Yuko Sekiya

Paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia

Acquired aplastic anemia is an immune-mediated disease characterized by severe defects in stem cell number resulting in hypocellular marrow and peripheral blood cytopenias. Minor paroxysmal nocturnal hemoglobinuria populations and a short telomere length were identified as predictive biomarkers of immunosuppressive therapy responsiveness in aplastic anemia. We enrolled 113 aplastic anemia patients (63 boys and 50 girls) in this study to evaluate their response to immunosuppressive therapy. The paroxysmal nocturnal hemoglobinuria populations and telomere length were detected by flow cytometry. Forty-seven patients (42%) carried a minor paroxysmal nocturnal hemoglobinuria population. The median telomere length of aplastic anemia patients was −0.99 standard deviation (SD) (range −4.01–+3.01 SD). Overall, 60 patients (53%) responded to immunosuppressive therapy after six months. Multivariate logistic regression analysis identified the absence of a paroxysmal nocturnal hemoglobinuria population and a shorter telomere length as independent unfavorable predictors of immunosuppressive therapy response at six months. The cohort was stratified into a group of poor prognosis (paroxysmal nocturnal hemoglobinuria negative and shorter telomere length; 37 patients) and good prognosis (paroxysmal nocturnal hemoglobinuria positive and/or longer telomere length; 76 patients), respectively. The response rates of the poor prognosis and good prognosis groups at six months were 19% and 70%, respectively (P<0.001). The combined absence of a minor paroxysmal nocturnal hemoglobinuria population and a short telomere length is an efficient predictor of poor immunosuppressive therapy response, which should be considered while deciding treatment options: immunosuppressive therapy or first-line hematopoietic stem cell transplantation. The trial was registered in www.umin.ac.jp with number UMIN000017972.

Recurrent MYB rearrangement in blastic plasmacytoid dendritic cell neoplasm

Lymphoproliferative Disorders Team, Centre de Recherche des Cordeliers, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France; Service d’Hématologie Biologique, GH Pitié-Salpêtrière, Paris, France; Service de Biostatistique et Informatique Médicale, Hôpital Saint Louis, Paris, France.; Département de génétique, GH Pitié-Salpêtrière, Paris, France; INSERM U1170, Institut Gustave Roussy, Villejuif, France; Département d’Hématologie, Hôpital Becquerel, Rouen, France; Pôle d’Hématologie, Hôpital Brabois, Vandoeuvre-les-Nancy, France; Service d’Hématologie Clinique, Hôpital d’Argenteuil, Argenteuil, France and Karyopharm Therapeutics, Newton, MA, USA E-mail: santos.susin@crc.jussieu.fr or florence.nguyen-khac@psl.aphp.fr These authors contributed equally to this work. Senior coauthorship.

Successful treatment of pulmonary hypertension with beraprost and sildenafil after cord blood transplantation for infantile leukemia

Pulmonary hypertension (PH) is an infrequently reported complication after hematopoietic stem cell transplantation, and its etiology and therapeutic strategies, especially in infants, remain unclear. We report a case of severe PH that developed in an infant with acute leukemia following administration of busulfan as a preconditioner for cord blood transplantation; the case was successfully treated with sildenafil and beraprost, which to our knowledge is the first reported successful use of this regimen in PH following transplantation for infantile leukemia. From a review of all previous reports, use of busulfan in infants may raise the risk of developing PH, and unlike definitive pulmonary veno-occlusive disease, PH in this subgroup may be reversible by early detection and treatment.

Clinical utility of next-generation sequencing-based minimal residual disease in paediatric B-cell acute lymphoblastic leukaemia

We assessed the clinical utility of next‐generation sequencing (NGS)‐based monitoring of minimal residual disease (MRD) in a uniformly treated cohort of 79 patients with paediatric B‐cell acute lymphoblastic leukaemia. Bone marrow samples were collected at the time of diagnosis, days 33 and 80, pre‐ (4–5 months) and post‐ (24 months) maintenance therapy time points, and at relapse. We identified leukaemia‐specific CDR3 sequences in 72 of 79 patients (91%) and detected MRD in 59 of 232 samples. Although MRD was detected in 28 of 55 samples (51%) on day 33, the frequencies of MRD detection decreased to 25% (16/65) at day 80, 19% (11/58) at 4–5 months and 7·4% (4/54) at 24 months. In a univariate analysis, positive MRD results on day 80 [relative risk (RR) 95% confidence interval (CI) = 7·438 (2·561–21·6), P < 0·001], at 4–5 months [RR (95% CI) = 10·24 (3·374–31·06), P < 0·001], and at 24 months [RR (95% CI) = 19·26 (4·974–74·59), P < 0·001] exhibited statistically significant associations with inferior leukaemia‐free survival; this was confirmed using a Cox proportional hazard model. Our study suggests the promising potential of NGS‐MRD for patients with B‐cell ALL.

