low as 1%). The TGS approach detected more mutations than NGS (27 vs. 21) and accurately distinguished more complex mutation patterns, including multiple compound or polyclonal mutations. It detected five mutations in the NGS mutation-negative group. The VAF obtained in mutation analysis was inconsistent compared to NGS, indicating that the mutations and mutation frequencies detected by TGS are more in line with the true situation and significant for continuous monitoring. Conclusion Through comparative analysis with the NGS project, the novel TGS approach shows the advantages of longer reads, higher sensitivity and accuracy, and the ability to complete the scheme from library construction to data output within 12 hours. It may provide the clinic with faster and more accurate results of in-cis BCR::ABL1-KD resistance mutation analysis with higher sensitivity and guide the clinic to quickly change treatment plans, thereby improving therapeutic outcomes.

Background : Emerging evidence suggests that viral infections play a crucial role in the pathogenesis of certain leukemias, with viral integration and its interaction with the human genome potentially contributing to disease onset and progression. Since Astolfi et al. identified the first case of TTMV::RARA-APL in a 6-year-old child, more than ten cases have been identified in the past two years. However, no other viruses have been reported to form fusion genes with human genes in leukemia. Our study leverages a vast repository of poly(A) RNA-seq data from AML patients to develop a sensitive bioinformatics workflow. This workflow aims to uncover the molecular footprints left by viruses in the leukemic transcriptome and discover novel viral-human fusion events that may have been previously overlooked. Methods : The cDNA library construction and sequencing were performed as previously described (Chen, Blood Cancer J 2021). Raw RNA sequencing data were initially processed using Fastp for adapter trimming, polyA/T trimming, and low-quality reads filtering. The high-quality reads were then mapped to the human reference genome (GRCH38) using the STAR aligner (version 2.7.10b). Samtools was used to sort the aligned BAM files by chromosome position and extract reads that were properly paired. A coustom Perl scripts was written to extract the reads without proper paired alignment. SPAdes was used to assemble the unmapped reads into scaffolds, which were then annotated by Blast against the NCBI Refseq Virus database, using an expected value (E-value) cutoff of less than 1e-10 and an alignment percent more than 80%. Due to the limited data available for TTV and TTMV in the NCBI RefSeq Virus database, we enhanced the accuracy of our annotations by incorporating sequences of TTV and TTMV from the NCBI Nucleotide database into our reference database. Reads with soft clipping were identified from the BAM alignment and grouped by the genome positions where soft clipping occurred. Cap3 was then used to assemble these soft clipping reads into consensus sequences (contigs) for each group. The soft-clipped contigs were aligned to the unmapped scaffolds using Blast to reconstruct the fusion transcripts. Results : A cohort of 712 AML cases was enrolled, including 154 children and 556 adults (divided by ≥18 years), with an age range of 0-89 years (median 42 years), and a male-to-female ratio of 375:337. Virus sequences were identified in 523 cases (73.46%), with an average of 4.25 viruses in one sample. Duplodnaviria was found in 414 cases (58.15%), Riboviria in 220 cases (30.90%), Varidnaviria in 38 (5.34%), Monodnaviria in 14 (1.97%) and other virus in 68 cases (9.55%). Alphatorquevirus (Torque Teno Viruses, TTVs) and betatorquevirus (Torque Teno mini Viruses, TTMVs) compose mostly of the Anelloviridae fraction, with TTV was found in 101 cases (14.19%) and TTMV in 30 cases (4.21%). Human herpesvirus 1 (HHV-1) was identified in 297 cases (41.71%), Cytomegalovirus (CMV) in 239 cases (33.57%), Epstein-Barr virus (EBV) in 25 cases (3.51%), and Human adenovirus C (HAdV-C) in 24 cases (3.37%), these were the most frequently identified pathogens in this cohort. Using this analytical approach, we detected one case with the TTMV::RARA fusion in this cohort. We re-analyzed the poly(A) transcriptome sequencing data from several cases of TTMV::RARA previously reported by our center using this analytical process. The re-analysis confirmed that the TTMV::RARA fusion could be successfully identified using this protocol. No other virus-human coding gene fusion was found in this cohort, underscoring the rarity of virus-human fusion genes. Conclusions : We present an analytical workflow for the detection of viral gene transcripts and viral-human gene fusions, providing a reference for future studies of tumor-associated viruses using poly(A) transcriptome sequencing data. This workflow facilitates the discovery of viral integration events and the identification of expressed viral transcripts in various diseases.

Two recent guidelines, the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) and the International Consensus Classification (ICC), were published to refine the diagnostic criteria of acute myeloid leukemia (AML). They both consider genomic features more extensively and expand molecularly defined AML subtypes. In this study, we compared the classifications of 1135 AML cases under both criteria. According to WHO-HAEM5 and ICC, the integration of whole transcriptome sequencing, targeted gene mutation screening, and conventional cytogenetic analysis identified defining genetic abnormalities in 89% and 90% of AML patients, respectively. The classifications displayed discrepancies in 16% of AML cases after being classified using the two guidelines, respectively. Both new criteria significantly reduce the number of cases defined by morphology and differentiation. However, their clinical implementation heavily relies on comprehensive and sophisticated genomic analysis, including genome and transcriptome levels, alongside the assessment of pathogenetic somatic and germline variations. Discrepancies between WHO-HAEM5 and ICC, such as the assignment of RUNX1 mutations, the rationality of designating AML with mutated TP53 as a unique entity, and the scope of rare genetic fusions, along with the priority of concurrent AML-defining genetic abnormalities, are still pending questions requiring further research for more elucidated insights.

