Gene expression prognostic of early relapse risk in low-risk B-cell acute lymphoblastic leukaemia in children.

ETV6::RUNX1 is the most common fusion gene in childhood acute lymphoblastic leukaemia (ALL) and is associated with favorable outcomes, especially in low-risk children. However, as many as 10% of children relapse within 3 years, and such early relapses have poor survival. Identifying children at risk for early relapse is an important challenge. We interrogated data from 87 children with low-risk ETV6::RUNX1-positive B-cell ALL and with available preserved bone marrow samples (discovery cohort). We profiled somatic point mutations in a panel of 559 genes and genome-wide transcriptome and single-nucleotide variants. We found high TIMD4 expression (> 85th-percentile value) at diagnosis was the most important independent prognostic factor of early relapse (hazard ratio [HR] = 5.07 [1.76, 14.62]; p = 0.03). In an independent validation cohort of low-risk ETV6::RUNX1-positive B-cell ALL (N = 68) high TIMD4 expression at diagnosis had an HR = 4.78 [1.07, 21.36] (p = 0.04) for early relapse. In another validation cohort including 78 children with low-risk ETV6::RUNX1-negative B-cell ALL, high TIMD4 expression at diagnosis had an HR = 3.93 [1.31, 11.79] (p = 0.01). Our results suggest high TIMD4 expression at diagnosis in low-risk B-cell ALL in children might be associated with high risk for early relapse.

Early Detection of Molecular Residual Disease and Risk Stratification for Children with Acute Myeloid Leukemia via Circulating Tumor DNA.

PURPOSE: Patient-tailored minimal residual disease (MRD) monitoring based on circulating tumor DNA (ctDNA) sequencing of leukemia-specific mutations enables early detection of relapse for pre-emptive treatment, but its utilization in pediatric acute myelogenous leukemia (AML) is scarce. Thus, we aim to examine the role of ctDNA as a prognostic biomarker in monitoring response to the treatment of pediatric AML. EXPERIMENTAL DESIGN: A prospective longitudinal study with 50 children with AML was launched, and sequential bone marrow (BM) and matched plasma samples were collected. The concordance of mutations by next-generation sequencing-based BM-DNA and ctDNA was evaluated. In addition, progression-free survival (PFS) and overall survival (OS) were estimated. RESULTS: In 195 sample pairs from 50 patients, the concordance of leukemia-specific mutations between ctDNA and BM-DNA was 92.8%. Patients with undetectable ctDNA were linked to improved OS and PFS versus detectable ctDNA in the last sampling (both P < 0.001). Patients who cleared their ctDNA post three cycles of treatment had similar PFS compared with persistently negative ctDNA (P = 0.728). In addition, patients with >3 log reduction but without clearance in ctDNA were associated with an improved PFS as were patients with ctDNA clearance (P = 0.564). CONCLUSIONS: Thus, ctDNA-based MRD monitoring appears to be a promising option to complement the overall assessment of pediatric patients with AML, wherein patients with continuous ctDNA negativity have the option for treatment de-escalation in subsequent therapy. Importantly, patients with >3 log reduction but without clearance in ctDNA may not require an aggressive treatment plan due to improved survival, but this needs further study to delineate.

Clonal evolution dissection reveals that a high MSI2 level promotes chemoresistance in T-cell acute lymphoblastic leukemia.

ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer with resistant clonal propagation in recurrence. We performed high-throughput droplet-based 5' single-cell RNA with paired T-cell receptor (TCR) sequencing of paired diagnosis-relapse (Dx_Rel) T-ALL samples to dissect the clonal diversities. Two leukemic evolutionary patterns, "clonal shift" and "clonal drift" were unveiled. Targeted single-cell DNA sequencing of paired Dx_Rel T-ALL samples further corroborated the existence of the 2 contrasting clonal evolution patterns, revealing that dynamic transcriptional variation might cause the mutationally static clones to evolve chemotherapy resistance. Analysis of commonly enriched drifted gene signatures showed expression of the RNA-binding protein MSI2 was significantly upregulated in the persistent TCR clonotypes at relapse. Integrated in vitro and in vivo functional studies suggested that MSI2 contributed to the proliferation of T-ALL and promoted chemotherapy resistance through the posttranscriptional regulation of MYC, pinpointing MSI2 as an informative biomarker and novel therapeutic target in T-ALL.

Integration of Transcriptomic Features to Improve Prognosis Prediction of Pediatric Acute Myeloid Leukemia With KMT2A Rearrangement.

