Background and Aims

We recently reported the importance of CD9 in pediatric ALL but its role in AML remains unknown. In this disease context, we pursued to characterize its prognostic significance, elucidate its regulation and function, and identify its role in leukemia immunosurveillance.

Methods

Patients were stratified based on CD9 status for comparison of long-term survival. Epigenetic control of CD9 was investigated by ChIP-sequencing and confirmed by HDACi treatment. Impact of CD9 on leukemia aggressiveness was measured by competition and colony formation assays. Influence of CD9 on leukemia progression was evaluated in xenograft models, coupled with single-cell transcriptomics to dissect the underlying mechanisms. Immune-related CD9 interactors were identified by IP-MS, followed by proof-of-function experiments in an immune-reconstituted mouse model.

Results

CD9 expression of AML patients was significantly lower than normal BM donors (13.2% vs. 48.4%). Among 81 AML cases, blasts of 32 patients (39.5%) were CD9+. The 5-year RFS rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%). A marked decrease of H3K9/27Ac occupancy of the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold), and strongly correlated with CD9 repression (r=0.585-0.719). Panobinostat exposure elevated CD9 expression (3.1-32.2-fold) and restored activating histone marks in AML samples. Overexpression of CD9 reduced proliferation and clonogenicity of AML cell lines. NOD/SCID mice receiving CD9+ AML exhibited a drastic reduction of leukemic load by 70.7-91.8%. Mechanistically, CD9 promoted basal and cytokine-induced MHC I/II expression through the JAK-STAT axis, and regulated their intracellular trafficking by physical binding. Preliminary data showed that CD9 could enhance BM infiltration of cytotoxic T cells and thereby mount an effective immunity against AML in humanized NSG mice.

Conclusions

Our data established CD9 as a novel tumor suppressor and immune regulator in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.

Background and Aims

KIT mutations (KIT+) are common in CBF-AML and have been associated with varying prognostic significance. We sought to define the clinical significance of distinct KIT mutations in this subgroup.

Methods

Children less than ≤15-years of age with CBF-AML diagnosed between January-2017 and December-2019 were included. KIT mutation was detected by Sanger-Sequencing (till December-2017) and subsequently by panel based next generation sequencing. For the later, computational analysis was done using open-source tools according to the GATK best practices. Treatment consisted of four cycles of intensive chemotherapy followed by maintenance therapy for 1 year. Clinical outcomes were analysed according to mutation status [KIT+ vs. wild-type KIT(KIT−)] and mutation location.

Results

KIT data was available for 90(87%) of 103 patients with CBF-AML. KIT+ were detected in 37(41%) of 90 patients; 26(70%) involved only exon-17, 6(16%) only exon-8, 3(8%) both exons, and 2(6%) alternative exons. The incidence of KIT+ was 38%(n=28) and 56%(n=9) amongst patients with t(8,21) and Inv16 respectively. Baseline demographics was comparable between the 2 groups. The incidence of induction failure [1(3.4%) vs 2(4%), p=0.9], post induction-I MRD >0.01% [10(34%) vs 19(58%), p=0.7] and toxic deaths [6(18%) vs 7(14%), p=0.5] were comparable between KIT+ and KIT- patients respectively. After a median follow-up of 21(95%CI:18.1-23.9) months, KIT+ CBF-AML had a poorer 2-year overall survival (52.3±9.1% vs 76.3±6.0%: P = 0.042) and a higher 2-year cumulative incidence of relapse (CIR) (40±10% vs 15.8±5.5%; P = 0.022) to those with KIT−. Amongst KIT+, relapse was higher for exon-17 mutation alone as compared to other exon mutations (2-year CIR 47.2±12% vs 16.7%±15.2%; p=0.180). On multivariate analysis, KIT+ was associated with inferior survival [HR:3.4(95%CI:1.3-9.1; p=0.014].

Conclusions

Our findings indicate that KIT+, especially at position exon-17 is a poor prognostic factor in pediatric CBF-AML. The role of tyrosine kinase inhibitors and bone-marrow transplantation needs exploration.

Background and Aims

Together with a rising number of paediatric cancer survivors emerges an increased incidence of late effects, such as therapy-related acute myeloid leukaemia (t-AML). Here, we aim to increase understanding of the origin and clonal evolution of paediatric t-AML.

Methods

We studied mutation accumulation and mutational patterns in 17 paediatric patients with t-AML by whole genome sequencing (WGS) of both t-AML blasts and normal haematopoietic stem and progenitor cells (HSPCs) at different timepoints between the first cancer diagnosis and the t-AML.

