Search

Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and a susceptibility to leukemia. There are at least 8 complementation groups (A through H). Extensive analyses of the FA group C gene FANCC in Western countries revealed that 10% to 15% of FA patients have mutations of this gene. The most common mutation is IVS4 + 4 A to T (IVS4), a splice mutation in intron 4, which has been found only in patients of Ashkenazi Jewish ancestry. When we screened 29 Japanese patients (20 unrelated patients and 4 families) using polymerase chain reaction-single strand conformation polymorphism, we found 8 unrelated patients homozygous for IVS4. This is apparently the first non-Ashkenazi-Jewish population for whom this mutation has been detected. The Ashkenazi Jewish patients homozygous for IVS4 have a severe phenotype, in comparison with other FA patients. Our analyses of Japanese patients indicate no significant difference between IVS4 homozygotes and other patients with regard to severity of a clinical phenotype. Thus, ethnic background may have a significant effect on a clinical phenotype in FA patients carrying the same mutation. (Blood. 2000;95:1493-1498)

Fanconi anemia (FA) is a severe hereditary disorder with defective DNA damage response and repair. It is characterized by phenotypes including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. Recent studies in mice have suggested that the FA proteins might counteract aldehyde-induced genotoxicity in hematopoietic stem cells. Nearly half of the Japanese population carries a dominant negative allele (rs671) of the aldehyde-catalyzing enzyme ALDH2 (acetaldehyde dehydrogenase 2), providing an opportunity to test this hypothesis in humans. We examined 64 Japanese FA patients, and found that the ALDH2 variant is associated with accelerated progression of BMF, while birth weight or the number of physical abnormalities was not affected. Moreover, malformations at some specific anatomic locations were observed more frequently in ALDH2-deficient patients. Our current data indicate that the level of ALDH2 activity impacts pathogenesis in FA, suggesting the possibility of a novel therapeutic approach., 「アルデヒド分解酵素遺伝子」ALDH2が解き明かす難病の謎. 京都大学プレスリリース. 2013-09-13

Survival after stem cell transplantation (SCT) in children with acquired bone marrow failure (aBMF) has improved over decades. However, we have experienced a certain number of patients who presented with bone marrow aplasia with full donor chimerism after SCT, especially in the last decade. We named the complication “donor-type aplasia”, and now, this is one of the main causes of treatment failure after SCT in children with aBMF. Because fludarabine (FLU)-based conditioning regimen has been often used for Japanese children with aBMF since the 2000s, use of FLU was suspected to associate with donor-type aplasia. On the other hand, when FLU was introduced in the regimen for children with aBMF, the dose of cyclophosphamide (CY) was reduced by half, to reduce the toxicity. Given these, we previously investigated the risk factors for donor-type aplasia, and reported that the dose reduction of CY rather than the use of FLU in itself was relevant to the recent increase of donor-type aplasia (Yoshida N, et al. ASH 2012). To reduce the risk for donor-type aplasia, the conditioning regimen for children with aBMF needs to be reconsidered. The purpose of the present study is to evaluate the outcomes of SCT using FLU/melphalan (MEL)-based conditioning instead of the standard FLU/CY-based regimen in children with aBMF. We retrospectively reviewed the clinical data of 488 patients ( The 5-year overall survival and event free survival of all patients who received the FLU/MEL-based regimen was 88%. Engraftment was achieved in 27 out of 28 patients (96%) and secondary graft failure including donor-type aplasia was not observed. Regarding the MEL dose, the favorable survival in patients who received 120 mg/m2 or more was observed (100% vs. 62%; P=0.006). Notably, all patients who received bone marrow transplantation (BMT) were alive without any complication. We then compared the outcomes in the setting of BMT with the FLU/MEL-based regimen (n=19) to those with the FLU/CY-based regimen (n=219). The event free survival was inferior in patients treated with the FLU/CY-based regimen (85% vs. 100%), although this difference was not statistically significant. With the FLU/CY-based regimen, engraftment was achieved in 214 patients (98%), whereas secondary graft failure including donor-type aplasia was seen in 18 patients. In conclusion, the FLU/MEL-based conditioning regimen provided excellent outcomes especially in the setting of BMT. To validate whether this regimen can reduce the risk of donor-type aplasia, a larger study is necessary. Therefore, a prospective study on SCT with this conditioning for aBMF children with high risk of donor-type aplasia is now planned by the Japan Childhood Aplastic Anemia Study Group. Disclosures No relevant conflicts of interest to declare.