TTMV::RARA is a recently reported fusion gene associated with acute promyelocytic leukemia (APL), caused by the integration of torque teno mini virus (TTMV) genomic fragments into the second intron of the RARA gene. Currently, there have been only six documented cases, with clinical presentations showing significant variability. Although initial responses to all-trans retinoic acid (ATRA) treatment may be observed in patients with TTMV::RARA-APL, the overall prognosis remains unfavorable among infrequent reported cases. This article presents a pediatric case that manifested as PML::RARA-negative APL with central nervous system involvement at onset. The patient experienced both intramedullary and extramedullary relapse one year after undergoing allogeneic hematopoietic stem cell transplantation. Upon identification as TTMV::RARA-APL and subsequent administration of two rounds of ATRA-based treatment, the patient rapidly developed multiple RARA ligand-binding domain mutations and demonstrated extensive resistance to ATRA and various other therapeutic interventions. Additionally, the patient experienced ARID1A mutant clone expansion and progressed MYC-targeted gene activation. This case represents the first documentation of extramedullary involvement at both the initial diagnosis and relapse stages, emphasizing the intricate clinical features and challenges associated with the rapid accumulation of multiple ATRA-resistant mutations in TTMV::RARA-APL, characterizing it as a distinct and complex sub-entity of atypical APL.

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: All-trans retinoic acid (ATRA) and arsenic have been used with great success in the treatment of PML::RARA fusion gene (FG) positive acute promyelocytic leukemia (APL). There are still PML::RARA negative cases manifest like APL, that is, variant APL (APLv). These cases often carry rare FGs involved in RARA, RARB, or RARG. So far, 24 RARG-FG positive APLv (RARG-APL) cases have been reported, increasing year by year, thanks to the application of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all reported cases tried ATRA treatment, but all evaluable cases were resistant. On the contrary, cells transfected with artificial X::RARG fusion protein responded well to ATRA in vitro. Aims: To decipher the undiscovered traits of RARG-FGs and the critical molecular abnormalities in RARG-APL. Methods: The characteristics of RARG-FGs were analyzed by WGS, RNA-seq, and RT-PCR. Sequence-specific primers were designed to determine whether two fusion sites were located on the same cistron. The biological effects and responsiveness to ATRA of fusion proteins were evaluated in vitro. Results: In 2022, we admitted one APLv case (#1) who was resistant to ATRA and chemotherapy. In this case, we identified a trinary fusion of PRPF19-e1_4::RARG-e4_9::PRPF19-e5_16 (e, abb. of exon). The RARG 3’ and 5’ fusions were confirmed to be in the same cistron. That is, the fusion is the insertion of RARG-e4_9 between PRPF19-e4 and e5, rather than forming two transcripts of PRPF19-e1_4::RARG-e4_10 and RARG-e1_9::PRPF19-e5_16. This case reminds us of a case (#2) with NPM1::RARG::NPM1 fusion that we reported [Br J Cancer.2019;120:1023-1025], the only case reported as RARG trinary fusion till now. We then performed analysis on archived samples of one more APLv case (#3) and identified HNRNPC:: RARG::HNRNPC fusion. We also reanalyzed one case (#4) reported by other groups for which RNA-seq data was available [Hematology.2022;27:518-522]. This case was reported with the RARG-e9::HNRNPM-e3 fusion, but our ancillary analysis further confirmed the HNRNPM-e12::RARG-e1 splicing. The untranslated region (UTR) of RARG-e1_3 confers a derived coding sequence, and the reading frame of RARG-e4_9 was retained in this trinary fusion. The involvement of UTR caused bioinformatic analysis failure to report in-frame fusion, resulting in missed detection. RARG-e10 deletion was a strictly recurrent event in all 4 cases. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in response to ATRA [Structure.2019;27:1270-1285.e6]. In principle, the RARG trinary fusion protein with LBD truncation will lose responsiveness to ATRA. Our in vitro experiments also confirmed that cells transfected with chimeric protein with intact RARG LBD responded well to ATRA, while chimeric proteins with RARG LBD-H11_12 truncation were unresponsive to ATRA even at high concentrations. Summary/Conclusion: We revealed that trinary fusion and LBD truncation as recurrent and crucial molecular features in RARG-APL and render unresponsiveness to ATRA. RARG 5’ translocation leads to aberrant expression, with variation in the splicing site (intron 3 or further upstream). RARG 3’ splicing is highly recurrent at exon 9, resulting in LBD truncation, which plays a pivotal role in leukemogenesis and renders unresponsiveness to ATRA. This trait was ignored previously due to the complexity of trinary fusion, and the inertial only focus on RARG 5’ splicing. We are now recruiting multicenter RNA-seq data to further validate this finding.Keywords: Gene fusion, AML, Retinoic acid receptor (RAR), APL

