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.

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.

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.

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.

DNTT encodes the most template-independent DNA polymerases TdT. The canonical function of TdT is to boost the diversity of immunoglobulin and T cell receptors by incorporating non-templated nucleotides (NTN) to their variable regions via RAG1/2 mediated DNA breaks and non-homologous end joining (NHEJ) rearrangement process. This study aimed to investigate the relationship between aberrant DNTT expression and illegitimately TdT-aided microhomology-mediated replication-dependent recombination (MMRDR) with the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and their prognosis relevance.

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.