Prospective validation of the provisional entity of refractory cytopenia of childhood, proposed by the World Health Organization.

In 2008, the World Health Organization proposed a new entity of childhood myelodysplastic syndrome (MDS), which was referred to as refractory cytopenia of childhood (RCC). However, whether this morphological classification reflects clinical outcomes remains unclear. We performed a prospective evaluation of bone marrow morphology in 252 children with acquired bone marrow failure between 2009 and 2013. Of 252 patients, 63 were diagnosed with aplastic anaemia (AA), 131 with RCC without multilineage dysplasia (RCC-w/o-MLD) and 58 with RCC with MLD (RCC-MLD). One patient with AA, three with RCC-w/o-MLD and nine with RCC-MLD presented with chromosomal abnormalities at diagnosis (P = 0·001). The response rates to immunosuppressive therapy (IST) at 6 months and the cumulative incidence of clonal evolution at 5 years did not significantly differ among the three groups. A multivariate analysis revealed that the morphological classification of RCC-MLD was a significant risk factor for secondary graft failure after haematopoietic cell transplantation (HCT) (P = 0·003). In view of these findings, RCC could be divided into two categories, RCC-w/o-MLD and RCC-MLD, because children with this condition exhibited a distinct morphology, frequent chromosomal abnormalities at diagnosis and a high frequency of secondary graft failure after HCT.

[Morphological diagnosis of childhood bone marrow failure syndromes].

In 2008, the World Health Organization proposed a provisional entity of childhood myelodysplastic syndrome (MDS) without a blasts increase, which was referred to as the refractory cytopenia of childhood (RCC). We performed a central review of bone marrow morphology in 252 children with acquired bone marrow failure syndromes to clarify the clinical relevance of the RCC. The RCC was divided two categories, namely, RCC without multilineage dysplasia (MLD) and RCC with MLD, which is similar to MDS with MLD in adult MDS. Furthermore, the clinical outcomes were investigated for cases diagnosed with aplastic anemia, RCC without MLD, and RCC with MLD. The response rates to immunosuppressive therapy and the incidence of the development of the new chromosomal aberration did not significantly differ among the three groups. The RCC with MLD can be adopted in childhood MDS since children with this condition exhibited a frequent chromosomal aberration at the time of diagnosis and a high frequency of secondary graft failure after a hematopoietic cell transplantation.

[Diagnosis and treatment of childhood myelodysplastic syndrome].

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic disorders characterized by peripheral cytopenia and morphological abnormalities in hematopoietic cells, i.e., myelodysplasia. Aging-related somatic variants acquired in the hematopoietic cells are associated with MDS pathogenesis in adults. However, pediatric MDS often occurs because of germline predispositions. Myelodysplasia can be observed in not only MDS but also other hematopoietic and non-hematopoietic disorders, such as infections and primary immunodeficiencies. Therefore, careful differential diagnosis between MDS and other diseases is necessary. The bone marrow histopathology should be evaluated for accurate differentiation of MDS without excess blasts from aplastic anemia and MDS with excess blasts from acute myeloid leukemia. The treatment strategy for childhood MDS differs based on disease subtypes. The clinical courses of pediatric MDS without excess blasts are heterogeneous; therefore, it is crucial to assess the prognostic values of clinical and cytogenetic findings. In contrast, allogeneic hematopoietic cell transplantation should be considered as the only curative option for pediatric MDS with excess blasts.

Clinical impact of dysplastic changes in acquired aplastic anemia: A systematic study of bone marrow biopsies in children and adults.

