ALBERT

Description: Transient abnormal myelopoiesis (TAM) occurs in about 10% of neonates with Down syndrome. While TAM is generally considered as self-limiting disease, substantial number of patients suffer from serious complications including liver fibrosis or pericardial effusion and eventually leads to fatal clinical outcomes. Low-dose cytosine arabinoside (CA) therapy has been reported to be effective for clinical symptoms of TAM patients, but it is still controversial as to which patient should be treated. We have retrospectively reviewed the clinical outcomes of TAM patients in our institution to confirm the efficacy as well as the safety of low-dose CA therapy by comparing the clinical outcomes before and after introduction of CA. Before introduction of CA, we have experienced 20 TAM patients of 13 male and 7 female between September 1992 and November 2008. The median gestational age of those was 37w4d (range, 31w5d - 39w2d) , median peak value of white blood cell (WBC) count was 48×109/L (range, 19.8 - 399×109/L), and median peak direct bilirubin(DB) level was 0.9 mg/dL (range, 0.1 - 18.4 mg/dL), respectively. Sixteen of twenty patients (80%) had various complications including dyspnea, hepatosplenomegaly, pericardial effusion, and ascites. Since 2009, we have introduced low dose CA (0.4 - 1.0mg/kg/day) for those with high WBC count (〉100×109/L) until WBC count decrease to 10×109/L . Three patients received chemotherapy, but one patient with high WBC count did not, because consent was not obtained. The median gestational age of those was 36w4d (range, 33w6d - 39w2d) , median peak value of WBC count was 106×109/L (range, 101 - 267×109/L), and median peak DB level was 1.95mg/dL (range, 1.0 - 11.0 mg/dL), respectively. All of them had various complications including dyspnea, hepatosplenomegaly, pericardial effusion, liver fibrosis and ascites. Before introduction of low dose CA therapy, five of six patients (83%) with high WBC count (〉100×109/L) died of liver failure(4 of 5 patients) or AML, and one of 14 patients (7%) with low WBC count (

Description: Introduction: Bone marrow-derived mesenchymal stromal cells (MSCs) exhibit in vitro immunomodulatory properties and have been effectively used to treat patients with acute GVHD following hematopoietic stem cell transplantation (HSCT). Expansion of MSCs involves the use of fetal bovine serum (FBS), although regulatory guidelines recommend that alternatives be used. Therefore, expansion of MSCs where FBS has been replaced by platelet lysates (PL) has been reported. Previous phase I studies of PL expanded third party, bone marrow derived MSCs have been reported showing no acute toxicity and 66 to 71% overall response rate. Although a uniform mechanism has not yet discovered, the available data have revealed immunomodulatory effects of MSCs therapy on T lymphocytes, B lymphocytes, NK cells, dendritic cells and macrophages. Therefore, we planned comprehensive evaluation of cytokines in patients who received PL expanded MSC therapy for steroid refractory acute GVHD. Materials and Methods: We conducted the phase 1 clinical trial of PL expanded MSC therapy for steroid resistant acute GVHD in children and young adults. Steroid resistance was defined as lack of clinical improvement after 7days treatment of methylprednisolone (2mg/kg/day) or GVHD progression within 3 days from steroid onset. MSCs were harvested frombone marrow donors and were expanded in 5% PL-containing medium. MSCs product had to satisfy all the release criteria including absence of bacteria, fungi, mycoplasma, endotoxin level 〈 5 EU/kg, 〉 70% viability, 〉 70% positivity for CD44, CD73 and CD105, and 〈 10% contamination by CD14, CD34, and CD45 hematopoietic cells. MSCs were injected intravenously immediately after thawing. Each MSC infusions aimed at reaching 1-2 x 10(6) cells/kg recipient body weight. Further MSC administrations could be provided with one week interval. Serum samples were collected before steroid treatment, before MSCs therapy, 1, 7, 14, 21, 28 days after MSCs therapy. A panel of 64 different cytokines was measured using Milliplex map Human Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA) to quantify the serum cytokine levels. The assay was performed according to the manufacturer's instructions. Results: Four patients (aged 2, 5, 15, 25 years) received intravenous (i.v.) MSCs for grade III-IV acute GVHD (liver: two cases, gut: two cases) on two occasions following HLA haploidentical HSCT or unrelated HLA matched SCT. The median dose of infusions was 1.2 x 10(6)/kg (range: 0.7-2.7 x 10(6)/kg). No acute side effects after infusions were observed. Clinical response was recognized within 7days and complete response was obtained in all four patients within 3 weeks from the administration of MSCs. Relapse of GVHD was not observed. Reactivation of cytomegalovirus (n = 3), Epstein-Barr virus (n = 