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Introduction Graft‐versus‐host disease (GVHD) is frequent and fatal complication following allogeneic hematopoietic stem cell transplantation (HSCT) and characteristically involves skin, gut, and liver. Macrophages promote tissue regeneration and mediate immunomodulation. Macrophages are divided into two different phenotypes, classically activated M1 (pro‐inflammatory or immune‐reactive macrophages) and alternatively activated M2 (anti‐inflammatory or immune‐suppressive macrophages). The anti‐inflammatory effect of M2 macrophage led us to test its effect in the pathophysiology of GVHD. Methods GVHD was induced in lethally irradiated BALB/c mice. M2 macrophages derived from donor bone marrow (BM) were administered intravenously, while controls received donor BM‐mononuclear cells and splenocytes. Animals were monitored for clinical GVHD and analyzed. Results We confirmed that administering donor BM‐derived M2 macrophages attenuated GVHD severity and prolonged survival after HSCT. Moreover, donor BM‐derived M2 macrophages significantly suppressed donor T cell proliferation by cell‐to‐cell contact in vitro. Conclusions We showed the protective effects of donor‐derived M2 macrophages on GVHD and improved survival in a model of HSCT. Our data suggest that donor‐derived M2 macrophages offer the potential for cell‐based therapy to treat GVHD., Adoptive transferred M2 macrophages prolong the survival of GVHD mice.

BACKGROUND The quality of end-of-life (Q-EOL) care is influenced by various factors such as resources for palliative care (PC). We introduced a multi-professional expert team (MET) in 2014, which provides home-based care for children and adolescents with incurable cancer. This study investigated the impacts of the outreach activities by the MET on Q-EOL care of pediatric oncology patients. METHODS This observational study retrospectively examined 112 patients receiving end-of-life care between 1989 and 2018 at a pediatric cancer center in Japan. Some of the indicators of Q-EOL care before and after the introduction of the outreach activities by the MET were compared. The subjects were 92 in pre-MET and 20 in post-MET periods. RESULTS The median number of days for which the patients stayed at home during the final seven or 30 days were significantly prolonged in the post-MET period (0.0 vs 1.5 days, P = 0.020, 3.0 vs 12.0 days, P = 0.042). The change was more significant in hematologic malignancies than solid and central nervous system tumors. Patients receiving longer PC before their deaths could stay at home longer during the last 7 days. The ratio of patients receiving PC for more than 2 months was significantly increased in post-MET period (60.9 vs 90.0%, P = 0.014). More patients also greeted their deaths at home in the post-MET period (3.3 vs 25.0%, P < 0.001). CONCLUSIONS The activities of the MET transformed the end-of-life care of children and adolescents with incurable cancer. Earlier transitions to PC from curative treatment were associated with longer home-based care and more deaths at home.

Myeloid leukemia of Down syndrome (ML-DS) is associated with good response to chemotherapy, resulting in favorable outcomes. However, no universal prognostic factors have been identified to date. To clarify a subgroup with high risk of relapse, the role of minimal residual disease (MRD) was explored in the AML-D11 trial by the Japanese Pediatric Leukemia/Lymphoma Study Group. MRD was prospectively evaluated at after induction therapy and at the end of all chemotherapy, using flow cytometry (FCM-MRD) and GATA1-targeted deep sequencing (GATA1-MRD). A total of 78 patients were eligible and 76 patients were stratified to the standard risk (SR) group by morphology. In SR patients, FCM-MRD and GATA1-MRD after induction were positive in 5/65 and 7/59 patients, respectively. Three-year event-free survival (EFS) and overall survival (OS) rates were 93.3% and 95.0% in the FCM-MRD-negative population, and 60.0% and 80.0% in the positive population. Three-year EFS and OS rates were both 96.2% in the GATA1-MRD-negative population, and 57.1% and 71.4% in the positive population. Adjusted hazard ratios for associations of FCM-MRD or GATA1-MRD with EFS were 10.98 (p = 0.01) and 27.68 (p < 0.01), respectively. Detection of MRD by either FCM or GATA1 after initial induction therapy represents a significant prognostic factor for predicting ML-DS relapse.

Li-Fraumeni syndrome (LFS) is a rare inherited disease characterized by a high and early-onset cancer risk. A cancer surveillance program is important to reduce cancer-related morbidity and mortality in individuals with LFS. We report 2 pediatric cases with LFS-related malignancy in a family. Eight-year-old elder brother was diagnosed with adrenocortical carcinoma and was found to have a heterozygous missense germline mutation c.736A>G: p.Met246Val in the TP53 gene. Cancer screening led to the diagnosis of rhabdomyosarcoma at a curable stage in his 2-year-old younger brother. Comprehensive surveillance resulted in early tumor detection and improved survival.

Patient: Female, 14 Final Diagnosis: Nasopharyngeal carcinoma Symptoms: Fever • neck swelling • trismus Medication: — Clinical Procedure: Biopsy Specialty: Oncology Objective: Challenging differential diagnosis Background: Clinical presentation of nasopharyngeal carcinoma (NPC) is correlated with the extent of primary and nodal disease. Hence, depending on the anatomical structures affected, the clinical presentation varies accordingly, ranging from non-specific symptoms of epistaxis, unilateral nasal obstruction, and auditory complaints, to cranial nerve palsies. Nodal metastasis in the neck is a frequent clinical finding in nasopharyngeal carcinoma. Case Report: A female was admitted to the hospital because of fever and trismus with painful swelling in the right neck. Computed tomography (CT) revealed a mass in the nasopharynx with heterogeneous enhancement and multiple swollen lymph nodes in the corresponding neck. Initial biopsies of nasopharyngeal mass and lymph node of the neck revealed nonspecific lymphoid hyperplasia; we administered antibiotics with the provisional diagnosis of bacterial infection, including Lemierre syndrome that is typically defined by the constellation of septic internal jugular vein thrombophlebitis, pulmonary and other septic emboli, and sterile site bacterial infection. However, the patient was refractory to antibiotics over a month of treatments. The third biopsy of the throat lesion revealed NPC and bacterial cultures using the biopsy specimen were negative. She received intensity-modulated radiation therapy and chemotherapy for NPC stage II (TNM staging: T2N1M0). She never developed Lemierre syndrome-like symptoms after chemoradiotherapy. Conclusions: We report a unique case of NPC presenting with Lemierre syndrome-like symptoms, including prior sore throat, trismus, painful swollen neck, and high fever. Since these symptoms have not been reported in NPC, we included NPC as a differential diagnosis.