This study aimed to investigate the relationship between anomalous DNA nucleotidylexotransferase ( DNTT ) activation and the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and the relevance of their prognosis in antithymocyte globulin (ATG)‐based regimen allogeneic hematopoietic stem cell transplantation (allo‐HSCT). A cohort of 578 AML cases was enrolled. Next‐generation sequencing was performed to screen mutations of 86 leukemia driver genes. RNA‐seq was used to analyze gene expression. Prognostic analysis was investigated in 239 AML cases who underwent ATG‐based regimen allo‐HSCT. We report a refined subtyping algorithm of LMs (type I–IV) based on sequence anatomy considering the TdT‐aided mutagenesis mechanism. GC content adjacent to LM junctions, inserted nontemplate nucleotide bases, and DNTT expression analysis supported the DNTT activation and TdT‐aided mutagenesis in type II/III LMs in the total AML cohort. Both single‐variate and multivariate analyses showed a better overall survival of FLT3 type III compared to type I in a subset of ATG‐based regimen allo‐HSCT cases. The novel LM subtyping algorithm not only deciphers the etiology of the mutagenesis of LMs but also helps to fine‐tune prognosis differentiation in AML. The possible prognostic versatility of this novel LM subtyping algorithm in terms of chemotherapy, targeted therapy, and allo‐HSCT merits further investigation.

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML) which is characterized by specific clinical and biological features. Typical APL cases are caused by PML::RARA fusion gene and are exquisitely sensitive to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Rarely, APLs are caused by atypical fusions involving RARA or, in fewer cases still, fusions involving other members of the retinoic acid receptors (RARB or RARG). To date, seven partner genes of RARG have been reported in a total of 18 cases of variant APL. Patients with RARG fusions showed distinct clinical resistance to ATRA and had poor outcomes. Here, we report PRPF19 gene as a novel partner of RARG and identify a rare interposition-type gene fusion in a variant APL patient with a rapidly fatal clinical course. The incomplete ligand-binding domain of RARG in the fusion protein may account for the clinical ATRA resistance in this patient. These results broaden the spectrum of variant APL associated molecular aberrations. Accurately and timely identification of these rare gene fusions in variant APL is essential to guide therapeutic decisions.

Introduction Two guidelines were recently published to refine the diagnostic criteria of acute myeloid leukemia (AML): the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors (WHO-HAEM5, PMID: 35732831) and the International Consensus Classification (ICC; PMID: 35767897), and both expanded molecularly defined AML subtypes. In this study, we utilized a cohort of 1135 AML cases to examine their performance. Methods From Sep 1, 2018 to Dec 31, 2022, a consecutive cohort of 1135 cases diagnosed with de novo AML according to WHO-HAEM4R (PMID: 27069254) were enrolled, with a median follow-up of 20 months. All underwent high-throughput sequencing (PMID: 34135310, 29932212). Results When classified according to WHO-HAEM4R, 55% of the 1135 cases were diagnosed as AML with recurrent genetic abnormalities, 16% were AML with myelodysplasia-related changes (AML-MRC), 2% were therapy-related AML, 4% were AML with germline predisposition, and 24% fell into AML not otherwise specified (AML, NOS). Major changes in WHO-HAEM5 include the inclusion of AML subtypes of KMT2A rearrangement (KMT2A-r), MECOM-r, and NUP98-r; adding AML with other defined genetic alterations (AML-ODGA) as a basket entity; and annulling the entity of AML with mutated RUNX1 (AML-RUNX1). The entity of AML with CEBPA mutation (AML-CEBPA) has changed to include not only biallelic (biCEBPA) but also single bZIP (smbZIP-CEBPA) mutations. AML-MRC in WHO-HAEM4R is redefined as AML, myelodysplasia-related (AML-MR), with a mutation-based definition based on an 8-gene panel (without RUNX1). When comparing WHO-HAEM4R and WHO-HAEM5 (Fig. 1), AML with KMT2A-r increased from 2% to 10% because WHO-HAEM5 no longer restricts the partner genes of KMT2A. AML with NUP98-r and AML-ODGA in WHO-HAEM5 account for 6% and 4%, respectively. AML-MR increased from 12% AML-MRC to 21% benefited from the 8-gene panel-based definition. AML-RUNX1 has been annulled in WHO-HAEM5 and 63% cases of this subtype were reclassified as AML-MR, and 30 cases with RUNX1 mutations as the only basis for classification were reclassified as AML defined by differentiation. In total, AML without defining genetic abnormalities dropped from 24% to 11%. When comparing WHO-HAEM4R and ICC (Fig. 1), AML with mutated TP53 (AML-TP53) is a new category in ICC and accounted for 4% of AML, 79% of them were previously classified as AML-MRC in WHO-HAEM4R. AML-MR in WHO-HAEM5 is divided into two entities in ICC: AML with MR gene mutations (including RUNX1) and AML with MR cytogenetic abnormalities, comprising 13% and 5% of cases, respectively. ICC only includes in-frame bZIP CEBPA mutations. Thus 6 cases classified as AML-biCEBPA based on WHO-HAEM4R fell into AML, NOS under ICC since their CEBPA mutations were not in bZIP. Conversely, 21 cases with smbZIP-CEBPA and diagnosed as AML, NOS according to WHO-HAEM4R were classified as AML-CEBPA under ICC. Cases classified as AML-CEBPA based on WHO-HAEM5 tended to have a better overall survival (OS) than cases classified as AML-biCEBPA under WHO-HAEM4R because AML-smbZIP-CEBPA was added while cases with MR gene mutations which had a worse prognosis fell into AML-MR under WHO-HAEM5. Despite ICC only included in-frame bZIP-CEBPA mutations, the prognosis of AML-CEBPA diagnosed under ICC was not superior to that under WHO-HAEM5. This might be because cases with both MR cytogenetic abnormalities and CEBPA mutations were reclassified as AML-CEBPA under ICC's hierarchical principle. ICC distinguished AML with KMT2A::MLLT3 from AML with KMT2A-r, while WHO-HAEM5 only included AML with KMT2A-r entity. Survival analysis showed significantly better OS in AML with KMT2A::MLLT3 than AML with KMT2A-r. ICC separated AML-TP53 from AML-MR due to the notorious prognosis of TP53 defects, and cases classified as AML-TP53 showed the worst OS in this cohort. But prognostic analysis showed no difference between AML-TP53 and cases with concurrent TP53 mutation and other AML-defining genetic abnormalities. The rationality of considering AML-TP53 as a distinct entity remains debatable. Conclusions Both new classification systems consider genomic features more extensively and enhance the precise classification of AML. Discrepancies between WHO-HAEM5 and ICC, like classifying AML with RUNX1 mutations and the rationality of defining AML-TP53 as a unique entity, remain open questions that require further research for clearer answers.