Background: Dyskeratosis congenita (DKC) is an inherited bone marrow failure syndrome typified by reticulated skin pigmentation, nails dystrophy, and mucosal leukoplakia. Hoyeraal-Hreidarsson syndrome (HHS) is considered to be a severe form of DKC. Unconventional forms of DKC, which develop slowly in adulthood without physical anomalies characteristic to DKC, have been reported. Clinical and genetic features of DKC have been investigated in Caucasian, Black, and Hispanic populations, but never in Asian populations. Therefore, the present study aimed to determine the clinical and genetic features of DKC, HHS, and cryptic DKC among Japanese patients. Methods: We analyzed 16 patients diagnosed with DKC, 3 patients with HHS, and 21 patients with cryptic DKC between 2003 and 2014 in Japan. Telomere length was measured by Southern blot and/or flow-fluorescence in situ hybridization methods. Mutation analyses were performed using direct sequencing for DKC1, TERC, TERT, NOP10, NHP2, and TINF2. In some patients, we also analyzed the exon sequence and genome copy number using a next-generation sequencer. Results: Age at diagnosis was significantly older in the following order: HHS, DKC, and cryptic DKC (p Conclusions: The present study is the first to address DKC, HHS, and cryptic DKC in Japanese people, an Asian race. We found marked reductions in platelet counts in DKC patients in blood tests at diagnosis, a high prevalence of TINF2 mutations as the causative genetic mutation, and the existence of DKC patients with large deletions in the TERT gene and cryptic DKC patients with RTEL1 mutations on both alleles. Disclosures No relevant conflicts of interest to declare.

Introduction: Anthracyclines (Atc) are the key drugs in the treatment of acute promyelocytic leukemia (APL) combined with all-trans retinoic acid (ATRA), however, dose-dependent risk of long-term cardiac toxicity is sometimes problematic especially in pediatric patients. To evaluate efficacy of the treatment with reduced cumulative doses of Atc in childhood APL, we conducted a nationwide non-randomized prospective study, AML-P05, in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Patients & Methods: Between April 2006 and March 2011, forty-six children with newly diagnosed APL were enrolled in this study. All patients received at least 3-day precedence of ATRA monotherapy (45mg/m2/d for 35 days in total), followed by chemotherapeutic agents consisted of cytarabine (200mg/m2/d for 7 days) and daunorubicin (DNR, 45mg/m2/d for 3 days) as the first induction therapy. The second induction therapy and subsequent 3 courses of consolidation therapy consisted of ATRA, either high- or intermediate-dose of cytarabine, triple intrathecal injection, and single dose of Atc; 10mg/m2 of mitoxantrone (MIT) in the first 2 courses and 45mg/m2 of pirarubicin (THP) in the last 2 courses. Finally, patients received one-year maintenance therapy with ATRA for 15 days every 3 months. Results: Among the 46 patients registered in this study, 3 were excluded because of PML/RARA negativity, and the remaining 43 patients including 2 with molecular variants were evaluated. The median age at diagnosis was 9 years (range, 11 months to 15 years old). The median follow up period was 4.47 years (0.00-7.45 years). FLT3-ITD was positive in 6 out of 40 patients examined. Two patients died during induction therapies; one of coagulopathy in first course and the other of infection in second course. Two patients developed differentiation syndrome but resolved with temporary cessation of ATRA and supportive therapies. Three patients did not achieve complete remission (CR) after 2 courses of induction therapy, and overall CR rate was 85.7% (36/42, 95%CI: 71.5-94.6%). Neither cardiac adverse events nor treatment-related death were observed during consolidation and maintenance therapies. Three patients exhibited relapse during or after maintenance therapy, and another one developed secondary acute myeloid leukemia. Consequently, the 3-year event-free (EFS) and overall survival rate were 83.6% (95%CI: 68.6-91.8%) and 90.7% (95%CI: 77.1-96.4%), respectively. Age less than 4 years old at diagnosis and non-M1 marrow after first induction therapy were associated with lower EFS. High WBC count (>10,000/μL), low platelet count ( Conclusions: According to the JPLSG criteria of Atc equivalents, cumulative Atc dose in this study was converted to 246mg/m2 of doxorubicin (for 1:0.83 for DNR, 1:4 for MIT, and 1:0.6 for THP). The dose was much lower than that in recent US or European protocols, especially in those designed primarily for adult APL patients where more than 600mg/m2 of Atc were used. Comparing with these studies, we have achieved equivalent good results with minimum does of Atc. Therefore, single administration of Atc in each consolidation phases seems sufficient in the treatment of childhood APL. This trial is registered with UMIN Clinical Trials Registry (UMIN-CTR, URL: http://www.umin.ac.jp/ctr/index.htm), number UMIN000000645 Disclosures No relevant conflicts of interest to declare.