Transient hypogammaglobulinemia of infancy may be associated with chromosome del (16)(p11.2) that has reportedly been associated with other forms of primary immunodeficiency (Clin Immunol, 2009, 131, 24; J Allergy Clin Immunol, 2015;135, 1569)., Transient hypogammaglobulinemia of infancy may be associated with chromosome del (16)(p11.2) that has reportedly been associated with other forms of primary immunodeficiency1,2.

Patients with SMID (severe motor and intellectual disabilities) have severe limb disorders and severe mental disabilities. More than half of their deaths are due to respiratory disorders. Therefore, respiratory rehabilitation is important. The effect of respiratory rehabilitation is generally determined by measuring respiratory volume and rate with an expired gas analyzer. However, the equipment is expensive and requires direct contact, making it difficult to use. The purpose of this research is to develop a non-contact measurement system for respiratory function to assess the effect of respiratory rehabilitation in patients with SMID. The proposed method detects respiration by depth change of the abdomen measured using a three-dimensional camera designed to identify body tremor /motion and respiration based on respiratory parameters and individually adapted parameters. Finally, we verify the rehabilitation effect of an RTX respirator on patients with SMID and the effectiveness of the proposed method in an experiment.

BACKGROUND The prognosis of children with acute myeloid leukemia (AML) has improved with the efficacy of hematopoietic cell transplantation (HCT) as a second-line therapy and improvements in supportive care following anthracycline- and cytarabine-based chemotherapy; however, the outcomes of children with relapsed AML still remain unsatisfactory. PROCEDURE In order to identify prognostic factors and improve their prognosis, we analyzed 111 patients who relapsed after treatment with the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol and who were registered in the retrospective JPLSG AML-05R study. RESULTS The 5-year overall survival rate was 36.1%. The major determinant of survival was duration from the diagnosis to relapse. The mean duration in the nonsurviving group (10.1 ± 4.1 months) was shorter than that in the surviving group (16.3 ± 8.3 months) (P

The variability in myelosuppression after chemotherapy for acute myeloid leukaemia (AML) can affect its prognosis; however, the underlying mechanism remains controversial. In the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 study, we showed that prolonged neutropenia was associated with high overall survival (P = 0·011) and low frequency of relapse (P = 0·042) in patients without granulocyte-colony stimulating factor (G-CSF) who completed the indicated treatment protocol. Our data indicate that predisposition to prolonged neutropenia after chemotherapy is correlated with a better outcome of AML treatment. Our results promote the usage of individualised drug dosing strategies to improve the therapeutic outcome in AML patients.

Acute myeloid leukemia harboring internal tandem duplication of FMS-like tyrosine kinase 3 (AMLFLT3-ITD) is associated with poor prognosis. We evaluated the results of the AML-05 study, in which all AMLFLT3-ITD patients were assigned to receive hematopoietic stem cell transplantation (HSCT) in the first remission (1CR). We also investigated the effects of additional genetic alterations on FLT3-ITD. The 5-year overall survival (OS) and event-free survival (EFS) rates among the 47 AMLFLT3-ITD patients were 42.2 and 36.8%, respectively. The 5-year disease-free survival rate among 29 patients without induction failure was 58.4%. We defined the allelic ratio (AR) of FLT3-ITD to WT > 0.7 as high. Significant differences were found in OS (AR-high, 20% vs. AR-low, 66%, p 0.7 and expression of the NUP98-NSD1 chimera strongly impacted OS and EFS. Although all the AMLFLT3-ITD patients received HSCT at 1CR, the treatment outcome of AMLFLT3-ITD patients did not improve compared with those in a previous study. Heterogeneity was observed among AMLFLT3-ITD patients.

Although prognosis in patients with localized osteosarcoma has been dramatically improved by the introduction of multiple chemotherapy agents known as combination chemotherapy, there is growing concern about the development of secondary malignant neoplasms. We report the case of a 13-year-old girl in whom the diagnosis of Ewing sarcoma of bone localized on the shaft of left femur was made 2 years after successful treatment without radiotherapy for osteosarcoma of right proximal femur. EWS-FLI1 fusion gene was detected by reverse transcriptase-polymerase chain reaction. To our knowledge, this is the first case with Ewing sarcoma of the bone as a secondary malignant neoplasm developed in osteosarcoma survivor. We collected 15 cases, included this case, with secondary Ewing sarcoma family of tumor by utilizing the PubMed search and might consider the causes of this secondary cancer.

