Encephalitozoon hellem infection after haploidentical allogeneic hematopoietic stem cell transplantation in children: a case report.

Background: Encephalitozoon hellem (E. hellem) infection is a zoonotic disease, rarely observed in individuals, causing various clinical manifestations including diarrhea, keratoconjunctivitis, cystitis, etc. E. hellem infection after hematopoietic stem-cell transplantation (HSCT) is a rare, serious complication. Case presentation: Herein, we present a case of E. hellem infection developing during HLA-haploidentical HSCT in a 9-year-old boy who suffered from aplastic anemia. On 15 days after HSCT, the patient developed recurrent and prolonged fever, diarrhea and hematuria. It is challenging to differentiate whether the symptoms mentioned in this case are caused by graft-versus-host disease (GVHD) or a specific infection. Based on the result of metagenomic next-generation sequencing (mNGS) and clinical observation, the patient was diagnosed as E. hellem infection, and received albendazole and decreased the immunosuppressive treatment. Finally, he had recovered. Conclusion: We should pay attention to the uncommon disease caused by the E. hellem infection after HSCT, especially in cases with immune reconstitution unrecovered. Among those rare infection, mNGS can be performed for better understanding the source of infection and targeted therapy, which can benefit the patients.

Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

Direct identification of pathogens via microbial cellular DNA in whole blood by MeltArray.

Rapid identification of pathogens is critical for early and appropriate treatment of bloodstream infections. The various culture-independent assays that have been developed often have long turnaround times, low sensitivity and narrow pathogen coverage. Here, we propose a new multiplex PCR assay, MeltArray, which uses intact microbial cells as the source of genomic DNA (gDNA). The successive steps of the MeltArray assay, including selective lysis of human cells, microbial cell sedimentation, microbial cellular DNA extraction, target-specific pre-amplification and multiplex PCR detection, allowed the detection of 35 major bloodstream infectious pathogens in whole blood within 5.5 h. The limits of detection varied depending on the pathogen and ranged from 1 to 5 CFU/mL. Of 443 blood culture samples, including 373 positive blood culture samples and 70 negative blood culture samples, the MeltArray assay showed a sensitivity of 93.8% (350/373, 95% confidence interval [CI] = 90.7%-96.0%), specificity of 98.6% (69/70, 95% CI = 91.2%-99.9%), positive predictive value of 99.7% (95% CI = 98.1%-99.9%), and negative predictive value of 75.0% (95% CI = 64.7%-83.2%). The MeltArray detection results of 16 samples differed from MALDI-TOF and were confirmed by Sanger sequencing. Further testing of 110 whole blood samples from patients with suspected bloodstream infections using blood culture results revealed that the MeltArray assay had a clinical sensitivity of 100% (9/9, 95% CI = 62.8%-100.0%), clinical specificity of 74.5% (70/94, 95% CI = 64.2%-82.7%), positive predictive value of 27.3% (95% CI = 13.9%-45.8%), and negative predictive value of 100.0% (95% CI = 93.5%-100.0%). Compared with metagenomic next-generation sequencing, the MeltArray assay displayed a positive agreement of 85.7% (6/7, 95% CI = 42.0%-99.2%) and negative agreement of 100.0% (4/4, 95% CI = 39.6%-100.0%). We conclude that the MeltArray assay can be used as a rapid and reliable tool for direct identification of pathogens in bloodstream infections.

Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.

Genotype-guided model significantly improves accuracy of tacrolimus initial dosing after liver transplantation.