Results

Our data confirm that paediatric t-AML is mainly driven by gene fusions, in specific MLL-rearrangements (67% of our patients), while few other driving mutations are identified. In contrast to HSPCs from de novo paediatric AML, normal HSPCs in t-AML patients show an increased clonal mutational load compared to our previously established healthy baseline. Mutational signatures SBS5 and HSPC, which act in a clock-like manner in healthy blood, had expected contributions to the spectra of these HSPCs. However, the large contribution of treatment-related signatures in most patients indicates that different chemotherapies can leave a distinct mutational footprint in DNA of both normal HSPCs and t-AML blasts. Specifically, thiopurine and cisplatin treatment caused a clear mutational pattern, with thiopurine-caused mutations more frequently present in t-AML blasts than normal HSPCs. Moreover, we identified four novel signatures, with a thus far unknown cause. Finally, in two t-AML cases, we identified a rare type of MLL-rearranged cells with stem-like properties which were closely related to the t-AML blasts but phenotypically similar to normal HSPCs.

Conclusions

The variance in mutational effects of different chemotherapies could indicate that, in children, not all cancer treatments have large mutagenic effects. These differences might affect a patient’s risk to develop t-AML and could, in the future, influence the development of treatment regiments in paediatric oncology.

Background and Aims

Recently discovered activating germline heterozygous SAMD9 and SAMD9L mutations cause a severe blood disorder with high potential for the development of myelodysplastic syndrome (MDS) in need of better understanding and guidelines for diagnosis and therapy. In November 2019, 40 international clinical and basic science experts discussed current knowledge and management strategies. A major aim for this collaboration was to define the disease from its biological roots to its clinical presentation, improving accuracy of diagnosis and timely detection of complications.

Methods

A steering committee initially formulated questions to be addressed by meeting attendees. Identified subspecialty fields provided multidisciplinary expert opinion including Hematology, Oncology, Bone Marrow Transplant, Endocrinology, Neurology, and Genetics. The meeting consisted of literature review, review of current models to study disease, and international experience with SAMD9/9L families. Moderators then summarized each question followed by debate with attendees. Following the meeting, surveys were distributed to all attendees to ensure a 75% consensus for finalized consensus statements. This transparent methodology to develop expert opinion was utilized given the novelty of disease and lack any clinical trial data.

Results

Statements were drafted on the following subjects: (1) Suspicion of diagnosis, (2) Diagnostic evaluation (3) Hematologic and multidisciplinary evaluation, and (4) Management of patients with SAMD9/9L mutations. Statements underwent surveys leading to finalized consensus statements. A recommended comprehensive initial evaluation and ongoing disease surveillance was decided upon. Standardized treatment approaches for most clinical manifestations of SAMD9/9L syndromes have been recommended. No clear recommendation could be made yet for patients with MDS and monosomy 7 with stable counts.

Conclusions

Recommendations are derived from expert opinion gathered from the SAMD9/9L symposium. This evolving collaboration represents an attempt to close the critical knowledge gap that exists in identifying, diagnosing and treating patients with germline SAMD9/9L mutations

Background and Aims

Introduction of Post-Transplant Cyclophosphamide (PTCy) as graft-versus-host disease (GVHD) prophylaxis has made haploidentical hematopoietic stem cell transplantation (HSCT) a standard approach in adults, but pediatric experience is limited. Based on adult data, decreased donor availability and need to cryopreserve cells during the COVID19 pandemic, our center has increasingly used peripheral blood stem cells (PBSCs) from haploidentical donors (HaD) utilizing PTCy. Here we compare outcomes of traditional donor choices including matched sibling donor (MSD) and HLA matched unrelated donor (MUD) with those receiving HaD PBSC.

Methods

Retrospective single-center study. GVHD-free relapse-free survival (GRFS, defined as absence of acute GVHD grade III-IV, relapse, death or chronic GVHD requiring systemic therapy), overall survival (OS), relapse free survival (RFS), incidence of acute and chronic GVHD (cGVHD) were analyzed.

Results

Of 104 allogeneic transplants for leukemia/MDS from January 2014-December 2020 26 patients underwent HaD transplants with PBSC, 31 patients MSD, and 47 patients MUD bone marrow transplants. Demographic/HSCT characteristics were not significantly different, apart from remission status, with HaD cohort having more patients in ≥CR3 prior to HSCT (p<0.01). Median follow-up time of entire cohort-573 days.

GRFS at 18 months for HaD was 61% (95CI 43.3%-85.9%), for MUD 44.6% (95CI 31.8%-62.5%) and for MSD 62.1% (95CI 45.7-84.3%), p=0.26.

OS and RFS at 18 months for HaD were 80.7% (95CI 61.7%-100%) and 73.8% (95CI 55.5%-98.1%), for MUD 83.4% (95CI 72.8%-95.5%) and 70.3% (95CI 57.9%-85.3%) and for MSD 80.9% (95CI 66.9%-97.7%) and 66.5% (95CI 50.5%-87.5%), respectively. Incidence of chronic GVHD was higher with MUD 31.7% versus 10% and 9.2% in the MSD and HaD cohorts (p=0.02).

Conclusions

Haploidentical PBSC transplant with PTCy had comparable outcomes to MSD and MUD BM and less cGvHD compared to MUD BM. Logistical advantages and lower resource burden make Haploidentical HSCT with PBSC an alternative to MUD donors in children. Further prospective studies are needed.