Aplastic anemia (AA) is a rare disorder characterized by suppression of bone marrow function, which can progress to myelodysplastic syndrome (MDS) or to acute myeloid leukemia (AML). To determine if there are characteristics in bone marrow biopsies in children and adults previously diagnosed with acquired AA, which could predict progression to MDS, we evaluated 118 hypocellular bone marrow biopsies from adults (76 patients) and children (42) diagnosed initially with acquired AA previously to any treatment. Histology was reviewed according to a detailed protocol including Bennett and Orazi criteria for hypocellular myelodysplastic syndrome (h-MDS) and Bauman et al. criteria for refractory cytopenia of childhood (RCC). Twelve patients (10.2%; 6 children and 6 adults) progressed to MDS after a median time of 56 months. Criteria described by Bennett and Orazi suggestive of h-MDS in bone marrow biopsies were detected in 16 cases (13.5%; 8 adults and 8 children), and none in patients that progressed to MDS/AML. Twenty adults' biopsies (26.3%) had the histological criteria used for the diagnosis of pediatric RCC, and none showed MDS/AML evolution. Ten children (23.8%) were reclassified morphologically as RCC, and only one progressed to MDS. In this population with acquired aplastic anemia (AAA), no histological/immunohistochemical (H/IHC) bone marrow findings could discriminate patients with higher risk for myeloid clonal progression, which questions the diagnosis of h-MDS/RCC based only on the finding of dysplasia in the cases without increased blasts and/or the characteristic genetic abnormalities.

Differences in the bone marrow histology between childhood myelodysplastic syndrome with multilineage dysplasia and refractory cytopenia of childhood without multilineage dysplasia.

AIMS: Refractory cytopenia of childhood (RCC) is subdivided into myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) and RCC without (w/o) multilineage dysplasia (RCC without MLD). Although RCC is a histomorphological distinct entity, the bone marrow (BM) histology of RCC is not yet characterised in relation to multilineage dysplasia. We investigated the BM histological features of RCC to clarify the characteristics of BM histology of MDS-MLD in childhood compared to RCC without MLD. METHODS AND RESULTS: The BM histology and cytology in 60 RCC patients from the nationwide registry of Japanese Childhood AA-MDS Study Group were reviewed retrospectively. Although a thorough genetic assessment, including GATA2 and/or SAMD9, was not performed, inherited BM failure disorders were excluded by a cytogenetic test, a chromosome fragility test and a telomere length measurement along with careful clinical assessments. Among the 60 patients, 20 (33%) of MDS-MLD and 40 (67%) of RCC w/o MLD were classified according to their BM cytology. We then investigated the BM histological features and compared them between the two groups. The BM cellularity, distribution pattern of haematopoiesis, frequency of left-shifted granulopoiesis, numbers of micromegakaryocytes and p53 immunostaining-positive cells were significantly different between the groups. The BM histology of MDS-MLD in childhood showed higher cellularity, the more common occurrence of diffuse distribution pattern, more frequently left-shifted granulopoiesis and more micromegakaryocytes and p53 immunostaining-positive cells than RCC without MLD. CONCLUSIONS: Our results showed that MDS-MLD in childhood had a characteristic BM histology compared to RCC without MLD. The clinical relevance of MDS-MLD in childhood needs to be evaluated.

Bone marrow failure in childhood: central pathology review of a nationwide registry.

Refractory cytopenia of childhood (RCC) was proposed as a provisional entity in the 2008 WHO classification of myelodysplastic syndromes (MDS). It is defined as a childhood MDS featuring persistent cytopenia without increase blasts in bone marrow (BM) or peripheral blood (PB). Because the majority of RCC cases feature hypocellularity and pancytopenia, it is quite challenging to differentiate RCC from acquired aplastic anemia (AA) and many kinds of inherited bone marrow failure syndromes (IBMFS). Diagnosis of RCC requires BM histology of characteristic features such as isolated erythroid islet with left shift, abnormal localization and micromegakaryocytes. The Japanese Society of Pediatric Hematology/Oncology has opened the central registry review system since 2009 to evaluate childhood cases of bone marrow failure (BMF). It has reviewed cytology and BM pathology of all registered BMF cases, which number more than 1,700. In the evaluation of BMF, BM pathology is important to assess the mechanism of hematopoiesis. Pathological dysplasia should be differentiated from cytological dysplasia. A central review system is important for rare diseases, such as pediatric BMF. Standardization of pathological diagnosis should be established upon consensus findings, descriptions, and diagnostic approaches. In this review, the pathology of pediatric BMF syndromes is summarized.

A Cytokine-Based Diagnostic Program in Pediatric Aplastic Anemia and Hypocellular Refractory Cytopenia of Childhood.

