Biallelic inactivation of the NF1 tumour suppressor gene in juvenile myelomonocytic leukaemia: Genetic evidence of driver function and implications for diagnostic workup.

Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF-1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild-type allele. Here we examined the two-hit concept in leukaemic cells of 25 patients with JMML and NF-1. Ten patients with JMML/NF-1 exhibited a NF1 loss-of-function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound-heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF-1 and no variation in the JMML-associated driver genes PTPN11, KRAS, NRAS or CBL (JMML-5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound-heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF-1 reliably indicates a NF1-driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF-1.

Clinical evolution, genetic landscape and trajectories of clonal hematopoiesis in SAMD9/SAMD9L syndromes.

Germline SAMD9 and SAMD9L mutations (SAMD9/9Lmut) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a clinically annotated pediatric MDS cohort (n = 669) to define the prevalence, genetic landscape, phenotype, therapy outcome and clonal architecture of SAMD9/9L syndromes. In consecutively diagnosed MDS, germline SAMD9/9Lmut accounted for 8% and were mutually exclusive with GATA2 mutations present in 7% of the cohort. Among SAMD9/9Lmut cases, refractory cytopenia was the most prevalent MDS subtype (90%); acquired monosomy 7 was present in 38%; constitutional abnormalities were noted in 57%; and immune dysfunction was present in 28%. The clinical outcome was independent of germline mutations. In total, 67 patients had 58 distinct germline SAMD9/9Lmut clustering to protein middle regions. Despite inconclusive in silico prediction, 94% of SAMD9/9Lmut suppressed HEK293 cell growth, and mutations expressed in CD34+ cells induced overt cell death. Furthermore, we found that 61% of SAMD9/9Lmut patients underwent somatic genetic rescue (SGR) resulting in clonal hematopoiesis, of which 95% was maladaptive (monosomy 7 ± cancer mutations), and 51% had adaptive nature (revertant UPD7q, somatic SAMD9/9Lmut). Finally, bone marrow single-cell DNA sequencing revealed multiple competing SGR events in individual patients. Our findings demonstrate that SGR is common in SAMD9/9Lmut MDS and exemplify the exceptional plasticity of hematopoiesis in children.

Supportive Care During Pediatric Hematopoietic Stem Cell Transplantation: Prevention of Infections. A Report From Workshops on Supportive Care of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT).

Specific protocols define eligibility, conditioning, donor selection, graft composition and prophylaxis of graft vs. host disease for children and young adults undergoing hematopoietic stem cell transplant (HSCT). However, international protocols rarely, if ever, detail supportive care, including pharmaceutical infection prophylaxis, physical protection with face masks and cohort isolation or food restrictions. Supportive care suffers from a lack of scientific evidence and implementation of practices in the transplant centers brings extensive restrictions to the child's and family's daily life after HSCT. Therefore, the Board of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) held a series of dedicated workshops since 2017 with the aim of initiating the production of a set of minimal recommendations. The present paper describes the consensus reached within the field of infection prophylaxis.

Current Practices on Diagnosis, Prevention and Treatment of Post-Transplant Lymphoproliferative Disorder in Pediatric Patients after Solid Organ Transplantation: Results of ERN TransplantChild Healthcare Working Group Survey.

(1) Background: Post-transplant lymphoproliferative disease (PTLD) is a significant complication of solid organ transplantation (SOT). However, there is lack of consensus in PTLD management. Our aim was to establish a present benchmark for comparison between international centers and between various organ transplant systems and modalities; (2) Methods: A cross-sectional questionnaire of relevant PTLD practices in pediatric transplantation was sent to multidisciplinary teams from 17 European center members of ERN TransplantChild to evaluate the centers' approach strategies for diagnosis and treatment and how current practices impact a cross-sectional series of PTLD cases; (3) Results: A total of 34 SOT programs from 13 European centers participated. The decision to start preemptive treatment and its guidance was based on both EBV viremia monitoring plus additional laboratory methods and clinical assessment (61%). Among treatment modalities the most common initial practice at diagnosis was to reduce the immunosuppression (61%). A total of 126 PTLD cases were reported during the period 2012-2016. According to their histopathological classification, monomorphic lesions were the most frequent (46%). Graft rejection after PTLD remission was 33%. Of the total cases diagnosed with PTLD, 88% survived; (4) Conclusions: There is still no consensus on prevention and treatment of PTLD, which implies the need to generate evidence. This might successively allow the development of clinical guidelines.