We previously reported that risk-stratified therapy and intensive postremission chemotherapy (PRC) contributed to the improved survival of childhood acute myeloid leukemia (AML) in the AML99 study, which led us to consider a reduction in the number of PRC courses with more restrictive indications for stem cell transplantation (SCT) in the successor AML-05 study. We here report the outcome of AML patients without core-binding factor mutation (non-CBF AML) in the AML-05 study. Two-hundred eighty-nine children (age < 18 years old) with non-CBF AML were eligible. Patients with unfavorable cytogenetics and/or poor bone marrow response to the first induction course were candidates for SCT in the AML-05 study. After two courses of induction, a further three courses of PRC were given in AML-05, while four courses were given in the AML99 study. The 3-year event-free survival (EFS) rate in the AML-05 study (46.7%, 95% CI: 40.6-52.6%) was comparable to that of non-CBF AML in the AML99 study (51.5%, 95% CI: 42.7-59.6%) (P = .16). However, the 3-year overall survival (OS) rate in the AML-05 study (62.9%, 95% CI: 56.3-68.8%) was slightly lower than that in the AML99 study (71.6%, 95% CI: 63.2-78.5%) (P = .060), mainly due to decreased remission induction rate and increased nonrelapsed mortality. In conclusion, reductions in the number of PRC courses from four to three, together with repetitive cycles of high-dose cytarabine, were acceptable for non-CBF childhood AML.

Inversion of chromosome 16 [inv(16)] has a good prognosis in acute myeloid leukemia (AML), but additional genetic aberrations influence the outcome. We herein describe the case of a 15-year-old Japanese boy with inv(16) harboring a low-allelic burden internal tandem duplication of FLT3 (FLT3-ITD) and KIT mutations. Conventional chemotherapy eradicated a clone with a low-allelic burden FLT3-ITD mutation, although another clone with a KIT mutation occurred 17 months later. Further investigation is necessary to identify AML with inv(16) conferring poor prognosis, to facilitate appropriate treatment with additional drugs, such as dasatinib or gemtuzumab ozogamicin.

Children with severe motor and intellectual disabilities experience chronic pain but cannot communicate verbally. However, no Japanese tool currently exists for assessing pain in this population. This study aimed to develop and evaluate the reliability and validity of a Japanese version of the Paediatric Pain Profile, which is a behavioral rating scale to assess pain in children with severe neurological disabilities. The sample comprised 30 children with severe motor and intellectual disabilities at three hospitals in Japan. Three specialist nurses rated low and high pain video scenes of the children (twice at 1-week intervals) using the Face, Legs, Activity, Cry, Consolability behavioral scale and a translated Japanese version of the Paediatric Pain Profile. On the basis of their ratings, we calculated the internal consistency, test–retest reliability, and intra- and inter-observer reliabilities of the Paediatric Pain Profile. Additionally, we assessed concurrent validity using the Face, Legs, Activity, Cry, Consolability behavioral scale and construct validity using low versus high pain scenes. Both internal consistency (low pain: alpha = 0.735; high pain: alpha = 0.928) and test–retest reliability (r = 0.846) of the Japanese version of the Paediatric Pain Profile were good. Intra-observer reliability was substantial (r = 0.748), whereas inter-observer reliability was only moderate (r = 0.529). However, the concurrent validity with Face, Legs, Activity, Cry, Consolability scores was good (r = 0.629) and construct validity was confirmed (p < 0.001). We confirmed the validity of the Japanese version of the Paediatric Pain Profile, but reliable pain assessment may require repeated ratings by the same person. To accurately assess pain in children with severe motor and intellectual disabilities, healthcare staff must be properly trained and become more skilled in using the Japanese version of the Paediatric Pain Profile.

Background:The current event-free survival rates of children with acute myeloid leukemia (AML) approach 60%, with hematopoietic cell transplantation (HCT) as an effective second-line therapy and improvements in supportive care; however, the overall survival rates of children with relapsed AML still remains 70%, which is an unsatisfactory situation that urgently needs to be solved. Patients & Methods:In order to identify prognostic factors and improve their prognosis, we analyzed 111 patients with relapsed disease after treatment with the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol, who were registered in the retrospective JPLSG AML-05R study. We collected the following data: age at relapse, time from the diagnosis to relapse, site of relapse, FAB classification, chromosomal analysis results, reinduction chemotherapy regimen, rate of achieving a second complete remission (CR2) after initial reinduction therapy, detailed information on HCT, outcome and cause of death. Moreover, ninety-three leukemic samples obtained from 111 patients at the time of their diagnosis were available for a genetic analysis. Mutational analyses ofFLT3-ITD,KIT, N-andK-RAS, NPM1,WT1,andKMT2A-partial tandem duplication were performed. Screening ofNUP98-NSD1was also performed. The high or low expression ofMECOMandPRDM16were determined based on theABL1ratio. Results:The 5-year overall survival rate was 36.1%. t(8;21) was found in 27 patients and inv(16) was found in 5 patients.KMT2Arearrangement was found in 23 patients (24.7%).KITmutations were found in 17 patients (17.5%). The following mutations were also detected:FLT3-ITD (n=10),N-RAS(n=10),WT1(n=7),K-RAS(n=4),NUP98-NSD1(n=2),KMT2A-PTD (n=2), andNPM1(n=2). The high expression ofMECOMandPRDM16was found in 24 (25.8%) and 32 (34.4%) patients, respectively. A genetic analysis revealed the prognostic significance ofFLT3-ITD as a poor prognostic marker (p=0.04) and core binding factor-AML, t(8;21) and inv(16), as a good prognostic marker (p Conclusions:Achieving CR2 prior to HCT by intensive reinduction chemotherapy, ECM, or FLAG, is important for improving the prognosis of patients with relapsed pediatric AML. Liposomal daunorubicin or 3 fractionated doses of gemtuzumab ozogamicin is an attractive option which is under consideration for addition to FLAG. Moreover, recent molecular targeted therapeutics, such as FLT3 inhibitors, may contribute to improving the prognosis of these patients. Larger prospective investigations are needed in order to establish individualized treatment strategies for patients with relapsed childhood AML. Disclosures No relevant conflicts of interest to declare.

