Single-cell sequencing revealed the recurrence causes of ETV6::RUNX1 fusion-positive B-ALL in children.

Objective To analyze the immunophenotypes of B-cell acute lymphoblastic leukemia(B-ALL) and correlation between the expression of CD13 and clinical, hematologic and biological parameters in patients with Philadelphia chromosome-negative(Ph-) B-ALL. Methods Multiparameter flow cytometry was used to analyze the immunophenotypes of the bone marrow blasts in 111 cases of adult and 32 cases of childhood B-ALL. Results The positive rates of CD13 and CD33 in childhood B-ALL patients were no differences compared with those in adult patients [31.3%(10/32) vs 19.8%(22/111), 18.8%(6/32) vs 29.7%(33/111), both P>0.05]. In de novo Ph- B-ALL, the frequency of CD13 in childhood patients was higher than that in adult patients [32.3% (10/31) vs 9.4% (6/64), P 0.05). Difference on time of relapse was found between CD13+ and CD13- groups in childhood Ph- B-ALL patients[(111±16) d vs (683±57) d, P<0.000 1]. The time of relapse between childhood and adult CD13+ Ph- B-ALL cases had statistical difference [(111±16) d vs (319±16) d,P<0.000 1]. Conclusions The positive rate of CD13 is higher in childhood Ph- B-ALL patients than that in adult Ph- B-ALL patients. CD13+ Ph- B-ALL patients, in particular, childhood Ph- CD13+ patients are more likely to relapse. Key words: Leukemia, B lymphoblastic, acute; Multiparameter flow cytometry; Immunophenotyping; CD13

Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Among the CDKs that control cell cycle progression, cyclin D-dependent kinases CDK4 and CDK6 are considered important oncogenic drivers in many cancers. CDK6 is a direct target of MLL rearranged B-cell acute lymphoblastic leukemia (B-ALL), and this prompted us to investigate if pharmacologic inhibition of CDK4/CDK6 could increase the efficiency of chemotherapy and overcome drug resistance. Although many reports described CDK4/CDK6 inhibitors against a broad range of carcinomas, few studies have been performed on leukemia. Deletion and methylation of CDK4/CDK6 inhibitor CDKN2a are frequently observed in B-ALL and gene expression analysis performed in a cohort of childhood patients showed that cyclin D1, CDK4 and CDK6 are highly expressed. Moreover, Reverse Phase Protein Array (RPPA) analysis showed that cyclin D1 expression is higher in high risk-MRD patients. These results suggest specific inhibition of cyclin D/CDK4/CDK6 axis as an attractive strategy to improve the effect of common chemotherapy on B-ALL patients. Standard treatment for newly diagnosed childhood B-ALL patients include chemotherapy treatment, and although glucocorticoids (GC) are the most important drugs used in the treatment of acute lymphoblastic leukemia, the molecular basis of GC sensitivity and resistance remains largely unknown; understanding the molecular mechanisms related to GC cytotoxicity is crucial for modulating GC resistance. The aim of this study was to evaluate the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated two B-ALL dexamethasone-resistant cell lines, SEM and RCH-ACV, and two B-ALL dexamethasone-sensitive cell lines, RS4;11 and NALM6, with ribociclib, a highly specific CDK4/6 dual inhibitor. As expected, treatment with ribociclib induced a strong cell cycle arrest in G1 phase in a time-dose dependent manner along with a dose-dependent decrease in phosphorylated retinoblastoma (Rb); we investigated whether treatment with ribociclib could induce growth inhibition of B-ALL cell lines. We observed, in SEM and RCH-ACV, a strong growth inhibition at all drug concentration after continuous treatment. Washout of the inhibitor from the medium after 48 hours of treatment showed that both cell lines did not restore cell growth. These results show that ribociclib could have an irreversible activity. Moreover, a strong dose-dependent reduction of clonogenic potential was observed in SEM cell line, by CFU assay. Ribociclib exposure strongly synergizes (CI&amp;lt;1) with dexamethasone in SEM and RCH-ACV resistant cell lines with a strong decrease of proliferation and a significant increase of apoptotic cell death. Immunoblot analysis showed a decrease in phosphorylated Rb and Cyclin D1 starting from 48 hours of cotreatment and an increase in p27. Preliminary experiments showed a modest increase in glucocorticoid receptor (GR) after ribociclib and combination treatment in SEM and RCH-ACV cells. To further confirm the synergistic effect we evaluated mRNA level of GR targets, and we found that GR, BTG1, and GILZ are overexpressed in combination treatment in SEM and RCH-ACV. The synergistic effect of ribociclib-dexamethasone combination was also confirmed on primary cultures derived from pediatric patients affected by B-acute lymphoblastic leukemia. Our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance. Citation Format: Elena Mattiuzzo, Roberta Bortolozzi, Benedetta Accordi, Luca Trentin, Giuseppe Basso, Giampietro Viola. CDK4/CDK6 inhibition in childhood B-ALL: A new strategy to mediate glucocorticoid sensitivity [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 46.

