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.

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.

Favorable outcomes of hematopoietic stem cell transplantation in children and adolescents with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia associated with congenital abnormalities and cancer predisposition. Allogeneic hematopoietic stem cell transplantation (HSCT) can correct the hematological phenotype and is indicated in transfusion-dependent patients. In 70 children reported to the German DBA and French HSCT registries, HSCT was performed from 1985 to 2017. Median age at HSCT was 5.5 years (range, 0.9-17.3 years). Two-thirds of patients (64%) were transplanted from a matched sibling donor (MSD), and most procedures were performed after the year 1999 (73%). Primary engraftment was achieved in all patients. One patient developed secondary graft failure. Cumulative incidence of acute graft-versus-host disease (GVHD) was 24% for °II-IV (95% confidence interval [CI], 16% to 37%) and 7% for °III-IV (95% CI, 3% to 17%); cumulative incidence of chronic GVHD was 11% (95% CI, 5% to 22%). The probability of chronic GVHD-free survival (cGFS) was 87% (95% CI, 79% to 95%) and significantly improved over time (<2000: 68% [95% CI, 47% to 89%] vs ≥2000: 94% [95% CI, 87% to 100%], P < .01). cGFS was comparable following HSCT from a MSD and an unrelated donor (UD). Of note, no severe chronic GVHD or deaths were reported following MSD-HSCT after 1999. The difference of cGFS in children transplanted <10 years of age compared with older patients did not reach statistical significance (<10 years: 90% [95% CI, 81% to 99%] vs 10-18 years 78% [95% CI, 58% to 98%]). In summary, these data indicate that HSCT is efficient and safe in young DBA patients and should be considered if a MSD or matched UD is available. HSCT for transfusion dependency only must be critically discussed in older patients.

Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents.

Germline GATA2 mutations cause cellular deficiencies with high propensity for myeloid disease. We investigated 426 children and adolescents with primary myelodysplastic syndrome (MDS) and 82 cases with secondary MDS enrolled in 2 consecutive prospective studies of the European Working Group of MDS in Childhood (EWOG-MDS) conducted in Germany over a period of 15 years. Germline GATA2 mutations accounted for 15% of advanced and 7% of all primary MDS cases, but were absent in children with MDS secondary to therapy or acquired aplastic anemia. Mutation carriers were older at diagnosis and more likely to present with monosomy 7 and advanced disease compared with wild-type cases. For stratified analysis according to karyotype, 108 additional primary MDS patients registered with EWOG-MDS were studied. Overall, we identified 57 MDS patients with germline GATA2 mutations. GATA2 mutations were highly prevalent among patients with monosomy 7 (37%, all ages) reaching its peak in adolescence (72% of adolescents with monosomy 7). Unexpectedly, monocytosis was more frequent in GATA2-mutated patients. However, when adjusted for the selection bias from monosomy 7, mutational status had no effect on the hematologic phenotype. Finally, overall survival and outcome of hematopoietic stem cell transplantation (HSCT) were not influenced by mutational status. This study identifies GATA2 mutations as the most common germline defect predisposing to pediatric MDS with a very high prevalence in adolescents with monosomy 7. GATA2 mutations do not confer poor prognosis in childhood MDS. However, the high risk for progression to advanced disease must guide decision-making toward timely HSCT.

LIN28B overexpression defines a novel fetal-like subgroup of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive stem cell disease of early childhood. RAS activation constitutes the core component of oncogenic signaling. In addition, leukemic blasts in one-fourth of JMML patients present with monosomy 7, and more than half of patients show elevated age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care and results in an event-free survival rate of 50% to 60%, indicating that novel molecular-driven therapeutic options are urgently needed. Using gene expression profiling in a series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. LIN28B overexpression was significantly correlated with higher HbF levels, whereas patients with monosomy 7 seldom showed enhanced LIN28B expression. This finding gives a biological explanation of why patients with monosomy 7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Lastly, high LIN28B expression was associated with poor clinical outcome in our JMML patient series but was not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified elevated LIN28B expression as a hallmark of a novel fetal-like subgroup in JMML.

Morphological differentiation of severe aplastic anaemia from hypocellular refractory cytopenia of childhood: reproducibility of histopathological diagnostic criteria.