To the editor: Capillary leak syndrome (CLS) is a severe complication of allogeneic stem cell transplantation (SCT) characterized by weight gain, generalized edema, hypotension, and hypoalbuminemia.[1][1] The main CLS pathogenesis is injury of the capillary endothelium resulting in a loss of

Abstract 648 Introduction Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination. Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes. It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussion In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study. To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway. To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues. Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. Disclosures: No relevant conflicts of interest to declare.

Abstract 2583 Background: The 2008 WHO classification expanded the category of acute myeloid leukemia (AML) with multilineage dysplasia of the 2001 WHO classification into AML with myelodysplasia-related changes (AML-MRC). Although AML with multilineage dysplasia (AML-MLD) is diagnosed based on morphological features alone, AML-MRC includes AML with a previous history of myelodysplastic syndrome or myeloproliferative neoplasms and AML with a myelodysplasia-related cytogenetic abnormality as well as AML-MLD. Since this category of AML has been believed to be rare in children, its clinical impact on children remains unknown. We developed a comprehensive and prospective central review system in a clinical trial for pediatric AML, in which unexpected high incidence of AML-MRC was found. Thus, we evaluated the clinical characteristics and prognostic significance of AML-MRC in children. Patients & Methods: JPLSG AML-05, registered at http://www.umin.ac.jp/ctr/as UMIN000000511, is a nation-wide multi-institutional study for children (age Results: Among 447 eligible patients recruited, a total of 93 patients (20.8%) were diagnosed as AML-MRC; 34 from morphological features, 65 from myelodysplasia-related cytogenetic abnormalities and six from the both. The cases with AML-MRC included 48 boys and 45 girls with a median age of 3 years (range; 0–17 years). Ten patients with AML-MRC had FLT3-internal tandem duplication (ITD). Three-year probability of event-free survival (3y-pEFS) and 3-year probability of overall survival (3y-pOS) at the 93 patients was 37.1% (95%CI, 26.9 – 47.3%) and 56.8% (95%CI, 45.2 – 66.8%), respectively. The median follow-up of alive patients as of May 2012 was 3.4 years (range;1.3–5.3 years). Comparison of the clinical outcome of AML-MRC with AML, not otherwise specified (AML-NOS)(n=111), showed that AML-MRC had a lower complete remission rate after two induction courses(67.7% vs 85.6%, p Conclusions: Our study suggests that AML-MRC is not rare in children. AML-MRC had adverse prognostic significance in children partly due to the low induction rate. Induction chemotherapy awaits further improvement for children with AML-MRC. Disclosures: No relevant conflicts of interest to declare.

