Paper Details

ABSTRACT

Our previous studies demonstrated that BCR::ABL1-positive (BCR::ABL1+/Ph+) acute lymphoblastic leukaemia (ALL) comprises two distinct biological patterns - “Typical Ph+ ALL” and “CML-like” - differing in the association between the BCR::ABL1 fusion and the dominant ALL clone. This distinction was established by integrating quantitative monitoring of clonal IG/TR rearrangements (characterising the “fully leukaemic” clone) and BCR::ABL1 fusion. CML-like cases are characterised by discordant levels with BCR::ABL1 exceeding IG/TR, indicating that the fusion is present beyond the main ALL clone, resembling clonal haematopoiesis. Our findings were reflected in the International Consensus Classification (ICC) of leukaemias, where subtypes representing CML-like and Typical Ph+ ALL were re-named as BCR::ABL1+ leukaemias with “multilineage” and “lymphoid-only” involvement, respectively.

Recently, WTS (whole transcriptome sequencing)-based approaches identified separate clusters of BCR::ABL1+ ALL and some studies claimed that these represent the ICC-defined subtypes. However, whether the clusters indeed mirror the CML-like/Typical Ph+ ALL subtypes was never validated. To reveal the relationship between the original and the WTS-defined subtypes, we performed WTS in children with BCR::ABL1+ B-cell precursor ALL (n=78), chronic phase CML (CML-CP; n=12), and CML in lymphoid blast crisis (CML-LBC; n=3). In addition, we established a DNA-based single-cell approach for parallel detection of patient-specific BCR::ABL1 genomic fusions, clonal IG/TR rearrangements, and other clonal markers (IKZF1del, CDKN2Adel, PAX5del, etc.). This approach was applied to four cellular compartments (leukaemic blasts and non-leukaemic myeloid cells/B-cells/T-cells) from eight patients (1 CML-LBC, 3 Typical Ph+ ALL, 4 CML-like ALL) to resolve lineage involvement and clonal architecture.

Unsupervised UMAP analysis of WTS data separated CML-CP from BCR::ABL1+ ALL, and the ALL samples were distributed into two clusters, clearly corresponding to the claimed multilineage and lymphoid-only subtypes (as confirmed by the previously established multilineage/lymphoid WTS predictor). However, instead of CML-like being assigned to the multilineage cluster and Typical Ph+ ALL to the lymphoid cluster, the original subtypes were distributed across the two clusters independently. Strikingly, all CML-LBC were located in the lymphoid cluster. Moreover, analysis of eight paired diagnosis-relapse samples revealed that in two CML-like patients relapsing from distinct IG/TR clones while retaining the same BCR::ABL1 genomic fusion, diagnoses clustered within the lymphoid cluster, whereas relapses shifted to the multilineage cluster, further highlighting that the WTS-based clustering does not reflect the underlying origin of the BCR::ABL1+ ALL. Single-cell genomic analysis provided a biological explanation for these differences. CML-like cases showed an expected pattern, with a subset of BCR::ABL1+ cells outside the dominant ALL clone (i.e. in IG/TR-negative cells) carrying BCR::ABL1 as the only detectable leukaemia-associated marker, resembling clonal haematopoiesis. In contrast, in Typical Ph+ ALL, BCR::ABL1 was consistently detected only in the same cells as clonal IG/TR rearrangements. This remained true even in cases with partial myeloid features (CD13 and/or CD33 expression, or multilineage WTS classification), where BCR::ABL1+ cells can be found in myeloid (seemingly non-leukaemic) compartment, but in the case of Typical Ph+ ALL, the BCR::ABL1-positivity is always accompanied by clonal IG/TR rearrangement, thus demonstrating lineage plasticity of these fully leukaemic BCR::ABL1+ cells, rather than a clue to the fusion origin.

In conclusion, our findings demonstrate that the original CML-like vs. Typical Ph+ ALL assignment and the transcriptome-based classifications are complementary but not interchangeable, as they capture distinct biological dimensions of BCR::ABL1+ ALL. Integrating these approaches provides a more comprehensive view of disease evolution and lineage involvement in this high-risk leukaemia subtype. Importantly, to identify the clinically relevant CML-like subgroup, single-cell analysis and/or minimal residual disease monitoring of both BCR::ABL1 and IG/TR are still the only options.