PurposeThis study screened serum proteins to identify potential biomarkers for childhood B-cell and T-cell acute lymphoblastic leukemia (ALL).Patients and methodsSerum collected from 20 newly diagnosed B-cell ALL, 20 T-cell ALL and 20 healthy children. The peptides from these samples were subjected to iTRAQ. Differentially expressed proteins (DEPs) were further validated by ELISA in 24 B-ALL, 24 T-ALL, and 24 healthy children.ResultsBioinformatics analysis revealed several pathways, including atherosclerosis signaling, interleukin signaling and production in macrophages and clathrin-mediated endocytosis signaling, that were closely related to childhood T-cell ALL. Furthermore, four selected proteins, namely LRG1, S100A8, SPARC and sL-selectin, were verified by ELISA. These results were consistent with the results of the proteomics analysis.ConclusionSerum S100A8 may serve as new diagnostic biomarkers in childhood B-cell ALL and T-cell ALL.

BackgroundA20 is a dual inhibitor of NF-κB activation and apoptosis in the tumor necrosis factor receptor 1 signaling pathway, and both are related to tumorigenesis. A20 is frequently inactivated by deletions and/or mutations in several B and T cell lymphoma subtypes; however, knowledge of the role of A20 in B-cell acute lymphoblastic leukemia (B-ALL) remains limited. In this study, we characterized the A20 gene expression pattern, the expression level of its upstream regulating factor MALT1, and its downstream target NF-κB in adult B-ALL.MethodsThe expression level of MALT1, A20 and NF-κB1 was detected in peripheral blood mononuclear cells (PBMCs) from 20 patients with adult B-ALL (including 12 de novo B-ALL and 8 refractory/relapse B-ALL cases), and nine patients with B-ALL in complete remission (CR) using real-time PCR. Sixteen healthy individuals served as controls.ResultsSignificant A20 overexpression was found in the B-ALL (median: 13.489) compared with B-ALL CR (median: 3.755) (P = 0.003) patients and healthy individuals (median: 8.748) (P = 0.002), while there was no significant difference in A20 expression between B-ALL CR patients and healthy individuals (P = 0.107). Interestingly, the A20 expression level in the B-ALL samples was relatively different with approximately 50% of the B-ALL cases showing a relatively high A20 expression level, while the remaining 50% cases demonstrated slight upregulation or a similar expression level as the healthy controls. However, there was no significant difference in the A20 expression level between de novo B-ALL (median 12.252) and refractory/relapse B-ALL patients (median 21.342) (P = 0.616). Similarly, a significantly higher expression level of NF-κB1 was found in the B-ALL (median 1.062) patients compared with healthy individuals (median 0.335) (P < 0.0001), while the NF-κB1 expression level was downregulated in the B-ALL CR group (median 0.339), which was significantly lower than that in those with B-ALL (P = 0.001). Moreover, the MALT1 expression level in B-ALL was upregulated (median 1.938) and significantly higher than that in healthy individuals (median 0.677) (P = 0.002) and B-ALL CR patients (median 0.153) (P = 0.008). The correlation of the expression levels of all three genes was lost in B-ALL.ConclusionsWe found that MALT1-A20-NF-κB is overexpressed in adult B-ALL, which may be related to the pathogenesis of B-ALL, and this pathway may be considered a potentially attractive target for the development of B-ALL therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0222-0) contains supplementary material, which is available to authorized users.

