Abstract Recently, studies in adults with acute promyelocytic leukemia (APL) showed high cure rates in low‐risk patients treated with all‐ trans retinoid acid (ATRA) and arsenic trioxide (ATO), while toxicities were significantly reduced compared to the standard treatment with ATRA and chemotherapy. Here we report about first experience with 11 pediatric patients with low‐risk APL treated with ATRA and ATO. All patients stayed in molecular remission. All suffered from hyperleukocytosis. Two patients experienced reversible severe side effects. One suffered from osteonecroses at both femurs, seizures, as well as posterior reversible encephalopathy syndrome, the other patient had an abducens paresis.
10524 Background: Dependence on the prosurvival protein B-cell lymphoma 2 (BCL-2) occurs in certain pediatric solid tumors, resulting in tumorigenesis and resistance to therapies. Venetoclax (VEN), an orally administered BCL-2-selective inhibitor, has preclinical anticancer activity in human-derived neuroblastoma models. Reported here are preliminary results from pediatric patients (pts) with recurrent or refractory (R/R) solid tumors treated with VEN monotherapy or VEN with cyclophosphamide and topotecan (Cy-Topo). Methods: This phase 1 open-label, 2-part study (NCT03236857) enrolled pts < 25 yr old with R/R malignancies; we report only on pts with solid tumors. Following a dose ramp-up, pts received 800 mg VEN (age/weight-adjusted adult equivalent) once daily for the first 8 wk; Cy-Topo was added optionally after wk 8. Dose-limiting toxicities (DLTs) were assessed during the first 21 days of VEN therapy or cycle 1 of VEN-Cy-Topo. Objectives included safety, toxicity, and preliminary efficacy. Results: As of Dec 17, 2019, 11 solid tumor pts were enrolled: neuroblastoma (n = 6), rhabdomyosarcoma (n = 2), Wilms’ tumor, Carney-Stratakis syndrome, and low-grade fibromyxoid sarcoma (n = 1 each). Median age was 11 yr (range 3–22); median time on study was 6.9 mo (range 1.2–17.8). All pts experienced ≥1 treatment-emergent adverse event (TEAE); vomiting (72%; all grades) was most common. Grade ≥3 TEAEs were reported in 82% of pts; febrile neutropenia (64%), decreased blood cell count, and neutropenia (36% each) were the most common. Seven pts received 800-mg monotherapy for 8 wk; 3 of these pts did not receive Cy-Topo after monotherapy. Of the 7 pts who received VEN-Cy-Topo, 3 pts received 400 mg VEN with Cy-Topo. DLTs of grade 4 neutropenia/thrombocytopenia with delayed count recovery occurred in 2 pts on 800 mg VEN-Cy-Topo, necessitating a dose de-escalation (to 400 mg VEN). Grade 4 neutropenia occurred in 2 pts on 400 mg VEN with Cy-Topo, leading to the addition of myeloid growth factor to the therapy regimen. The best response after 8 wk of VEN monotherapy was stable disease (SD). Six pts were evaluable for tumor response with VEN-Cy-Topo; 1 neuroblastoma pt had a complete response after 5 cycles of 400 mg VEN, 4 pts had SD (3 on 800 mg and 1 on 400 mg VEN) and 1 (800 mg VEN) had progressive disease as best response. Conclusions: Continuous dosing of VEN with Cy-Topo was not tolerated due to cytopenias in 4/7 pts with solid tumors. Discontinuous dosing of VEN with Cy-Topo is being explored. Clinical trial information: NCT03236857.
Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Juvenile myelomonocytic leukemia (JMML) is a rare and highly aggressive myeloid neoplasia of early childhood. Due to excessive proliferation and organ infiltration of monocytic and granulocytic progenitor cells, patients are experiencing massive hepatosplenomegaly, respiratory distress, diarrhea and skin rash. JMML is driven by constitutive activation of the RAS signaling pathway induced by mutations in PTPN11 (coding for SHP2), KRAS, NRAS, NF1 and CBL. For most patients, the only curative treatment is hematopoietic stem cell transplantation (HSCT), and Graft versus Leukemia (GvL) effects were shown to be relevant for cure. Especially for patients with PTPN11 mutation, risk of relapse after HSCT remains high. Aims: We hypothesized that relapse after HSCT is caused by immune escape mechanisms allowing JMML cells to circumvent immune control. Methods: We focused on the most aggressive, i.e. PTPN11-mutated JMML subtype and used human naïve JMML cells as well as a genetically modified mouse model expressing Ptpn11D61Y in the hematopoietic system (mx-cre;Ptpn11D61Y/+). Results: Phenotypical analysis of immune escape molecules expressed by naïve JMML cells using mass cytometry revealed strong expression of various immunoregulatory molecules on leukemic myeloid cells, including CD39, CD47, Siglec7, Siglec9, NOX2 and PD-L1. In line with this finding, splenic monocytes and granulocytes of mx-cre;Ptpn11D61Y/+ mice expressed high levels of PD-1, PD-L1, PD-L2, CD39, CD73 and VISTA. Of those, only CD73 was downregulated by the MEK inhibitor trametinib indicating direct regulation by the oncogenic SHP2. Together with CD39, CD73 is a known ectonucleotidase that converts extracellular ATP to adenosine and thus induces an anti-inflammatory state. To test whether the checkpoint molecules affect T cell proliferation, we co-cultured wildtype (WT) T cells with Ptpn11D61Y/+ leukemic monocytes or granulocytes. Indeed, T cell proliferation was inhibited by leukemic monocytic cells. Proliferation could be restored by the CD39-inhibitor, POM-1. In contrast, inhibitors or monoclonal antibodies for CD73, PD1 and VISTA did not affect the inhibitory effect of leukemic cells on T cell proliferation. Summary/Conclusion: We conclude that the purinergic network is responsible for the immune escape of JMML cells and might be an attractive therapeutic target to prevent JMML relapse after HSCT. Other immune escape molecules such as PD1 and VISTA seem to play only minor roles in JMML. Keywords: T cell response, JMML, Immunosuppression