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Attribute to the application of all-trans retinoic acid (ATRA) and arsenic, acute promyelocytic leukemia (APL) positive for PML::RARA fusion gene (FG) has achieved a high cure rate. There are still cases manifest like APL but negative for PML::RARA, that is, variant APL (APLv). So far, fifteen RARA fusion partners have been reported in APLv, and all RARA-FGs were reported as X::RARA (X stands for 5’ partner genes). Unlike PML::RARA-positive APL, most other X::RARA cases were resistant to ATRA, suggesting molecular mechanisms remain to be revealed. Here we report the finding of transposable element participates in composing STAT3::RARA::LTR40a-RC (RC, abb. of reverse complement) trinary fusion in APLv and confer ATRA resistance via RARA ligand binding domain (LBD) truncation. Aims: To analyze the structural traits of the RARA-FG in APLv and to explore its sensitivity to ATRA treatment. Methods: The FG was analyzed by transcriptome sequencing (RNA-seq), whole genome sequencing (WGS), and optical genome mapping (OGM). The biological effects and responsiveness to ATRA of fusion proteins were evaluated in vitro. Results: A 35-year-old female came with 2 months of skin ecchymosis and 2 days of lower abdominal pain and diarrhea. Laboratory tests showed moderate anemia and abnormal coagulation function. Morphological examination showed 79% myeloblasts and immunophenotype analysis showed APL phenotype. RT-PCR screening for PML::RARA was negative. The karyotype showed inv(17)(p12q21). The patient received ATRA and arsenic combination therapy, but the response was poor. Unfortunately, the patient died from non-medical reasons 20 days after starting treatment. We identified a special form of STAT3::RARA::LTR40a trinary fusion, which is spliced in tandem with 3 fragments of STAT3 exon 1_21, RARA exon 3 to truncated exon 9, and LTR40a-RC sequence together with its 5’ adjacent 151bp sequence. Both long-read WGS, OGM, and RT-PCR confirmed that these 3 fragments spliced within the same cistron. RNA-seq data showed the gene expression pattern of this case was like classic APL, and the aberrant activation expression of the transposon element LTR40a. The 3’ fusion sequence confers a short derived 3 amino acids coding sequence with a swift termination. Importantly, LTR40a-RC confers a poly(A) signal sequence, which is necessary for a qualified mRNA. The RARA exon 9 truncation led to the deletion of helix 12 (H12) of its LBD, which plays a pivotal role in response to ATRA [Structure.2019;27:1270-1285.e6]. In principle, the RARA protein with LBD-H12 truncation will lose responsiveness to ATRA. Our in vitro experiments confirmed that cells transfected with chimeric protein with intact RARA LBD responded well to ATRA. In contrast, chimeric proteins with RARA LBD-H12 truncation were unresponsive to ATRA, even at high concentrations. Summary/Conclusion: This is the first report of pathological trinary fusion, the first report of transposable element participates in composing pathological fusion gene, and the first report of the RARA LBD truncation confers ATRA resistance in APLv. Moreover, in the two previously reported STAT3::RARA cases, the authors also observed breakpoints of exon 9 at the genomic level but did not conduct further analysis [Blood. 2018;131(8):935- 939.]. It strongly suggests that RARA LBD truncation might be the recurrent event in STAT3::RARA fusion or even other APLv cases. It may be due to the inertial only focusing on RARA 5’ splicing and the complexity of transposon sequence analysis, so RARA 3’ splicing was previously unobserved.Keywords: AML, Retinoic acid receptor (RAR), APL, Gene fusion

