A Non-Canonical Lymphoblast in Refractory Childhood T-Cell Acute Lymphoblastic Leukemia
Authors: Lim Bram S.J. , Whitfield Holly J. , Trinh Mi K. , Bloye Gianna , Thomas Rebecca , Anderson Nathaniel D. , Wenger Anna , Hodder Angus , Treger Taryn D. , Lee-Six Henry , Coorens Tim H.H. , Parks Conor , Ogbonnah Toochi , Pölönen Petri , Mullighan Charles G. , Teachey David T. , Hagleitner Melanie M , Kester Lennart A. , van Leeuwen Frank N. , Beattie Gordon , Mansour Marc R. , Williams Owen , Bartram Jack , Adams Stuart , Jardine Laura , Behjati Sam , O'Connor David 
Published: December 2024

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy which carries a worse clinical prognosis than its B-cell counterpart (B-ALL). Despite extensive genomic characterization, no robust genetic or phenotypic marker has been identified for accurate clinical risk stratification of T-ALL at the point of diagnosis, unlike in B-ALL. Instead, the earliest reliable indicator of high-risk disease is failure to respond to four weeks of induction chemotherapy (minimal residual disease ≥5%), limiting the potential for early intervention. Children with induction failure (i.e. refractory disease) make up 10% of childhood T-ALL cases and constitute a very high-risk group, with only half surviving beyond 5 years.

Here, we studied the transcriptional and phylogenetic profiles of refractory T-ALL. We collected diagnostic bone marrow aspirates from 21 children with T-ALL, including 8 who responded to induction and 13 who were refractory. Crucially, for 8 of the children with induction failure, we collected post-induction bone marrows, allowing analysis of the true refractory blast population. We applied high-throughput single-cell mRNA sequencing to all samples and identified a distinct blast population that was consistently associated with induction failure cases and almost entirely absent in responsive cases. Expression analysis identified a common gene set enriched in this population, spearheaded by the ZBTB16 gene, a transcription factor key to lymphocyte differentiation. Presence of ZBTB16+ blasts at diagnosis heralded refractory disease across independent bulk transcriptome datasets and was associated with survival in the largest reported T-ALL cohort of ~1,300 individuals from the Children's Oncology Group AALL0434 trial. Additionally, we confirmed the ability to detect the ZBTB16 protein using intracellular flow cytometry, demonstrating its potential utility as a biomarker of high-risk disease.

Previous studies have associated refractory T-ALL with particular T-cell differentiation states, including the early T-cell precursor (ETP). We found that the non-canonical ZBTB16+ blasts have a distinct profile which broadly resembles innate-like lymphocytes such as innate lymphoid cells and natural killer cells as well as unconventional T cells, in strong contrast to responsive T-ALL blasts which resemble conventional T cells and their developing stages. ZBTB16+ blasts exhibited varied T-cell receptor rearrangement status, suggesting that they can arise from maturation block at multiple stages across T-cell development, as opposed to a single fixed point. Moreover, the detection of ZBTB16+ blasts in bulk transcriptomes was able to risk stratify children with T-ALL more reliably than ETP status determined by immunophenotyping or transcriptome profiling.

Refractory cancers may arise either through the acquisition of resistance mechanisms by cancer cells or represent a distinct entity that is intrinsically resistant. In the majority of cases, ZBTB16+ blasts were clearly seen at both diagnostic and post-induction timepoints. However, for one individual, by projecting somatic mutations onto single-cell transcriptomes, we showed that ZBTB16+ blasts were detectable at diagnosis as a minor subclone (~1% of blasts), but expanded dramatically during induction to become the dominant clone, thereby affirming that ZBTB16+ blasts are intrinsically resistant lymphoblasts that are already present at diagnosis.

Overall, our study presents refractory T-ALL as a distinct leukemia that is associated with a previously unknown lymphoblast variant. We believe the presence of ZBTB16+ blasts has the potential to be a powerful clinical predictor at diagnosis of high-risk T-ALL. We therefore suggest that our findings warrant urgent investigation in prospective clinical cohorts.