B-1a cells are distinguishable from conventional B cells, which are designated B-2 cells, on the basis of their developmental origin, surface marker expression, and functions. In addition to the unique expression of the CD5 antigen, B-1a cells are characterized by the expression level of CD23. Although B-1a cells are considered to be independent of T cells and produce natural autoantibodies that induce the clinical manifestations of autoimmune diseases, there is much debate on the role of B-1a cells in the development of autoimmune diseases. We examined the involvement of B-1a cells in autoimmune-prone mice with the lpr gene. MRL/lpr and B6/lpr mice exhibited lupus and lymphoproliferative syndromes because of the massive accumulation of CD3+CD4-CD8-B220+ T cells. Interestingly, the B220+CD23-CD5+ (B-1a) cell population in the peripheral blood and peritoneal cavity increased with age and disease progression. Ninety percent of B-1a cells were CD3 positive (CD3+ B-1a cells) and did not produce tumor necrosis factor alpha, interferon gamma, or interleukin-10. To test the possible involvement of CD3+ B-1a cells in autoimmune disease, we tried to eliminate the peripheral cells by hypotonic shock through repeated intraperitoneal injections of distilled water. The fraction of peritoneal CD3+ B-1a cells decreased, and symptoms of the autoimmune disease were much milder in the distilled water-treated MRL/lpr mice. These results suggest that CD3+ B-1a cells could be mediators of disease progression in autoimmune-prone mice.

The prognosis of paediatric acute myeloid leukaemia (AML) with primary induction failure (PIF) is extremely poor, and effective treatment strategies have not been established. We investigated the clinical and biological features of paediatric AML patients with PIF registered to the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 study. The 3-year overall survival rate of the 41 PIF patients was 19.0%. High leucocyte count, M7 morphology, and unfavourable genetic aberrations, such as FLT3-internal tandem duplication, NUP98-NSD1 and high MECOM or PRDM16 expression, were risk factors for PIF. More effective treatment strategies based on leukaemia biology need to be urgently explored.

Recently, a new disease of lymphocyte homeostasis caused by somatic mosaicism for the RAS mutation has been discovered (known as RALD, RAS-associated leukoproliferative disorder). Since few cases have been reported in literature, the prognosis and standard treatment for autoimmune diseases associated with RALD remain poorly understood. Standard rituximab therapy (375 mg/m for 4 wk) is effective in patients with autoimmune diseases, but early recurrences are common. We highlight the potential for monthly administration of rituximab in a patient with autoimmune thrombocytopenia and hemolytic anemia associated with RALD. RALD was diagnosed in an 11-year-old girl following a 9-year history of severe hepatosplenomegaly and autoimmune cytopenias. Genetic analyses confirmed somatic mosaicism for the G13C KRAS mutation without an autoimmune lymphoproliferative syndrome-related gene mutation. Rituximab therapy was used because of the refractory character of the autoimmune cytopenias which failed to respond to steroids and other immunosuppressive agents. Her treatment consisted of weekly infusions of rituximab for 4 weeks followed by monthly rituximab for 11 months. She maintained her response in hematologic parameters for 2 years after monthly rituximab was ceased and her scores representing quality of life were improved. Rituximab could improve clinical responses and quality of life of the patients with RALD.

Background On the basis of results of previous Japanese trials for myeloid leukemia in Down syndrome (ML-DS), the efficacy of risk-oriented therapy was evaluated in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D05 study. Procedure All patients received induction chemotherapy that consisted of pirarubicin, intermediate-dose cytarabine, and etoposide. Patients who achieved complete remission (CR) after initial induction therapy were stratified to the standard risk (SR) group and received four courses of reduced-dose intensification therapy. Patients who did not achieve CR were stratified to the high risk (HR) group and received intensified therapy that consisted of continuous or high-dose cytarabine. Results A total of 72 patients were eligible and evaluated. One patient died of sepsis during initial induction therapy. Sixty-nine patients were stratified to SR and two patients to HR. No therapy-related deaths were observed during intensification therapy. The 3-year event-free and overall survival rates were 83.3% ± 4.4% and 87.5% ± 3.9%, respectively. Age at diagnosis less than 2 years was a significant favorable prognostic factor for risk of relapse (P = 0.009). Conclusions The attempt of risk-oriented prospective study for ML-DS was unsuccessful, but despite the dose reduction of chemotherapeutic agents, the overall outcome was good, and further dose reduction might be possible for specific subgroups.

As past studies of adolescent and young adults (AYA) with acute myeloid leukemia (AML) reported conflicting results, we conducted a retrospective analysis using data from three Japanese pediatric AML studies. Among the 782 patients with de novo AML, 44 were classified as AYA (age ≥15 years at diagnosis), 164 as infants (0-1 year), 413 as younger children (2-11 years), and 161 as older children (12-14 years). While the 5-year event-free survival rate of AYA was not different among the groups, the five-year survival rate (54.7 %) was significantly lower than that of the other three groups (P = 0.019): 68.7 % for infants, 73.2 % for younger children, and 75.5 % for older children. No difference in the 5-year cumulative incidence of relapse was observed, but treatment-related death (TRD) of AYA was significantly higher (29.4 %) than that in infants (14.8 %), younger children (10.2 %), and older children (13.8 %). Multivariate analysis showed age ≥15 years old at diagnosis was associated with both poor survival rate and high TRD. Adolescents with AML had inferior survival due to a higher incidence of TRD, especially after failure of initial frontline treatment.

