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.

Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7.

Familial myelodysplastic syndromes arise from haploinsufficiency of genes involved in hematopoiesis and are primarily associated with early-onset disease. Here we describe a familial syndrome in seven patients from four unrelated pedigrees presenting with myelodysplastic syndrome and loss of chromosome 7/7q. Their median age at diagnosis was 2.1 years (range, 1-42). All patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without an increase of blasts. Genomic studies identified constitutional mutations (p.H880Q, p.R986H, p.R986C and p.V1512M) in the SAMD9L gene on 7q21, with decreased allele frequency in hematopoiesis. The non-random loss of mutated SAMD9L alleles was attained via monosomy 7, deletion 7q, UPD7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21. Incomplete penetrance was noted in 30% (3/10) of mutation carriers. Long-term observation revealed divergent outcomes with either progression to leukemia and/or accumulation of driver mutations (n=2), persistent monosomy 7 (n=4), and transient monosomy 7 followed by spontaneous recovery with SAMD9L-wildtype UPD7q (n=2). Dysmorphic features or neurological symptoms were absent in our patients, pointing to the notion that myelodysplasia with monosomy 7 can be a sole manifestation of SAMD9L disease. Collectively, our results define a new subtype of familial myelodysplastic syndrome and provide an explanation for the phenomenon of transient monosomy 7. Registered at: www.clinicaltrials.gov; #NCT00047268.

Molecular profile of inflammatory and megakaryocytic factors in pediatric myelodysplastic syndrome with acute myelofibrosis.

Pediatric fibrotic myelodysplastic syndromes (ped-MDS-MF) and pediatric primary myelofibrosis (ped-PMF) are rare, and the molecular changes which mediate fibrosis have never been investigated. Histology and gene expression profile of 119 fibrosis/angiogenesis/inflammation/megakaryopoiesis-related factors in bone marrow biopsies were performed (two ped-MDS-MF and one ped-PMF). In one progressive ped-MDS, comparison of MF grade 0 (no myelofibrosis) and MF grade 2 (dense network of reticulin fibres) after 4 months showed that expression of fibrosis-related transcripts increased and dysplastic megakaryocytes formed a dense net of CD42b+ proplatelets. These changes were not observed in another ped-MDS-MF, whereas ped-PMF showed a similar proplatelet pattern. These findings indicate that fibrotic changes in ped-MDS may involve proplatelet-related and unrelated pathways.

Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood.

Refractory cytopenia of childhood is the most common type of childhood myelodysplastic syndrome. Because the majority of children with refractory cytopenia have a normal karyotype and a hypocellular bone marrow, differentiating refractory cytopenia from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia can be challenging. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable diagnostic tool in differentiating myelodysplastic syndrome from non-clonal cytopenias in adults. Here, we performed the first comprehensive flow cytometric analysis of immature myeloid, lymphoid cells and erythroid cells, and granulocytes, monocytes, and lymphoid cells in bone marrow obtained from a large prospective cohort of 81 children with refractory cytopenia. Children with refractory cyotopenia had a strongly reduced myeloid compartment, but not as severe as children with aplastic anemia. Furthermore, the number of flow cytometric abnormalities was significantly higher in children with refractory cytopenia than in healthy controls and in children with aplastic anemia, but lower than in advanced myelodysplastic syndrome. We conclude that flow cytometric immunophenotyping could be a relevant addition to histopathology in the diagnosis of refractory cytopenia of childhood. (The multi-center studies EWOG-MDS RC06 and EWOG-MDS 2006 are registered at clinicaltrials.gov identifiers 00499070 and 00662090, respectively).

Hematopoietic stem cell transplantation in children and young adults with secondary myelodysplastic syndrome and acute myelogenous leukemia after aplastic anemia.

Secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/sAML) are the most serious secondary events occurring after immunosuppressive therapy in patients with aplastic anemia. Here we evaluate the outcome of hematopoietic stem cell transplantation (HSCT) in 17 children and young adults with sMDS/sAML after childhood aplastic anemia. The median interval between the diagnosis of aplastic anemia and the development of sMDS/sAML was 2.9 years (range, 1.2 to 13.0 years). At a median age of 13.1 years (range, 4.4 to 26.7 years), patients underwent HSCT with bone marrow (n = 6) or peripheral blood stem cell (n = 11) grafts from HLA-matched sibling donors (n = 2), mismatched family donors (n = 2), or unrelated donors (n = 13). Monosomy 7 was detected in 13 patients. The preparative regimen consisted of busulfan, cyclophosphamide, and melphalan in 11 patients and other agents in 6 patients. All patients achieved neutrophil engraftment. The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) was 47%, and that of chronic GVHD was 70%. Relapse occurred in 1 patient. The major cause of death was transplant-related complication (n = 9). Overall survival and event-free survival at 5 years after HSCT were both 41%. In summary, this study indicates that HSCT is a curative therapy for some patients with sMDS/sAML after aplastic anemia. Future efforts should focus on reducing transplantation-related mortality.

Spliceosomal gene aberrations are rare, coexist with oncogenic mutations, and are unlikely to exert a driver effect in childhood MDS and JMML.

Somatic mutations of the spliceosomal machinery occur frequently in adult patients with myelodysplastic syndrome (MDS). We resequenced SF3B1, U2AF35, and SRSF2 in 371 children with MDS or juvenile myelomonocytic leukemia. We found missense mutations in 2 juvenile myelomonocytic leukemia cases and in 1 child with systemic mastocytosis with MDS. In 1 juvenile myelomonocytic leukemia patient, the SRSF2 mutation that initially coexisted with an oncogenic NRAS mutation was absent at relapse, whereas the NRAS mutation persisted and a second, concomitant NRAS mutation later emerged. The patient with systemic mastocytosis and MDS carried both mutated U2AF35 and KIT in a single clone as confirmed by clonal sequencing. In the adult MDS patients sequenced for control purposes, we detected previously reported mutations in 7/30 and a novel SRSF2 deletion (c.284_307del) in 3 of 30 patients. These findings implicate that spliceosome mutations are rare in pediatric MDS and juvenile myelomonocytic leukemia and are unlikely to operate as driver mutations.

Juvenile-like (inflammatory/hyperplastic) mucosal polyps of the gastrointestinal tract in neurofibromatosis type 1.

AIMS: Diffuse neurofibromatosis/ganglioneuromatosis, solitary/plexiform neurofibroma, periampullary carcinoids and gastrointestinal stromal tumour (GIST) are the main gastrointestinal manifestations of neurofibromatosis type 1 (NF-1, von Recklinghausen disease). Inflammatory (juvenile-like) polyps have not been recognised to date as specific gastrointestinal (GI) manifestations of NF-1. METHODS AND RESULTS: We describe four males aged 23-65 years with NF-1 and inflammatory (juvenile-like) gastrointestinal polyps, and review the literature for similar cases. Two patients had single polyps (sigmoid colon and antrum, respectively), one had two polyps (left colon), and one had three polyps (distal oesophagus and colon). Histological appearances were variable, ranging from juvenile-like to granulation tissue-rich, predominantly inflammatory and hyperplastic. Three lesions showed obliterative vasculopathic changes. None had neurofibromatous or ganglioneuromatous polyps. A review of the literature disclosed 11 similar cases. Most patients presented with severe gastrointestinal symptoms and/or anaemia. CONCLUSIONS: NF-1-associated inflammatory polyps probably represent specific GI manifestations of this disorder, and should be considered, particularly in patients with GI symptoms. They should be distinguished from inflammatory fibroid polyps and from juvenile-like changes associated with ganglioneuroma/ganglioneuromatosis and neurofibroma/neurofibromatosis. Their aetiology remains obscure, but different mechanisms, including NF-1 inactivation, NF-1-associated vasculopathy, and localised mucosal prolapse/damage caused by motility disorders, might be involved.

Comparison of horse and rabbit antithymocyte globulin in immunosuppressive therapy for refractory cytopenia of childhood.

