Advances in B-ALL Diagnosis and Classification: A Comparative Analysis of Who-HAEM4R, Who-HAEM5 and International Consensus Classification
Authors: Chen Xue , Yuan Lili , Wang Fang , Zhang Yang , Ma Xiaoli , Cao Panxiang , Fang Jiancheng , Wang Tong , Chen Jiaqi , Zhou Xiaosu , Liu Hongxing 
Published: December 2024

Introduction
Recently, two guidelines were published to refine the diagnosis and classification of B-cell lymphoblastic leukemias (B-ALL): the 5th edition of the World Health Organization Classification of Lymphoid Neoplasms (WHO-HAEM5) and the International Consensus Classification (ICC). These updates introduce new genetic subtypes and refine existing categories based on recent advancements in gene expression and sequencing studies. This study aims to compare the diagnostic and classification outcomes of B-ALL cases using these guidelines.
Methods
We retrospectively analyzed 1,015 cases diagnosed with B-ALL according to WHO-HAEM4R in our hospital from Sep 1, 2018, to Apr 30, 2024. The patients ranged from 8 months to 94 years old, with a median age of 17 years. Among them, 554 were children (≤18 years), and 461 were adults (&gt;18 years). Each case was re-evaluated using WHO-HAEM5 and ICC guidelines. In addition to karyotyping and FISH, high throughput sequencing, including whole transcriptome sequencing (WTS), was performed in all cases to detect gene fusions, mutations, and gene expression profiles. Written informed consent was obtained from all cases or guardians per the Declaration of Helsinki.
Results
Based on the WHO-HAEM4R, a total of 563 cases (55%) were classified with recurrent genetic abnormalities. The distribution of these subtypes, in descending order of prevalence, was as follows: 172 cases (17%) of B-ALL with t(9;22)(q34.1;q11.2)/BCR-ABL1; 128 cases (13%) of B-ALL with hyperdiploidy; 73 cases (7%) of B-ALL, BCR-ABL1-like; 71 cases (7%) of B-ALL with t(v;11q23.3)/KMT2A-rearranged; 60 cases (6%) of B-ALL with t(12;21)(p13.2;q22.1)/ETV6-RUNX1; 38 cases (4%) of B-ALL with t(1;19)(q23;p13.3)/TCF3-PBX1; 17 cases (2%) of B-ALL with hypodiploidy; and 1 case of B-ALL with t(5;14)(q31.1;q32.3)/IL3-IGH. Additionally, 3 cases exhibited both ploidy changes (hyperdiploidy or hypodiploidy) and recurrent fusion genes, making their classification indeterminate.
Upon reclassification using WHO-HAEM5, the distribution was refined by incorporating new genetic subtypes and gene expression features. 219 out of the 452 B-ALL, not otherwise specified (NOS) cases (48%) were classified into new genetic entities. These included 60 cases (6%) with ZNF384 rearrangement, 50 cases (5%) with DUX4 rearrangement, 42 cases (4%) with MEF2D rearrangement, 10 cases (1%) with HLF rearrangement, 3 cases with MYC rearrangement, and 2 cases with NUTM1 rearrangement. Additionally, 43 cases (4%) were classified as PAX5alt, and 9 cases (1%) were classified as PAX5 p.P80R. Overall, after reclassification using WHO-HAEM5, cases diagnosed as B-ALL, NOS was reduced from 45% to 23%.
Compared to WHO-HAEM5, ICC included two additional subtypes: B-ALL with CDX2/UBTF and B-ALL with IKZF1 N159Y, which were identified in 18 and 3 cases, respectively. ICC also listed 4 provisional entities that require gene expression clustering studies for definitive identification. These include 13 cases of ETV6::RUNX1-like, 4 cases of ZNF384 rearranged-like, 3 cases of KMT2A rearranged-like, and 8 cases of B-ALL with mutated ZEB2 (p.H1038R)/IGH::CEBPE. Additionally, the ICC separates “B-ALL, BCR::ABL1-like” into 3 subtypes: ABL1-class rearranged, JAK-STAT activated, and NOS. In this study, these subtypes were identified in 23, 49, and 2 patients, respectively. Furthermore, in ICC, B-ALL with hypodiploidy is formally separated into 2 categories: “B-ALL, low hypodiploid” (32-39 chromosomes), and “B-ALL, near haploid” (24-31 chromosomes). Thus, a case initially diagnosed as hypodiploid B-ALL under WHO-HAEM5 with high hypodiploidy (40-43 chromosomes) was reclassified as B-ALL, NOS under ICC. Overall, after reclassification using ICC guidelines, the number of cases diagnosed as B-ALL, NOS was further reduced to 185 cases (18%).
Conclusion
The WHO-HAEM5 and ICC guidelines introduce significant updates to B-ALL classification, including new genetic subtypes and refined categories. Although overlapping, each guideline offers unique enhancements in the precision of diagnosis and the effectiveness of targeted therapy. Our comparative analysis of 1,015 B-ALL cases demonstrates that these guidelines can lead to different classification outcomes, influencing treatment decisions and prognostication. Comprehensive molecular diagnostics are crucial for accurate B-ALL classification and management.