Hematopoietic stem cell transplantation in children and adolescents with GATA2-related myelodysplastic syndrome.

GATA2 deficiency is a heterogeneous multi-system disorder characterized by a high risk of developing myelodysplastic syndrome (MDS) and myeloid leukemia. We analyzed the outcome of 65 patients reported to the registry of the European Working Group (EWOG) of MDS in childhood carrying a germline GATA2 mutation (GATA2mut) who had undergone hematopoietic stem cell transplantation (HSCT). At 5 years the probability of overall survival and disease-free survival (DFS) was 75% and 70%, respectively. Non-relapse mortality and relapse equally contributed to treatment failure. There was no evidence of increased incidence of graft-versus-host-disease or excessive rates of infections or organ toxicities. Advanced disease and monosomy 7 (-7) were associated with worse outcome. Patients with refractory cytopenia of childhood (RCC) and normal karyotype showed an excellent outcome (DFS 90%) compared to RCC and -7 (DFS 67%). Comparing outcome of GATA2mut with GATA2wt patients, there was no difference in DFS in patients with RCC and normal karyotype. The same was true for patients with -7 across morphological subtypes. We demonstrate that HSCT outcome is independent of GATA2 germline mutations in pediatric MDS suggesting the application of standard MDS algorithms and protocols. Our data support considering HSCT early in the course of GATA2 deficiency in young individuals.

Organ sparing total marrow irradiation compared to total body irradiation prior to allogeneic stem cell transplantation.

OBJECTIVES: Total body irradiation (TBI) is commonly used prior to hematopoietic stem cell transplantation (HSCT) in myeloablative conditioning regimens. However, TBI may be replaced by total marrow irradiation (TMI) at centres with access to Helical TomoTherapy, a modality that has the advantage of delivering intensity-modulated radiotherapy to long targets such as the entire bone marrow compartment. Toxicity after organ sparing TMI prior to HSCT has not previously been reported compared to TBI or with regard to engraftment data. METHODS: We conducted a prospective observational study on 37 patients that received organ sparing TMI prior to HSCT and compared this cohort to retrospective data on 33 patients that received TBI prior to HSCT. RESULTS: The 1-year graft-versus-host disease-free, relapse-free survival (GRFS) was 67.5% for all patients treated with TMI and 80.5% for patients with matched unrelated donor and treated with TMI, which was a significant difference from historical data on TBI patients with a hazard ratio of 0.45 (P = .03) and 0.24 (P < .01). Engraftment with a platelet count over 20 [K/µL] and 50 [K/µL] was significantly shorter for the TMI group, and neutrophil recovery was satisfactory in both treatment cohorts. There was generally a low occurrence of other treatment-related toxicities. CONCLUSIONS: Despite small cohorts, some significant differences were found; TMI as part of the myeloablative conditioning yields a high 1-year GRFS, fast and robust engraftment, and low occurrence of acute toxicity.

The combination of clofarabine, etoposide, and cyclophosphamide shows limited efficacy as a bridge to transplant for children with refractory acute leukemia: results of a monitored prospective study.

Clofarabine has been shown to effectively induce remission in children with refractory leukemia. We conducted a prospective trial (clinicval.trials.gov NCT01025778) to explore the use of clofarabine-based chemotherapy as a bridge-to-transplant approach. Children with refractory acute leukemia were enrolled to receive two induction courses of clofarabine, etoposide, and cyclophosphamide (CloEC). Responding patients were scheduled for T-cell depleted haploidentical hematopoietic stem cell transplantation (HSCT). The primary objective was to improve survival by achieving sufficient disease control to enable stem cell transplantation. Secondary objectives were to evaluate safety and toxicity. Seven children with active disease entered the study. Two children responded to induction courses and underwent transplantation. Five children did not respond to induction: one died in progression after the first course; two received off-protocol chemotherapy and were transplanted; and two succumbed to progressive leukemia. All transplanted children engrafted and no acute skin graft-versus-host disease > grade I was observed. One child is alive and well 7.5 years after the first CloEC course. One child developed fulminant adenovirus hepatitis and died in continuous complete remission 7 months after start of induction. Two children relapsed and died 6.5 and 7.5 months after enrollment. Infection was the most common toxicity. CloEC can induce responses in some patients with refractory acute leukemia but is highly immunosuppressive, resulting in substantial risk of life-threatening infections. In our study, haploidentical HSCT was feasible with sustained engraftment. No clinically significant organ toxicity was observed. Also, repeating CloEC probably does not increase the chance of achieving remission.