Background: The initial dose of tacrolimus after liver transplantation (LT) is critical for rapidly achieving the steady state of the drug concentration, minimizing the potential adverse reactions and warranting long-term patient prognosis. We aimed to develop and validate a genotype-guided model for determining personalized initial dose of tacrolimus. Methods: By combining pharmacokinetic modeling, pharmacogenomic analysis and multiple statistical methods, we developed a genotype-guided model to predict individualized tacrolimus initial dose after LT in the discovery (n = 150) and validation cohorts (n = 97) respectively. This model was further validated in a prospective, randomized and single-blind clinical trial from August, 2021 to February, 2022 (n = 40, ChiCTR2100050288). Findings: Our model included donor's and recipient's genotypes, recipient's weight and total bilirubin, which achieved an area under the curve of receiver operating characteristic curve (AUC of ROC) of 0.88 and 0.79 in the discovery and validation cohorts, respectively. We found that patients who were given tacrolimus within the recommended concentration range (RCR) (4-10 ng/mL), the new-onset metabolic syndromes are lower, especially for new-onset diabetes (p = 0.043). In the clinical trial, compared to those in experience-based (EB) group, patients in the model-based (MB) group were more likely to achieving the RCR (75% vs 40%, p = 0.025) with a more variable individualized dose (0.023-0.096 mg/kg/day vs 0.045-0.057 mg/kg/day). Moreover, significantly fewer medication adjustments were required for the MB group than the EB group (2.75 ± 2.01 vs 6.05 ± 3.35, p = 0.001). Interpretation: Our genotype-based model significantly improved the initial dosing accuracy of tacrolimus and reduced the number of medication adjustments, which are critical for improving the prognosis of LT patients. Funding: National Natural Science Foundation of China, Shanghai three-year action plan, National Science and Technology Major Project of China.

Phenotypic and genotypic correlation evaluation of 148 pediatric patients with Fanconi anemia in a Chinese rare disease cohort.

BACKGROUND: Fanconi anemia (FA) is a rare autosomal recessive, X-linked or autosomal dominant disease. Few large-scale FA investigations of rare disease cohorts have been conducted in China. METHODS: We enrolled 148 patients diagnosed with FA according to evidence from the clinical phenotype, family history, and a set of laboratory tests. Next, the clinical manifestations and correlation between the genotype and phenotype of FA pediatric cases were investigated. RESULTS: The most common FA subtype in our cohort was FA-A (51.4 %), followed by FA-D2 and FA-P. Finger (26 %) and skin (25 %) deformities were the most common malformations. Based on family history, blood system diseases (51 %) had the highest incidence rate, followed by digestive system tumours. A set of new or prognosis-related mutation sites was identified. For example, c.2941 T > G was a new most common missense mutation site for FANCA. FANCP gene mutation sites were mainly concentrated in exons 12/14/15. The mutations of FANCI/FANCD2 were mainly located at the α helix and ß corners of the protein complex. FA-A/D1 patients with splicing or deletion mutations showed more severe disease than those with missense mutations. Chromosome 1/3/7/8 abnormalities were closely linked to the progression of FA to leukemia. CONCLUSION: Our study investigated the clinical features and genotype/phenotype correlation of 148 Chinese pediatric FA patients, providing new insight into FA.

Genetic factors underlying tacrolimus intolerance after liver transplantation.

Background: Tacrolimus (FK506) is the cornerstone of immunosuppression after liver transplantation (LT), however, clinically, switching from FK506 to cyclosporine (SFTC) is common in LT patients with tacrolimus intolerance. The aim of this study was to investigate the genetic risk of patients with tacrolimus intolerance. Methods: A total of 114 LT patients were enrolled in this retrospective study. SNPs were genotyped using Infinium Human Exome-12 v1.2 BeadChip, and genome-wide gene expression levels were profiled using Agilent G4112F array. Results: SFTC was a potential risk factor of dyslipidemia (OR=4.774[1.122-20.311], p = 0.034) and insulin resistance (IR) (OR=6.25[1.451-26.916], p = 0.014), but did not affect the survival of LT patients. Differential expression analysis showed donor CYP3A5, CYP2C9, CFTR, and GSTP1, four important pharmacogenetic genes were significantly up-regulated in the tacrolimus intolerance group. Twelve SNPs of these four genes were screened to investigate the effects on tacrolimus intolerance. Regression analysis showed donor rs4646450 (OR=3.23 [1.22-8.60] per each A allele, p = 0.01), donor rs6977165 (OR=6.44 [1.09-37.87] per each C allele, p = 0.02), and donor rs776746 (OR=3.31 [1.25-8.81] per each A allele, p = 0.01) were independent risk factors of tacrolimus intolerance. Conclusions: These results suggested that SFTC was a potential risk factor for dyslipidemia and IR after LT. Besides, rs4646450, rs6977165, and rs776746 of CYP3A5 might be the underlying genetic risks of tacrolimus intolerance. This might help transplant surgeons make earlier clinical decisions about the use of immunosuppression.

