ALBERT

Description: 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

Description: 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 (

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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