Matched versus Haploidentical Hematopoietic Stem Cell Transplantation as Treatment Options for Primary Immunodeficiencies in Children.

Primary immunodeficiencies (PIDs) are inherited disorders of the immune system with allogeneic hematopoietic stem cell transplantation (HSCT) as the only curative treatment in some of them. In case an HLA-matched donor is not available, HSCT from a haploidentical family donor may be considered. We compared the outcomes of HSCT from HLA-matched unrelated or related donors (MUDs or MRDs) and mismatched related haploidentical donors (MMRDs) in patients with a variety of PIDs in 2 centers. A total of 44 pediatric patients were evaluated. We reviewed the outcomes of 25 children who underwent transplantation with HLA-matched grafts (MRD, n = 13; MUD, n = 12) and 19 patients receiving haploidentical stem cells. Bone marrow (BM) was transplanted in 85% (MRD) and 75% (MUD) of the matched cohort and peripheral blood stem cells (PBSCs) in 15% (MRD), 25% (MUD), and 100% (MMRD). All but 9 patients (MRD, n = 6; MMRD, n = 3) with severe combined immunodeficiency (SCID) received a chemotherapy-based conditioning regimen. Immune reconstitution of T, B, and natural killer cells was comparable for all groups with an advantage of recipients of MRD grafts in early CD4 reconstitution. However, deaths due to viral infections occurred more often in the haploidentical cohort. The disease-free survival was 91.7% (MRD), 66.7% (MUD), and 62.7% (MMRD), respectively. Grade II to IV acute graft-versus-host disease (GVHD) occurred in 15% (MRD), 8% (MUD), and 21% (MMRD) of the patients. Only 1 patient had severe grade IV GVHD in the MRD group, whereas no grade >II GVHD was observed in the MUD or MMRD cohort. These data indicate that in the absence of a suitable HLA-identical family donor, haploidentical HSCT may be a viable option for patients with life-threatening disease and urgent need of HSCT.

Correction: Supportive care during pediatric hematopoietic stem cell transplantation: beyond infectious diseases. A report from workshops on supportive care of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Supportive care during pediatric hematopoietic stem cell transplantation: beyond infectious diseases. A report from workshops on supportive care of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT).

Hematopoietic stem cell transplantation (HSCT) is currently the standard of care for many malignant and nonmalignant blood diseases. As several treatment-emerging acute toxicities are expected, optimal supportive measurements critically affect HSCT outcomes. The paucity of good clinical studies in supportive practices gives rise to the establishment of heterogeneous guidelines across the different centers, which hampers direct clinical comparison in multicentric studies. Aiming to harmonize the supportive care provided during the pediatric HSCT in Europe, the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) promoted dedicated workshops during the years 2017 and 2018. The present paper describes the resulting consensus on the management of sinusoidal obstructive syndrome, mucositis, enteral and parenteral nutrition, iron overload, and emesis during HSCT.

Low incidence of hemorrhagic cystitis following ex vivo T-cell depleted haploidentical hematopoietic cell transplantation in children.

Hemorrhagic cystitis (HC) is a debilitating complication following allogenic hematopoietic cell transplantation (HCT). HLA disparity and T-cell depletion have been implicated as risk factors for HC. However, reports on the incidence and risk factors for HC in ex vivo T-cell depleted haploidentical HCT (haploHCT) in children are lacking. We studied 96 haploHCT procedures performed in 83 children between 2002 and 2017. Sixty-three patients were diagnosed with a malignant disease and 20 with nonmalignant disease. All but three patients with SCID underwent myelotoxic and/or lymphotoxic conditioning therapy. Grafts were CD3+ (36.5%) or TcRαß+ (63.5%) depleted to prevent graft versus host disease (GvHD). Fourteen patients (14.6%) were diagnosed with HC; 12 (12.5%) had clinically significant stage II-IV HC. All patients with HC had BK viruria and/or viremia. Increasing age and chemotherapeutic treatment prior to conditioning were identified as risk factors for HC. Immune recovery did not significantly differ between patients with and without HC. Thus, we report a low incidence of HC in pediatric haploHCT using ex vivo T-cell depletion. The combination of a reduced toxicity conditioning regimen, and typically absent pharmaceutical post-HCT GvHD prophylaxis in our patients might have contributed to the decreased the risk of HC, despite HLA disparity.