In this manuscript, we report torque teno mini virus (TTMV) as a cause of acute promyelocytic leukemia (APL) lacking PML::RARA in a 3-year-old boy. Astolfi et al. firstly identified partial integration of the TTMV genome into RARA intron 2, which resulted in in-frame TTMV::RARA fusion in two APL-like pediatric cases without PML::RARA in November 2021. This fascinating report identified an unexpected exogenous genetic cause of APL and could be of great importance for diagnosing and managing APL. Here we report the third childhood APL-like case caused by TTMV integration and investigate the location and structure of the integrated TTMV sequence. These findings suggest TTMV::RARA is a recurrent cause of APL lacking PML::RARA. Considering the widespread prevalence of TTMV in the population, more TTMV::RARA positive APL-like cases might remain to be identified. Establishing a bioinformatic analysis strategy optimized for the highly variable TTMV genome sequence may facilitate the identification of TTMV::RARA by whole transcript sequencing. An effective PCR protocol to identify TTMV::RARA based on a profound analysis of the conservation of TTMV segments in the fusion transcript is also expected. Also, further investigation is needed to elucidate the oncogenic mechanisms of TTMV integration and the clinical features of TTMV::RARA positive patients.

Objective:To investigate the molecular genetic and clinical characteristics of MEF2D-BCL9 fusion gene-positive acute B-cell lymphoblastic leukemia (B-ALL), and to provide the reference for the diagnosis and treatment of the disease.Methods:The medical record and experimental examination data of a 18-year-old female MEF2D-BCL9 fusion gene-positive B-ALL patient were retrospectively analyzed. The clinical manifestations and biological characteristics of MEF2D-BCL9 fusion gene-positive B-ALL were summarized.Results:This 18-year-old female patient was treated in a local hospital in December 2018 and was diagnosed as B-ALL. She achieved complete remission after chemotherapy and recurred at 6 months after the initial onset, and then she was admitted to Hebei Yanda Ludaopei Hospital in the 9 months after the initial onset.MEF2D-BCL9 fusion gene was detected through RNA-sequencing (RNA-seq) and verified by using polymerase chain reaction and Sanger sequencing. Bone marrow cell morphology was similar to mature B cells with vacuoles but without characteristic chromosome karyotype abnormalities. The patient achieved remission after VLD regimen chemotherapy, chimeric antigen receptor T-cell (CAR-T) therapy and bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT). She has maintained complete remission for 2 years at the last follow-up in February 2022.Conclusions:MEF2D-BCL9 fusion gene-positive B-ALL is characterized with high risk, early relapse and poor prognosis. These patients may benefit from CAR-T and allo-HSCT. It further emphasizes the importance of taking MEF2D-BCL9 fusion gene into the detection or identification by using RNA-seq, particularly for those newly diagnosed B-ALL patients in children and adolescents with specific bone marrow morphology.

Myelodysplastic syndrome (MDS) represents a group of neoplasms with extensive heterogeneity. Recurrent mutations in dozens of driver genes have been identified in over 90% of MDS cases, although fusion genes are rarely seen. We first report the competitive evolved sub‐clonal breakpoint cluster region (BCR)::ABL1 and novel MSI2::PC fusion gene in MDS with del(5q) in initial diagnosis that underwent dismal progression. However, the BCR::ABL1 clone vanished while the MSI2::PC clone rose to the major one with disease progression. A novel MSI2::PC fusion transcript was identified in initial diagnosis and disease progression of the patient through transcriptome sequencing (RNA‐seq) and Quantitative reverse transcription polymerase Chain Reaction (PCR) showed MSI2::PC/ABL1 expression at initial diagnosis and disease progression. In addition, mutation screening of 300 leukemia driver genes identified ARID2 c.5046del/p.F1682Lfs*19 and ZNF292 c.4565A > G/p.Q1522R mutation in bone marrow sample at initial diagnosis and disease progression. In conclusion, the dynamic process of the two fusion and phenotype manifestations may help to understand further the molecular significance of the anomalies of BCR::ABL1, MSI2, and PC in oncogenesis.