Refractory cytopenia of childhood is the most common subtype of myelodysplastic syndrome in children. In this study, we compared the outcome of immunosuppressive therapy using horse antithymocyte globulin (n=46) with that using rabbit antithymocyte globulin (n=49) in 95 patients with refractory cytopenia of childhood and hypocellular bone marrow. The response rate at 6 months was 74% for horse antithymocyte globulin and 53% for rabbit antithymocyte globulin (P=0.04). The inferior response in the rabbit antithymocyte globulin group resulted in lower 4-year transplantation-free (69% versus 46%; P=0.003) and failure-free (58% versus 48%; P=0.04) survival rates in this group compared with those in the horse antithymocyte globulin group. However, because of successful second-line hematopoietic stem cell transplantation, overall survival was comparable between groups (91% versus 85%; P=ns). The cumulative incidence of relapse (15% versus 9%; P=ns) and clonal evolution (12% versus 4%; P=ns) at 4 years was comparable between groups. Our results suggest that the outcome of immunosuppressive therapy with rabbit antithymocyte globulin is inferior to that of horse antithymocyte globulin. Although immunosuppressive therapy is an effective therapy in selected patients with refractory cytopenia of childhood, the long-term risk of relapse or clonal evolution remains. (ClinicalTrial.gov identifiers: NCT00662090).

Apoptotic cells and clonally expanded cytotoxic T cells in bone marrow trephines of patients with myelodysplastic syndrome.

AIMS: There is increasing evidence that autoimmunity is involved in the pathogenesis of myelodysplastic syndromes (MDS). We examined the number of apoptotic cells, and analysed the T cells and the T cell receptor gene rearrangements in bone marrow trephines of patients with low-grade MDS [refractory anaemia (RA), refractory anaemia with ringed sideroblasts (RAS) and refractory cytopenia with multilineage dysplasia (RCMD)] to investigate the correlation between T cells and apoptosis. METHODS AND RESULTS: Bone marrow trephines from 30 patients with RA, seven patients with RCMD, four patients with RAS and 11 normal bone marrow donors were stained for CD3 and for apoptotic cells using immunohistochemistry and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate (dUTP) nick end labelling (TUNEL) technique, respectively. The positive cells were quantified by computer-assisted image analysis. In addition, CD 8 and T cell-restricted intracellular antigen-1 (TIA-1)-positive cells were analysed by single staining and evaluated semiquantitatively by light microscopy. Junctional diversity of the T cell receptor (TCR) α-, ß- and γ-chains were analysed in 24 cases of RA and RCMD by reverse transcription-polymerase chain reaction (RT-PCR). In all cases of RA, RCMD and RAS an increase of apoptotic cells was accompanied by an increase of T cells, when compared to normal donors (P < 0.001). Expression of TIA-1 was found in 33 of 41 patients with low-grade MDS. In contrast, normal controls showed either no or only very weak expression. Furthermore, 14 of 24 cases with low-grade MDS showed clonal TCR gene rearrangement. CONCLUSION: These findings provide evidence that increased apoptosis in low-grade MDS correlates with increased numbers of cytotoxic T cells. A considerable proportion of the MDS cases showed clonal TCR rearrangement suggesting an antigen-driven selection of the T cells. We therefore speculate that cases of MDS can be accompanied by a presumably autoreactive T cell-mediated apoptosis induction in bone marrow cells.

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%.

Response to upfront azacitidine in juvenile myelomonocytic leukemia in the AZA-JMML-001 trial.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for most children with juvenile myelomonocytic leukemia (JMML). Novel therapies controlling the disorder prior to HSCT are needed. We conducted a phase 2, multicenter, open-label study to evaluate the safety and antileukemic activity of azacitidine monotherapy prior to HSCT in newly diagnosed JMML patients. Eighteen patients enrolled from September 2015 to November 2017 were treated with azacitidine (75 mg/m2) administered IV once daily on days 1 to 7 of a 28-day cycle. The primary end point was the number of patients with clinical complete remission (cCR) or clinical partial remission (cPR) after 3 cycles of therapy. Pharmacokinetics, genome-wide DNA-methylation levels, and variant allele frequencies of leukemia-specific index mutations were also analyzed. Sixteen patients completed 3 cycles and 5 patients completed 6 cycles. After 3 cycles, 11 patients (61%) were in cPR and 7 (39%) had progressive disease. Six of 16 patients (38%) who needed platelet transfusions were transfusion-free after 3 cycles. All 7 patients with intermediate- or low-methylation signatures in genome-wide DNA-methylation studies achieved cPR. Seventeen patients received HSCT; 14 (82%) were leukemia-free at a median follow-up of 23.8 months (range, 7.0-39.3 months) after HSCT. Azacitidine was well tolerated and plasma concentration--time profiles were similar to observed profiles in adults. In conclusion, azacitidine monotherapy is a suitable option for children with newly diagnosed JMML. Although long-term safety and efficacy remain to be fully elucidated in this population, these data demonstrate that azacitidine provides valuable clinical benefit to JMML patients prior to HSCT. This trial was registered at www.clinicaltrials.gov as #NCT02447666.