BACKGROUND: Distinguishing hypocellular refractory cytopenia of childhood (RCC) from aplastic anemia (AA) is challenging. Thus far, no studies have compared the cytokine profiles in patients with AA to those with hypocellular RCC. In the present study, we addressed whether thrombopoietin (TPO) and interleukin 17 (IL-17) plasma levels are useful for differentiating between the two diseases. METHODS: We measured the endogenous plasma concentrations of TPO and IL-17 in 29 patients with AA, 34 patients with hypocellular RCC, and 31 healthy controls using sensitive enzyme-linked immunosorbent assays. RESULTS: The TPO and IL-17 plasma levels were significantly lower in patients with hypocellular RCC than in patients with AA (P < 0.001 and P = 0.007, respectively). The multivariate logistic regression analysis identified moderate disease severity, TPO levels of <1,369.8 pg/ml (TPO-low group, n = 32; odds ratio (OR), 13.40; 95% confidence intervals (CI), 3.001-51.254; P < 0.001), and IL-17 levels of <22.2 pg/ml (IL-17-low group, n = 33; OR, 4.11; 95% CI, 1.033-19.404; P = 0.031) as independent factors discriminating hypocellular RCC from AA. Importantly, 25 (78.1%) of 32 patients in the TPO-low group and 25 (75.8%) of 33 patients in the IL-17-low group were diagnosed as having hypocellular RCC. Moreover, 22 (71%) of 31 patients in the TPO-high group and 21 (70%) of 30 patients in the IL-17-high group were diagnosed as having AA. CONCLUSIONS: TPO and IL-17 levels are useful for differentiating hypocellular RCC from AA. Prospective studies are required to confirm our findings.

Hematopoietic stem cell transplantation in children with refractory cytopenia of childhood: single-center experience using high-dose cytarabine containing myeloablative and aplastic anemia oriented reduced-intensity conditioning regimens.

Refractory cytopenia of childhood (RCC) is the most common subtype of myelodysplastic syndrome in children, and the clinical course of RCC is heterogeneous. A certain proportion of RCC patients need allogeneic hematopoietic stem cell transplantation (HSCT); however, data on HSCT outcomes are not abundant, and the optimal intensity of a preparative conditioning regimen remains uncertain. In this study, we evaluated the outcomes of HSCT in 24 patients with RCC. Eleven patients received myeloablative conditioning (MAC) consisting of high-dose cytarabine, cyclophosphamide, and either total body irradiation (TBI) or busulfan. Nine patients (38%) received a reduced-intensity conditioning (RIC) regimen; of these, 7 received low-dose TBI and cyclophosphamide (200 mg/kg) with or without antithymocyte globulin or fludarabine, and 2 patients received low-dose TBI, fludarabine, and melphalan (140 mg/m(2)). The remaining 4 patients had disease progression before HSCT and received the MAC regimen. With a median follow-up of 91 months (range, 6 to 263), the probability of overall survival at 5 years was 81.1% (95% CI, 57.0 to 92.5). The 5-year overall survival for the 15 patients who received MAC was 73.3% (95% CI, 43.6 to 89.1), and all 9 patients with RIC are alive without any events. Further study is needed to evaluate the efficacy of RIC for children with RCC with an expectation for reduction of late effects such as growth retardation and infertility.

Clinical characteristics and treatment outcome in 65 cases with refractory cytopenia of childhood defined according to the WHO 2008 classification.

This study analysed 65 children who were prospectively registered between 1999 and 2008 and fulfilled the World Health Organization 2008 criteria of refractory cytopenia of childhood (RCC). First-line therapy was determined by the treating physicians: 25 patients received immunosuppressive therapy (IST), 12 patients received haematopoietic stem cell transplantation (HSCT) and one patient received intensive chemotherapy. The remaining 27 patients were followed without treatment for more than 2 years (watch and wait; WW). In the WW group, 18 patients had stable disease without further intervention. Thirteen of 29 patients (45%) who ended up receiving IST showed response. The combination of ciclosporin and antithymocyte globulin was not shown to be superior to ciclosporin alone with regard to response rate or survival. Of 28 patients who ended up undergoing HSCT, 17 patients are alive in complete remission, whereas nine patients died mostly due to transplantation-related mortality. The 5-year overall survival for all patients was 82 ± 5%. Eight patients suffered from disease progression. Patients with monosomy 7 or multilineage-dysplasia had a significantly higher incidence of disease progression. This analysis revealed heterogeneity in the clinical course of RCC, varying from those who remained stable for long periods to those who progressed to advanced disease.