Low burden of minimal residual disease prior to transplantation in children with very high risk acute lymphoblastic leukaemia: The NOPHO ALL2008 experience.

The population-based Nordic/Baltic acute lymphoblastic leukaemia (ALL) Nordic Society for Paediatric Haematology and Oncology (NOPHO) ALL2008 protocol combined minimal residual disease (MRD)-driven treatment stratification with very intense first line chemotherapy for patients with high risk ALL. Patients with MRD ≥5% at end of induction or ≥10-3 at end of consolidation or following two high risk blocks were eligible for haematopoietic cell transplantation (HCT) in first remission. After at least three high risk blocks a total of 71 children received HCT, of which 46 had MRD ≥5% at end of induction. Ten patients stratified to HCT were not transplanted; 12 received HCT without protocol indication. Among 69 patients with evaluable pre-HCT MRD results, 22 were MRD-positive, one with MRD ≥10-3 . After a median follow-up of 5·5 years, the cumulative incidence of relapse was 23·5% (95% confidence interval [CI]: 10·5-47·7) for MRD-positive versus 5·1% (95% CI: 1·3-19·2), P = 0·02) for MRD-negative patients. MRD was the only variable significantly associated with relapse (hazard ratio 9·1, 95% CI: 1·6-51·0, P = 0·012). Non-relapse mortality did not differ between the two groups, resulting in disease-free survival of 85·6% (95% CI: 75·4-97·2) and 67·4% (95% CI: 50·2-90·5), respectively. In conclusion, NOPHO block treatment efficiently reduced residual leukaemia which, combined with modern transplant procedures, provided high survival rates, also among pre-HCT MRD-positive patients.

Haploidentical Stem Cell Transplantation for Refractory/Relapsed Neuroblastoma.

Pediatric patients with refractory or relapsed metastatic neuroblastoma (NBL) have a poor prognosis despite autologous stem cell transplantation (SCT). Allogeneic SCT from a haploidentical donor has a remarkable alloreactive effect in patients with leukemia; thus, we evaluated this approach in children with very high-risk NBL. We analyzed data from 2 prospective phase I/II trials. A total of 26 patients with refractory (n = 5), metastatic relapsed (n = 20), or locally relapsed MYCN-positive (n = 1) NBL received a median of 17 × 106/kg T/B cell-depleted CD34+ stem cells with 68 × 103/kg residual T cells and 107 × 106/kg natural killer cells. The conditioning regimen comprised melphalan, fludarabine, thiotepa, OKT3, and a short course of mycophenolate mofetil post-transplantation. Engraftment occurred in 96% of the patients. Event-free survival and overall survival at 5 years were 19% and 23%, respectively. No transplantation-related mortality was observed, and the single death was due to progression/subsequent relapse. The median duration of follow-up was 8.1 years. Patients in complete remission before SCT had a significantly better prognosis than those with residual tumor load (P < .01). All patients with progressive disease before SCT relapsed within 1 year. Grade II and grade III acute graft-versus-host disease (GVHD) occurred in 31% and 12% of the patients, respectively. Chronic limited and extensive GVHD occurred in 28% and 10%, respectively. Our data indicate that haploidentical SCT is a feasible treatment option that can induce long-term remission in some patients with NBL with tolerable side effects, and may enable the development of further post-transplantation therapeutic strategies based on the donor-derived immune system.

Ataxia-pancytopenia syndrome with SAMD9L mutations.