1), adenovirus (n = 1), BK virus (n = 1), and fungal infection (n = 1) were observed in the four patients following the administration of MSCs. At a median follow-up of 21 months (range: 10-38 months), three of the four patients survived although one patient died of relapse of leukemia. The serum concentrations of the interleukins (IL)-6, interferon (IFN)g and Flt-3L were significantly elevated in all four patients before steroid therapy. Although Flt-3L level was decreased after steroid therapy in all the patients, IL-6 and IFNg were kept in high in two patients with liver GVHD grade 4. Serum level of IL-6 and IFNg were significantly decreased on day7 and day14 after MSCs therapy in both patients concomitant with 52.6-54.1% and 81.7-86.5% reduction of total bilirubin level on day7 and day14 respectively. Conclusion: This study supports the safety and efficacy of PL-expanded MSCs in the treatment of steroid-resistant GVHD in children and young adults. Reduction of inflammatory cytokines such as IL-6 and IFNg on day7 after MSCs infusion and observation of clinical response within 7days suggested the immunomodulatory effects of MSCs therapy on activated T lymphocytes as therapeutic mechanism of MSCs for acute GVHD rather than differentiation potential and regenerative effects of MSCs on damaged tissues. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Pearson syndrome (PS) was originally reported as a sideroblastic anemia in infancy with vacuolization of marrow precursors and exocrine pancreas dysfunction. It is now recognized as a fatal multisystem mitochondrial disorder caused by single mitochondrial DNA deletions (SLSMDs) presenting with anemia. PS, Kearns-Sayre Syndrome (KSS) and progressive external ophthalmoplegia (PEO) form a continuous spectrum of disease associated with SLSMDs. There have been only a few systematic studies on PS. Methods: We retrospectively reviewed hematological features and clinical course of 25 children with PS diagnosed between 1987 and 2019. Results: Patients presented with normo/macrocytic transfusion-dependent anemia (n=25), failure to thrive (n=3), diarrhea (n=1), acidosis (n=1) and/or omphalocele/esophageal atresia (n=1) at a median age of 5 (0-31) months. A median hemoglobin, platelet count and neutrophil count were 6.5 (1.9-9.8) g/dl, 104 (31-300) G/L, and 0.9 (0.1-2.4) G/L, respectively. Bone marrow (n=24) was normo- (n=15) or hypocellular (n=9). Vacuoles in erythroid and myeloid precursors were observed in all patients, but ring sideroblasts were present in only 16 of 23 patients examined. Morphology can resemble Diamond-Blackfan anemia (DBA) because of erythroid hypoplasia (15/21). Dysplastic features are often observed including micromegakaryocytes. Lactic acid was elevated in most patients examined (14/18). Exocrine pancreas insufficiency at diagnosis was documented in 5 patients only. The detection of SLSMDs confirmed the diagnosis of PS in all patients. The median age at the time of the last follow-up was 47 (7 - 183) months. Among 11 patients with hematological follow-up for more than 3 years after diagnosis, 8 had spontaneous resolution of anemia at a median age of 28 (12-67) months, and 3 died at the age of 3, 6 or 8 years without hematological recovery. Clinical course was highly heterogeneous and various organ dysfunctions appeared. Renal tubulopathy/Fanconi syndrome (n=7) and cardiomyopathy/arrhythmia (n=5) were often fatal complications which developed at the median age of 32 and 45 months, respectively. Failure to thrive/short stature (n=13) and muscle hypotonia (n=9) were commonly observed. Other complications included pancreas insufficiency (n=7), liver dysfunction (n=4), endocrine dysfunctions (n=7), hearing loss (n=1), ophthalmoplegia (n=1), retinitis pigmentosa (n=1), cataract (n=1), ataxia (n=2) and encephalopathy (n=1). Thirteen patients died of acute metabolic acidosis with/without other complications (n=7), arrhythmia (n=2), respiratory failure (n=3) and liver/renal failure (n=1) at the median age of 50 (14-183) months. Two patients developed KSS and PEO-like phenotypes at the age 92 months and 19 months, respectively. Summary: Anemia is generally the only presenting syndrome of PS. While the bone marrow morphology can resemble DBA or myelodysplastic syndrome, recognition of vacuolated myeloid/erythroid precursors lead to the correct diagnosis of PS in all cases. Other classical signs of PS, ring-sideroblasts and pancreas insufficiency, are often missing. Anemia spontaneously resolves in most patients surviving early childhood. However, PS is unexceptionally fatal (Figure), most patients succumb to metabolic acidosis and various forms of multi-organ failure. Since there is no effective therapy, the diagnosis of PS is one of the saddest news that pediatric hematologists have to break to parents of an anemic infant. Figure Disclosures Niemeyer: Celgene: Consultancy.