BMP2/SMAD pathway activation in JAK2/p53-mutant megakaryocyte/erythroid progenitors promotes leukemic transformation.

Leukemic transformation (LT) of myeloproliferative neoplasm (MPN) has a dismal prognosis and is largely fatal. Mutational inactivation of TP53 is the most common somatic event in LT; however, the mechanisms by which TP53 mutations promote LT remain unresolved. Using an allelic series of mouse models of Jak2/Trp53 mutant MPN, we identify that only biallelic inactivation of Trp53 results in LT (to a pure erythroleukemia [PEL]). This PEL arises from the megakaryocyte-erythroid progenitor population. Importantly, the bone morphogenetic protein 2/SMAD pathway is aberrantly activated during LT and results in abnormal self-renewal of megakaryocyte-erythroid progenitors. Finally, we identify that Jak2/Trp53 mutant PEL is characterized by recurrent copy number alterations and DNA damage. Using a synthetic lethality strategy, by targeting active DNA repair pathways, we show that this PEL is highly sensitive to combination WEE1 and poly(ADP-ribose) polymerase inhibition. These observations yield new mechanistic insights into the process of p53 mutant LT and offer new, clinically translatable therapeutic approaches.

Immune microenvironment infiltration landscape and immune-related subtypes in prostate cancer.

Background: The tumor microenvironment (TME) primarily comprises cancer cells, cancer-infiltrating immune cells, and stromal cells. The tumor cells alter the TME by secreting signaling molecules to induce immune tolerance. The immune cell infiltrating the TME influences the prognosis of patients with cancers. However, immune cell infiltration (ICI) in the TME of patients with prostate cancer (PC) has not yet been studied. Methods: In this study, we used Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) and Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) algorithms to identify three ICI clusters based on 1,099 genes associated with ICI in the TME. The patients were classified into three distinct ICI gene clusters based on overlapping differentially expressed genes in ICI clusters. Furthermore, the ICI scores were calculated using principal component analysis. Results: The results revealed that patients with high ICI scores had poor prognoses and reduced expression of immune-checkpoint genes and immune-related genes. Furthermore, the transforming growth factor-beta (TGF-ß) and WNT-ß signaling pathways were enriched in the high ICI score subgroup, which suggests that suppression of T cells could contribute to poor prognosis of patients with PC. A positive correlation was observed between the high-ICI-score subgroup and the high tumor mutation burden (TMB) value. Patients with low ICI scores could benefit from immunotherapy, indicating that the ICI score could be used to predict the efficacy of immunotherapeutic response. Conclusions: In summary, we provide a comprehensive overview of the landscape of ICI in PC, which could aid in designing the strategies for immunotherapy for patients with PC.

MSI2 deficiency in ILC3s attenuates DSS-induced colitis by affecting the intestinal microbiota.

Background: The etiology and pathogenesis of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are generally believed to be related to immune dysfunction and intestinal microbiota disorder. However, the exact mechanism is not yet fully understood. The pathological changes associated with dextran sodium sulfate (DSS)-induced colitis are similar to those in human UC. As a subgroup of the innate immune system, group 3 innate lymphoid cells (ILC3s) are widely distributed in the lamina propria of the intestinal mucosa, and their function can be regulated by a variety of molecules. Musashi2 (MSI2) is a type of evolutionarily conserved RNA-binding protein that maintains the function of various tissue stem cells and is essential for postintestinal epithelial regeneration. The effect of MSI2 deficiency in ILC3s on IBD has not been reported. Thus, mice with conditional MSI2 knockout in ILC3s were used to construct a DSS-induced colitis model and explore its effects on the pathogenesis of IBD and the species, quantity and function of the intestinal microbiota. Methods: Msi2flox/flox mice (Msi2fl/fl ) and Msi2flox/floxRorcCre mice (Msi2ΔRorc ) were induced by DSS to establish the IBD model. The severity of colitis was evaluated by five measurements: body weight percentage, disease activity index, colon shortening degree, histopathological score and routine blood examination. The species, quantity and function of the intestinal microbiota were characterized by high-throughput 16S rRNA gene sequencing of DNA extracted from fecal samples. Results: MSI2 was knocked out in the ILC3s of Msi2ΔRorc mice. The Msi2ΔRorc mice exhibited reductions in body weight loss, the disease activity index, degree of colon shortening, tissue histopathological score and immune cells in the peripheral blood compared to those of Msi2fl/fl mice after DSS administration. The 16S rRNA sequencing results showed that the diversity of the intestinal microbiota in DSS-treated Msi2ΔRorc mice changed, with the abundance of Firmicutes increasing and that of Bacteroidetes decreasing. The linear discriminant analysis effect size (LEfSe) approach revealed that Lactobacillaceae could be the key bacteria in the Msi2ΔRorc mouse during the improvement of colitis. Using PICRUST2 to predict the function of the intestinal microbiota, it was found that the functions of differential bacteria inferred by modeling were mainly enriched in infectious diseases, immune system and metabolic functions. Conclusions: MSI2 deficiency in ILC3s attenuated DSS-induced colonic inflammation in mice and affected intestinal microbiota diversity, composition, and function, with Lactobacillaceae belonging to the phylum Firmicutes possibly representing the key bacteria. This finding could contribute to our understanding of the pathogenesis of IBD and provide new insights for its clinical diagnosis and treatment.

