Comprehensive Fusion Gene Landscape in B-Cell Lymphoblastic Leukemia: Insights from Transcriptome Sequencing of 1015 Cases

Background

B-cell lymphoblastic leukemia (B-ALL) is a heterogeneous hematologic malignancy where fusion genes (FGs) play a crucial role in its pathogenesis. These genetic alterations drive oncogenesis and influence disease prognosis and treatment. In 2021, we published the FG map of acute leukemia revealed by whole transcriptome sequencing (WTS) of 1000 cases, including 365 B-ALL patients (Blood Cancer Journal. PMID: 34135310). Building upon this initial study, we have now expanded our research to include 1015 B-ALL cases, aiming to provide a comprehensive map of FGs in B-ALL, revealing novel fusions and assessing their clinical relevance, thereby enhancing our understanding of B-ALL's molecular landscape and informing targeted therapies.

Methods

We enrolled 1015 B-ALL cases diagnosed in our hospital from Sep. 2018 to Apr. 2024, with high-quality WTS data. Among them, 554 (55%) were children (≤18 years, median age 8 years), and 461 (45%) were adults (>18 years, median age 35 years). We used bone marrow samples from 100 healthy donors as controls. Written informed consent was obtained from all participants or guardians per the Declaration of Helsinki.

RNA quality assessment, sequencing library preparation, paired-end sequencing, and FGs detection were conducted as we previously reported.

High-confidence in-frame FGs detected by WTS were classified into 4 tiers based on pathogenicity. (A) pathogenic: well-known FGs or new members of common FG families (FG-FMs) with definite pathogenicity in hematological malignancies. (B) likely pathogenic: FGs reported in hematologic malignancies without functional verification, or novel FGs involving genes associated with these malignancies. (C) uncertain significance: novel FGs involving genes without known associations with these malignancies. (D) non-pathogenic: FGs found in normal samples.

Results

A total of 330 different FGs were detected in 717 cases, including 122 tier A, 77 tier B, 127 tier C, and 4 tier D FGs. Tier D FGs were excluded from further analysis.

881 fusion events (647 tier A, 95 tier B, and 139 tier C) were detected in 709 (70%) cases. Most patients (n=564; 80%) carried only one FG. Additionally, 125 cases had 2 FGs, 13 cases had 3 FGs, and 7 cases had 4 FGs. Only 26 patients had coexisting tier A FGs, accounting for 2.6% of all cases and 3.7% of all positive cases.

We found 52 kinds of recurrent FGs that occurred at least twice, including 37 tier A, 9 tier B, and 6 tier C FGs, respectively. The positivity rate of FGs presented a typical long-tail distribution, with only 11 FGs having a positivity rate >1%: BCR::ABL1 (16.9%), ETV6::RUNX1 (6.1%), KMT2A::AFF1 (4.6%), DUX4::IGH (3.8%), TCF3::PBX1 (3.7%), EP300::ZNF384 (3.1%), MEF2D::BCL9 (2.5%), UBTF::ATXN7L3 (1.8%), TCF3::ZNF384 (1.4%), MEF2D::HNRNPUL1(1.0%), TCF3::HLF (1.0%).

Notably, a considerable number of so-far unreported FGs were detected in this study. Totally, 236 kinds of novel FGs were discovered (40 tier A, 69 tier B, and 127 tier C), accounting for 72% of all FGs. However, only 13 of them were recurrent.

We classified the 199 distinct tier A and tier B FGs into FG-FMs, which involve one protagonist gene and various fusion partners. More than half of them (130/199, 65%) could be classified into 25 FG-FMs, such as PAX5-FM, JAK2-FM, KMT2A-FM, and ZNF384-FM. When we focused on tier A FGs, 93% (114/122) could be clustered into FG-FMs, indicating the central involvement of key genes in B-ALL pathogenesis.

Conclusion

This analysis of 1015 B-ALL cases using WTS expands our understanding of the FG landscape in B-ALL. We identified FGs in 70% of cases, revealing a map that differed from previous expectations. Some FGs or FG-FMs were underestimated before due to their cryptic nature. For example, the positivity rate of ZNF384-FM is second only to BCR::ABL1, surpassing the well-known ETV6::RUNX1 and TCF3::PBX1 fusions in B-ALL. Classification of FGs into FG-FMs reveals the central involvement of key genes in pathogenic FGs. These findings highlight the role of FGs in B-ALL pathogenesis and their potential as biomarkers for diagnosis, prognosis, and targeted therapy. The long-tail distribution of FGs suggests that comprehensive genomic profiling is essential. This study enhances the FG map of B-ALL and provides a resource for future research and clinical applications, aiding in the development of precision medicine and improving patient outcomes.