Abstract 4899 [Background] The WHO classification has been widely accepted among physicians who are engaged in treating pediatric AML patients. In 2008, the revised WHO classification has expanded the two categories in AML; AML with recurrent genetic abnormalities and AML with myelodysplasia-related changes. The epidemiology and prognostic significance of these refined categories remains to be explored in children. [Methods] JPLSG AML-05 is a nationwide clinical trial for children with de novo AML, excluding acute promyelocytic leukemia and myeloid leukemia with Down syndrome, which was conducted between November 2006 and December 2010 in Japan. A central review of diagnosis based on the WHO classification was prospectively performed on each case soon after morphological, cytogenetical and immunological data were submitted to data center. Regarding the cases with discrepant results among these parameters, further diagnostic tests including FISH and chimera gene analyses were underwent to confirm the diagnoses. [Results] Four hundred and eighty four patients were enrolled in the study. Thirty patients did not meet the criteria of AML. We could not collected suitable data for diagnosis in 6 patients. Regarding the rest 448 patients, diagnoses based on the WHO classification 2001 and 2008 were determined. According to the 2001 version, 227 (50.6%) had AML with recurrent genetic abnormalities:124 (27.7%) of AML with t(8;21)(q22;q22);(AML1/ETO ), 32 (7.1%) of AML with inv(16)(p13q22); (CBFβ/MYH11), 38 (8.5%) of AML with t(9;11)(p22;q23), and 33 (7.4%) of AML with the other11q23 (MLL) abnormalities, 36 (8.0%) had AML with multilineage dysplasia, and 185 (41.3%) had AML, not otherwise categorized. According to 2008 version, 235 (52.5%) had AML with recurrent genetic abnormalities: 124 (27.7%) of t(8;21)(q22;q22);(AML1/ETO ), 32 (7.1%) of AML with inv(16)(p13q22); (CBFβ/MYH11), 38 (8.5%) of AML with t(9;11)(p22;q23), 33 (7.4%) of AML with the other11q23 (MLL) abnormalities,4 of AML with t(6;9)(p23;q34);DEK-NUP214,2 of AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI13, and 2 of AML with t(1;22)(p13;q13);RBM15-MKL, 88 (19.6.7%) had AML with myelodysplasia-related changes (29 from morphological features of myelodysplasia and 59 from myelodysplasia-related cytogenetic abnormalities), 119 (26.6%) had AML, not otherwise categorized and 7(1.6%) had mixed phenotype acute leukemia (6 of T/myeloid and 1 of B/myeloid). [Discussion] Our comprehensive approach for diagnosis was a useful modality for precise diagnosis of uncertain cases, which might have been assigned to the category of AML, with not otherwise categorized, previously. As a result, the present study shows an increased prevalence of AML with recurrent genetic abnormalities or AML with myeloid dysplasia-related changes among pediatric patients with previously untreated AML. Analysis of the AML-05 trial will elucidate the prognostic impact of these categories. Disclosures: No relevant conflicts of interest to declare.