Evans syndrome after unrelated bone marrow transplantation for refractory cytopenia of childhood.

Post-transplant ES, which is often resistant to therapies, has seldom been described. This report describes a case of ES after UBMT for RCC. A five-yr-old boy developed RCC with no evidence of monosomy 7. Because no matching family donors were available for SCT and immunosuppressive therapy was ineffective, UBMT was performed when he was six yr old. The conditioning regimen included TAI (3 Gy) and administration of FLU, CY, and rabbit antithymocyte globulin. The recovery of blood cells was good. He displayed grade II acute GVHD involving only the skin. ES developed on day 66, with positive results for Epstein-Barr virus DNA and HHV 6. Cytopenia was resolved with treatment with RTX, GCV, an escalated dose of steroids, high-dose gammaglobulin, and romiplostim. No relapse has occurred since discontinuing steroids on day 177 and romiplostim on day 268. Post-SCT ES after UBMT is rare, and the risk factors and therapies are unclear. Prospective analysis and collection of cases from multiple centers are required for clarification.

Recent advances in the diagnosis and treatment of pediatric acquired aplastic anemia.

Acquired aplastic anemia (AA) in children is a rare bone marrow failure that requires several special considerations for its diagnosis and treatment compared with that in adults. The most common issue is the differential diagnosis with refractory cytopenia of childhood and inherited bone marrow failure syndromes, which is crucial for making decisions on the appropriate treatment for pediatric AA. In addition to detailed morphological evaluation, a comprehensive diagnostic work-up that includes genetic analysis using next-generation sequencing will play an increasingly important role in identifying the underlying etiology of pediatric AA. When discussing treatment strategies for children with acquired AA, the long-term sequelae and level of hematopoietic recovery that affect daily or school life should also be considered, although the overall survival rate has reached 90% after immunosuppressive therapy or hematopoietic cell transplantation (HCT). Recent advances in HCT for pediatric patients with acquired AA have been remarkable, with the successful use of upfront bone marrow transplantation from a matched unrelated donor, unrelated cord blood transplantation or haploidentical HCT as salvage treatment, and fludarabine/melphalan-based conditioning regimens. This review discusses current clinical practices in the diagnosis and treatment of acquired AA in children based on the latest data.

Haploidentical Hematopoietic Stem Cell Transplantation in Pediatric Patients with Acquired Hypocellular Bone Marrow Failure.

Children with acquired hypocellular bone marrow failure of unknown cause (AHBMF) are usually diagnosed either with severe aplastic anemia (SAA) or refractory cytopenia of childhood (RCC). Patients with AHBMF who lack a matched donor and who failed or relapsed after immunosuppressive therapy (IST) need alternative therapies. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) offers a curative treatment for these patients. We report a multicenter Spanish experience with haplo-HSCT in pediatric patients with AHBMF. Eleven pediatric patients (SAA, n = 9; RCC, n = 2) underwent haplo-HSCT with different lymphodepletion strategies. Most patients (10 of 11) had previously failed to respond or relapsed after IST. The conditioning regimen was reduced intensity in SAA and myeloablative in RCC. Patients with SAA received low-dose radiotherapy as part of their conditioning regimen. All patients engrafted. Viral reactivation was common (8 of 11). Acute GVHD grade ≥II was seen in 5 patients. Chronic GVHD was diagnosed in 4 of the long-term survivors. Transplantation-associated microangiopathy was a frequent complication in SAA patients and was related to worse outcome. Two patients died of transplantation-related complications. Overall survival was 81%, with a median follow-up of 36 months. Haplo-HSCT can be a successful salvage curative treatment for pediatric patients with AHBMF, but with significant toxicities that must be addressed. Transplantation-associated microangiopathy was the most critical complication.