Background The anti-cancer effect of vitamin C (VC) has long been speculated, but studies yielded inconsistency. Recent studies reported that supraphysiological concentration of VC have therapeutic or prevention effects for myeloid malignancies with certain mutation signatures. There was a notable proportion of DAT (i.e., DNMT3A, ASXL1, and TET2) and dozens of other genes that mutate in age-related clonal hematopoiesis (ARCH). Methods and results Through analyzing the plasma VC concentration and mutations of 21 genes in 215 senior volunteers, we revealed that ARCH is significantly associated with dietary plasma VC concentrations, especially TET2 mutations and non-DAT mutations. Conclusion This study firstly disclosed the significant association between VC inadequacy and ARCH in the senior population. It provides evidence that physiological VC concentration has ARCH prevention effect. It will illuminate future explorations on the oral VC supplement in maintaining sound hematopoiesis, reversal ARCH, adjuvant therapy for myeloid malignancies, and prevention of other ARCH related comorbidities.

Abstract Introduction B-cell acute lymphoid leukemia (B-ALL) with t(17;19)(q22;p13)/TCF3-HLF is very rare and has a dismal prognosis even with the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Due to the rarity of this fusion, only a few cases have been described in the literature. In this study, we retrospectively analyzed 24 cases with TCF3-HLF from a large cohort of B-ALL, which constituted the largest cohort of TCF3-HLF-positive ALL reported to date and reported in detail the laboratory characteristics and prognoses of this group of patients. Methods From Apr. 2012 to Feb. 2020, a total of 3287 cases were diagnosed with B-ALL in our hospital. All of them underwent afusion gene screening test including TCF3-HLF through multiplex-nested reverse transcription-PCR. Whole transcriptome sequencing (WTS) was performed using RNA extracted from the bone marrow (BM) samples by HiSeq 2500. The gene expression signature for TCF3-HLF-positive B-ALL was investigated by comparing with healthy controls, B-ALL with TCF3-PBX1/TCF3-ZNF384, and B-ALL negative for pathogenic fusion genes (Chen X et al., Blood Cancer J 2021). Results A total of 24 cases with TCF3-HLF were identified, accounting for 0.73% of all B-ALL cases. Among them, 22 (91.67%) were children (≤18 years), and 2 (8.33%) were adults. Length of follow-up varied. The endpoint of the follow-up was Jul. 1st, 2020. Overall survival (OS) was defined as the time from diagnosis to death or the time of the last follow-up. Of the 24 cases with TCF3-HLF, 8 had type I, 12 had type II, 4 had both type II and III chimera isoforms. Karyotype was available in 22 cases, 19 showed abnormal karyotypes and most of them (12/19, 63%) harbored further structural and/or numerical aberrations besides t(17;19)(q22;p13) translocation. Gene mutation screening of 58 genes was performed on 17 cases at the time of diagnosis. Eight (47%) of them showed mutations and 7 of them had mutations involving RAS signaling pathway genes (NRAS, KRAS, FLT3, and PTPN11). Immunophenotypic examination showed 12 (50%) and 19 (79%) patients exhibited aberrant expression of CD13 and CD33, only 4 patients (17%) were negative of both CD13 and CD33. Gene expression clustering revealed apparent separation of TCF3-HLF-positive cases from TCF3-PBX1/TCF3-ZNF384-positive cases and those without pathogenic fusions. The differential expression of 535 genes (up: 207, down: 328) was identified in TCF3-HLF-positive cases compared to TCF3-PBX1-positive cases. The differential expression of 471 genes (up: 281, down: 190) was identified in TCF3-HLF-positive cases compared to TCF3-ZNF384-positive cases. TCF3-HLF-positive patients displayed a significantly up-regulated expression of HLF, which was almost not expressed in other cases (Figure 1). The median OS of the 24 patients was 18.5 months (range 6-75 months). Thirteen of them underwent allogeneic HSCT (allo-HSCT) and the median OS was 23 months (range 13-75 months). Eight of them were in complete remission (CR) until the last follow-up; 2 of them relapsed after a first allo-HSCT and survived in CR after a second allo-HSCT; 3 of them died (2 died of relapse and 1 died of lung infection under CR). Eleven cases did not receive allo-HSCT, and the median OS was 9 months (range 6-29 months). Seven of them died (6 died after relapse, 1 died without achieving remission); 3 of them relapsed and re-induction was failed; only one case has survived in CR for 19 months till the last follow-up. Twelve cases underwent chimeric antigen receptor T-cells (CAR-T) therapy. Nine of them achieved CR after CAR-T therapy and bridged to allo-HSCT; one case achieved CR after CAR-T therapy but relapsed and lost opportunity for allo-HSCT; the other 2 patients achieved CR after the first application of CAR-T therapy but failed to achieve CR again by CAR-T therapy when relapsed. Conclusions We provide systematic insights into the laboratory characteristics and prognoses of B-ALL cases with TCF3-HLF in a large cohort. TCF3-HLF-positive B-ALL has a characteristic gene expression profile that differs markedly from TCF3-PBX1 and TCF3-ZNF384-positive B-ALL and shows a dismal prognosis. TC3F-HLF-positive ALL remains an incurable disease, although CAR-T therapy and allo-HSCT can improve the prognosis to some extent. Advanced therapeutic approaches, including novel drug discovery and development, are urgently required to improve the outcome of this ALL subtype. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