AIMS: To evaluate the reproducibility and reliability of the histomorphological criteria differentiating severe aplastic anaemia (SAA) and hypoplastic refractory cytopenia of childhood (RCC), the most frequently acquired hypocellular bone marrow conditions of childhood. METHODS AND RESULTS: We performed a double-blind interobserver study of 100 different cases of SAA and RCC among seven haematopathologists of the European Working Group of MDS in Childhood (EWOG-MDS) and the German SAA study. Cases with foci of typical myelodysplastic syndrome (MDS) morphology, such as patchy erythropoiesis with defective maturation, in an otherwise highly hypocellular or adipocytic bone marrow were classified as having RCC. Bone marrow samples without a patchy distribution, few scattered myeloid cells or haematopoietic aplasia were diagnosed as SAA. In only four of 100 cases did the reference pathologists not reach agreement regarding classification as SAA or RCC. The kappa index was 0.79. CONCLUSIONS: Our results show that the vast majority of SAA and RCC cases can be reliably differentiated by morphological means alone. A clear differentiation between SAA and RCC at presentation is mandatory for optimizing therapy strategies, and might be responsible for the fact that, in the German childhood SAA study, the probability of developing clonal disease after immunosuppressive therapy has dropped to 3%.

Complex karyotype newly defined: the strongest prognostic factor in advanced childhood myelodysplastic syndrome.

To identify cytogenetic risk factors predicting outcome in children with advanced myelodysplastic syndrome, overall survival of 192 children prospectively enrolled in European Working Group of Myelodysplastic Syndrome in Childhood studies was evaluated with regard to karyotypic complexity. Structurally complex constitutes a new definition of complex karyotype characterized by more than or equal to 3 chromosomal aberrations, including at least one structural aberration. Five-year overall survival in patients with more than or equal to 3 clonal aberrations, which were not structurally complex, did not differ from that observed in patients with normal karyotype. Cox regression analysis revealed the presence of a monosomal and structurally complex karyotype to be strongly associated with poor prognosis (hazard ratio = 4.6, P < .01). Notably, a structurally complex karyotype without a monosomy was associated with a very short 2-year overall survival probability of only 14% (hazard ratio = 14.5; P < .01). The presence of a structurally complex karyotype was the strongest independent prognostic marker predicting poor outcome in children with advanced myelodysplastic syndrome.

Performance of Interleukin-6 and Interleukin-8 serum levels in pediatric oncology patients with neutropenia and fever for the assessment of low-risk.

BACKGROUND: Patients with chemotherapy-related neutropenia and fever are usually hospitalized and treated on empirical intravenous broad-spectrum antibiotic regimens. Early diagnosis of sepsis in children with febrile neutropenia remains difficult due to non-specific clinical and laboratory signs of infection. We aimed to analyze whether IL-6 and IL-8 could define a group of patients at low risk of septicemia. METHODS: A prospective study was performed to assess the potential value of IL-6, IL-8 and C-reactive protein serum levels to predict severe bacterial infection or bacteremia in febrile neutropenic children with cancer during chemotherapy. Statistical test used: Friedman test, Wilcoxon-Test, Kruskal-Wallis H test, Mann-Whitney U-Test and Receiver Operating Characteristics. RESULTS: The analysis of cytokine levels measured at the onset of fever indicated that IL-6 and IL-8 are useful to define a possible group of patients with low risk of sepsis. In predicting bacteremia or severe bacterial infection, IL-6 was the best predictor with the optimum IL-6 cut-off level of 42 pg/ml showing a high sensitivity (90%) and specificity (85%). CONCLUSION: These findings may have clinical implications for risk-based antimicrobial treatment strategies.

Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre-hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously analyzed peripheral blood and bone marrow samples demonstrate that blood can be monitored for residual disease. Importantly, these assays provide a sensitive strategy to evaluate molecular responses to new therapeutic strategies.

Interferon-gamma sensitizes resistant Ewing's sarcoma cells to tumor necrosis factor apoptosis-inducing ligand-induced apoptosis by up-regulation of caspase-8 without altering chemosensitivity.