Childhood Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) in children is rare, accounting for < 5% of all childhood hematologic malignancies. With the advent of next-generation sequencing, the etiology of many childhood MDS (cMDS) cases has been elucidated with the finding of predisposing germline mutations in one-quarter to one-third of cases; somatic mutations have also been identified, indicating that cMDS is different than adult MDS. Herein, cMDS classification schema, clinical presentation, laboratory values, bone marrow histology, differential diagnostic considerations, and the recent molecular findings of cMDS are described.

Wilms' tumor 1 mRNA expression: a good tool for differentiating between myelodysplastic syndrome and aplastic anemia in children?

Objectives: To evaluate the value of Wilms' tumor 1 mRNA (WT1) expression in the differential diagnosis of childhood myelodysplastic syndrome (MDS) and aplastic anemia (AA). Methods: This study compared WT1 expression levels in children of MDS and AA to evaluate its value in differential diagnosis. Results: WT1 overexpression rate and mean WT1 expression level were significantly higher in MDS compared to AA (P = 0.000 and P = 0.013, respectively). Patients with RCC and normal cytogenetics exhibited significantly greater portion of patients exposing WT1 overexpression, compared to all AA subtypes (P = 0.001, P = 0.000 and P = 0.001, respectively). ROC curve analysis revealed that WT1 expression could differentiate between RCC with normal cytogenetics and non-severe AA. Based on a cut-off value of 1.45%, WT1 expression provided a sensitivity of 23.2% and a specificity of 100%. Discussion: In the present study, WT1 overexpression rate was gradually decreased in RAEB group, RCC group and AA subtypes, and the mean WT1 expression level of the MDS patients was significantly higher than that of the AA group. It is very difficult to differentiate between RCC with normal cytogenetics and NSAA in children. Our results showed significant differences in WT1 overexpression rate between these two groups. When we set the cut-off value as 1.45%, WT1 expression levels could be used to differentiate between cases of RCC with normal cytogenetics and NSAA in children. Conclusion: WT1 expression might be useful for distinguishing between myelodysplastic syndrome and aplastic anemia in children.

The histopathology of bone marrow failure in children.

Bone marrow failure (BMF) is a rare but life-threatening disorder that usually manifests as (pan)cytopenia. BMF can be caused by a variety of diseases, but inherited BMF (IBMF) syndromes are a clinically important cause, especially in children. IBMF syndromes are a heterogeneous group of genetic disorders characterized by BMF, physical abnormalities, and predisposition to malignancy. An accurate diagnosis is critical, as disease-specific management, surveillance, and genetic counselling are required for each patient. The major differential diagnoses of IBMF syndromes are acquired aplastic anemia (AA) and refractory cytopenia of childhood (RCC). These diseases have overlapping features, such as BM hypocellularity and/or dysplastic changes, which make the differential diagnosis challenging. RCC has been defined as a histomorphologically distinct entity. Therefore, understanding the BM histopathology of these diseases is essential for the differential diagnosis. However, the BM histopathological features have not been characterized in detail, as descriptions of BM histopathology are very limited due to the rarity of the diseases. This review provides a detailed description of the BM histopathology in cases of RCC, AA, and the four most common IBMF syndromes: Fanconi anemia (FA), dysketatosis congenita (DC), Diamond-Blackfan anemia (DBA), and Shwachman-Diamond syndrome (SDS). An overview, including the clinical features and diagnosis, is also provided.

The current perspective of low-grade myelodysplastic syndrome in children.

Myelodysplastic syndrome (MDS) without increased blasts, i.e., low-grade MDS, is the most common subtype of pediatric MDS and has characteristics different from adult form of the disease. Although histological findings of bone marrow (BM) biopsies suggest that low-grade MDS is a morphologically distinctive entity, a subset of pediatric low-grade MDS may clinically overlap with aplastic anemia (AA), such as high likelihood of hypocellular marrow and normal karyotype. In addition, children with low-grade MDS are as likely to respond to immunosuppressive therapy as those with AA, which indicates that a part of these disorders might share a common pathogenesis, that is, T cell-mediated inhibition of hematopoiesis. In contrast, a small part of children with low-grade MDS experience disease progression to advanced MDS. Given that the clinical courses of pediatric low-grade MDS are heterogeneous, assessing prognostic values of clinical, morphological, histological and cytogenetic findings is critical. Thus far, monosomy 7 and multilineage dysplasia have been suggested as prognostic factors that could predict disease progression. Treatment strategy will be optimized based on more precise prognostic factors. In the future, molecular findings may also help prognostification in children with hypoplastic BM disorders.