Therapeutic Efficacy of Combined JAK1/2, Pan-PIM, and CDK4/6 Inhibition in Myeloproliferative Neoplasms.

PURPOSE: The JAK1/2 inhibitor ruxolitinib has demonstrated significant benefits for patients with myeloproliferative neoplasms (MPN). However, patients often lose response to ruxolitinib or suffer disease progression despite therapy with ruxolitinib. These observations have prompted efforts to devise treatment strategies to improve therapeutic efficacy in combination with ruxolitinib therapy. Activation of JAK-STAT signaling results in dysregulation of key downstream pathways, notably increased expression of cell-cycle mediators including CDC25A and the PIM kinases. EXPERIMENTAL DESIGN: Given the involvement of cell-cycle mediators in MPNs, we sought to examine the efficacy of therapy combining ruxolitinib with a CDK4/6 inhibitor (LEE011) and a PIM kinase inhibitor (PIM447). We utilized JAK2-mutant cell lines, murine models, and primary MPN patient samples for these studies. RESULTS: Exposure of JAK2-mutant cell lines to the triple combination of ruxolitinib, LEE011, and PIM447 resulted in expected on-target pharmacodynamic effects, as well as increased apoptosis and a decrease in the proportion of cells in S-phase, compared with ruxolitinib. As compared with ruxolitinib monotherapy, combination therapy led to reductions in spleen and liver size, reduction of bone marrow reticulin fibrosis, improved overall survival, and elimination of disease-initiating capacity of treated bone marrow, in murine models of MPN. Finally, the triple combination reduced colony formation capacity of primary MPN patient samples to a greater extent than ruxolitinib. CONCLUSIONS: The triple combination of ruxolitinib, LEE011, and PIM447 represents a promising therapeutic strategy with the potential to increase therapeutic responses in patients with MPN.

Sfxn1 is essential for erythrocyte maturation via facilitating hemoglobin production in zebrafish.

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one­carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.

DNAH2 facilitates the homologous recombination repair of Fanconi anemia pathway through modulating FANCD2 ubiquitination.

Fanconi anemia (FA), an X-linked genetic or autosomal recessive disease, exhibits complicated pathogenesis. Previously, we detected the mutated Dynein Axonemal Heavy Chain 2 (DNAH2) gene in 2 FA cases. Herein, we further investigated the potential association between DNAH2 and the homologous recombination repair pathway of FA. The assays of homologous recombination repair, mitomycin C (MMC) sensitivity, immunofluorescence, and ubiquitination modification were performed in U2OS and DR-U2OS cell lines. In MMC-treated U2OS cells, the downregulation of the DNAH2 gene increased the sensitivity of cells to DNA inter-strand crosslinks. We also observed the reduced enrichment of FANCD2 protein to DNA damage sites. Furthermore, the ubiquitination modification level of FANCD2 was influenced by the deficiency of DNAH2. Thus, our results suggest that DNAH2 may modulate the cell homologous recombination repair partially by increasing the ubiquitination and the enrichment to DNA damage sites of FANCD2. DNAH2 may act as a novel co-pathogenic gene of FA patients.