AIM:This study aimed to investigate the regularity of gene mutations in patients with myelodysplastic syndrome (MDS) and in those that progressed to acute myeloid leukemia (MDS/AML).PATIENTS & METHODS:High-throughput sequencing technology was used to detect gene mutations in 99 newly diagnosed patients with MDS or MDS/AML.RESULTS:Gene mutations were detected in 88 patients. The mutation incidence in the MDS/AML group was significantly higher than that in the MDS group. Statistically significant differences were observed between the MDS with refractory anemia (MDS-RA) and MDS-RA with excess blasts groups and between the MDS/AML and MDS-RA groups.CONCLUSION:Our data demonstrate that there is a cumulative accumulation of gene mutations, especially in transcription factor genes, during disease progression in MDS and MDS/AML.

Whether Fanconi anemia (FA) heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is a crucial but unsettled issue in cancer prevention and family consulting. We retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 patients with aplastic anemia (AA) and hematologic malignancy. Sixty-eight variants were identified in 66 patients (8.38%). FANCA was the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20). Compared with that of the ExAC East Asian dataset, the overall frequency of rare PSVs was higher in our cohort (P = 0.016). BRCA2 PSVs showed higher frequency in acute lymphocytic leukemia (P = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (P = 0.020; P = 0.008). FA-PSV-positive MDS/AML patients had a higher tumor mutation burden, higher rate of cytogenetic abnormalities, less epigenetic regulation, and fewer spliceosome gene mutations than those of FA-PSV-negative MDS/AML patients (P = 0.024, P = 0.029, P = 0.024, and P = 0.013). The overall PSV enrichment in our cohort suggests that heterozygous mutations of FA genes contribute to hematopoietic failure and leukemogenesis.

Objective:To detect fusion gene with pathological significance in a patient with refractory and relapsed acute B cell lymphoblastic leukemia (B-ALL) and to explore its laboratory and clinical characteristics.Methods:Transcriptome sequencing was used to detect potential fusion transcripts. Other laboratory results and clinical data of the patient were also analyzed.Results:The patient was found to harbor TCF3 exon 17- ZNF384 exon 7 in-frame fusion transcript. The minimal residual disease (MRD) has remained positive after multiple chemotherapy protocols including CD19-, CD22- targeted chimeric antigen receptor T cells immunotherapy. The patient eventually achieved complete remission and sustained MRD negativity after allogeneic hemopoietic stem cell transplantation (allo-HSCT). Conclusion:Transcriptome sequencing can effectively detect potential fusion genes with clinical significance in leukemia. TCF3-ZNF384 positive B-ALL has unique laboratory and clinical characteristics, may not well respond to chemotherapy and immunotherapy, and is more likely to relapse. Timely allo-HSCT treatment may help such patients to achieve long-term disease-free survival. TCF3-ZNF384 positive B-ALL is not uncommon in pediatric patients but has not been effectively identified.

Chromosome translocation t(12;22)(p13;q12)/MN1-ETV6 and MN1 overexpression confer a subset of adverse prognostic AML but so far lack in-depth research. We focused on the clinical course and comprehensive genetic analysis of eight cases with t(12;22)(p13;q12) and one with t(12;17;22) (p13;q21;q13) to elucidate their molecular etiology and outcomes of allogeneic hemopoietic stem cell transplantation (allo-HSCT). The total incidence of t(12;22)(p13;q12) and related translocations was 0.32% in myeloid neoplasms. These patients were confirmed to have dismal prognosis when treated only with chemotherapy, and we firstly provided evidence that they can significantly benefit from timely allo-HSCT. Five cases were MN1-ETV6 positive, and a novel MN1-STAT3 fusion was identified in the patient with triadic translocation. Significant MN1 overexpression was observed in all three MN1-fusion-negative cases. Genetic analysis highlighted the evidence of an ectopic super-enhancer associated orchestrated mechanism of MN1 overexpression and ETV6 haploinsufficiency in t(12;22)(p13;q12) myeloid neoplasms, rather than the conventional thought of MN1-ETV6 fusion formation. We also disclosed the high concomitance of trisomy 8 and 531 Kbps focal 8q duplication in t(12;22)(p13;q12) cases. The new perspective about this entity of disease will enlighten further research to define the mechanism of tumorigenesis and discover effective treatments for MN1-driven malignancies.