Lysine methyltransferase 2A-rearranged acute myeloid leukemia (KMT2A-r AML) is a special entity in the 2022 World Health Organization classification of myeloid neoplasms, characterized by high relapse rate and adverse outcomes. Current risk stratification was established on the treatment response and translocation partner of KMT2A. To study the transcriptomic feature and refine the current stratification of pediatric KMT2A-r AML, we analyzed clinical and RNA sequencing data of 351 patients. By implementing least absolute shrinkage and selection operator algorithm, we identified 7 genes (KIAA1522, SKAP2, EGFL7, GAB2, HEBP1, FAM174B, and STARD8) of which the expression levels were strongly associated with outcomes. We then developed a transcriptome-based score, dividing patients into 2 groups with distinct gene expression patterns and prognosis, which was further validated in an independent cohort and outperformed the LSC17 score. We also found cell cycle, oxidative phosphorylation, and metabolism pathways were upregulated in patients with inferior outcomes. By integrating clinical characteristics, we proposed a simple-to-use prognostic scoring system with excellent discriminability, which allowed us to distinguish allogeneic hematopoietic stem cell transplantation candidates more precisely. In conclusion, pediatric KMT2A-r AML is heterogenous on transcriptomic level and the newly proposed scoring system combining clinical characteristics and transcriptomic features can be instructive in clinical routines.

Pediatric acute myeloid leukemia and hyperleukocytosis with WBC count greater than 50 × 109/L.

BACKGROUND: Acute myeloid leukemia (AML) and hyperleukocytosis have an unfavorable prognosis, but the impact of hyperleukocytosis on the prognosis of pediatric AML remains uncertain. We investigated the clinical characteristics and prognosis of pediatric AML with hyperleukocytosis, defined as WBC ≥ 50 × 109/L. METHODS: A total of 132 patients with newly diagnosed childhood AML with hyperleukocytosis were consecutively enrolled at our center from September 2009 to August 2021 to investigate prognostic factors and clinical outcomes. RESULTS: Hyperleukocytosis occurred in 27.4% of AML patients. Pediatric patients with hyperleukocytosis had similar CR and OS rates to those without hyperleukocytosis, but had a lower EFS rate. In our study, rates of CR1, mortality, relapsed/refractory disease, and HSCT were comparable between AML patients with WBC counts of 50-100 × 109/L and ≥ 100 × 109/L. AML patients with a WBC count of 50-100 × 109/L had a similar 5-year OS rate to patients with a WBC count ≥ 100 × 109/L (74.6% vs. 75.4%, P = 0.921). Among all patients with hyperleukocytosis, the FAB M5 subtype was associated with significantly inferior survival, and the prognosis of CBF-AML was good. CONCLUSIONS: Pediatric AML patients with hyperleukocytosis have the similar prognosis regardless of whether their WBC count is 50-100 × 109/L or ≥ 100 × 109/L.

Pan-HDAC inhibitors augment IL2-induced proliferation of NK cells via the JAK2-STAT5B signaling pathway.

BACKGROUND: Natural killer (NK) cells are a subtype of lymphocytes with the ability to quickly and efficiently identify and eliminate tumor cells. In the presence of IL2, NK cells can divide rapidly but in limited numbers. According to previous studies, in vivo treatment with histone deacetylase (HDAC) inhibitors did not impair NK-cell function. This study aimed to investigate the effect of HDAC inhibitors on NK-cell proliferation and the underlying regulatory mechanism. METHODS: NK92 cells, primary NK (pNK) cells, and CD19-CAR-NK92 cells were treated with low concentrations of pan-HDACi Dacinostat (Dac) and Panobinostat (Pan) in the presence of IL2, and Cell Counting Kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays were used to assess cell proliferation and apoptosis. The expression of granzyme B was detected by immunofluorescence, and the expression of CD107a and NKG2D was determined by flow cytometry. The downstream regulatory genes were identified by RNA-seq, and the "JAK-STAT signaling pathway"- and "Cell cycle signaling pathway"-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. The JAK2V617F mouse model was constructed to simulate the upregulation of the JAK2 signaling pathway in vivo, and the NK proliferation was evaluated by flow cytometry. A tumor-bearing nude mouse model was constructed to determine the anti-tumor efficacy of NK92 cells following Dac treatment. RESULTS: In the presence of IL2, the proliferation rate of NK92 cells, pNK cells, and CD19-CAR-NK92 cells treated with pan-HDACi Dac and Pan at low nanomolar doses was significantly increased, although cell function was unaffected. Low doses of Dac upregulated the JAK-STAT signaling pathway and enhance the cell cycle via that pathway. In addition, the in vivo experiment in nude mice showed that the capacity of Dac treated NK92 cells to eliminate tumor cells was unaffected. CONCLUSION: Low nanomolar doses of Pan-HDACi enhanced IL2-induced NK cell proliferation without compromising the functioning of NK cells.