Are micromegakaryocytes specific for refractory cytopenia of childhood (RCC)? A study of 38 pediatric patients with thrombocytopenia unrelated to RCC.

BACKGROUND: Micromegakaryocytes (microMKs) are considered the most reliable dysplastic feature for myelodysplastic syndrome (MDS), particularly refractory cytopenia of childhood (RCC); there is no minimal threshold for the diagnosis of RCC. Since most RCC patients present with thrombocytopenia, the presence of microMKs should raise concern for MDS/RCC. This study attempted to investigate the prevalence of microMKs and associated marrow fibrosis in patients with thrombocytopenia unrelated to MDS/RCC and the need for establishing a threshold for microMKs for the diagnosis of MDS/RCC. DESIGN: Bone marrow biopsies of pediatric patients with thrombocytopenia unrelated to RCC were examined for microMKs and fibrosis by CD61 immunohistochemical and reticulin stains respectively. RESULT: Thirty eight patients (1-18 years old) were included: 33 immune thrombocytopenia (ITP), 3 chronic thrombocytopenia, and 2 inherited macrothrombocytopenia. Fourteen cases (37%) had microMKs; four cases showed increased marrow fibrosis associated with microMKs (two had ITP and two had macrothrombocytopenia). All patients are alive and none developed MDS (follow up: 3months to 4 years). CONCLUSION: MicroMKs can be seen in pediatric patients with thrombocytopenia unrelated to RCC. Hence the mere presence of microMKs is insufficient for the diagnosis of RCC in the pediatric population, and a quantitative threshold needs to be established.

Reversible Cerebral Vasoconstriction Syndrome Promptly Diagnosed with Magnetic Resonance Imaging Including Magnetic Resonance Angiography During Immunosuppressive Therapy in a 16-Year-Old Girl with Refractory Cytopenia of Childhood.

Reversible cerebral vasoconstriction syndrome (RCVS) is a syndrome characterized by severe headache with segmental vasoconstriction of the cerebral arteries that resolves within 12 weeks. A 16-year-old girl with refractory cytopenia of childhood, who was receiving the immunosuppressant cyclosporine, developed severe headache and was diagnosed with RCVS using magnetic resonance imaging, including magnetic resonance angiography (MRA). MRA is a non-invasive and very effective technique for diagnosing RCVS. MRA should be performed at the onset of severe headache during immunosuppressant administration for children with hematological disorders and may prevent sequelae such as posterior reversible encephalopathy syndrome or ischemic attack.

TET2 expression level and 5-hydroxymethylcytosine are decreased in refractory cytopenia of childhood.

Myelodysplastic syndromes (MDS) are myeloid malignancies characterized by ineffective hematopoiesis, dysplasia, peripheral cytopenia and increased risk of progression to acute myeloid leukemia. Refractory cytopenia of childhood (RCC) is the most common subtype of pediatric MDS and has overlapping clinical features with viral infections and autoimmune disorders. Mutations in TET2 gene are found in about 20-25% of adult MDS and are associated with a decrease in 5-hydroxymethylcytosine (5-hmC) content. TET2 deregulation and its malignant potential were reported in adult but not in pediatric MDS. We evaluated the gene expression and the presence of mutations in TET2 gene in 19 patients with RCC. TET2 expression level was correlated with 5-hmC amount in DNA and possible regulatory epigenetic mechanisms. One out of 19 pediatric patients with RCC was a carrier of a TET2 mutation. TET2 expression and 5-hmC levels were decreased in patients when compared to a disease-free group. Lower expression was not associated to the presence of mutation or with the status of promoter methylation, but a significant correlation with microRNA-22 expression was found. These findings suggested that TET2 downregulation and low levels of 5-hmC are inversely related to miR-22 expression. The existence of a regulatory loop between microRNA-22 and TET2 may play a role in MDS pathogenesis.