[The Correlation of Minimal Residual Disease with Prognosis in TCF3-PBX1+ Acute Lymphoblastic Leukemia in Children].

OBJECTIVE: To investigate the consistency between FCM and PCR on the detecting of MRD in TCF3-PBX1+ ALL, and to investigate the prognosis value of these 2 methods. METHODS: 55 cases of paediatric TCF3-PBX1+ ALL patients from April 2008 to April 2015 were enrolled and analyzed. The FCM and PCR was used to detect the MRD in 239 bone marrow samples of 55 patients. All statistical analyses were carried out by using SPSS software version 16. RESULTS: Among the 55 children with TCF3-PBX1+ ALL, there were 30 male and 25 female. The median age was 5 (1-14) years. 20 patients relapsed during follow-up. The MRD results from PCR and FCM showed a strong correlation between both methods (K=0.774, P<0.001). There was no significant difference in 5-years DFS and OS between the patients in PCR+ and PCR- groups on day 15 or day 33. The 5 year DFS rate between the patients in FCM- and FCM+ was 63.9%±7.0% and 0; the 5 year OS rate was 66.5%±7.9% and 0. Combined with the result of FCM and PCR, at the d 33 of treatment, the 5-year DFS rate in FCM-/PCR- and single positive group was 65.4%±7.2% and 25.0%±15.3% (P<0.01). CONCLUSION: The detection result of MRD in TCF3-PBX1 detect by FCM and PCR shows better consistency. MRD positivity detected by FCM at the end of induction therapy (day 33) predicts a high risk of relapse in TCF3-PBX1 ALL patients.

[Clinical features and gene mutations of children with Shwachman-Diamond syndrome and malignant myeloid transformation].

OBJECTIVE: To study the clinical features and genetic mutations of children with Shwachman-Diamond syndrome (SDS) and malignant myeloid transformation. METHODS: Next-generation sequencing was used to analyze the gene mutations in 11 SDS children with malignant myeloid transformation, and their clinical features and genetic mutations were analyzed. RESULTS: Of the 11 children with SDS, 9 (82%) presented with refractory cytopenia of childhood (RCC), 1 (9%) had myelodysplastic syndrome with excess blasts (MDS-EB), and 1 (9%) had acute myeloid leukemia with myelodysplasia-related changes (AML-MRC). The median age of onset of malignant myeloid transformation was 48 months (ranged 7 months to 14 years). Of the 11 children, 45% had abnormalities in the hematological system alone. Mutations of the SBDS gene were detected in all 11 children, among whom 5 (45%) had c.258+2T>C homozygous mutation and 3 (27%) had c.184A>T+c.258+2T>C compound heterozygous mutation. The new mutations of the SBDS gene, c.634_635insAACATACCTGT+c.637_638delGA and c.8T>C, were rated as "pathogenic" and "possibly pathogenic" respectively. The 3-year predicted overall survival rates of children transformed to RCC and MDS-EB/AML-MRC were 100% and 0% respectively (P=0.001). CONCLUSIONS: SDS children may have hematological system symptoms as the only manifestation, which needs to be taken seriously in clinical practice. The type of malignant transformation is associated with prognosis.

[Clinical features and gene mutation spectrum in children with sideroblastic anemia].

OBJECTIVE: To study the clinical features and gene mutation spectrum of children with sideroblastic anemia (SA) and the clinical value of targeted next-generation sequencing in the molecular diagnosis of children with SA. METHODS: Clinical data were collected from 36 children with SA. Targeted next-generation sequencing was used to detect mutations in SA-related pathogenic genes and genes associated with heme synthesis and mitochondrial iron metabolism. The association between genotype and clinical phenotype was analyzed. RESULTS: Of the 36 patients, 32 had congenital sideroblastic anemia (CSA) and 4 had myelodysplastic syndrome with ring sideroblasts (MDS-RS). Mutations in CSA-related genes were detected in 19 children (19/36, 53%), among whom 9 (47%) had ALAS2 mutation, 4 (21%) had SLC25A38 mutation, and 6 (32%) had mitochondrial fragment deletion. No pathogenic gene mutation was detected in 4 children with MDS-RS. Among the 19 mutations, 89% (17/19) were known mutations and 11% (2/19) were novel mutations. The novel mutation of the ALAS2 gene c.1153A>T(p.I385F) was rated as "possibly pathogenic" and the novel mutation of the SLC25A38 gene c.175C>T(p.Q59X) was rated as "pathogenic". CONCLUSIONS: ALAS2 and SLC25A38 gene mutations are commonly seen in children with CSA, but mitochondrial gene fragment deletion also accounts for a relatively high proportion. For children with hypoplastic anemia occurring in infancy, mitochondrial disease should be considered.

