Subtype Classification of BCP-ALL Dic(9;20)

Introduction: The dic(9;20)(p11-13;q11) chromosomal aberration is a rare occurrence in acute lymphoblastic leukemia (ALL), more commonly identified in pediatric BCP-ALL (2-3%) than in adults (<2%). With the integration of BCP-ALL subtype classification by gene expression analysis into standard diagnostic protocols, as endorsed by WHO 2022 and ICC 2022, the precise classification of dic(9;20) cases remains to be elucidated. This necessity arises from inconsistent subtype assignments of dic(9;20) cases in the literature, highlighting the need for a more refined understanding of its clinical implications. Aim: Genetic characterization of BCP-ALL patients with dic(9;20). Methods: We analyzed 12 BCP-ALL patients (pts) with dic(9;20) by CBA, immunophenotyping, WTS (cluster >50 mio) and amplification-free WGS (median coverage > 90x). Ten pts were analyzed by FISH for deletions/rearrangements in PAX5 and CDKN2A. Patient stratification based on WTS data was done with ALLCatchR. Genes with known relevance to hematological malignancies were analyzed (n=47). The following variant callers were used: Strelka2 (mutations and VAF), Pindel (for FLT3-ITD), HadoopCNV (CN-LOH), GATK4 (CNV WGS), and RNAseqCNV (CNV WTS). Results: Gene expression profiling led to the high-confidence classification of 8/12 pts into the PAX5 P80R subgroup. The presence of the PAX5 mutation was confirmed by DNA mutation analysis. Additionally, 3/12 pts were categorized into the PAX5alt subgroup (PAX5 T75A, PAX5 P35L, PAX5::C20orf112), and one patient, exhibiting a NUP214::ABL1 fusion transcript, was accordingly classified as BCR::ABL1-like. Analysis of key downstream PAX5 target genes (i.e. BLK, EBF1, FLT3), revealed similar expression levels for all dic(9;20) cases. Interestingly, FGFR1 and MED12L showed significantly increased expression (log fold change of 2.62 and 2.81, respectively; p-value < 0.0001) in dic(9;20) cases compared to other BCP-ALL subgroups. Despite these findings, and as indicated by the high-confidence subgroup assignments, dic(9;20) cases do not possess a unique gene expression signature but rather mirror the profiles of established subgroups as outlined above. This implies that the dic(9;20) chromosomal aberration may not be a primary event in leukemogenesis. Further analysis revealed downregulation of genes on chromosome 9p, consistent with the partial loss of this chromosome arm. Integrated CNV data from WGS and FISH confirmed the deletion of chromosome 9p in 11 out of 12 cases, affecting key genes such as CDKN2A (6 heterozygous, 5 homozygous, 1 n.a.) and PAX5. The sole exception was a patient with a PAX5::C20orf112 fusion transcript. CNV data from WGS revealed that in 9/12 cases the 9p deletion comprised the bands p24 to p13 (del(9)(p24p13)), in 2 cases it was even larger including almost the whole chromosome arm (del(9)(p24p11)). Analysis of CBA and WGS data showed only a low number of additional chromosomal aberrations with monosomy 7 as the sole recurrent co-alteration in 2 cases. Immunophenotyping data was available for 9/12 pts, all of whom were classified as common BCP-ALL. Among these, 2 pts were CD20 bright (WTS subgroup: PAX5alt) and 1 patient was CD20 dim (WTS subgroup: BCR::ABL1-like). Notably, dic(9;20) cases assigned to the PAX5 P80R subgroup were consistently CD20neg. These findings align with known association between elevated CD20 expression and the presence of high-risk genetic markers, suggesting a potential link to adverse prognostic outcomes. WGS was performed in 11/12 cases, revealing by SNV analysis that 10 pts had at least one mutation in a RAS pathway gene, suggesting avenues for targeted therapeutic strategies. The most common mutations identified were PAX5 (82%, 9/11 cases), KRAS (45%, 5/11), and both NRAS and FLT3 (27%, 3/11 each). Conclusion: Our findings indicate that dic(9;20) cases do not constitute a distinct subgroup but can be further classified into PAX5 P80R, PAX5alt, or even BCR::ABL1-like. Here, dic(9;20) may contribute to leukemogenesis via PAX5 wild-type allele deletion, particularly in cases with PAX5 mutation. Although the presence of PAX5 deletion can be detected using FISH, a combined approach of molecular genetic analyses and gene expression profiling is essential for accurate subgroup assignment, which in turn facilitates precise diagnosis, risk stratification, and tailoring of treatment strategies.