Acute lymphoblastic leukemia (ALL), cancer of the white blood cells, is a heterogeneous disease that mainly occurs due to the malignant cloning of original and naive lymphocytes. The aim of this study was to explore the immunophenotype, the percentage of CD4(+)CD25(+) regulatory T cells (Tregs) and the expression of cytokines interleukin (IL)-2, IL-10 and TGF-β in patients with ALL. The immunophenotype and levels of CD4(+)CD25(+) Tregs were detected using flow cytometry in the peripheral blood of 35 ALL patients, with 18 healthy individuals being selected as controls. The results suggested that 22 patients had B cell ALL (B-ALL) and 13 had T cell ALL (T-ALL) among the 35 ALL patients. In B-ALL patients, the surface antigen CD19 was most commonly expressed; in T-ALL patients, CD7 was most common. Furthermore, the percentage of CD4(+)CD25(+) Treg cells in the peripheral blood of B-ALL and T-ALL patients was higher compared to that of healthy individuals (P<0.05). Additionally, IL-10 and TGF-β levels in cell culture supernatants from B-ALL and T-ALL patients were higher compared to those in the controls (P<0.05); IL-2 levels were lower in ALL patients. No significant differences were observed in the levels of CD4(+)CD25(+) Treg cells, IL-2, IL-10 or TGF-β in B-ALL versus T-ALL patients. The authors concluded that CD19 and CD7 may serve as diagnostic markers of B-ALL and T-ALL, respectively. The increased presence of CD4(+)CD25(+) Treg cells and the altered levels of secreted cytokines are indicative of an immunosuppressive mechanism in the pathogenesis of ALL.

The bone marrow microenvironment plays a critical role in B-cell acute lymphoblastic leukemia (B-ALL) progression, yet its cellular heterogeneity remains poorly understood. Using single-cell RNA sequencing on patient-derived bone marrow aspirates from pediatric B-ALL patients, we identified two distinct mesenchymal stromal cell (MSC) populations: early mesenchymal progenitors and adipogenic progenitors. Spatial transcriptomic analysis further revealed the localization of these cell types and identified a third stromal population, osteogenic-lineage cells, exclusively present in the bone biopsy. Functional ex vivo assays using sorted stromal populations derived from B-ALL patient bone marrow aspirates demonstrated that both early mesenchymal and adipogenic progenitors secrete key niche-supportive factors, including CXCL12 and Osteopontin, and support leukemic cell survival and chemoresistance. Transcriptomic profiling revealed that B-ALL cells interact differently with stromal subtypes. Notably, adipogenic progenitors, but not early mesenchymal progenitors, provide support to leukemic cells through interleukin-7 and VCAM1 signaling. Stromal cells from B-ALL patients exhibited an enhanced adipogenic differentiation capacity compared to healthy controls. Moreover, co-culture experiments showed that B-ALL cells induce adipogenic differentiation in healthy MSCs through a cell contact-dependent mechanism. Adipogenic progenitors were also enriched in relapse samples, implicating them in disease progression. These findings highlight the complexity of the B-ALL microenvironment and identify different specialized stromal niches with which the leukemic cells can engage.

BackgroundImmune regulation is crucial for the pathogenesis of B-cell acute lymphoblastic leukemia (B-ALL). It has been reported that Th17 cells as a newly identified subset of CD4+ T cells are involved in the pathogenesis of several hematological disorders. However, the role of Th17 cells in the pathophysiology of B-ALL is still unclear.MethodsThe frequencies of T cells were determined by flow cytometry in the peripheral blood and bone marrow of 44 newly diagnosed B-ALL patients and 25 age-matched healthy donors. The cell viability and apoptosis were determined by CCK-8 assay and Annexin V staining, respectively. Western blot was applied to identify the level of Akt and Stat3 phosphorylation.ResultsWe assessed and observed a significantly increased frequency of Th17 cells and a drastically decreased frequency of Th1 cells in peripheral blood mononuclear cells and bone marrow mononuclear cells from newly diagnosed B-ALL patients compared with healthy donors. Furthermore, increased levels of Th17-related cytokines including IL-17, IL-21, IL-23, IL-1β, and IL-6 were presented in between blood and marrow in B-ALL patients. Both IL-17A and IL-21, two Th17-secreted cytokines, induced the proliferation of B-ALL cell line Nalm-6 and patient B-ALL cells isolated from B-ALL patients, herein either cytokine led to the phosphorylation of Akt and Stat3. Additionally, IL-17A promoted resistance to daunorubicin via activation of Akt signaling and the PI3K/Akt inhibitor LY294002 or perifosine almost completely rescued daunorubicin-induced cell death in B-ALL cells.ConclusionsOur findings suggest that elevated Th17 cells secrete IL-17A by which promotes the proliferation and resistance to daunorubicin in B-ALL cells through activation of Akt signaling. Th17 cells may represent a novel target to improve B-ALL immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0894-9) contains supplementary material, which is available to authorized users.

