High CX3CR1 expression predicts poor prognosis in paediatric acute myeloid leukaemia undergoing hyperleukocytosis

Introduction

Paediatric AML patients with hyperleukocytosis have a poor prognosis and higher early mortality. Therefore, more studies are needed to explore relevant prognostic indicators and develop effective prevention strategies for this type of childhood AML.

Methods

All original data were obtained from the TARGET database. First, we explored meaningful differentially expressed genes (DEGs) between the hyperleukocytosis group and the non-hyperleukocytosis group. Next, we screened and identified valuable target genes using univariate Cox regression, Cytoscape software, and Kaplan–Meier survival curves. Finally, the coexpressed genes, functional networks, and immune-related activities associated with the target gene were deeply analysed by the GeneMANIA, LinkedOmics, GEPIA2021, TISIDB, and GSCA databases.

Results

We selected 1229 DEGs between the hyperleukocytosis group and the non-hyperleukocytosis group in paediatric AML patients. Among them, 495 DEGs were significantly linked with the overall survival of paediatric AML patients. Further, we discovered that CX3CR1 was a promising target gene. Meanwhile, we identified CX3CR1 as an independent prognostic predictor. Besides, we showed that CX3CR1 had strong physical interactions with CX3CL1. Additionally, functional network analysis suggested that CX3CR1 and its coexpressed genes modulated immune response pathways. Subsequent analysis found that immune cells with a high median value of CX3CR1 were monocytes, resting NK cells and CD8 T cells. Finally, we observed that CX3CR1 expression correlated with infiltrating levels of immune cells and immune signatures.

Conclusion

Elevated CX3CR1 expression may be an adverse prognostic indicator in paediatric AML patients undergoing hyperleukocytosis. Moreover, CX3CR1 may serve as an immunotherapeutic target for AML with hyperleukocytosis in children.     

CBL mutations do not frequently occur in paediatric acute myeloid leukaemia

RAS‐pathway mutations, causing a proliferative advantage, occur in acute myeloid leukaemia (AML) and MLL‐rearranged leukaemia. Recently, mutations in the Casitas B lineage lymphoma (CBL) gene were reported to be involved in RAS‐pathway activation in various myeloid malignancies, but their role in paediatric AML is still unknown. We performed mutation analysis of 283 newly diagnosed and 33 relapsed paediatric AML cases. Only two mutant cases (0·7%) were identified in the newly diagnosed paediatric AML samples, of which one was MLL‐rearranged. Both mutant cases showed CBL mRNA expression in the range of the non‐mutated cases. Phosphorylated extracellular signal‐regulated kinase (pERK) was not correlated with CBL protein expression (n = 11). In conclusion, we report a very low CBL mutation frequency in paediatric AML, which, together with the lack of difference in protein and mRNA expression, illustrates the limited role of CBL in paediatric AML.     

Phase II study of 2-chlorodeoxyadenosine plus idarubicin for children with acute myeloid leukaemia in first relapse: a paediatric oncology group study

Relapse remains the leading cause of death in patients with acute myeloid leukaemia (AML). Relatively few new chemotherapy agents have been proven to be effective in this population. We report on a Phase 2 clinical trial using the novel combination of 2-chlorodeoxyadenosine (2-CDA) (8 mg/m2 per d x 5 d) plus idarubicin (Ida) (10 mg/m2 per d x 3 d). The study involved 109 paediatric patients with AML at first relapse, of whom 104 were available for analysis. The overall response rate was 51% (complete response [CR] + partial response) with a CR rate of 46%. 2-year event-free survival (EFS) and overall survival (OS) were 20% and 26%. The only significant variable in determining response, EFS and OS was duration of initial remission, with patients who had an initial remission >1 year having much worse outcomes overall (response rate 74% vs. 25%, EFS 8% vs. 37% and OS of 16% vs. 39%, P < 0.01 for all). There was an acceptable toxicity profile with one neurological event and no cardiac events observed. The most common grade 3-4 toxicities observed were neutropenia (59%) and thrombocytopenia (68%). This study demonstrated that the novel combination of 2-CDA/Ida was effective and should be considered for incorporation in front line therapy for children with AML.     

Whole‐exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole‐exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence.     

High bax expression is a good prognostic indicator in acute myeloid leukaemia

Most cytotoxic drugs kill cells by instigating the process of apoptosis and it has been suggested that apoptotic markers may provide an indication of tumour chemosensitivity. The aim of this study was to determine if such a relationship exists in acute myeloid leukaemia (AML). The levels of spontaneous apoptosis, bcl-2 and bax were evaluated in 56 newly diagnosed AML patients to determine if they correlated with a response to cytotoxic therapy. Spontaneous apoptosis was lower, but bcl-2, bax and the bcl-2/bax ratio were higher in AML compared with normal individuals. AML patients with high bax expression at diagnosis had significantly better prognosis for disease-free survival, event-free survival and overall survival (P = 0.016). In the standard risk group, high bax expression was in keeping with significantly improved survival. Multivariate analysis revealed bax to be an independent predictor of survival. There was a significant reduction in bcl-2 and bax expression when AML patients entered complete remission and also in relapsed AML patients who entered a second remission. This study suggests that bax is a useful prognostic indicator in AML and may assist with therapeutic decision-making for patients in the standard risk category.     

Pneumocystis jirovecii pneumonia in patients with acute myeloid leukaemia

The association of Pneumocystis jirovecii pneumonia (PJP) and acute myeloid leukaemia (AML) is not clearly defined. In our experience of 291 patients with AML, 20 (14 males and 6 females, median age 56) developed PJP (incidence 6.8%). Thirteen patients (65%) survived until discharge from hospital. We conclude that PJP is not uncommon among patients with AML. In clinical care of AML, awareness of PJP should be heightened and prophylaxis should be considered.     

RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv(16) subtype

Correlative and functional studies support the involvement of the RUNX gene family in haematological malignancies. To elucidate the role of epigenetics in RUNX inactivation, we evaluated promoter DNA methylation of RUNX1, 2, and 3 in 23 leukaemia cell lines and samples from acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL) and myelodysplatic syndromes (MDS) patients. RUNX1 and RUNX2 gene promoters were mostly unmethylated in cell lines and clinical samples. Hypermethylation of RUNX3 was frequent among cell lines (74%) and highly variable among patient samples, with clear association to cytogenetic status. High frequency of RUNX3 hypermethylation (85% of the 20 studied cases) was found in AML patients with inv(16)(p13.1q22) compared to other AML subtypes (31% of the other 49 cases). RUNX3 hypermethylation was also frequent in ALL (100% of the six cases) but low in MDS (21%). In support of a functional role, hypermethylation of RUNX3 was correlated with low levels of protein, and treatment of cell lines with the DNA demethylating agent, decitabine, resulted in mRNA re‐expression. Furthermore, relapse‐free survival of non‐inv(16)(p13.1q22) AML patients without RUNX3 methylation was significantly better (P = 0·016) than that of methylated cases. These results suggest that RUNX3 silencing is an important event in inv(16)(p13.1q22) leukaemias.