Background: JMML is a chemotherapy‐resistant neoplasia of early childhood. Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is the only curative therapy, being able to cure approximately 50% of these children. Relapse is the major cause of treatment failure, with chemotherapy prior to HSCT being notably unsuccessful. Novel therapies controlling JMML prior to HSCT are urgently needed. Aims: We conducted a phase 2, multicenter, open‐label study to evaluate pharmacodynamics, safety, and antileukemia activity of azacitidine monotherapy prior to HSCT in patients with newly diagnosed JMML. Methods: Azacitidine was administered at 75 mg/m 2 /day intravenously on Days 1–7 of a 28‐day cycle for 3 to 6 cycles. The primary endpoint was the number of patients achieving clinical complete remission or clinical partial remission (cPR) at Cycle 3 Day 28 (C3D28); secondary endpoints included overall survival following HSCT. Results: Between September 2015 and November 2017, 18 JMML patients (13 PTPN11 ‐, 3 NRAS ‐, 1 KRAS ‐, 1 NF1‐mutated), aged 0.2–7.0 years, were enrolled in the study. Median (range) white blood cell (WBC) count, platelet count, and spleen size were: 19.7 (4.3–59.0) × 10 9 /L, 28 (7–85) × 10 9 /L, and 4 (2–14) cm below the costal margin, respectively. DNA methylation class (Lipka et al. Nat Comm . 2017;8:2126; n = 18) was high, intermediate, or low in 11, 5, and 2 patients, respectively. Sixteen patients completed 3 cycles of therapy and 5 of them completed 6 cycles. Two patients discontinued treatment before completing 3 cycles due to disease progression. Six patients (33%) experienced ≥1 grade 3 or 4 manageable adverse event, consistent with the known azacitidine safety profile. Eleven patients (61%) achieved cPR at C3D28 and 7 had progressive disease either at C3D28 or prior. Importantly, 8 of the 15 patients who needed platelet transfusions before or shortly after treatment initiation did not require transfusions at the time of HSCT. Seven of these 8 platelet responders had normalized their platelet count (≥130 × 10 9 /L). Palpable spleen size decreased in 11 responders by a median of 3.5 cm after 3 cycles and ranged from 0 to 2 cm below the costal margin after 6 cycles. Seventeen patients received allo‐HSCT from a human leukocyte antigen‐compatible related or unrelated donor following a busulfan‐ (n = 15) or treosulfan‐based (n = 2) preparative regimen after a median of 57 days (36–112) from last azacitidine dose. Fourteen out of 16 transplanted patients were leukemia‐free at median follow‐up of 15.6 months (0.8–22.5) after HSCT. Two children (both high‐methylation class) given HSCT relapsed after the allograft. Sixteen of the 18 patients were alive at a median follow‐up of 22.2 months (14.2–33.1). One patient who discontinued treatment before Cycle 3 died from disease progression, and 1 non‐responder child died from graft failure. Summary/Conclusion: This study showed azacitidine monotherapy was well tolerated in children with newly diagnosed JMML. Although the long‐term advantage of azacitidine therapy remains to be fully assessed, both decrease in spleen size and significant platelet responses observed demonstrate that the drug was effective in JMML and provided clinical benefit to patients with JMML in this study. This clinical trial has shown that azacitidine therapy prior to HSCT may be considered for patients with JMML.
Abstract Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50–60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs ( lnc-THADA-4 , lnc-ACOT9-1 and NRIR ) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4 , lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.
Juvenile myelomonocytic leukemia (JMML) stands out among malignant neoplasms of childhood in several ways. First, JMML is a model condition to elucidate the relevance of deregulated Ras signal transduction in human cancer. Second, the identification of Ras pathway mutations in JMML has informed the field of germline cancer predisposition and advanced the understanding of molecular mechanisms underlying the progression from predisposition to neoplasia. Third and not least, genomic DNA methylation was discovered to play a salient role in the classification and prognostication of the disease. This article discusses the evolution of epigenetic research on JMML over the past years and reviews the relevance of aberrant DNA methylation in the diagnosis, concept, and clinical decision-making of JMML.
A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.