Choreito formula for BK virus-associated hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

Therapy for BK virus (BKV)-associated hemorrhagic cystitis (BKV-HC) is limited after hematopoietic stem cell transplantation (HSCT). We examined whether choreito, a formula from Japanese traditional Kampo medicine, is effective for treating BKV-HC. Among children who underwent allogeneic HSCT between October 2006 and March 2014, 14 were diagnosed with BKV-HC (median, 36 days; range, 14 to 330 days) after HSCT, and 6 consecutive children received pharmaceutical-grade choreito extract granules. The hematuria grade before treatment was significantly higher in the choreito group than in the nonchoreito group (P = .018). The duration from therapy to complete resolution was significantly shorter in the choreito group (median, 9 days; range, 4 to 17 days) than in the nonchoreito group (median, 17 days; range, 15 to 66 days; P = .037). In 11 children with macroscopic hematuria, the duration from treatment to resolution of macroscopic hematuria was significantly shorter in the choreito group than in the nonchoreito group (median, 2 days versus 11 days; P = .0043). The BKV load in urine was significantly decreased 1 month after choreito administration. No adverse effects related to choreito administration were observed. Choreito may be a safe and considerably promising therapy for the hemostasis of BKV-HC after HSCT.

Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia

The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and 2010. During the follow‐up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10‐year probability of clinical PNH development was 10·2% (95% confidence interval, 3·6–20·7%). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development.

JAK2, MPL, and CALR mutations in children with essential thrombocythemia

hypothesized that these differences might be attributable to different frequencies of CALR mutation, and performed a mutational analysis of pediatric patients with ET. Seventeen pediatric patients with ET (median 8-yearsold at diagnosis, range 0–12) were examined under approval of the ethics committee at Nagoya University Graduate School of Medicine. This cohort includes nine patients reported in previous studies by our group. Clinical diagnosis was made following 2008 WHO criteria. Patient characteristics were listed in Table S1. Genomic DNA was extracted from peripheral blood mononuclear cells. Sanger sequencing was performed for JAK2 exon 14, MPL exon 10, and CALR exon 9 (for primer sequences, see Table S2. Figure S1 shows the representative electropherogram for CALR). Fisher’s exact test and Chi-square test were used to determine statistical significance. Mutational analysis identified five patients (29 %) with the JAK2 p.V617F mutation (Fig. 1). No MPL or CALR mutations were detected among the remaining 12 patients (71 %). The distribution of mutations in Essential thrombocythemia (ET) is an intractable clonal stem cell disorder characterized by a marked increase in platelet count [1]. It is extremely rare among children. Somatic mutations in JAK2 and MPL cause ET, and recent comprehensive genomics studies of adult ET have identified CALR, which encodes calreticulin, as a novel causative gene. Subsequent study revealed that genotype defines the clinical characteristics of ET [2]; patients with JAK2 mutations are at higher risk of thrombosis than those with CALR mutations. These findings highlight the clinical importance of molecular diagnosis. The clinical presentation and genetic determinants of ET may differ between adult and pediatric cases [1]. Pediatric patients with ET have a lower risk of vascular complications and transformation to myelofibrosis than do adult patients. Several groups have reported that frequencies of JAK2 and MPL mutations are lower among children. We

Correlation of rabbit antithymocyte globulin serum levels and clinical outcomes in children who received hematopoietic stem cell transplantation from an alternative donor.

We analyzed the correlation between rabbit ATG (rATG) serum levels and clinical outcomes in 37 children who received rATG at a total dose of 10 or 15 mg/kg during HSCT conditioning from an alternative donor. Fourteen patients had advanced malignant diseases, 13 had severe AA, and 10 had inherited disorders. Complete engraftment was achieved in all patients, and no rejection occurred. The cumulative incidence of grades II–IV acute GVHD and extensive chronic GVHD was 27% (95% CI, 12.5–39.6%) and 8.1% (95% CI, 0–23.1%), respectively. Multivariate analysis identified lower rATG levels at week 4 as an independent risk factor in the development of grades II–IV acute GVHD (p = 0.037). Serious infections were not observed in any patient following HSCT. No correlation was found between EBV reactivation and rATG levels at week 2 and week 4 after HSCT. Furthermore, no correlation was found between relapse and rATG levels two and four wk post‐transplantation. The probability of five‐yr OS among patients was 70.3% (95% CI, 59.8–79.2%). Our results suggest that targeted rATG administration may protect patients from severe acute GVHD without increasing the risk of EBV reactivation or relapse.

Danaparoid reduces transplant-related mortality in stem cell transplantation for children

In SCT, death from transplant‐related complications is the major obstacle hindering improvement of transplant outcomes, and proper supportive care is essential to reduce TRM. The transplant outcomes of 210 pediatric patients with malignant and non‐malignant disorders who consecutively underwent SCT in our institution from 2000 to 2013 were analyzed. The transplant years were divided into three periods: A (2000‐2004), B (2005‐2008), and C (2009‐2013), and an improvement in 5‐year OS and a decrease in 5‐year TRM were observed over these time periods; that is, OS was 61.5%, 60.3%, and 79.5% (P = .062), and TRM was 19.9%, 7.9%, and 0.0% (P < .001) in periods A, B, and C, respectively. On multivariate analysis, the prognostic factor for TRM for all patients was administration of danaparoid (HR = 0.109, 95% CI = 0.033‐0.363, P < .001), and for patients with hematological malignancies in allogeneic SCT, the prognostic factors were danaparoid (HR = 0.046, 95% CI = 0.006‐0.326, P = .002) and advanced disease at SCT (HR = 4.802, 95% CI = 1.734‐13.30, P = .003). A reduction in TRM after SCT was observed over the time periods, and supportive care with danaparoid was found to be significantly effective in reducing TRM in SCT for children.

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.