Prognostic factors of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children: report of the Japan Histiocytosis Study Group.

BACKGROUND: Despite several advances in the treatment of Epstein-Barr virus (EBV) in recent years, patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) do not always show satisfactory outcomes. We here conducted a nationwide survey in Japan to identify prognostic factors of EBV-HLH in children with this disease in an effort to improve the management and the outcomes of these patients. PROCEDURE: Between January 2003 and June 2008, we enrolled 98 children younger than 18 years of age who were diagnosed with EBV-HLH. We then studied the clinical characteristics and laboratory findings at the time of diagnosis with the aim to identify prognostic factors for EBV-HLH. RESULTS: The mean age of onset of EBV-HLH was 3.9 ± 2.8 years. Most of our patients presented with fever, hepatosplenomegaly, lymphadenopathy, and hemophagocytosis of bone marrow. Sixty-two percent of patients showed T cell clonality, and 97% had EBV infection in either T or natural killer cells. Most patients (60%) were treated with a multi-agent chemotherapeutic regimen, including corticosteroid, etoposide, and cyclosporine. After initial treatment, 90.3% of patients were in remission, and 7 patients (8.2%) experienced recurrence of EBV infection. Among several prognostic factors, patients with both hyperbilirubinemia (>1.8 mg/dl) and hyperferritinemia (>20,300 ng/ml) at the time of diagnosis had significantly poorer outcomes than those with low serum bilirubin and ferritin levels. CONCLUSIONS: These findings suggest that the therapeutic strategy for children with EBV-HLH could be tailored according to the laboratory findings at diagnosis.

Assessment of corticosteroid-induced osteonecrosis in children undergoing chemotherapy for acute lymphoblastic leukemia: a report from the Japanese Childhood Cancer and Leukemia Study Group.

Steroid-induced osteonecrosis (ON) is a challenging complication encountered during modern chemotherapy for childhood acute lymphoblastic leukemia (ALL). We retrospectively assessed the incidence of ON and its risk factors in a total of 1095 patients enrolled in 3 consecutive Japanese Children's Cancer and Leukemia Study Group ALL studies (ALL941 [1994 to 2000], n=464; ALL2000 [2000 to 2004], n=305; and ALL2004 [2004 to 2010], n=326). ON was diagnosed in 16 patients, of whom 15 were symptomatic. The cumulative incidence of ON was 0.76% in ALL941, 0.35% in ALL2000, and 3.6% in ALL2004. The incidence of ON in ALL941/2000, in which only prednisolone was administered as a steroid, was significantly lower than that in ALL2004, in which dexamethasone was used as a partial substitute for prednisolone (P<0.01). In ALL2004, sex and age were significantly correlated with the incidence of ON (1.3% in boys vs. 6.7% in girls, P=0.0132; 0.42% for age <10 y vs. 15.6% for age ≥10 y, P<0.0001), suggesting that girls aged 10 years and above are at a greater risk of ON onset. These results indicate that the risk of ON should be considered when administering dexamethasone as part of ALL protocol treatment in girls aged 10 years and above.

Neonatal herpes encephalitis caused by a virologically confirmed acyclovir-resistant herpes simplex virus 1 strain.

A neonate with herpes simplex virus 1 encephalitis was treated with intravenous acyclovir. During the course of therapy, the infection became intractable to the treatment and a mutation in the viral thymidine kinase gene (nucleotide G375T, amino acid Q125H) developed. This mutation was demonstrated in vitro to confer acyclovir resistance.

Clinical characteristics and outcomes of chédiak-Higashi syndrome: a nationwide survey of Japan.