Abstract 1074 Introduction: Transient leukemia (TL), which is also referred to as transient myeloproliferative disorder or transient abnormal myelopoiesis, occurs in approximately 10% of infants with Down syndrome (DS). This disorder is characterized by the appearance of blast cells with megakaryoblastic and/or erythroblastic characteristics in the peripheral blood. While most TL patients have a favorable clinical course and the blast cells disappear spontaneously, vital organ failure and early death occurs in some patients. In this group of patients, liver failure with hepatic fibrosis and cardiopulmonary failure are the major causes of death. Recently, low-dose cytosine arabinoside (Ara-C) therapy has been reported to be effective for improving the clinical outcome in TL patients with severe liver or cardiopulmonary disease. However, because the disease resolves spontaneously in most instances, it is not generally recommended that all patients receive antileukemic therapy. It is unknown whether the survival rate of certain TL patients could be improved by low-dose Ara-C. This study was conducted to clarify the safety and efficacy of low-dose Ara-C therapy for TL patients with DS. Patients and Methods: A retrospective questionnaire survey of patients diagnosed with DS between 2003 and 2009 was conducted to identify all DS neonates with TL. The eligibility criteria for the analysis were infants with DS who were younger than 3 months and had circulating blast cells in the peripheral blood. A total of 153 patients (81 male, 72 female) were identified. Results: Thirty-four of the 153 patients (22%) died. Early death occurred in 31 of 153 patients (20%); the median age at death was 47 days (range, 9–241 days). The covariates that were significantly correlated with early death were examined further. On univariate analysis, the following covariates were identified: early estimated gestational age, WBC ≥ 100 × 109/L, severe bleeding, and anasarca. On multivariate analysis, it was confirmed that WBC ≥ 100 × 109/L and anasarca were independent risk factors for early death. Twenty of the 121 patients (16.5%) who survived for more than 9 months after birth subsequently developed acute myeloid leukemia at a median age of 16 months (range, 3–45 months). All 21 patients were diagnosed as having acute megakaryoblastic leukemia (AMKL) and received low intensity chemotherapy specific for AMKL in DS patients. Eighteen of 21 (86%) patients achieved a complete remission, and they are alive and well; whereas 3 patients who had refractory or relapsed leukemia died. Twenty-eight of the 153 patients (18%) received low-dose Ara-C therapy. The median dose and duration of Ara-C were 0.95 mg/kg/day (range, 0.4–3.1 mg/kg/day) and 7 days (range, 2–15 days), respectively. The median duration of neutropenia ( While the median WBC count of early treated patients was significantly higher than that of other patients (133.5 × 109/L; range, 16.0–356.9 □L 109/L vs 31.9 × 109/L; range, 4.4–341.5 × 109/L.; p < 0.001), the probability of 1-year overall survival of patients who received low-dose Ara-C within 10 days after the initial diagnosis of TL was similar to that of patients who received Ara-C later or who did not receive Ara-C (69.4 ± 12.9%; n = 16 vs. 79.7 ± 3.5%; n = 137; p = 0.614). By subgroup analysis of patients with WBC ≥ 100 × 109/L, the probability of 1-year overall survival of patients who received early Ara-C treatment was significantly higher than that of others (66.1 ± 13.9%; n = 13 vs. 33.3 ± 8.6%; n = 30; p = 0.035). Conclusion: In summary, we found that low-dose Ara-C therapy for patients with TL and DS has a tolerable toxicity profile, and early intervention with this therapy could improve the clinical outcome of patients whose WBC count exceeds 100 × 109/L. We plan to confirm the efficacy of low dose Ara-C treatment in a prospective clinical trial for high-risk patients with DS and TL. Disclosures: No relevant conflicts of interest to declare.