Description: Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm (MPN) that occurs during childhood and has a poor prognosis. Somatic or germline mutations in canonical RAS pathway genes, i.e., PTPN11, NF1, NRAS, KRAS, and CBL, are reported be detected in approximately 85% patients. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for JMML. Although spontaneous remission is occasionally observed in others with supportive therapy, some patients show aggressive disease progression despite HSCT. Recent studies have identified several additional genetic events in an array of genes, including SETBP1 and JAK3, but the relationship between genetic alterations and clinical outcomes remains unclear. Patients and Methods: A total of 131 patients (88 boys, 43 girls) with JMML were enrolled in the study. The median age was 15 months (range, 1-160 months). Eighty-two of 131 patients underwent HSCT, and 36 patients died (disease related, n = 27, transplantation-related complications, n = 16, infection, n = 5, unknown, n = 3). We performed comprehensive genetic analyses of the 131 JMML patients using whole-exome sequencing (n = 68, 52%) or targeted deep sequencing (n = 92, 70%), and assessed the impact of genetic alterations on clinical outcomes in 119 patients, excluding 12 patients with Noonan syndrome-related myeloproliferative disorder (NS/MPD). Results: We identified canonical RAS pathway gene mutations in 115 of 131 patients (88%). Although most RAS pathway mutations were mutually exclusive, coexisting secondary RAS pathway mutations were found in nine patients (8%). In addition, 28 patients harbored secondary genetic alterations in other genes, including SETBP1 (n = 10), JAK3 (n = 12), ASXL1 (n = 6), SH3BP1 (n = 1), RRAS2 (n = 2), and SOS1 (n = 3). In total, 34 of 131 patients harbored secondary genetic mutations. In univariate analysis, patients with secondary genetic mutations showed poorer survival rates than patients without these mutations [5-year transplantation-free survival (TFS) (95% CI) = 8.8% (2.3%-21.1%) vs. 24.1% (15.2%-34.1%), p = 0.007; 5-year overall survival (OS) (95% CI) = 49.6% (32.0%-65.0%) vs. 62.3% (50.8%-71.8%), p = 0.135]. On the basis of the dominant canonical RAS pathway mutations classification, patients with PTPN11 and NF1 mutations were significantly associated with the presence of secondary genetic mutations compared to patients with other RAS pathway gene mutations (PTPN11 (20 of 43, 47%), NF1 (5 of 7, 71%), NRAS (2 of 18, 11%), KRAS (4 of 20, 20%), CBL (1 of 17, 6%), p 〈 0.001). Consistent with previous reports, patients with PTPN11 and NF1 mutations had inferior survival rates than other JMML patients [5-year TFS (95% CI) = 0% vs. 32.7% (21.5%-44.3%), p 〈 0.001; 5-year OS (95% CI) = 45.3% (31.1%-58.5%) vs. 68.1% (55.2%-78.0%), p = 0.006]. Multivariate survival analysis identified the RAS pathway mutations (i.e., patients with PTPN11 and NF1 mutations vs. others) [TFS: HR (95% CI) = 3.732 (2.382-5.847), p 〈 0.001; OS: HR (95% CI) = 1.983 (1.117-3.521), p 〈 0.019] and low platelet count (

Description: Purpose Next-generation sequencing (NGS)-based monitoring of minimal residual disease (MRD) was developed to increase the sensitivity and specificity of standard MRD detection methods. However, few published studies have tested the clinical utility of this novel technique. We assessed the clinical utility of NGS-MRD in a uniformly treated cohort of patients with pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). PATIENTS AND METHODS We enrolled 79 unselected patients with pediatric BCP-ALL. Bone marrow samples were collected at the time of diagnosis, on days 33 and 80, at pre- and post-maintenance therapy time points (4-5 and 24 months, respectively), and upon relapse. Genomic DNA was extracted from frozen bone marrow mononuclear cells at each time point. We used diagnostic samples to define the immunoglobulin heavy chain (IGH), complementarity-determining region 3 (CDR3), and T-cell receptor gamma chain (TCRG) loci. From these samples, we detected leukemia-specific CDR3 sequences in 〉5.0% of all sequence reads. In addition, we performed a multiplex polymerase chain reaction (PCR) to determine the IGH, CDR3, and TCRG loci and subsequently assessed MRD using NGS. The result was considered positive for NGS-MRD if the leukemia-specific CDR3 sequence was detected. The resulting positive MRD values were categorized as "low positive" (

