Nowadays, thanks to improvements in diagnostics and treatment protocols, the outcome for paediatric patients with acute leukaemia is quite favourable. Especially in the case of B-ALL, about 80% of children go through a full recovery. However, there are cases of relapses in which standard therapies are ineffective, leading to a poor prognosis. On the other hand, paediatric T-cell leukaemias often have a poorer prognosis due to their aggressiveness and resistance to many standard treatments (35). It is indeed crucial to identify the disease correctly and early enough to allow an accurate and timely choice of the treatment protocol most appropriate for the patient's clinical situation. This could be a tricky decision to make since childhood leukaemias are heterogeneous diseases. The advent of NGS has strongly boosted the identification of new biomarkers for use in diagnosis and/or therapy. Yet, these advances have been rapid but uneven. While some aspects have been studied in detail, such as cell surface protein and protein-coding genes that could be targeted in therapy protocols ), other aspects have been overlooked, such as the molecular non-coding footprint underlying the disease. Our work aims to contribute to bridge this gap and finely characterise the lncRNA landscape of paediatric acute leukaemias. LncRNAs are a class of biomarkers of crescent interest in the haematologic and oncologic field (19, 29–31). They do not encode proteins and have been reported by several studies to modulate gene expression at the transcriptional, post-transcriptional, and epigenetic levels (20). In particular, due to their involvement in vital oncogenic processes such as differentiation, proliferation, migration, angiogenesis, and apoptosis, lncRNAs have attracted much attention as potential diagnostic and prognostic biomarkers in leukaemia (39, 40).
Starting from NGS transcriptome analyses of B-ALL and T-ALL patients in comparison with B and T lymphocytes from cord blood, we identified a specific lncRNAs signature able to discriminate B-ALL and T-ALL not only from healthy subjects but also between the two types of leukaemia. We selected some candidate lncRNAs that have never been associated with ALL and tested their expression in a larger cohort of patients. For most of them, this experiment confirmed the expression absence in the healthy patient and a significant upregulation in a specific type of ALL, hinting at a potential diagnostic application in clinical practice.
Furthermore, we showed that the lncRNA landscape is specific not only for the two paediatric lymphoblastic leukaemias (B-ALL and T-ALL) but also for myeloid ones. Interestingly, the T-ALL lncRNA signature is somewhat more related to AML than B-ALL, despite the great etiopathological difference between the two diseases. This finding was unexpected; however, it is important to consider that both T-ALL and AML may have common traits in the case of leukemic transformation. Specifically, in the case of AML transformation, it is more likely to find the ectopic expression of T-cell-associated antigens ( such as CD2, CD5 and CD7) than B-cell ones (CD19, CD20) (41). In particular, the CD7 antigen was found to be expressed in 30% of de novo AML and some authors proposed to use the ectopic expression of this antigen for planning AML blasts specific CAR-T therapy (42). Identifying common traits between AML and T-ALL in terms of lncRNA could open a novel scenario to investigate on altered pathways leading to leukemogenesis and characteristics of aggressiveness.
In the final part of this work, since the role of many lncRNAs involved in childhood acute lymphoblastic leukaemia is still unknown, especially in T-ALL, we performed correlation analyses to try to identify the potential role of the lncRNAs in this pathology. Our in silico analyses revealed a gene ontology enrichment in the key pathways for T-cell differentiation for those genes positively correlating with AC247036.1, suggesting its potential role in the modulation of some genes involved in these processes associated with leukemogenesis. Further, we found that lncRNA PCAT18 was associated with the expression of CD3D antigen, a well-known T-cell marker used in diagnostics for monitoring minimal residual disease by flow cytometry. This finding is new and confirmed the relationship between the PCAT18 lncRNA expression and T cell lineage commitment, however additional functional experiments are needed to evaluate the role of PCAT18 in sustaining leukemic growth. Last, our data disclose a highly significant positive correlation between the lncRNA HHIP-AS1 and its relative sense protein-coding transcript HHIP (33). This probably happens because HHIP-AS1 is actively transcribed from a SHH-responsive bidirectional promoter shared with the SHH signalling intermediate HHIP. In SHH-driven tumours, the knockdown of HHIP-AS1 induces mitotic spindle deregulation and the consequential reduction of tumorigenicity in vitro and in vivo (43). Taken together, these data suggest HHIP-AS1 to be a suitable candidate for further functional studies to explore its possible role in enabling the pro-mitotic effects of SHH pathway activation in childhood T-ALL.
Ultimately, our work made available to the research community a comprehensive map of the lncRNA landscape of the various types of paediatric leukaemia, useful not only for diagnostic purposes but also, after appropriate ad hoc functional studies, for therapeutic purposes. In this respect, it is important to note that lncRNAs are easily detectable. Their identification could be included in normal clinical practice strengthening the diagnostic process and improving paediatric patient management. Increasing the patient cohort could help to correlate the expression of specific candidate lncRNAs identified by our study with clinical information, testing their potential prognostic effectiveness in stratifying patients according to their clinical characteristics. This aspect made our study an important resource for the scientific community, laying the foundations for future functional and clinical studies.