Cytokine Analysis in 154 Patients with Transient Abnormal Myelopoiesis: Jccg JPLSG TAM-10 Clinical Study

Introduction: Transient abnormal myelopoiesis (TAM), also known as transient leukemia or transient myeloproliferative disorder, is a unique clonal myeloproliferation characterized by immature megakaryoblasts occurring in 10% of neonates with Down syndrome. Although most patients show spontaneously resolution of TAM without therapeutic interventions, approximately 20% of TAM cases result in early deaths, i.e., within 9 months, and approximately 20% of the survivors develop acute megakaryoblastic leukemia (AMKL) within 4 years. A somatic GATA1 gene mutation that leads to the exclusive expression of a truncated GATA1 protein is shared by both TAM and AMKL cells. According to previous reports, cytokine levels are associated with liver failure, which is a cause of early death. Here, we analyzed 154 DS patients with TAM enrolled in the TAM-10 prospective observational study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group to determine the association between clinical characteristics and cytokine levels in such patients. Patients and Methods: A total of 167 neonates (89 boys and 78 girls) diagnosed with TAM were prospectively registered in the TAM-10 study between May 2011 and February 2014. We analyzed cytokine levels in 154 of the 167 enrolled patients whose samples were available. Somatic GATA1 gene mutations were confirmed in 151 (98%) of 154 patients using Sanger and/or next-generation sequencing. Using the Bio-Prex cytokine assay, serum concentrations of the following 27 cytokines were measured: interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins alpha and beta (MIP-1α and MIP-1β), eotaxin, interferon gamma-induced protein (IP-10), regulated upon activation, normal T-cell expressed and secreted, interleukin I receptor agonist (IL-RA), and 13 different interleukins (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, and IL-17). For all analyses, P values were two-tailed and a P value of <0.05 was considered statistically significant. Mann-Whitney test were used as appropriate for comparisons between groups. Moreover, the cumulative incidence for competing events was compared with the Gray test. Results: The median (range) white blood cell (WBC) count at diagnosis was 37.2 (2.4-478.7) × 10 9 cells/L. Forty-seven (31%) of 154 patients received low-dose cytarabine. When we compared 29 patients with a high WBC count (≥100 × 10 9 cells/L, known as a poor prognostic factor in TAM patients) to 125 patients without a high WBC count for 27 cytokine levels, the levels of 16 cytokines (IL-1b, IL-1ra, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, Eotaxin, PDGF-bb, basic FGF, G-CSF, GM-CSF, MCP-1b, and VEGF) were significantly higher in patients with a high WBC group. Early death occurred in 14 (9%) of 154 patients. Cytokine levels were compared between the early death group (n = 14) and remaining patients (n = 140) and it was observed that the levels of 10 cytokines (IL-1b [p = 0.016], IL-1ra [p < 0.001], IL-6 [p < 0.001], IL-7 [p = 0.009], IL-8 [p < 0.001], IL-10 [p = 0.014], IL-13 [p = 0.002], MCP-1 [p = 0.030], MIP-1b [p = 0.024], and TNF-α [p = 0.008]) were significantly higher in the early death group. When the patients were divided in two groups according to the median IL-1β concentration showing the lowest p-value, the early death rate in the IL-1β high group was significantly higher than that in the IL-1β low group (16% vs. 3%, p < 0.001). IL-1, IL-6, IL-8, and TNF-α are proinflammatory cytokines induced by MCP-1 and MIP1-b. Early death was strongly associated with hypercytokinemia, suggesting that therapeutic interventions (e.g., systemic steroid therapy) may be effective in patients with hypercytokinemia. However, there was no relation between the levels of 27 cytokines and leukemia development. Conclusion: Our findings suggested that measurement of cytokine levels may be a useful marker for predicting early death and an indicator of therapeutic interventions required in TAM patients. Disclosures No relevant conflicts of interest to declare.

Introduction of STAG2 Mutation in an iPSC Model of Transient Abnormal Myelopoiesis Mimics Down Syndrome Myeloid Leukemia