BACKGROUND: Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by immunodeficiency, neurological dysfunction, and oculocutaneous albinism. Recently, several clinical CHS phenotypes have been reported. Here, we report results of a nationwide survey performed to clarify clinical characteristics and outcomes of CHS patients in Japan. METHODS: Questionnaires were sent to 287 institutions to collect data regarding CHS patients diagnosed between 2000 and 2010, including results of lysosomal trafficking regulator (LYST) gene analysis. Cytotoxicity and degranulation activity of cytotoxic T lymphocytes were analyzed in available patient samples. RESULTS: A total of 15 patients diagnosed with CHS were eligible for enrollment in this study. Of these, 10 (67%) had recurrent bacterial infections, five (33%) developed life-threatening hemophagocytic lymphohistiocytosis (HLH), and one patient had complicated malignant lymphoma. Hematopoietic stem cell transplantation (HSCT) was performed for six patients including three with HLH, and 10 of the enrolled patients have survived at the time of this writing. LYST analysis was performed for 10 patients; seven different mutations were detected in seven patients, whereas no mutation was identified in three patients. Cytotoxicity and degranulation activity were impaired in patients with and without LYST mutation. DISCUSSION: Results of this survey indicate that one or two patients with CHS were newly diagnosed each year in Japan. The incidence of HLH was not as high as expected. Mutations of genes other than LYST were suspected in some cases. We conclude that determining indication for HSCT for CHS patients should be based on genetic and cytotoxic analysis.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia.

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

Clinical features and outcome of X-linked lymphoproliferative syndrome type 1 (SAP deficiency) in Japan identified by the combination of flow cytometric assay and genetic analysis.

OBJECTIVE: X-linked lymphoproliferative syndrome (XLP) type 1 is a rare immunodeficiency, which is caused by mutations in SH2D1A gene. The prognosis of XLP is very poor, and hematopoietic stem cell transplantation (HSCT) is the only curative therapy. We characterized the clinical features and outcome of Japanese patients with XLP-1. METHODS: We used a combination of flow cytometric analysis and genetic analysis to identify XLP-1 and reviewed the patient characteristics and survival with HSCT. RESULTS: We identified 33 patients from 21 families with XLP-1 in Japan. Twenty-one of the patients (65%) who did not undergo a transplant died of the disease and complications. Twelve patients underwent HSCT, and 11 of these (92%) survived. CONCLUSION: We described the clinical characteristics and outcomes of Japanese patients with XLP-1, and HSCT was the only curative therapy for XLP-1. The rapid and accurate diagnosis of XLP with the combination of flow cytometric assay and genetic analysis is important.

Hemophagocytic lymphohistiocytosis after hematopoietic stem cell transplantation in children: a nationwide survey in Japan.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is associated with hypercytokinemia in children. Although HLH can be also observed after hematopoietic stem cell transplantation (HSCT), the incidence and clinical features of HLH after HSCT remain obscure. PROCEDURE: The clinical features of HLH after HSCT (post-HSCT HLH) were investigated in children with malignancies, immune deficiencies, or aplastic anemia. The HLH/Langerhans Cell Histiocytosis (LCH) Committee of the Japanese Society of Pediatric Hematology (JSPH) sent questionnaires to hospitals with JPSH members asking for details of cases in which HLH occurred after HSCT between 1998 and 2008. RESULTS: Among 42 children who were diagnosed with post-HSCT HLH between 1998 and 2008 in Japan, 37 fulfilled our inclusion criteria; of these, 26 were classified as early-onset (onset <30 days after HSCT) and 11 were classified as late-onset (onset >30 days after HSCT). In the early-onset group, the presence of respiratory symptoms, high levels of total bilirubin, and triglycerides at onset and the lack of control of GVHD with tacrolimus were significantly associated with non-resolution of HLH (P < 0.05). The survival rate was significantly higher in patients with resolution of HLH than in those without resolution (59% vs. 14%, P < 0.05). CONCLUSIONS: These findings suggest that early-onset post-HSCT HLH is a specific entity of HLH, and appropriate diagnosis and prompt management need to be established.

Passenger lymphocyte syndrome with hemophagocytic syndrome after peripheral blood stem-cell transplantation from an HLA-matched full biological sibling: case report.

Massive hemolysis due to passenger lymphocyte syndrome (PLS) is rare after peripheral blood stem-cell (PBSC) transplantation with a minor ABO mismatch. We present, in a 16-year-old boy (group A Rh+), PLS with hemophagocytic syndrome (HPS) after PBSC transplantation from his HLA (human leukocyte antigens)-matched biological sister (group O Rh+). Mild-to-moderate hemolysis was evident from day +11 to day +15 after transplantation. HPS was diagnosed by bone marrow examination on day +16, while antibodies against the recipient's red blood cell antigens were detected on days +15 and +27. This hemolysis may have been due to PLS with HPS. Therefore, measurement of antibodies may provide a useful hallmark of immune hemolysis.