Ewing's sarcoma cells are highly susceptible to apoptosis via tumor necrosis factor apoptosis-inducing ligand (TRAIL). Resistance to TRAIL has been linked to deficient expression of caspase-8 in vitro. Here, we report on the status of caspase-8 expression in tumors from patients with Ewing's sarcoma, the effect of interferon-gamma on caspase-8 expression and apoptosis, and the role of caspase-8 for TRAIL- and chemotherapy-mediated apoptosis in Ewing's sarcoma. Using immunohistochemistry, we show that low expression of caspase-8 is seen in about 24% of tumors. Interferon-gamma induces expression of caspase-8 at concentrations achievable in humans and sensitizes cells to TRAIL. Transfection of wild type but not mutant caspase-8 into caspase-8-deficient Ewing's sarcoma cells restored sensitivity to TRAIL, indicating that up-regulation of caspase-8 is sufficient to restore TRAIL sensitivity. In contrast, no role for caspase-8 in chemotherapy-induced apoptosis was identified, because 1) transfection of caspase-8 or treatment with interferon-gamma did not alter the sensitivity of caspase-8-deficient cells to chemotherapeutics, 2) application of chemotherapy did not select for caspase-8-negative tumor cells in vivo, and 3) the caspase-8 status of tumors did not influence survival after chemotherapy-based protocols. In conclusion, our data provide a rationale for the inclusion of interferon-gamma in upcoming clinical trials with TRAIL.

Monosomy 7 and deletion 7q in children and adolescents with acute myeloid leukemia: an international retrospective study.

Monosomy 7 (-7) and deletion 7q \del(7q)] are rare in childhood acute myeloid leukemia (AML). We retrospectively collected data on 258 children with AML or refractory anemia with excess blasts in transformation (RAEB-T) and -7 or del(7q) with or without other cytogenetic aberrations \+/- other]. Karyotypes included -7 (n = 90), -7 other (n = 82), del(7q) (n = 21), and del(7q) other (n = 65). Complete remission (CR) was achieved in fewer patients with -7 +/- other compared with del(7q) +/- other (61% versus 89%, P < .001). Overall, the 5-year survival rate was 39% (SE, 3%). Survival was superior in del(7q) +/- other compared with -7 +/- other (51% versus 30%, P < .01). Cytogenetic aberrations considered favorable in AML \t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q22;q21), t(9;11)(p22;q23)] (n = 24) were strongly associated with del(7q) and a higher 5-year survival rate compared with del(7q) without favorable cytogenetics (75% versus 46%, P = .03). Patients with -7 and inv(3),-5/del(5q), or + 21 had a 5-year survival rate of 5%. Stem cell transplantation analyzed as a time-dependent variable had no impact on overall survival. However, patients not achieving CR had a 31% survival rate after stem cell transplantation. Childhood AML with chromosome 7 aberrations represents a heterogeneous group of disorders with additional cytogenetic aberrations having a major prognostic impact which should be reflected in future risk-group stratification.

Non-hematopoietic stem cell transplantation treatment of juvenile myelomonocytic leukemia: a retrospective analysis and definition of response criteria.

BACKGROUND: Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of infancy. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment modality, while the role of anti-leukemic therapy prior to HSCT is uncertain. A comparative evaluation of the efficacy of different clinical protocols and great variety of anti-neoplastic drugs applied pre-HSCT is hampered by the lack of uniform criteria of response. Classification schemas applied in other forms of leukemia are of little value, because in JMML therapy may result in divergent responses in solid organs compared to peripheral blood (PB). PROCEDURE: We therefore defined separate response criteria for white blood count (WBC), platelet count, liver size, and spleen size. We then retrospectively evaluated the efficacy of 129 treatment courses other than HSCT administered to 63 children with JMML. Treatment consisted of intensive therapy according to AML-type chemotherapy, maintenance-type combination therapy, and single agent therapy. To account for the variability observed in the natural course of disease, we also evaluated 32 episodes of "no therapy." RESULTS: Best responses within 3 months of initiation of therapy were highly variable for the four response criteria. In contrast to platelet count and liver size, there was a significant correlation between WBC or spleen size and therapy. Response rates for WBC and spleen size were best for purine analogs, etoposide, and cytarabine as single agents or for maintenance-type combination therapy. CONCLUSION: To rigorously test future therapeutic strategies in this rare disease an international consensus on the definition of response criteria will be helpful.