OBJECTIVE: We describe the neurologic, neuroradiologic, and ophthalmologic phenotype of 1 Swedish and 1 Finnish family with autosomal dominant ataxia-pancytopenia (ATXPC) syndrome and SAMD9L mutations. METHODS: Members of these families with germline SAMD9L c.2956C>T, p.Arg986Cys, or c.2672T>C, p.Ile891Thr mutations underwent structured interviews and neurologic and ophthalmologic examinations. Neuroimaging was performed, and medical records were reviewed. Previous publications on SAMD9L-ATXPC were reviewed. RESULTS: Twelve individuals in both families were affected clinically. All mutation carriers examined had balance impairment, although severity was very variable. All but 1 had nystagmus, and all but 1 had pyramidal tract signs. Neurologic features were generally present from childhood on and progressed slowly. Two adult patients, who experienced increasing clumsiness, glare, and difficulties with gaze fixation, had paracentral retinal dysfunction verified by multifocal electroretinography. Brain MRI showed early, marked cerebellar atrophy in most carriers and variable cerebral periventricular white matter T2 hyperintensities. Two children were treated with hematopoietic stem cell transplantation for hematologic malignancies, and the neurologic symptoms of one of these worsened after treatment. Three affected individuals had attention deficit hyperactivity disorder or cognitive problems. Retinal dysfunction was not previously reported in individuals with ATXPC. CONCLUSIONS: The neurologic phenotype of this syndrome is defined by balance or gait impairment, nystagmus, hyperreflexia in the lower limbs and, frequently, marked cerebellar atrophy. Paracentral retinal dysfunction may contribute to glare, reading problems, and clumsiness. Timely diagnosis of ATXPC is important to address the risk for severe hemorrhage, infection, and hematologic malignancies inherent in this syndrome; regular hematologic follow-up might be beneficial.

Transplantation of Haploidentical TcRaß-Depleted Hematopoietic Cells Allows for Optimal Timing and Sustained Correction of the Metabolic Defect in Children With Infantile Osteopetrosis.

In osteopetrosis, osteoclast dysfunction can lead to deafness, blindness, bone marrow failure, and death. Hematopoietic cell transplantation (HCT) is currently the only curative treatment, but outcome remains disappointing. Although a rapid progression toward HCT is detrimental to prevent further progress of disease manifestations, 70% of cases lack an HLA-matched sibling and require alternative stem cell sources. We present two cases of osteopetrosis that successfully received an HCT with haploidentical TcRαß-depleted cells from one of the parents. These cases showed no further disease progression, had restoration of functional osteoclasts, and illustrate this approach to enable prompt HCT with ready available parental donors and rapid and sustained hematological, including osteoclast, recovery. © 2016 American Society for Bone and Mineral Research.

Novel treatment of severe combined immunodeficiency utilizing ex-vivo T-cell depleted haploidentical hematopoietic stem cell transplantation and CD45RA+ depleted donor lymphocyte infusions.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment available for severe combined immunodeficiency (SCID); although, there is a high incidence of severe infections and an increased risk of graft-versus host-disease (GvHD) with HSCT. Early intervention is a crucial prognostic factor and a HLA-haploidentical parental donor is often available. Haploidentical HSCT protocols utilizing extensively ex vivo T-cell depleted grafts (CliniMACs system) have proven efficient in preventing GvHD, but cause a delay in early T-cell recovery that increases the risk of viral infections. Here, we present a novel approach for treating SCID that combines selective depletion of GvHD-inducing alpha/beta (α/ß) T-cells from the haploidentical HSCT graft with a subsequent donor lymphocyte infusion (DLI) enriched for CD45RO+ memory T-cells. RESULTS: Our patient was diagnosed with SCID (T-B + NK+ phenotype). At 9 months of age, he received a T cell receptor(TCR)α/ß-cell depleted graft from his haploidentical mother, following a reduced intensity conditioning regimen with no additional GvHD prophylaxis. Engraftment was rapid with complete donor chimerism and no signs of GvHD. However, at 12 weeks post HSCT, the patient was still T-cell lymphopenic with clinical symptoms of multiple severe viral infections. Consequently, therapeutic DLIs were initiated for enhanced anti-viral immunity. The patient was treated with CD45RA+ depleted haploidentical maternal donor lymphocytes enriched from unmobilized whole blood, and a total T-cell dose of no more than 25 x10(3) CD3+ cells/kg with >99.9% purity of CD3 + CD45RO+ memory T-cells was transferred. Following the DLI, a prompt increase in CD3 + CD4+ and CD3 + CD8+ counts was observed with a subsequent clearance of viral infections. No acute or chronic GvHD was observed. CONCLUSIONS: Automated depletion of CD45RA+ naïve T-cells from unmobilized whole blood is a simple and rapid strategy to provide unmanipulated DLIs, with a potentially broad repertoire of pathogen specific memory T-cells. In the haploidentical setting, CD45RA+ depleted DLIs can be safely administered at low T-cell doses for efficient enhancement of viral immunity and limited risk of GvHD. We demonstrate the successful use of this approach following TCR-α/ß-cell depleted HSCT for the treatment of SCID.