Pediatric non-Down's syndrome acute megakaryoblastic leukemia patients in China: A single center's real-world analysis.

Non-Down's syndrome acute megakaryocytic leukemia (non-DS-AMKL) is a subtype of childhood acute myeloid leukemia (AML), whose prognosis, prognostic factors and treatment recommendations have not yet to be defined in children. We conducted a retrospective study with 65 newly diagnosed non-DS-AMKL children from August 2003 to June 2020 to investigate the clinical impact of factors and clinical outcome. Among all 65 patients, 47 of them were treated at our center who received three different regimens due to time point of admission (CAMS-another, CAMS-2009 and CAMS-2016 protocol), and the efficacy were compared. Patients with newly diagnosed non-DS-AMKL accounted for 7.4% of pediatric AML cases. The median age of the patients was 18 months at diagnosis, and over 90% of them were under three-years-old. The overall survival (OS) rates were 33.3% ± 1.7%, 66.7% ± 24.4% and 74.2% ± 4.0% for three groups (CAMS-another, CAMS-2009 and CAMS-2016 regimen), respectively. In CAMS-2016 group, the complete remission (CR) rate after induction was 67.7% (21/31), while the total CR rate after all phases of chemotherapy was 80.6% (25/31). The 2-year survival probability did not significantly improve in patients underwent HSCT when compared with non-HSCT group (75.0% ± 4.7% vs. 73.9% ± 4.6%, p=0.680). Those who had a "dry tap" during BM aspiration at admission had significantly worse OS than those without "dry tap" (33.3% ± 8.6% vs. 84.0% ± 3.6%, p=0.006). Moreover, the results also revealed that patients with CD34+ had significantly lower OS (50.0% ± 6.7% vs. 89.5% ± 3.5%, p=0.021), whereas patients with CD36+ had significantly higher OS than those who were negative (85.0% ± 4.0% vs. 54.5% ± 6.6%, p=0.048). In conclusion, intensive chemotherapy resulted in improved prognosis of non-DS-AMKL children and subclassification may base on "dry tap" and immunophenotypic. Although some progress has been made, outcomes of non-DS-AMKL children remain unsatisfactory, especially in HSCT group, when compared with other AML types.

Elucidating minimal residual disease of paediatric B-cell acute lymphoblastic leukaemia by single-cell analysis.

Minimal residual disease that persists after chemotherapy is the most valuable prognostic marker for haematological malignancies and solid cancers. Unfortunately, our understanding of the resistance elicited in minimal residual disease is limited due to the rarity and heterogeneity of the residual cells. Here we generated 161,986 single-cell transcriptomes to analyse the dynamic changes of B-cell acute lymphoblastic leukaemia (B-ALL) at diagnosis, residual and relapse by combining single-cell RNA sequencing and B-cell-receptor sequencing. In contrast to those at diagnosis, the leukaemic cells at relapse tended to shift to poorly differentiated states, whereas the changes in the residual cells were more complicated. Differential analyses highlighted the activation of the hypoxia pathway in residual cells, resistant clones and B-ALL with MLL rearrangement. Both in vitro and in vivo models demonstrated that inhibition of the hypoxia pathway sensitized leukaemic cells to chemotherapy. This single-cell analysis of minimal residual disease opens up an avenue for the identification of potent treatment opportunities for B-ALL.

Minimal residual disease monitoring via AML1-ETO breakpoint tracing in childhood acute myeloid leukemia.

Relapse of childhood AML1-ETO (AE) acute myeloid leukemia is the most common cause of treatment failure. Optimized minimal residual disease monitoring methods is required to prevent relapse. In this study, we used next-generation sequencing to identify the breakpoints in the fusion gene and the DNA-based droplet digital PCR (ddPCR) method was used for dynamic monitoring of AE-DNA. The ddPCR technique provides more sensitive and precise quantitation of the AE gene during disease progression and relapse. Quantification of the AE fusion gene by ddPCR further contributes to improved prognosis. Our study provides valuable methods for dynamic surveillance of AE fusion DNA and assistance in determining the prognosis.