Eculizumab, terminal complement inhibitor, has become the frontline treatment for atypical hemolytic uremic syndrome (aHUS). However, the optimal treatment schedule has not yet been established. We describe here an aHUS patient with a mutation of C3 I1157T who achieved remission with eculizumab and suffered a recurrence after eculizumab discontinuation, a clinical situation that has not been previously described in patients with C3 mutation. A 9-year-old male experienced an onset of aHUS after viral gastroenteritis and was treated with hemodialysis. At 13 years of age he developed bacterial enterocolitis due to Campylobacter jejuni and experienced a recurrence of aHUS. Eculizumab was initiated on day 4 after disease onset resulting in recovering laboratory parameters. The patient received eculizumab for 5 months before its discontinuation. Second relapse induced by bacterial pharyngitis was confirmed 4 months after eculizumab discontinuation and prompt eculizumab reinitiation resulted in rapid remission. The patients carrying mutations in CFH or C3 have a high frequency of relapse and worse prognosis. More than 50% of aHUS relapses occurred during the first year after the onset. Therefore, long-term treatment with eculizumab is appropriate in patients with aHUS who have experienced a relapse or have mutations associated with poor prognosis.

Background The combination of fludarabine (Flu), high-dose cytarabine (Ara-C) and granulocyte colony-stimulating factor (G-CSF; FLAG), with anthracyclines has become standard chemotherapy for refractory acute myeloid leukemia (AML) in European children and adults. To clarify the efficacy and the safety of FLAG-idarubicin (IDA) for children prospectively, we planned a multicenter phase II study (AML-R11) by the Japanese Pediatric Leukemia/Lymphoma Study Group. Methods Patients with AML aged between 2 and 20 years old, who had the first bone marrow (BM) relapse or induction failure, were enrolled. The FLAG-IDA regimen consisted of Flu 30 mg/m2 for 5 days, Ara-C 2 g/m2 for 5 days, G-CSF (lenograstim) 5 μg/kg for 6 days and IDA 10 mg/m2 for 3 days. The primary endpoint was remission rate after therapy. Results Due to drug supply issues, the trial was suspended after the inclusion of seven eligible patients. There were six cases of early relapse within 1 year of the first remission. All seven patients completed the therapy and no early death was observed. Hematological toxicity was common, and one patient developed grade 4 non-hematological toxicity of bacterial meningitis. Although only one patient with late relapse achieved complete remission, minimal residual disease was positive on both flow cytometry and Wilms' tumor 1 mRNA. Two patients were alive in remission following hematopoietic stem cell transplantation, whereas the other five patients died of either the disease or treatment-related causes. Conclusion FLAG-IDA might be tolerable for children with refractory AML although the efficacy should be further investigated.

Background: Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy thus results in a favorable outcome. However, relapsed and refractory cases are rarely salvageable, regardless of receiving hematopoietic stem cell transplantation. Several factors such as certain chromosomal abnormalities and age at diagnosis are somewhat prognostic, but no universal prognostic factor has been found to date. In order to identify a subgroup with high risk of treatment failure, the role of minimal residual disease (MRD) with three methods were explored in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) trial AML-D11. Procedure: AML-D11 is a nationwide single-arm clinical trial for children (4 months to 17 years old) with ML-DS. All patients received an identical chemotherapy to the previous AML-D05 study (Taga T. Pediatr Blood Cancer 2016). MRD was evaluated at two time points, one after the induction therapy and another at the end of whole chemotherapy, using 3 different methods; flow cytometric MRD (FCM-MRD), deep sequencing MRD of mutant GATA1 (GATA1-MRD) and PCR MRD of WT1 mRNA expression (WT1-MRD). WT1-MRD was measured in both bone marrow (BM) and peripheral blood (PB) samples, while FCM- and GATA1-MRD were measured only in BM samples. Results: A total of 78 patients were eligible and followed-up with a median of 47.6 months (range, 8 to 68.8 months). Seventy-six patients were stratified to the standard risk (SR) and one patient to the high risk (HR) group by morphological response. One patient died of sepsis during initial induction therapy. Three-year event-free survival (EFS) and overall survival (OS) rates were 87.2% (95%CI, 77.5 to 92.9%) and 89.7% (95%CI, 80.5 to 94.7%), respectively. FCM-MRD and GATA1-MRD after initial induction therapy were positive in 5/65 and 7/59 patients, respectively, which were both significantly prognostic (Fig.1). Prognostic significance of WT1-MRD could not be evaluated due to a limited number of collected samples. Conclusions: MRD detections by FCM and targeted deep sequencing of GATA1 after initial induction therapy are both significant prognostic factors for predicting relapse. Risk stratification using FCM-MRD is currently incorporated in the on-going Japan Children's Cancer Group ML-DS trial (AML-D16; jrct.niph.go.jp, jRCTs041190047). Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction: Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome (DS) is characterized by the transient appearance of blast cells that harbor somatic GATA1 gene mutation. Although most patients show spontaneously resolution without therapeutic interventions, approximately 20% of TAM cases result in early deaths within 9 months and 20% of survivors develop acute megakaryoblastic leukemia (AMKL) within 4 years. Although the risk factors associated with early deaths are known, the definite clinical predictive indicators of AMKL onset in patients with TAM remain unclear. Therefore, we analyzed 167 TAM patients with DS enrolled in the TAM-10 prospective observational study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) to determine the clinical characteristics of TAM and predictive factors of leukemia development. Patients and Methods: Between May 2011 and February 2014, 167 neonates (89 boys and 78 girls) diagnosed with TAM were prospectively registered in the TAM-10 study. Somatic GATA1 gene mutations were confirmed in 163 (98%) patients using Sanger and/or next-generation sequencing. Minimal residual disease using flow cytometry (FCM-MRD; cut-off level, ≥0.1%) was monitored at 1 (n = 133) and 3 months (n = 104). Results: Median (range) gestational age, birth body weight, white blood cell (WBC) count, and percentage of blasts at diagnosis were 37 (29-40) weeks, 2,612 (1,066-3714) g, 38.3 (2.4-478.7) × 109 cells/L, and 37% (0.5%-95.5%), respectively. Systemic edema and organ hemorrhage was observed in 31/167 (19%) and 14/167 (8%) patients, respectively; 68/167 (41%) patients received some therapeutic interventions, including low-dose cytarabine (LDCA; n = 52), exchange blood transfusion (n = 20), and systemic steroid therapy (n = 31). Early death ( Among 145/167 patients without early death, 28 (19%) developed AMKL. FCM-MRD positivity at 1 month [positive, n = 107; negative, n = 26; cumulative incidence ratio (CIR) (95% CI) = 25.2% (17.3-33.9%) vs 3.8% (0.3%-16.8%), P = 0.022] and 3 months (positive, n = 20; negative, n = 84; CIR (95% CI), 45.0% (22.3%-65.4%) vs. 16.0% (9.0%-24.8%), P = 0.002] was significantly associated with leukemia development. However, other clinical covariates, including sex, birth weight, gestational age, WBC count, blast percentage, and GATA1 gene mutational types, could not predict AMKL development. Considering their severe clinical conditions, 13/31 (42%) patients who received systemic steroid therapy died before AMKL development; interestingly, none of the remaining 18 patients developed AMKL but they showed significantly lower CIR than those who did not receive this therapy [CIR (95% CI), 0% vs. 19.4% (10.9%-29.6%), P = 0.010]. Other therapeutic interventions, including LDCA and exchange blood transfusion, were not associated with AMKL development. Conclusion: FCM-MRD positivity at 1 month and 3 months might be a useful marker to predict leukemia development in patients with TAM. Although LDCA therapy significantly decreased the rate of early deaths, it did not suppress leukemia development. Interestingly, systemic steroid therapy might suppress leukemia development. These results pave the way to design clinical trials for developing MRD-directed leukemia prevention therapy for patients with TAM. Disclosures No relevant conflicts of interest to declare.