[Clinical features of Wiskott-Aldrich syndrome: an analysis of 13 cases].

OBJECTIVE: To study the clinical features of Wiskott-Aldrich syndrome (WAS) in children. METHODS: A retrospective analysis was performed for the clinical data of 13 children with WAS. RESULTS: All 13 children were boys, with a median age of onset of 3 months (range 1-48 months) and a median age of 24 months (range 1-60 months) at the time of diagnosis. Of the 13 children, only 3 had typical WAS and the remaining 10 children had X-linked thrombocytopenia (XLT). The mean WAS score was 2 (range 1-3), the mean platelet count was 20.5×109/L [range (13-46)×109/L], and the mean platelet volume was 8.1 fl (range 6.7-12.1 fl). Lymphocyte subsets and immunoglobulins were measured for 4 children, among whom 1 (25%) had a reduction in both the percentage of CD3+T cells per lymphocyte and lymphocyte per nuclear cells, 1(25%) had a reduction in CD3-CD56+ NK cells. Among these 4 children, 1 (25%) had an increase in IgG, 2 (50%) had a reduction in IgM, 1 (25%) had a reduction in IgA, and 4 (100%) had an increase in IgE. A total of 14 gene mutations belonging to 13 types were found in 13 children, among which there were 9 missense mutations (65%), 2 splicing mutations (14%), 2 nonsense mutation (14%), and 1 frameshift mutation (7%). The median follow-up time was 39 months (range 3-62 months), and all 13 children survived. CONCLUSIONS: Children with WAS often have a young age of onset, and most of them are boys. Major clinical features include thrombocytopenia with a reduction in platelet volume. Missense mutation is the main type of gene mutation.

[Value of multiparameter flow cytometry in the diagnosis and prognostic evaluation of childhood myelodysplastic syndrome].

OBJECTIVE: To investigate the value of multiparameter flow cytometry (MFC) and flow cytometric scoring system (FCSS) in the diagnosis and prognostic evaluation of childhood myelodysplastic syndrome (MDS). METHODS: A retrospective analysis was performed for the clinical data of 42 children who were diagnosed with MDS. MFC was performed to investigate the phenotype and proportion of each lineage of bone marrow cells. The correlations of FCSS score with MDS type, International Prognostic Scoring System (IPSS) score, and revised IPSS (IPSS-R) score were analyzed. RESULTS: Of all the 42 children, 20 (48%) had an increase in abnormal marrow blasts, 19 (45%) had a lymphoid/myeloid ratio of >1, 14 (33%) had abnormal cross-lineage expression of lymphoid antigens in myeloid cells, 8 (19%) had abnormal CD13/CD16 differentiation antigens, 5 (12%) had abnormal expression of CD56, 3 (7%) had reduced or increased side scatter of granulocytes, 3 (7%) had reduced expression of CD36 in nucleated red blood cells, 2 (5%) had reduced expression of CD71 in nucleated red blood cells, 1 (2%) had absent expression of CD33 in myeloid cells, 1 (2%) had reduced or absent expression of CD11b in granulocytes, and 1 (2%) had absent expression of CD56 and CD14 in monocytes. There were significant differences in the median overall survival time and event-free survival time among the low-, medium-, and high-risk FCSS groups (P<0.05). Among the low-, medium-, and high-risk FCSS groups, the low-risk FCSS group had the highest 2-year overall survival rate, while there was no significant difference between the medium- and high-risk FCSS groups (P>0.05). The three groups had a 2-year event-free survival rate of 95%, 60%, and 46% respectively (P<0.05). FCSS score was positively correlated with MDS type, IPSS score, and IPSS-R score (P<0.05). CONCLUSIONS: MFC and FCSS help with the diagnosis and prognostic evaluation of childhood MDS.