Hyper-eosinophilia in granular acute B-cell lymphoblastic leukemia with myeloid antigen expression.

Acute lymphoblastic leukemia with eosinophilia (ALLEo) is a rare but a distinctive clinical entity. Clinical features of idiopathic hyper-eosinophilic syndrome (HES) can be seen in patients with ALLEo. We report a 10-year-old girl, in whom HES was initially suspected but further investigation confirmed the diagnosis of acute B-cell lymphoblastic leukemia with myeloid antigen expression. Clinical response to chemotherapy was excellent with achievement of complete remission for 4 years. Serum interleukin-3 and -5 were elevated at presentation and normalized with disappearance of eosinophilia after induction therapy, supporting the reactive nature of eosinophilia in ALLEo. Hematologic malignancy should be considered in patients with hyper-eosinophilia, before attributing it to HES.

Continuous and high-dose cytarabine combined chemotherapy in children with down syndrome and acute myeloid leukemia: Report from the Japanese children's cancer and leukemia study group (JCCLSG) AML 9805 down study.

BACKGROUND: The aim of the JCCLSG AML 9805 Down study was to evaluate the effect of continuous and high-dose cytarabine combined chemotherapy on the survival outcome of acute myeloid leukemia (AML) with Down syndrome (DS). PROCEDURE: From May 1998 to December 2006, DS patients with newly diagnosed AML were enrolled. Remission induction therapy consisted of two courses of pirarubicin, vincristine, and continuous-dose cytarabine (AVC1). The patients who achieved complete remission (CR) after two courses of AVC1 were subsequently treated with mitoxantrone and continuous-dose cytarabine (MC), etoposide and high-dose cytarabine (EC) and pirarubicin, vincristine, and continuous-dose cytarabine (AVC2). RESULTS: Twenty-four patients were enrolled. All patients were younger than 4 years and diagnosed as having acute megakaryoblastic leukemia. Twenty-one patients achieved CR. Three patients died during remission induction therapy due to serious infection. No toxic deaths were observed during remission. All but one patient maintained CR without serious complications. The 5-year overall and event-free survivals were 87.5% ± 6.8% and 83.1% ± 7.7%, respectively. CONCLUSIONS: Continuous and high-dose cytarabine combined chemotherapy with reduced intensity would be effective in DS children with AML.

Minimal residual disease-based augmented therapy in childhood acute lymphoblastic leukemia: a report from the Japanese Childhood Cancer and Leukemia Study Group.

BACKGROUND: The majority of minimal residual disease (MRD)-positive patients with acute lymphoblastic leukemia (ALL) have poor outcomes. The ALL2000 study was performed to evaluate the efficacy of augmented chemotherapy based on MRD-restratification in childhood ALL. PROCEDURE: Between 2000 and 2004, 305 eligible patients with precursor B or T-cell ALL were enrolled in the ALL2000 study. The ALL941-based therapy protocol utilized PCR MRD assays using Immunoglobulin and T-cell receptor gene rearrangements. They were initially stratified into three risk-groups according to leukocyte count and age, and MRD levels were measured at weeks 5 (TP1) and 12 (TP2) for a second stratification. From week 14, patients with MRD levels ≥ 10(-3) received an increase in therapy (one risk group higher), while the remainder continued to receive the initial risk-adapted therapy. RESULTS: The overall 5-year event-free survival (EFS) rate for ALL2000 was 79.7 ± 2.4%. MRD stratification was feasible for 234 of 301 patients (77%) who achieved complete remission. The EFS rate of the MRD stratifiable (MRD) group was 82.5 ± 2.6%, considerably superior to the 74.7 ± 5.7% of MRD non-stratifiable (Non-MRD) group (P = 0.084) and the 74.4 ± 2.1% for ALL 941 (P = 0.012). MRD-positive patients at TP2 showed inferior outcomes as compared with MRD-negative cases, but the difference did not reach a statistically significant level in any risk groups or immunophenotypes. CONCLUSIONS: These results suggest that augmented therapy for MRD-positive patients at TP2 contributed to better outcomes of the ALL2000 study.