Abstract 4833 Diagnosis in accordance with the WHO classification provides important information for prognosis and risk stratification in the clinical trials of therapy for AML. However, each of the diagnostic parameter contained in the WHO classification, such as morphologic, cytochemical, immunophenotypic, cytogenetic, and genetic features, requires standardization and accuracy to ensure the validity of such clinical trials. In addition, discrepancies among the results of these parameters are encountered in some patients. Based on these considerations, we attempted to develop a comprehensive and rapid system for the central review of AML diagnosis in a nationwide clinical trial conducted by a multi-institutional cooperative study group. JPLSG AML-05 is a study that was conducted on children with de novo AML, excluding APL and myeloid leukemia associated with Down syndrome. Morphological diagnosis and flowcytometric immunophenotyping were performed through central laboratory systems. Cytogenetic analyses were carried out in regional laboratories, and the reports were reviewed centrally. Expression of chimeric genes including AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF9, MLL-AF6, MLL-ELL, FUS-ERG, and NUP98-HOXA9, and mutations of FLT3-ITD were also examined for all patients at a single laboratory. In case where cellular bone marrow aspirate smears could not be obtained, we recommended bone marrow biopsy, for which the histology was subjected to central pathologic review. When all the data had been submitted to our data center, a central review of the diagnosis based on the 2001 edition of the WHO classification was performed for each individual case by multidisciplinary conferencing based on electronic mail discussion. Between November 2006 and July 2010, the diagnoses of 383 patients enrolled in AML-05 were reviewed. The diagnosis was undetermined for seven patients because of inadequate submission. Nine patients did not meet the inclusion criteria for AML-05. Among the remaining 367 patients, 184 (48.8%) had AML with recurrent abnormalities, 32 (8.7%) had AML with multilineage dysplasia (AML-MLD), and 151 (41.1%) had AML, not otherwise categorized. Among the 184 patients with AML with recurrent abnormalities, 104 had AML with t(8;21)(q22;q22)/AML1-ETO. A higher prevalence of AML with t(8;21)(q22;q22)/AML1-ETO in our study, compared with those in previous reports from European countries, suggests a difference in genetic background. Two patients with t(8;21)(q22;q22)/AML1-ETO and one patient with inv(16)(p13q22) were assigned to each category through FISH analysis, because their cytogenetic analyses indicated a normal karyotype or suboptimal quality. Among 55 patients who were suspected of having AML with 11q23 (MLL) abnormalities by chromosome or chimeric gene screening tests, 30 had concordant results between chromosomes and chimeric genes. The remaining 25 patients were assigned to AML with 11q23 (MLL) abnormalities by either FISH for MLL or other chimeric gene tests involving MLL (MLL-AF10, MLL-ENL,MLL-AVI1 or MLL-TET1). The prevalence of AML-MLD in this study was higher than that (2.6%) in a previous report for Japanese children with AML, where a central review system of morphology had not been established (Adachi S., et al. Int J Hematol 86;358-63,2007). Patients with AML-MLD had a higher frequency of unfavorable cytogenetics (6/32) and/or FLT3-ITD mutations (7/32) in comparison with other types of AML. One patient with acute megakaryoblastic leukemia was diagnosed on the basis of bone marrow histology in our central pathologic review because the percentage of blasts on the smear did not meet the criteria for AML. We have successfully developed a rapid system for the integrated central review of AML diagnosis based on the WHO classification, which will enable us to evaluate trial data on the basis of standardized diagnosis and risk classification. Disclosures: No relevant conflicts of interest to declare.

Abstract 4292 INTRODUCTION Radiation induced cavernous hemangioma (RICH) is a late complication of cerebral radiation therapy. An increased number of long term surviving blood and marrow transplantation (BMT) recipients have recovered from their primary disease but are at risk of RICH. METHODS We investigated 66 patients who underwent BMT during childhood or adolescence. We evaluated RICH numbers, size, location and their annual changes. Furthermore we developed scoring system of RICH in order to classify severity. MRI of the brain was performed annually for 5 to 27 years after BMT, including gradient-echo sequence (T2* weighted image). RICH SCORE 1-4 is designated as mild, 5-9 as moderate and 10 or more as severe. RESULTS Twenty-five patients (37.9%) was diagnosed RICH. The age at the time of the diagnosis was 11-40 years old (median 27 years old). The age at the time of BMT was 1-22 years old (median 9 years old). The period from BMT to diagnosis was 10-24 years (median 16 years). All cases received TBI as conditioning of BMT and/or cranial radiation (CR) prior to BMT as treatment of primary disease. RICH was found in 25/48 (52%) who received TBI and/or CR, and was not found in any of 18 patients without radiation therapy to the brain. Total dose to the brain was 10-36 Gy. Clinical manifestations were present only in four cases. RICH SCORE ranged 1-18 points (median 4 points). Small RICHs tended to be recognized only by T2* weighted image, but not by routine imaging methods. Classification of the severity was mild in 13 patients, moderate in 8 patients and severe in 4 patients. Severity was correlated with higher radiation dose and/or with younger ages at transplantation. RICH SCORE increased yearly in 7 of 25 patients. One case developed giant RICH more than 40mm as shown in the attached image. CONCLUSION High incidence of RICH was found in long term survivors who underwent BMT with radiation therapy. Since all of those patients did not show RICH before BMT and all positive patients had a history of radiation therapy to the brain, the cause of RICH in those patients was considered to be radiation. Careful and long term evaluation with MRI including T2* weighted image is necessary in BMT recipients who received radiation therapy prior and/or during BMT. Disclosures: Ando: Alexion: Research Funding.