Infants (

Gastric antral vascular ectasia (GAVE) leading to upper gastrointestinal bleeding is a heterogeneous disorder that is not commonly recognized in hematopoietic stem cell transplantation (HSCT). Protein-losing enteropathy (PLE) is noted as another gastrointestinal complication in the context of chronic graft-versus-host disease (GVHD) after HSCT. The possibility of a relationship between these two distinct gastrointestinal disorders, however, remains obscure. A 6-year-old boy with acute myelogenous leukemia developed severe hematemesis 4 months after myeloablative HSCT from a human leukocyte antigen-matched sibling donor. The diagnosis of GAVE was made by upper endoscopy and histological examination. The patient simultaneously developed frequent diarrhea and significant hypoproteinemia, consistent with a diagnosis of PLE. This co-occurrence of GAVE and PLE against a background of chronic GVHD was successfully treated with cyclosporin A and prednisolone. To our knowledge, this is the first report of GAVE concurrent with PLE following HSCT. The possible association of GAVE and PLE in chronic GVHD is discussed.

Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy and favorable prognosis. Because little research has been focused on refractory/relapsed (R/R) cases, we conducted a retrospective analysis for R/R ML-DS. Among ML-DS patients diagnosed between 2000 and 2010 in Japan, 26 relapsed (25 in the BM and 1 in the skin), and 3 refractory patients were enrolled. The male/female ratio was 18/11. The median age at initial diagnosis of ML-DS was 2 years, and the median time to relapse was 8.6 months. Each patient initially had been treated with ML-DS–specific protocols. Thirteen of the 26 patients achieved complete remission with various kinds of reinduction chemotherapies; 2 of 8 survived without further recurrence after receiving allogeneic hematopoietic stem cell transplantation, and 4 of 5 maintained complete remissions with chemotherapy alone. Treatment failures mostly were associated with disease progression rather than treatment-related toxicities. The 3-year OS rate was 25.9% ± 8.5%. A longer duration from initial diagnosis to relapse was a significant favorable prognostic factor (P < .0001). We conclude that clinical outcome for patients with R/R ML-DS generally are unfavorable, even in those receiving hematopoietic stem cell transplantation. Novel methods to identify poor prognostic factors for ML-DS are necessary.

Although there are National Institutes of Health consensus criteria for the global assessment of chronic graft-versus-host disease, no validated biomarkers have been established for this disease. Furthermore, whereas the role of T cells, B cells, and dendritic cells in chronic graft-versus-host disease has been established, the contribution of monocytes has not been clearly addressed. Using an enzyme-linked immunospot assay, we measured the spot-forming cells for interferon-γ, interleukin-4, interleukin-10, and interleukin-17 in unstimulated peripheral blood of patients following allogeneic hematopoietic stem cell transplantation. Other immunological examinations, including skin biopsy, were also done. Fifty-seven patients were enrolled. Interleukin-10 spot-forming cells were evaluable for therapeutic monitoring in 16 patients with chronic graft-versus-host disease. The number of interleukin-10 spot-forming cells in patients with active chronic graft-versus-host disease was significantly higher than the number in those with no or inactive chronic graft-versus-host disease. Interleukin-10 was predominantly produced by monocytes. CD29 expression on monocytes in patients with active chronic graft-versus-host disease was elevated. The level of plasma fibronectin, a ligand of CD29, correlated with the number of interleukin-10 spot-forming cells. Immunohistochemical analysis of the skin in active chronic graft-versus-host disease showed that infiltrating CD29(+) monocytes might produce interleukin-10. A novel biomarker, interleukin-10 spot-forming cells, shows promise as both a diagnostic and prognostic indicator for chronic graft-versus-host disease, and may allow for early intervention prior to the onset of the disease. Measurement of interleukin-10 spot-forming cells would be helpful in clinical trials and in patients' management.