Delineating the Role of Interplay between m6A Machinery Genes and IGF2BP Group of RNA-Binding Proteins in B-Cell Acute Lymphoblastic Leukemia (B-ALL)

This study aimed to investigate clinical characteristics and prognostic significance of activation-induced cytidine deaminase (AID) gene, miR-181b and miR-155 expression in de novo adult B-cell acute lymphoblastic leukemia (B-ALL) patients. Results showed that AID and miR-155 expression were higher in B-ALL patients than healthy controls, while miR-181b expression was lower in B-ALL patients. In addition, Ph+ B-ALLs had higher AID expression than Ph- B-ALLs, and its high expression was associated with BCR-ABL. Moreover, B-ALL patients with AIDhigh or miR-181blow expression had a shorter overall survival (OS). AIDhigh with miR-181blow, AIDhigh with miR-155low, miR-181blow, miR-155low, AIDhigh with miR-181blow and miR-155low expression were associated with shorter OS. Combination of the three molecules are more accurate predictors for unfavorable OS compared with univariate group. Therefore, AID, miR-181b and miR-155 provide clinical prognosis of adult de novo B-ALL patients and may refine their molecular risk classification.

BackgroundLittle is known about DNMT3A expression and its prognostic significance in childhood B cell acute lymphoblastic leukemia (B-ALL).MethodsWe determined DNMT3A mRNA expression in 102 children with B-ALL. Correlations with relapse-free survival (RFS) and common clinical characteristics were analyzed. DNMT3A was stably knocked out by CRISPR/Cas9 gene editing technology in Reh and 697 B-ALL cell lines. Cell proliferation activity after treated with daunorubicin (DNR) was determined by CCK8 assay in DNMT3A KO Reh and 697 cell lines.ResultsDNMT3A expression in B-ALL patients who were in continuous complete remission (CCR) was higher than in those who got relapse (P = 0.0111). Receiver operating characteristic curve showed prognostic significance of DNMT3A expression (P = 0.003). Low expression of DNMT3A (≤ 0.197) was significantly correlated with poor RFS (P < 0.001) in children with B-ALL. Knock-out of DNMT3A in Reh and 697 cell lines significantly increased IC50 of DNR (P = 0.0201 and 0.0022 respectively), indicating elevated resistance to DNR.ConclusionLow expression of DNMT3A associates with poor prognosis in children with B-ALL. Knock-out of DNMT3A confers resistance to DNR on leukemic cells.

PI3Kδ Inhibition Enhances Sensitivity of Primary High-Risk Childhood B-Cell Acute Lymphoblastic Leukemia Cells to Glucocorticoid Chemotherapy

Background: Detection of MRD is an important predictor of patient outcome following treatment of B-ALL; importantly, MRD is emerging as a useful tool to detect early relapse, which may fulfill a key previously unmet clinical need. Objective: To evaluate the potential of MRD to predict morphologic relapse in pediatric and AYA patients with B-ALL. Methods: Bone marrow (BM) and peripheral blood (PB) specimens at screening (pre-tisagenlecleucel infusion), post-infusion, and relapse from two B-ALL clinical trials (ELIANA [NCT02435849] and ENSIGN [NCT02228096]) were tested using immunoglobulin next-generation sequencing (IgNGS) and flow cytometry (FC). We assessed concordance between two MRD assays to determine which method could support early relapse detection and whether using PB with IgNGS was comparable with BM testing with FC. Results: IgNGS was performed in 300 samples from 88 patients. 237 samples from 83 patients also had FC MRD results available. Baseline samples, which had high disease burden, showed 100% MRD concordance between both assays. However, post-treatment, where the leukemic burden was dramatically reduced, IgNGS detected a greater number of MRD-positive samples vs FC at each sensitivity level tested (10-4, 10-5, and 10-6). At the highest sensitivity level of 10-6, IgNGS was able to detect 18% more MRD-positive post-treatment samples. IgNGS was able to detect MRD positivity 1-4 months ahead of clinical relapse in a small number of relapsed patients, whether relapse was CD19+ or CD19-. MRD burden in BM was higher than in PB using both FC and IgNGS. In patients with matching data available, IgNGS was able to detect more MRD-positive PB samples than FC MRD-positive BM samples. Patients who were MRD negative by both IgNGS and FC at the end of first month post-infusion had better progression-free survival (PFS) and overall survival (OS) compared with those with detectable MRD. Tumor clonality at baseline and clonal evolution following tisagenlecleucel treatment will be presented. Conclusion: MRD detection using IgNGS in PB may be used as a surrogate for FC assessment of MRD in BM. Patients who were MRD negative by IgNGS 1 month after infusion had improved PFS and OS vs those with detectable MRD; ongoing studies will provide further information on the applicability of IgNGS MRD detection and its association with long-term outcome in tisagenlecleucel-treated pediatric and AYA relapsed/refractory B-ALL patients. Citation Format: Michael A. Pulsipher, Xia Han, Máire Quigley, Gabor Kari, Susana Rives, Theodore W. Laetsch, Gary D. Myers, Hidefumi Hiramatsu, Gregory A. Yanik, Muna Qayed, Timothy Driscoll, Michael W. Boyer, Heather Stefanski, Jochen Buchner, Andre Baruchel, Peter Bader, Lan Yi, Creton Kalfoglou, Harlan Robins, Erik Yusko, Gullu Gorgun, Eric Bleickardt, Stephane Wong, Stephan A. Grupp. Potential utility of minimal residual disease (MRD) to identify relapse in pediatric and young adult (AYA) B-cell acute lymphoblastic leukemia (B-ALL) patients treated with tisagenlecleucel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT077.