The transfer of antigen-specific cellular immunity in human bone marrow transplantation (BMT) was studied in 49 donor-recipient pairs, using a varicella-zoster-virus (VZV) specific lymphoproliferative response (LPR) assay. Posttransplant VZV-LPR could be serially measured in 31 long-term surviving recipients. VZV-specific T-cell immunity was detected in the early posttransplant period in 4 of 16 recipients who were, and whose donors were, immune to VZV before BMT, but two of those positive responses diminished in the first 100 days posttransplant. No positive response was detected in the immediate posttransplant period when either only the recipient or the donor was immune to VZV pretransplant. Herpes zoster or chickenpox developed in the recipients depending on a history of pretransplant VZV infection when the VZV-LPR became negative, and recovery from VZV infection was always followed by quick conversion of VZV-LPR. Long-lasting positive VZV-LPR was observed in the two recipients who experienced VZV infection in the immediate pretransplant period and received marrow graft from an immune donor. Our results indicate that a simple or direct transfer of VZV-specific cellular immunity from a marrow donor to a recipient cannot be expected in usual clinical bone marrow transplantation and that there might be a collaboration or recruitment of immune responses involving both donor and recipient that permits the VZV-LPR to remain positive posttransplant.

Background The role of intensive chemotherapy in treatment of childhood MDS has not been clarified yet. We conducted a prospective trial which employed intensive AML-type chemotherapy for children with de novo MDS. Methods Fifteen children aged 1 to 15 years who met diagnostic criteria of the FAB refractory anemia with excess of blasts (RAEB) with more than 10% of blasts in the bone marrow (BM) (RAEB-II) and RAEB in transformation (RAEBT) were prospectively enrolled in the study. Abnormal karyotype was documented in 11 patients including 3 with monosomy 7, 2 with complex karyotype, 1 with trisomy 8. Induction therapy consisted of etoposide (150mg/m2, on days 1–5), cytarabine (200mg/m2/12hr, on days 6–12), and mitoxantrone (5mg/m2, on days 6–10) in all but 1 patient, who received another regimen of AML induction therapy. The use of granulocyte-colony stimulating factor was allowed. Allogeneic stem cell transplantation (SCT) was scheduled after remission induction therapy. The median follow-up for 13 surviving patients was 685 days (58 to 1,476 days). Findings Eleven patients (73%) including 3 with monosomy 7 achieved a complete remission after induction therapy at a median of 40 days (31 to 61 days). Remission was not obtained in the other 4 patients. There was no significant difference in the frequency and severity of toxicity between in patients with RAEB-II/RAEBT and in those with de novo AML. Allogeneic SCT was performed in 13 patients including 9 in the remission at a median of 4 months (2 to 6 months) after the beginning of the therapy. None of nine patients who underwent SCT in the remission relapsed. Although two patients died of the complications of SCT. Overall survival for all the 15 patients at 2 years was 66.8 ± 14.1% (s.e.). Interpretation AML-type induction therapy appears to be safe and effective for children with RAEB with more than 10% of blasts in the BM and RAEBT. Figure Figure