Description: Introduction Paraneoplastic pemphigus (PNP), an autoimmune disorder that can arise from malignancies, is fatal when complicated with bronchiolitis obliterans (BO), similar to that in patients receiving hematopoietic stem cell transplantation (HSCT). Autoantibodies to the desmosome proteins envoplakin and periplakin of epithelial cell adhesion molecules are essential for diagnosing PNP. BO after HSCT is an unfavorable complication that develops in association with chronic graft-versus-host disease (cGVHD). The effectiveness of rituximab on BO has been reported, which suggests that BO after HSCT is associated with autoimmunity similar to PNP. Here we analyzed anti-envoplakin and anti-periplakin antibodies that are associated with PNP in patients with and without BO complications who developed cGVHD after HSCT. Patients and Methods We analyzed anti-envoplakin and anti-periplakin antibodies in 21 patients (11 males and 10 females) with (n = 10) or without (n = 11) BO complications who developed cGVHD following HSCT between 1988 and 2015 at our institution. The median age at the time of HSCT in patients with and without BO was 10.8 (range, 1-21) and 7.5 (range, 1-14) years, respectively. The median duration from HSCT to BO onset was 261.6 (range, 41-825) days. We analyzed anti-envoplakin and anti-periplakin antibodies in cryopreserved sera collected before and after BO or cGVHD onset using immunoprecipitation (IPP) and enzyme-linked immunosorbent assay (ELISA) for detecting both autoantibodies. Longitudinal sera were obtained from a patient with cGVHD. Fifty-eight serum samples were collected from 21 patients. Biotinylated recombinant periplakin and envoplakin were produced from cDNAs using the transcription and translation (TnT) T7 Quick Coupled Transcription/Translation System (Promega) with rabbit reticulocyte lysates. IPP and ELISA were performed using in vitro TnT products as previously reported (Muro Y, et al. Rheumatology 2012;51:1508-13). Results On SDS-polyacrylamide gel electrophoresis, biotinylated recombinant periplakin and envoplakin showed protein bands with predicted size of 190 kDa and 210 kDa, respectively. The serum samples from the PNP patient and normal control serum were examined in an ELISA titration study. These titration curves showed the possibility of quantitatively measuring these autoantibodies. Both anti-envoplakin and anti-periplakin antibodies were positive in two of 10 patients with BO at BO onset but negative in all 11 patients without BO during cGVHD. In one of the two patients with plakin family autoantibodies, titers of anti-periplakin and anti-envoplakin antibodies gradually decreased after administering rituximab five times and repeating plasmapheresis seven times and subsequently decreased to below cut-off level. Although oxygenation substantially decreased consistent with the clearance of autoantibodies, the patient died of invasive pulmonary aspergillosis during steroid tapering. In the other patient, plakin family autoantibodies in the sera became negative after administering rituximab nine times. The patient is alive, and the spirometry findings were normal 6 years after the clearance of both autoantibodies. Conclusions Our findings indicate that plakin family autoantibodies may participate in BO onset in some patients receiving HSCT. The detection of these autoantibodies in patients with BO may be a good biomarker of cGVHD, providing a rationale and target for repeated rituximab therapy. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children. Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with 〉0.005% PNH granulocytes or 〉0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA. Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH. Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Description: Key Points Targetable ALK/ROS1 tyrosine kinase fusions were detected in JMML patients without canonical RAS pathway mutations. Genome-wide methylation analysis identified the hypermethylation profile associated with poor clinical outcome.