Detection of the Genomic Variants in the Paired De Novo-Relapsed Samples with Pan-Cancer Panel and Whole Exome-Seq in Adult B-Cell Acute Lymphoblastic Leukemia

BackgroundNicotinamide Adenine Dinucleotide (NAD) depletion is reported to be a potential treatment for B-cell Acute Lymphoblastic Leukemia (B-ALL), but the mechanism of NAD metabolism-related genes (NMRGs) in B-ALL relapse remains unclear.MethodsTranscriptome data (GSE3912), and single-cell sequencing data (GSE130116) of B-ALL patients were downloaded from Gene Expression Omnibus (GEO) database. NMRGs were sourced from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. Further, the differentially expressed NMRGs (DE-NMRGs) were selected from the analysis between initial diagnosis and relapse B-ALL samples, which further performed functional enrichment analyses. The biomarkers were obtained through random forest (RF) algorithm and repeated cross validation. Additionally, cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm was used to evaluate the immune cell differences between the initial diagnosis and relapse samples, and the correlations between biomarkers and gene markers of differential immune cells were analyzed. Furthermore, single cell RNA sequencing was conducted in the GSE130116 dataset to find key cell clusters. In addition, according to biomarkers expressions, cell clusters were categorized into high and low biomarker expression groups, and Gene Set Enrichment Analysis (GSEA) analysis was performed on them. Finally, the cell clusters with the highest expression of biomarkers were selected to explore the roles of biomarkers in different cell clusters and identify transcription factors (TFs) influencing biological markers.Results23 DE-NMRGs were screened out, which were mainly enriched in nucleoside phosphate metabolic process, nucleotide metabolic process, and Nicotinate and nicotinamide metabolism. Moreover, 3 biomarkers (NADSYN1, SIRT3, and PARP6) were identified from the machine learning. CIBERSORT results demonstrated that four types of immune cells (B Cells naive, Monocyte, Neutrophils, and T cells CD4 memory Activated) were significantly different between the initial diagnosis and the relapse B-ALL samples, and there were strong correlations between biomarkers and differential immune cells such as positive correlation between NADSYN1 and B Cells naive. The single cell analyses showed that the biomarkers were highly expressed in common myeloid progenitors (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) cell clusters. Gene set enrichment analysis (GSEA) results indicated that 55 GO terms and 3 KEGG pathways were enriched by the genes in high and low biomarker expression groups. It was found that TF CREB3L2(+) was significantly reduced in the high expression group, which may be the TF affecting biomarkers in the high expression group.ConclusionThis study identified NADSYN1, SIRT3, and PARP6 as the biomarkers of B-ALL, explored biological significance of NMRGs in the initial diagnosis and relapse of B-ALL, and revealed mechanism of biomarkers at the level of the single cell.

CRM1/XPO1-FOXO1 Pathway Is Associated with Clinical Outcome and Represents a Promising Target in B-Cell Acute Lymphocytic Leukemia