We evaluated the efficacy of treatment using reduced cumulative doses of anthracyclines in children with acute promyelocytic leukaemia (APL) in the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-P05 study. All patients received two and three subsequent courses of induction and consolidation chemotherapy respectively, consisting of all-trans retinoic acid (ATRA), cytarabine and anthracyclines, followed by maintenance therapy with ATRA. Notably, a single administration of anthracyclines was introduced in the second induction and all consolidation therapies to minimize total doses of anthracycline. The 3-year event-free (EFS) and overall survival rates for 43 eligible children were 83·6% [95% confidence interval (CI): 68·6-91·8%] and 90·7% (95% CI: 77·1-96·4%), respectively. Although two patients died of intracranial haemorrhage or infection during induction phases, no cardiac adverse events or treatment-related deaths were observed during subsequent phases. Patients not displaying M1 marrow after the first induction therapy, or those under 5 years of age at diagnosis, showed inferior outcomes (3-year EFS rate; 33·3% (95% CI: 19·3-67·6%) and 54·6% (95% CI: 22·9-78·0%), respectively). In conclusion, a single administration of anthracycline during each consolidation phase was sufficient for treating childhood APL. In younger children, however, conventional ATRA and chemotherapy may be insufficient so that alternative therapies should be considered.

Most patients who have undergone hematopoietic cell transplantation (HCT) lose specific immunity to measles. However, due to its immunosuppressive potential, it has been recommended that a live attenuated measles vaccination be administered two years following HCT. Measles virus (MV) glycoproteins including hemagglutinin (HA) are expressed on MV-infected dendritic cells (DCs), and they impair efficient antigen presentation between the DC and T cell. We produced a DC-based vaccine against MV by loading DCs with MV-infected autologous DCs. MV in the infected DCs was inactivated using ultraviolet-B. The DC-based vaccine neither expressed HA nor inhibited allogeneic T cell proliferation, while it induced the production of interferon-gamma (IFN-gamma) by autologous CD4 and CD8 naive T cells ex vivo. Importantly, the vaccine derived from patients who had undergone HCT also efficiently induced IFN-gamma producing cells. These findings indicate that our DC-based MV vaccine induces MV-specific immunity even in post-HCT patients without causing immunosuppression.

Infections may coexist and in certain circumstances aggravate acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation. Early detection of aGVHD is often difficult in patients with concurrent infections. Using an enzyme-linked immunospot assay that reflects ongoing immune status in vivo, we enumerated spot-forming cells (SFCs) for interferon (IFN)-gamma, interleukin (IL)-4, and IL-12 in peripheral blood from 56 patients with hematological disorders. Eleven patients had viral, fungal, or bacterial systemic infections during first 10 weeks posttransplant. Of these, six patients with grade 0-I aGVHD showed normal levels of IFN-gamma SFCs. On the other hand, IFN-gamma SFCs were elevated in five patients with grade II-IV aGVHD. These data indicate that increased IFN-gamma SFCs seemed to be correlated with clinically significant aGVHD, but not with infection itself. IL-4 and IL-12 SFCs increased in some patients with infections, irrespective of the presence of aGVHD. Thus, IFN-gamma SFCs may be used to distinguish systemic infections from aGVHD.

Background Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. It was hypothesized that type 1 cytokines promoted aGVHD and type 2 cytokines inhibited it. However, recent publications demonstrated contradictory results in murine models. Type 1/2 paradigm in aGVHD remains to be determined in human. Methods Using enzyme-linked immunospot assay that reflects ongoing immune status in vivo, we measured spot-forming cells (SFCs) for interferon (IFN)-gamma, interleukin (IL)-12, IL-4, and IL-10 in peripheral blood from 56 patients with hematological disorders who underwent allogeneic hematopoietic stem cell transplantation. Results The numbers of IFN-gamma and IL-4 SFCs in patients with grade II approximately IV aGVHD were significantly higher than those in patients with grade 0 approximately I aGVHD. The enumeration of cytokine SFCs predicted aGVHD approximately 4 days before it became clinically evident, since IFN-gamma SFCs in asymptomatic phase that later progressed into grade II approximately IV aGVHD were elevated in 8 out of 8 evaluable patients. Similarly, IL-4 SFCs were elevated in 6 of 8 patients. In addition, Type 1 cytokine SFCs contributed to the intestinal, but not skin and hepatic aGVHD. Conclusions Enzyme-linked immunospot assay is clinically useful for predicting aGVHD and detecting distinct end-organ targets following allogeneic hematopoietic stem cell transplantation.

The human polyomavirus BK (BKV)-associated hemorrhagic cystitis (HC) has been a frequent and, seldom life-threatening complication after bone marrow transplantation (BMT). The authors report a male with melodysplastic syndrome, who developed BKV-associated late-onset HC 12 days after HLA-matched unrelated BMT. His urine contained epithelial cells with intranuclear inclusion bodies suggestive of BKV infection and was positive for BKV in polymerase chain reaction. He did not respond to any treatment for HC. In addition, he developed BKV-associated acute renal failure on day 26, followed by hepatic veno-occlusive disease on day 42. This is the first case in which BKV may be associated with fatal progressive renal failure.