Background Children with Down syndrome (DS) have a high risk for acute myeloid leukemia (DS-ML). Genomic characterization of DS-ML blasts showed the presence of unique mutations in GATA1, an essential hematopoietic transcription factor, leading to the production of a truncated from of GATA1 (GATA1s). GATA1s together with trisomy 21 is sufficient to develop a pre-leukemic condition called transient abnormal myelopoiesis (TAM). Approximately thirty percent of these cases progress into DS-ML by acquisition of additional somatic mutations in a step-wise manner. We previously developed a model for TAM by introducing disease-specific GATA1 mutation in trisomy 21 induced pluripotent stem cells (iPSCs) leading to the production of N-terminally truncated short form of GATA1 (GATA1s) (Barwe et al., 2021). In this study, we introduced co-operating mutation in STAG2, a member of the cohesin complex recurrently mutated in DS-ML but not in TAM, and evaluated its effect on hematopoietic differentiation. Methods Two different iPSC lines with trisomy 21 with or without GATA1 mutation as described in Barwe et al., 2021, were used. CRISPR/Cas9 gene editing was performed to introduce STAG2 mutation to generate a knockout of STAG2. Hematopoietic differentiation of these iPSC lines was performed using STEMdiff Differentiation kit. ProteinSimple Wes system was used for western blot analysis. Multi-dimensional flow cytometry was used for immunophenotypic analysis of megakaryoblasts cultured in lineage expansion media for 5 days. Multi-lineage colony forming potential was assessed by Methocult colony forming assay using day 10 hematopoietic stem progenitor cells (HSPCs). Results Hematopoietic differentiation of GATA1 and STAG2 double mutants in two independent trisomy 21 iPSC lines confirmed GATA1s expression and the loss of functional STAG2 protein (Fig. 1A). GATA1s expressing HSPCs collected on day 12 post differentiation showed reduced erythroid (CD71+CD235+) and increased megakaryoid (CD34+CD41+ within CD41+ compartment) and myeloid (CD18+CD45+) population compared to disomy 21 HSPCs with wild-type GATA1, consistent with our previous study (Fig. 2B). STAG2 knockout HSPCs showed higher erythroid population (P=0.033 and 0.016 in T21-1S and T21-2S respectively) and reduced myeloid population while it had no significant effect on the megakaryoid population in both iPSC lines. The GATA1s/STAG2 knockout HSPCs showed reduced erythroid, but higher megakaryoid and myeloid population compared to wild-type HSPCs. Strikingly, the immature megakaryoid population was significantly higher in the double mutant HSPCs compared to single mutant alone in both iPSC lines (P=0.005 and 0.004 for T21-1GS and T21-2GS respectively), indicating that the STAG2knockout co-operated with GATA1s for increasing megakaryoid population. The trisomy 21 iPSC line with wild-type GATA1 developed CFU-GEMM (colony-forming unit granulocyte erythroid macrophage megakaryocyte), CFU-GM (CUF granulocyte-macrophage) and BFU-E (burst-forming unit erythroid) colonies in Methocult. GATA1 mutation, unlike STAG2 mutation, inhibited the formation of CUF-GEMM and BFU-E colonies. The number of CFU-GM colonies in T21-2GS was significantly reduced compared to T21-2G (Fig. 1C, p=0.002). Lineage expansion and immunophenotyping of these HSPCs in megakaryocyte-specific media showed that these cells expressed markers closely resembling DS-ML immunophenotype. Of note, the myeloid markers, CD13 and CD11b are the only two markers expressed on majority of DS-ML blasts compared to TAM blasts (Karandikar et al., 2001) (Yumura-Yagi et al., 1992). The percentage of CD13 and CD11b expressing cells was higher in megakaryoblasts expanded from iPSC lines with STAG2 GATA1 double mutant (Fig. 1D). The number of cells expressing CD117, a stem cell marker shown recently to be involved in DS-ML progression, were highest in T21-1GS and T21-2GS lines when compared to their respective isogenic family of GATA1 mutant lines. Conclusion GATA1s and STAG2 knockout co-operated to increase the megakaryoid population and the percentage of cells expressing DS-ML markers. We have developed a model system representing DS-ML, which can be used for understanding the individual and synergistic contribution of these gene mutations in disease initiation and progression. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Recommendations for Diagnosis and Treatment of Children with Transient Abnormal Myelopoiesis (TAM) and Myeloid Leukemia in Down Syndrome (ML-DS)

Children with Down syndrome are at a high risk of developing transient abnormal myelopoiesis (TAM; synonym: TMD) or myeloid leukemia (ML-DS). While most patients with TAM are asymptomatic and go into spontaneous remission without a need for therapy, around 20% of patients die within the first six months due to TAM-related complications. Another 20–30% of patients progress from TAM to ML-DS. ML-DS patients are particularly vulnerable to therapy-associated toxicity, but the prognosis of relapsed ML-DS is extremely poor – thus, ML-DS therapy schemata must strive for a balance between appropriate efficacy (to avoid relapses) and treatment-related toxicity. This guideline presents diagnostic and therapeutic strategies for TAM and ML-DS based on the experience and results of previous clinical studies from the BFM working group, which have helped reduce the risk of early death in symptomatic TAM patients using low-dose cytarabine, and which have achieved excellent cure rates for ML-DS using intensity-reduced treatment protocols.

Trisomy 21, transposition of the great arteries and abnormal myelopoiesis

Down syndrome is a well-recognised genetic condition associated with several comorbidities. Although CHD is common in Down syndrome, transposition of the great arteries is exceptionally rare. We describe a neonate with Down syndrome who presented with transient abnormal myelopoiesis and transposition of the great arteries. Down syndrome may accelerate pulmonary hypertension in transposition of the great arteries and is associated with poor outcomes.

Transient Abnormal Myelopoiesis with a Novel GATA1 Mutation in a Child with Down Syndrome: A Case Report and Brief Review

Transient abnormal myelopoiesis (TAM) is a unique entity seen in children with Down syndrome (DS) with 10 to 20% risk of developing myeloid leukemia in the first 5 years of life. We report a 2 months old male infant with DS detected to have hyperleukocytosis on routine preoperative workup for cyanotic congenital heart disease. Peripheral blood and bone marrow aspiration showed blasts, and next-generation sequencing detected a novel GATA1 mutation, and a diagnosis of TAM was confirmed in this child. This mutation has not been reported in TAM in the literature earlier to the best of our knowledge.