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.

Loss of the Fanconi anemia-associated protein NIPA causes bone marrow failure.

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.

Myelodysplastic syndrome in children and adolescents.

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and subsequent frequent development of acute myeloid leukemia (AML). In children and adolescents, MDS are uncommon disorders, accounting for less than 5% of hematopoietic malignancy, with great heterogeneity in presentation and clinical course. The genetic changes predisposing children to MDS are largely obscure. Monosomy 7 is the most common chromosomal abnormality, often occurring as a sole abnormality. The recent pediatric modification of the World Health Organization (WHO) classification has greatly facilitated the diagnostic process. Refractory cytopenia (RC) is the most common MDS subtype in children, occurring in about half of all MDS cases. There is consensus that the relationship between MDS with increased blast count and de novo AML is better defined by biological and clinical features than by blast count. Because monosomy 7 is the only chromosomal abnormality strongly suggestive of MDS, children presenting with a low blast count and other chromosomal aberrations or normal karyotype must be closely observed before a diagnosis of MDS can be established. With an increasing number of children surviving primary cancer with chemotherapy or radiation therapy, the incidence of secondary therapy-related MDS is rising. The MDS risk is also increased in patients with inherited bone marrow failure disorders; this relationship provides valuable insights into MDS biology. Allogeneic hematopoietic stem cell transplantation (HSCT) from a matched related or suitable unrelated donor is the choice for most children with MDS and can rescue a large proportion of patients.

Diagnosis and classification of myelodysplastic syndrome: International Working Group on Morphology of myelodysplastic syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts.

The classification of myelodysplastic syndromes is based on the morphological criteria proposed by the French-American-British (FAB) and World Health Organization (WHO) groups. Accurate enumeration of blast cells, although essential for diagnosis of myelodysplastic syndrome and for assignment to prognostic groups, is often difficult, due to imprecise criteria for the morphological definition of blasts and promyelocytes. An International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) of hematopathologists and hematologists expert in the field of myelodysplastic syndrome reviewed the morphological features of bone marrows from all subtypes of myelodysplastic syndrome and agreed on a set of recommendations, including recommendations for the definition and enumeration of blast cells and ring sideroblasts. It is recommended that (1) agranular or granular blast cells be defined (replacing the previous type I, II and III blasts), (2) dysplastic promyelocytes be distinguished from cytologically normal promyelocytes and from granular blast cells, (3) sufficient cells be counted to give a precise blast percentage, particularly at thresholds that are important for diagnosis or prognosis and (4) ring sideroblasts be defined as erythroblasts in which there are a minimum of 5 siderotic granules covering at least a third of the nuclear circumference. Clear definitions and a differential count of a sufficient number of cells is likely to improve precision in the diagnosis and classification of myelodysplastic syndrome. Recommendations should be applied in the context of the WHO classification.

Immunosuppressive therapy with anti-thymocyte globulin and cyclosporine A in selected children with hypoplastic refractory cytopenia.

It is currently unknown whether immunosuppressive therapy or hematopoietic stem cell transplantation is the most appropriate treatment strategy for children with refractory cytopenia and normal karyotype or trisomy 8. We report on 31 children with hypoplastic refractory cytopenia treated with immunosuppressive therapy consisting of antithymocyte globulin and cyclosporine. At 6 months, 22 of 29 evaluable patients had a complete or partial response; a total of ten patients achieved a complete response at varying time points. Six patients subsequently received a transplant because of non-response, progression to advanced myelodysplastic syndrome or evolution of monosomy 7. Overall and failure-free survival rates at 3 years were 88% and 57%, respectively.

TERC mutations in children with refractory cytopenia.

Mutations in the human telomerase RNA gene (TERC) cause autosomal dominant dyskeratosis congenita and have been detected in individuals with bone marrow failure. Here, we screened for TERC mutations in a cohort of 80 children with hypocellular myelodysplastic syndrome and detected TERC alterations in two of them.