Cord blood transplantation (CBT) has been increasingly used to treat patients with hematological diseases, but active immunizations for patients have not been described. Patients certainly need immunizations following CBT, since transplanted cord blood is naive. The authors previously reported successful hematopoietic reconstitution following cord blood transplantation from an HLA-matched sibling in a transfusion-dependent child with Diamond-Blackfan anemia. No graft-versus-host disease, either acute or chronic, has been observed so far. Here, the authors report that immunological recovery of the patient has been rapid shortly after CBT and immunization has been done successfully. Vaccines (diphtheria, pertussis, tetanus, rubella, measles, and BCG) were administered during 22-34 months post-transplant. Seroconversion to these vaccines was excellent without significant adverse effects. These results indicate that both toxoid and live vaccines have been safely administered in the patient who underwent related cord blood transplantation.

Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children

INTRODUCTION: Treatment outcome of pediatric acute myeloid leukemia (AML) patients has improved over the past few decades due to intensive chemotherapy. Primary induction failure (PIF), defined as failure to achieve complete remission after the second induction chemotherapy, occurs in around 10% of children with newly diagnosed AML. Effective treatment strategy for these patients is not established and their poor prognoses have been reported elsewhere. Here we investigated the clinical and biological features of pediatric AML patients with PIF who were registered in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Study (AML-05). METHODS: We conducted a retrospective survey of patients who were registered in the AML-05. We analyzed probabilities of overall survival (OS) according to gender, age at diagnosis, white blood cell count (WBC) at diagnosis, FAB classification, etc. Genetic analysis of primary leukemic blasts was performed. We screened fusion genes and gene mutations as follows: RUNX1-RUNX1T1, CBFB-MYH11, NUP98-NSD1, DEK-NUP214, MLL-PTD, WT1, NPM1, and MLL-rearrangements, gene mutations of KIT and FLT3-ITD. Furthermore, we evaluated the clinical and biological background of these PIF patients to define their prognostic factors statistically. RESULTS: Among the 443 patients registered in the AML-05 between November 2006 and December 2010, 43 patients (9.7%) had PIF. The observation period from the date of PIF was 5-78 months (median, 20 months). The age at diagnosis was 0.1-17 years (median, 6.1 years). There was no significant difference in gender (28 males vs15 females). The 3-year OS rate was 19.0% (95% confidence interval 1.0%-38.0%). This was extremely poor compared to the patients who achieved complete remission (CR): 66.7% for high-, 73.2% for intermediate-, and 92.6% for low-risk groups, respectively. Disease recurrence is the major cause of death (n=19, 46.3%). The mean WBC at diagnosis of PIF patients was 96 x 109/L: this is significantly higher than that of non-PIF patients (51 x 109/L, P CONCLUSIONS: The prognosis of pediatric AML patients with PIF was extremely poor. In our survey, unfavorable molecular biological background of leukemia was associated with PIF. It is necessary to explore more effective treatment strategies based on leukemia biology for these patients in the future. Disclosures No relevant conflicts of interest to declare.

Summary Acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT) is initiated by donor T lymphocytes that recognize histocompatibility antigens presented by recipient dendritic cells (DCs). Current approaches to reduce GVHD are focused on suppressing donor T lymphocyte responses to alloantigens. However, these strategies may be inadequate in the setting of allogeneic transplants (particularly histoincompatible transplants), may increase the risk of tumour relapse and are associated with high rates of opportunistic infections. We hypothesized that inhibition of recipient DCs might suppress GVHD. We recently demonstrated in vitro that azithromycin, a macrolide antibiotic, also acts as a nuclear factor (NF)-κB inhibitor of murine DCs and inhibits their maturation and functions, including allogeneic responses. We investigated whether azithromycin could prevent alloreactions in a murine histoincompatibility model. Oral administration of azithromycin to recipient mice for 5 days during major-histoincompatible BMT suppressed lethal GVHD significantly, whereas ex-vivo lymphocyte function was not affected by the drug. These data suggest that azithromycin has potential as a novel prophylactic drug for lethal GVHD.

The acute myeloid leukaemia (AML) 99 trial conducted previously in Japan for the treatment of de novo paediatric AML showed excellent results, with a 5-year overall survival (OS) and event-free survival (EFS) of 75·6% and 61·6%, respectively. To examine reproducibility of these results in another cohort, the outcome of 146 newly diagnosed AML paediatric patients prospectively registered in the Japan Association of Childhood Leukaemia Study (JACLS) from 2003 to 2006 was compared to that of 240 patients in the original AML 99 clinical trial. The 5-year EFS and OS achieved in the new cohort was 66·7 ± 4·0% and 77·7 ± 8·0% respectively, which were comparable to those obtained in the original AML 99 clinical trial, although less frequent core-binding factor (CBF) AML (29·5% vs. 37%) and an almost equal frequency of allogeneic haematopoietic stem cell transplantation (allo-HSCT) during first complete remission (16·5% vs. 19%) were observed. The 5-year EFS in patients with a normal karyotype (NK) (n = 35, 54·9 ± 15·1%) was inferior in the present cohort when compared to the original AML99 trial. This study confirmed the excellent outcome of the original AML99 protocol.

Lymphocytic intestinal leiomyositis is a rare entity, which causes chronic intestinal pseudo-obstruction (CIPO) in children. We present the first case of a boy who had pure red cell anemia 1 year before onset. Prolonged ileus developed after gastroenteritis and the patient was diagnosed using a biopsy of the intestinal wall. Findings from the present case indicate that there are three important factors for accurate diagnosis: history of enteritis, positive serum smooth muscle antibody, and lymphocyte infiltration with muscle destruction in the muscularis propria in the intestinal wall. Earlier diagnosis and induction of immunosuppressive therapy may be essential for a better outcome.