Single Cell Profiling of Ph-Positive Acute Lymphoblastic Leukemias Reveals Distinct Cells of Origin

Background

Philadelphia chromosome-positive (Ph+) B-cell acute lymphoblastic leukemia (ALL) represents a group of heterogenous leukemias driven by a common oncogenic fusion (BCR::ABL1). The heterogeneity comes from different isoforms of BCR::ABL1 (p190, p210) in addition to presumed cell/cell state of origin (lymphoid blast phase of chronic myeloid leukemia (CML) vs de novo Ph+ ALL). This has implications for treatment and response to treatment, as well as ability and modalities for track measurable residual disease (MRD) following therapy. Additionally, how Ph+ ALL compares with Ph-negative and Ph-like ALL at a single cell resolution is not well understood. Here we present a comprehensive interrogation of leukemic and microenvironmental cells from patients with Ph+ lymphoid leukemia, with a focus on Ph+ ALL.

Methods

We conducted scRNA-seq profiling from 26 BM and PB samples of 24 patients, including 15 with de novo Ph+ B-ALL (p190 n=10 and p210 n=5) and 2 with de novo B-lymphoid blast phase of CML, 4 with Ph-like B-ALL (3 CRLF2 rearranged and ABL1 fusion), and 3 with Ph- B-ALL. All samples were collected prior to the initiation of definitive induction therapy. Data analyses were performed in R with Seurat. Harmony was used for batch effect correction. Cell annotation was performed using canonical markers and was validated through reference mapping to healthy human bone marrow references. To infer presumed cell of origin of ALL cells, we mapped the ALL cells to a human B-cell development reference (Lacobucci, bioRxiv 2023). ALL cell of origin enrichment within each disease subtype was done using Fisher’s exact test and Benjamini-Hochberg false-discovery correction. Gene set enrichment analysis (GSEA) was conducted on differentially expressed genes using Hallmark gene set.

Results

Our cohort contains 24 BM and 2 PB samples collected prior to the initiation definitive induction therapy. The median age of the cohort was 42 years old. A total of 92% of patients were tested for mutations using 81-gene panel. Among these, 8 patients had identified mutations, with IKZF1 and ETV5 being the most common affecting 2 patients each.

Cell of origin analysis showed the most common lineage in Ph+, Ph-, and Ph-like was the pro-B cells with productive immunoglobulin heavy chain in 78%, 58%, and 87% of the total B-lineage, respectively. Compared with Ph- B-ALL, Ph+ ALL showed greater cell of origin enrichment in both early hematopoietic stem cells/multipotent progenitors (HSC/MPP) and mature B cells (p<0.001), whereas Ph- were predominately common lymphoid progenitors (CLP) and myelo-lymphoid progenitors (MLP) with p<0.001. GSEA showed inflammatory pathways including, IL6, TNFA, and IFNG signaling enriched in Ph+ and cell cycle pathways enriched in Ph-.

A similar trend was seen in comparing Ph+ and Ph-like, where Ph+ ALL cells mapped more to HSC and early lymphoid (MPP, LMPP, MLP, CLP) lineages (p<0.001), while Ph-like were mapped more to early-B lineages (Pre-Pro-B, Pro-B, Pre-B), p<0.001. Similar to the comparison with Ph- B-ALL, GSEA showed IL6/JAK/STAT3, NF-KB by TNF, and KRAS signaling enriched in Ph+ as compared with Ph-like ALL.

Within the Ph+ groups, comparison of p190 vs. p210 isoforms, revealed that p210 isoform to have more HSC and early lymphoid cell of origin projections (p<0.001) while p190 were more enriched in early B cell lineages (p<0.001). GSEA showed E2F pathway was enriched in p190 Ph+ ALL.

Finally, our comparison of lymphoid blast phase CML and Ph+ ALL demonstrated a significant enrichment of more mature B cell lineages in Ph+ ALL.

Conclusions

In our study, we used scRNA-seq profiling to compare the B-cell developmental states in ALL. Our finding revealed that the Pro-B VDJ cell of origin is the most common accounting for 78% of the total B-lineage in all types. We also found that early lymphoid were enriched in Ph+ (HSC/MPP) and Ph- (CLP and MLP), while early-B lineages were enriched in Ph-like. Additionally, GSEA showed inflammatory pathways enriched in Ph+ whereas, cell cycle and proliferative pathways enriched in Ph-. Within Ph+ group, we observed high enrichment of late-B cells in Ph+ ALL, while in lymphoid blast phase CML, HSC, early lymphoid, and early-B cells were enriched.