Introduction Fusion genes (FGs) are major molecular biological abnormalities in acute leukemia and have been used as molecular markers for the diagnosis, classification, risk stratification and targeted therapy of leukemia. We previously reported common FGs were presented in approximately 41% of acute myeloid leukemia (AML) cases (Chen X et al., Leuk Lymphoma 2019). The rapid development of sequencing technology and the decline of sequencing costs in recent years have made whole transcriptome sequencing (WTS) more accessible, which can not only analyze known FGs, but also has unique advantages in identifying unknown rare and variant FGs. We aimed to identify novel fusion transcripts with clinical relevance and delineate a comprehensive map of FGs in AML based on a large cohort using WTS. Methods We studied 400 consecutively diagnosed AML patients using WTS, 50 normal bone marrow (BM) samples from healthy donors were used as controls. Written informed consents were obtained from all patients and healthy donors or their guardians in accordance with the Declaration of Helsinki. WTS was performed using RNA extracted from the BM samples by HiSeq 2500. Reads were mapped and processed by Arriba (v1.0.1) to generate gene fusions. Only in-frame fusions of high confidence were retained. We applied FGs to a four-tier system as follows; (A) pathogenic: well-known FGs or new members of common fusion gene families (FG-FMs) with definite pathogenicity in hematological malignancies or other tumors. (B) likely pathogenic: rarely reported FGs or new members of rare FG-FMs in hematological malignancies or other tumors without functional verification. (C) uncertain significance: novel FGs and both genes have not been reported in tumors. (D) non-pathogenic: FGs detected in normal samples. Results Our analysis identified 342 high confidence in-frame FGs in 400 AML cases. We further classified the FGs into four ties based on pathogenicity and the 199 tier A and 87 tier B FGs were adopted to the final FG list for further analysis (Figure 1A). The 286 tier A and tier B FGs were identified in 243 (61%) samples (mean, 1.2 per sample), of which 101 were distinct events. Tier A FGs were detected in 197 (49%) cases while 46 (12%) cases had tier B FGs without tier A FGs. The remaining 157 (39%) cases had no tier A nor tier B FGs. We identified 37 cases with co-existence of at least two different FGs, accounting for 9% of all cases enrolled in this study and 15% of all positive cases (Figure 1B). Multiplex-nested RT-PCR which was designed to detect 41 common FGs (all belonged to tier A FGs) was performed in all 400 cases and only 166 (42%) cases were positive. We found 27 kinds of recurrent FGs which occurred at least twice, including 18 tier A and 9 tier B FGs, respectively. Of these, 7 recurrent FGs have never been previously reported. Furthermore, we classified the 101 distinct FGs found in the 243 cases according to FG-FMs, which referred to FGs that involve one protagonist gene and multiple fusion partners. Nearly half (47%) FGs could be classified into 18 FG-FMs, such as RUNX1-FM, KMT2A-FM, NUP98-FM, RARA-FM, ZNF292-FM, DDX5-FM, and NUP214-FM. The other 54 distinct FGs like CBFB-MYH11, CBFA2T3-GLIS2, and KAT6A-CREBBP could not be classified into any family. Most FGs which could not be clustered into FG-FMs occurred only once. All in all, 74% of the 286 tier A and tier B FGs could be classified into FG-FMs, the remaining 26% FGs mainly belonged to tier B and rarely recurred in different samples. When we focused on tier A FGs, 90% of them could be clustered into FG-FMs, while only 10% of them could not be classified into any FG-FM. Conclusions We described the map of FGs detected in a large cohort of AML and revealed FGs with clinical relevance that have not been previously recognized. Classifying FGs according to FG-FMs can better understand their pathological significance and suggest new classification patterns of acute leukemia. WTS is a valuable tool and should be widely used in the routine diagnostic workup of AML. Disclosures No relevant conflicts of interest to declare.

OBJECTIVE:To study the correlation between tumor-associated somatic gene mutation and age in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia transformed from myelodysplastic syndrome (MDS/AML).METHODS:A total of 111 patients primarily diagnosed as MDS or MDS/AML were selected. Bone marrow samples from patients were collected or bone marrow smears prepared at the initial diagnosis were used for detecting the somatic gene mutations of 58 genes related with hematologic tumors by high-throughput gene sequencing. And the correlation of gene mutations with the age of patients was analyzed.RESULTS:The positive rate of total gene mutation was 87.39% (97/111) in 111 patients, and 231 mutations in 28 different genes were detected in the patients with positive gene mutation. The patients of mutation-positive group were significantly older than that of the mutation-negative group (P＜0.001). Among the mutation-positive patients, the mutation rate in the senile group (≥60 years) was 100% (14/14), followed by 89.04% (74/85) in the adult group (15-59 years) and 75% (9/12) in the children group (≤14 years). The average number of mutations in the children group, the adult group and the senile group were respectively 1.44, 2.47 and 2.5; the number of mutations in the adult group was greater than that in the children group (P＜0.05).The most common mutations in the children group occurred in signal transduction gene (46.15%, 6/13); The most common mutations in both the adult group (22.40%, 41/183) and the senile group (34.29%, 12/35) occurred in epigenetic regulatory gene; the mutation rate of transcription factor gene in the senile group was higher than that in the children group (50.00% vs 8.33%) (P＜0.05); the mutation rates of the splicing factor gene in the adult group and the senile group were higher than that in the children group (44.71% vs 8.33%) (P＜0.05), (47.06% vs 8.33%) (P＜0.05).CONCLUSION:The tumor-associated somatic gene mutations in patients with MDS and MDS/AML are significantly different between the different age groups, especially the children group and the adults group as well as the senile group, suggesting that the occurrence of MDS in children may involve genetic factors that are significantly different from those of adults and the senile.