Role of cytarabine in paediatric acute promyelocytic leukemia treated with the combination of all-trans retinoic acid and arsenic trioxide: a randomized controlled trial.

BACKGROUND: The combination of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) has been suggested to be safe and effective for adult acute promyelocytic leukaemia (APL). As of 2010, the role of cytarabine (Ara-C) in APL was controversial. The aim of this study was to test the efficacy and safety of ATRA and ATO in paediatric APL patients. Also, we assessed whether Ara-C could be omitted in ATO and ATRA- based trials in children. METHODS: We performed a randomized controlled trial in paediatric APL patients (≤14 years of age) in our hospital from May 2010 to December 2016. All of the patients were assigned to receive ATRA plus ATO for induction followed by one course of idarubicin (IDA) and ATO (28 days). The patients were then randomly assigned to receive two courses of daunorubicin (DNR, no- Ara-C group) or DNR + Ara-C (Ara-C group). All of the patients were followed with maintenance therapy with oral ATRA, 6-mercaptopurine, and methotrexate for 1.5 years. RESULTS: Among the 66 patients, 43 were male and 23 were female. All of the patients achieved complete remission (CR) with the exception of one who gave up the treatment. During induction therapy, all toxicity events were reversed after appropriate management. Thirty patients in the Ara-C group underwent 57 courses of treatment, and 35 patients in the no-Ara-C group underwent 73 courses of treatment. No significant differences in age, genders, white blood cell counts, haemoglobin levels, and platelet counts were found between the Ara-C and no-Ara-c groups. Greater myelosuppression and sepsis were observed in the Ara-C group during the consolidation courses. No patient died at consolidation, and only one patient relapsed. No differences were found in event-free survival, disease-free survival and overall survival between the two groups. Additionally, our analysis of the arsenic levels in the plasma, urine, hair and nails of the patients indicated that no significant accumulation of arsenic occurred after ATO was discontinued for 12 months. CONCLUSIONS: Overall, ATO and ATRA are safe and effective for paediatric APL patients and Ara-C could be omitted when ATO is used for two courses. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT01191541 , retrospectively registered on 18 August 2010).

[Clinical characteristics of clonal evolution after immunosuppressive therapy in children with severe/very severe aplastic anemia].

OBJECTIVE: To evaluate the clinical characteristics and risk factors of clonal evolution after immunosuppressive therapy (IST) in children with severe/very severe aplastic anemia (SAA/VSAA). METHODS: The clinical data of 231 children with newly-diagnosed SAA/VSAA who received IST were retrospectively studied. The incidence and risk factors of clonal evolution after IST were analyzed. RESULTS: The 5-year overall survival rate of the 231 patients was 82.7%. Except for 18 cases of early deaths, 213 patients were evaluated for IST efficacy. Among the 231 patients, cytogenetic abnormalities for at least two chromosome metaphase were detectable in 14 (7.4%) patients, and PNH clones were detectable in either peripheral red blood cells or neutrophils for 95 patients. Among the 213 patients evaluated for IST efficacy, 15 patients experienced clonal evolution after IST. Five patients had PNH and trisomy 8 which were defined as favorable progressions, and ten patients experienced monosomy 7 and MDS/AML as unfavorable progressions. The 5-year accumulative incidence of favorable and unfavorable progression were (2.2±2.2)% and (4.8±3.3)%, respectively. Until the last follow-up, 100% (5/5) of patients with favorable progressions and 50% (5/10) of patients with unfavorable progressions survived. WBC>3.5×109/L, CD3+T cell percentage>80%, dosage of antithymocyte globulin >3.0 mg/(kg·d) and no response to IST were related to unfavorable progressions by univariate analysis. Cox multivariate analysis revealed that an increased CD3+T cell percentage (>80%) and no response to IST were independent risk factors for unfavorable progressions. CONCLUSIONS: The children with SAA/VSAA who have an increased CD3+T cell percentage at diagnosis or have no response to IST are in high risks of unfavorable progressions.