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with multiple complementation groups, characterized by genomic instability and predisposition to MDS and AML. Recent evidence indicates that multiple FA proteins are involved in DNA repair. Thus, increased genetic damage and secondary dysregulation of cell proliferation, differentiation and apoptosis are thought to play important roles in the development of bone marrow failure and subsequent progression to MDS/AML. However, little is known about molecular abnormalities responsible for these hematological disorders. Numerous studies indicated that epigenetic silencing of p15/INK4B, an inhibitor of cyclin-dependent kinases, plays an important role in the pathogenesis of MDS and AML. In the present study, we examined methylation status of 5′ CpG islands of the p15 gene in bone marrow mononuclear cells of FA patients, using methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Bone marrow samples were analyzed in 10 patients and serially studied in 4 of them. Hypermethylation of the p15 promoter region was detected in 5 patients (50%). This group included 3 patients with MDS: FA28-1 with refractory anemia (RA), FA87 with RAEB (RA with excess of blasts), and FA88 with later development of RA and progression to RAEB; whereas myelodysplasia was not observed in 2 patients (FA89, FA90). In two cases (FA88, FA90), p15 hypermethylation became negative during their courses, perhaps because of decreased myeloid cells. On the other hand, none of 5 patients without p15 hypermethylation had MDS. These results suggest that p15 hypermethylation is associated with development of MDS and occurs in the early phase of clonal evolution in the disease. Methylation status of p15 may be a useful prognostic factor of FA. Patient Age at onset (year old) Time from onset (month) Cytopenia MDS Cytogenetic abnormalities p15 methylation MSP b p15 methylation COBRA c a siblings, b MSP: methylation specific PCR, c COBRA: combined bisulfite restriction analysis, d ND: not determined FA28-1a 5 128 severe RA − − + 133 severe RA − + ++ FA87 8 252 severe RAEB + + +++ FA88 5 31 moderate − − + +++ 45 severe RA + − − 58 severe RAEB + + + FA89 5 49 mild − − + + 56 severe − − + + FA90 2 2 mild − − + ++ 31 severe − − − − FA28-2a 5 51 mild − − − NDd FA28-3a 3 12 mild − − − NDd FA47 3 15 mild − − − NDd FA68 5 46 moderate − − − NDd FA91 5 129 mild − − − NDd

An optimum conditioning regimen is indispensable in improving outcomes for patients with acquired aplastic anemia (AA) who receive bone marrow transplantation (BMT) from an alternative donor. Although high-dose total body irradiation (TBI) is effective in enhancing engraftment, intensive conditioning regimens are associated with increased regimen-related toxicity, which results in a decreased success rate. Currently, two different approaches are primarily used; a conditioning regimen including low-dose TBI and a fludarabin-based regimen without TBI. We attempt to clarify the efficacy and regimen-related toxicity of these two approaches. Ten patients (7 males, 3 females; median age 12y; range 9–17y) underwent an alternative donor transplant following a conditioning regimen with low-dose TBI (TBI group); patients received cyclophosphamide (50 mg/kg/d on days −5 to −2), antithymocyte globulin (Thymoglobulin, 2.5 mg/kg/d on days −5 to −2) and TBI (2.5 Gy x 2 on day −1). The donor was unrelated in 9 patients (3 were serologically HLA A or B antigen mismatched; 3 were serologically HLA DR antigen mismatched, 1 was both serologically and genotypically DR antigen mismatched; 1 was genotypically DRB1 mismatched; 1 was a genotypically complete match) and was a family member in 1 patient (serologically HLA A-mismatched father). Twelve patients (6 males, 6 females; median age, 11 y; range 2–20 y) received a conditioning regimen of fludarabine (25 mg/kg/d on days −5 to −2), cyclophosphamide (750 mg/m 2 /d on days −5 to −2), thymoglobulin (1.5 mg/kg/d on days −5 to −2) and thoracoabdominal irradiation (TAI, 3Gy x 1 on day −1) (TAI group). The donor was unrelated in 11 patients (8 were genotypically complete matches, and 3 were genotypically DRB1 antigen mismatched) and a family member in 1 patient (3 antigen-mismatched mother). The source of stem cells was unmanipulated bone marrow. Graft versus host disease (GVHD) prophylaxis consisted of tacrolimus (0.02 mg/kg from day −1) and short-term methotrexate. Rejection was seen in one patient in each group. The doses of infused nucleated marrow cells were less than 2.0 x 10 8 /kg in both patients, and both patients then received a second transplant and achieved full donor chimerism. Acute GVHD grade II-IV developed in 2/10 evaluable patients in the TBI group and in 3/12 patients in the TAI group. Chronic GVHD developed in 5/9 evaluable patients in TBI group and 3/10 evaluable patients in TAI group. Median follow up was 9 months (4–27 months) in the TBI group and 21 months (3–33 months) in the TAI group. Both conditioning regimens were well tolerated and effective even in HLA-mismatched unrelated donor transplantation. Long-term follow up is warranted in order to compare the late sequelae in both regimens.