Identification of severe combined immunodeficiency by T-cell receptor excision circles quantification using neonatal guthrie cards.

OBJECTIVE: To assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID). STUDY DESIGN: Real-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3). RESULTS: TRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study. CONCLUSION: TRECs quantification can be used as a neonatal mass screening for patients with SCID.

Quantitative PCR assay used to monitor serum Trichosporon asahii DNA concentrations in disseminated trichosporonosis.

A bone marrow transplant recipient with disseminated trichosporonosis was successfully treated with voriconazole. Quantitative PCR assay results for Trichosporon asahii DNA in the sera were well correlated with the patient's clinical course. Based on the in vitro susceptibility test, the organism was susceptible to voriconazole.

Evans syndrome in a patient with Langerhans cell histiocytosis: possible pathogenesis of autoimmunity in LCH.

We report a 1-year-old girl with Evans syndrome coexisting with histologically confirmed Langerhans cell histiocytosis (LCH) affecting the cervical lymph nodes, liver, and spleen. Anti-cardiolipin antibody, anti-SS-A antibody, and anti-SS-B antibody as well as a direct antiglobulin test and platelet-associated IgG were all positive at the onset, and these autoantibodies became negative with the resolution of LCH by chemotherapy. Serum T-helper-2 (Th2) cytokine levels such as those of interleukin (IL)-6 and IL-10 were high whereas those of Th1 cytokines such as IL-2 and interferon-gamma were low at the onset, and this cytokine imbalance was normalized during the resolution of LCH. These results suggest that cytokine imbalance due to LCH led to multiple autoimmune phenomena in the present patient.

Successful unrelated cord blood transplantation for a patient with CD40 ligand deficiency.

We report a CD40 ligand deficiency (CD40LD) patient who was successfully treated with unrelated cord blood transplantation (URCBT). Conditioning regimen was busulfan and cyclophosphamide. The clinical course was uneventful and durable engraftment was achieved. This successful case encourages the use of URCB as an alternative donor source for CD40LD patients.

Immune status of representative infectious diseases among Japanese female university students.

OBJECTIVE: To elucidate the immune status of representative infectious diseases among Japanese youth, we retrospectively investigated serum antibody levels in university students, partly comparing these to immunization records and infectious disease histories confirmed by the maternal and child health (MCH) handbooks. MATERIALS AND METHODS: In total, 168 Japanese female university students, aged 20-21 years, were included. Data were collected from examinations of antibody titers against measles, rubella, varicella-zoster (VZ), mumps, and hepatitis B (HB) and C (HC) viruses, and from QuantiFERON®-TB Gold tests, between 2011 and 2015. Records of immunization and infectious disease histories were available from MCH handbooks for students who agreed with the use of their data for this study (n=23). RESULTS: All students had positive antibodies, detected by enzyme immunoassay (EIA), against measles, rubella, VZ, and mumps; however, seroprevalences within the range of seroprotective antibody levels were 38.1% (64/168), 67.9% (114/168), 95.9% (141/147), and 89.8% (132/147), respectively. The students had probably not been infected with HB, HC, or tuberculosis at the time of the examinations. DISCUSSION: The study indicated that a two-dose vaccine for measles and rubella (MR) might not be sufficient to produce antibodies at seroprotective levels. Therefore, we propose that health care workers, including students, should receive an additional MR vaccine, even if they have received two doses of MR vaccine or if they have unknown histories of immunizations or infectious diseases. Further investigations in these areas will be needed.

Prevalence of lesbian, gay, bisexual, and transgender among Japanese university students: a single institution survey.

OBJECTIVE: To elucidate the prevalence of lesbian, gay, bisexual, and transgender (LGBT) among Japanese youth, we conducted a survey research that targeted university students. MATERIALS AND METHODS: Participants were first-year students (n=1597) at Ibaraki University, Japan (Phase 1 study) or second- to fourth-year students (n=944) at the university who were randomly preferred in the survey (Phase 2 study). Surveys measured gender identity and sexual orientation, partly using the gender identity scale (GIS). RESULTS: The prevalence of LGBT youth among university students were 2.7%, 0.5%, 5.3%, and 0.8% (Phase 1 study) or 1.4% (Phase 2 study), respectively. The GIS scores of the transgender group were significantly lower than those of the female, male, and LGB groups (p<0.01). DISCUSSION: This is the first to clarify the prevalence of LGBT among youth in Japan. It has been suggested that such individuals in Japan, as in other countries, are at risk for physical and mental health concerns, thereby necessitating social and medical intervention. Further investigation in these areas will be needed.

Rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus 6A/B, and human herpes virus 7) by multiplex PCR assay.

A multiplex PCR assay was developed that enabled the simultaneous detection of DNA from 6 types of human herpes virus, HSV-1/2, VZV, EBV, CMV, HHV-6A/B, and HHV-7, using appropriate primer sets and conventional PCR techniques and instruments, with PCR products for each type of virus designed to be easily distinguishable by size. Electropherograms obtained from conventional agarose gels showed that, for each type, the observed number of base pairs corresponded to the intended product and that bands were easily distinguishable from each other. A minimum of 20 copies of viral DNA in a reaction was sufficient to confirm the existence of each of the 6 types of human herpes virus. Comparison of the data obtained from this method and the data obtained from conventional TaqMan PCR using clinical specimens from various sources showed consistent results. The multiplex PCR method reported here for the detection and differentiation of human herpes viruses did not require special equipment or techniques such as hybridization analysis and sequencing analysis and, therefore, enabled us to easily and rapidly detect and identify the 6 types of human herpes virus using conventional methods.

Usefulness of procalcitonin serum level for the discrimination of severe sepsis from sepsis: a multicenter prospective study.

Procalcitonin serum level has been recommended as a new marker of bacterial infectious diseases. The aim of this prospective, multicenter study was to determine the clinical usefulness of procalcitonin in differentiating patients with sepsis from those with severe sepsis. Eighty-two patients were enrolled: 20 without systemic inflammatory response syndrome (SIRS), 9 with SIRS, 34 with sepsis, and 19 with severe sepsis. The patients with severe sepsis had significantly higher procalcitonin levels (median, 36.1 ng/ml) than those with sepsis (median, 0.6 ng/ml). With a procalcitonin cutoff value of 2.0 ng/ml, sensitivity for the detection of severe sepsis and specificity for the detection of sepsis were 94.7% and 78.1%, respectively. A good correlation was found between the serum procalcitonin level and the Sepsis-Related Organ Failure Assessment (SOFA) score (r = 0.680), although no correlation was found between the C-reactive protein (CRP) level and the SOFA score. In conclusion, the procalcitonin serum level may be useful not only for aiding the diagnosis of sepsis but also for discriminating between sepsis and severe sepsis.

Bone marrow transplantation from a pediatric donor with a high frequency of cytomegalovirus-specific T-cells.

A recent study reported that quantitation of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in the graft and monitoring of these T cells might identify hematopoietic stem cell transplantation-recipients at the risk for progressive CMV infection. A 6-year-old girl underwent bone marrow transplantation from an HLA-identical sibling with a very high frequency of CMV specific tetramer-positive CD8+ T-cells. CMV-specific T-cell immunity was prospectively evaluated using a peptide (HLA-A2, NLVPMVATV). Tetramer assay showed that the frequency of CMV-specific CD8+ T cells of the donor in the peripheral blood was 5.3%, higher than average amongst young children. The frequency of CMV-specific CD8+ T cells of the donor in the graft was 3.7% of CD8+ T-cells. Before transplantation, the frequency of CMV specific CD8+ T cells of the recipient was 0.1% in the peripheral blood. Surprisingly, the frequency of CMV specific CD8+ T cells increased up to 30% of CD8+ T-cells at day 27 after transplantation. IFN-gamma enzyme-linked immunospot assay showed the recipient-T cells had strong responses to the A2-specific NLVPMVATV peptide. Although the phenotypic pattern of the CMV-specific T cells of the recipient was different from those of the donor before transplantation, the phenotype of the donor-derived cells retained their original phenotype in the recipient after transplantation. These finding suggested that active transferred immunity from the graft with a high frequency of CMV-specific CTL could induce a rapid reconstitution of CMV-specific T-cell mediated immunity in pediatric HLA-identical allogenetic bone marrow transplantation. The screening of peripheral blood using HLA-peptide tetramer staining might be beneficial to select donors.