ALBERT

Description: Background: Hematopoietic stem cell transplantation (HSCT) recipients carry a high risk of primary varicella and varicella-zoster virus (VZV) reactivation. VZV infections can be fatal, and VZV reactivation can be complicated by postherpetic neuralgia, which worsens the recipients’ post-HSCT quality of life. VZV vaccines may prevent such infections; however, international vaccination guidelines do not recommend VZV vaccination of HSCT recipients because few data regarding safety among HSCT recipients are available. In Japan, we use a high-titer Oka stain vaccine for varicella vaccination and zoster prevention in the elderly; the plaque forming unit (PFU) value of this vaccine is as high as the zoster vaccine used in the U.S. Data regarding the safety of zoster vaccine in HSCT recipients is limited; therefore, we report our experience with this high-titer zoster-equivalent varicella vaccine in pediatric allogeneic HSCT recipients. Patients and Methods: Forty-seven pediatric allogeneic HSCT recipients who underwent transplantation at the Saitama Children’s Medical Center from 2000-2011 were vaccinated with live vaccines and their antibody titers were evaluated after vaccination. Among the 47 recipients, the Japanese high-titer varicella vaccine was administered to 32 recipients. Since establishing the live vaccine initiation criteria in 2009, allogeneic HSCT recipients were considered as recipients for live vaccines, including the varicella vaccine, if 24 months had passed after HSCT without active chronic GVHD or required immunosuppression. In addition, recipients were required to have a lymphocyte count 〉1500/μl or CD4 cell count 〉700/μl if they were 1000/μl or CD4 cell count 〉500/μl if they were 〉6 years, normal phytohemagglutinin response, and serum IgG level 〉500 mg/dL. Before 2009, time for vaccination depended on each doctor’s judgment. Patients were given a single dose of the Biken varicella vaccine containing a minimum of 23,000 PFUs. We use EIA to measure the antibody titer and defined seropositivity as a titer 〉6.0. The time to evaluate the antibody titer was not fixed. We retrospectively collected the clinical data of recipients and laboratory data by reviewing the medical records. Results: The 32 recipients, who received the Japanese varicella vaccine, underwent HSCT at a median age of 5.15 years (range: 0.5-16.4 years) and were vaccinated at a median of 20.65 months (range: 4.8-112.1 months) after HSCT. Twenty and 11 patients had received myeloablative and nonmyeloablative conditioning, respectively. Nine patients received related-donor transplant, of which eight were bone marrow and one was peripheral blood. Twenty-three patients received unrelated-donor transplant, of which 14 were bone marrow and nine were cord blood. Antithymocyte globulin was administered to four patients. Eighteen of the 32 patients (56.3%) were seropositive after vaccination. Of the 26 patients, whose vaccination histories and pre-transplantation histories were available, eight were affected by natural disease, six had been immunized, and 12 had not been immunized. At vaccination, the median lymphocyte count was 3201/μl (range: 817-6630/μl) and the median IgG value was 880 mg/dL (range: 233-1461 mg/dL). There was no significant risk factor associated with vaccine failure. We experienced one case of varicella because of wild type VZV and no cases of zoster after vaccination during a median follow-up period of 4.8 years (range: 0.4-11.3 years); the single patient developed varicella 13 days after vaccination, immediately after an influenza virus infection. Whereas, two cases of varicella and one case of zoster developed before vaccination. Of the other 15 recipients, who did not receive varicella vaccine, three varicella and two zoster developed in three recipients. Conclusion: We safely vaccinated pediatric allogeneic HSCT recipients with the Japanese high-titer varicella vaccine. This finding could encourage the use in the U.S. of zoster vaccines such as Zostavax@, which contains similarly high PFUs, and low-titer varicella vaccines such as Varivax@. However, the efficacy of high-titer varicella vaccines for preventing VZV reactivation in this population remains unclear. Further studies are warranted to elucidate the efficacy and difference between low-titer and high-titer varicella and zoster vaccines. Disclosures No relevant conflicts of interest to declare.

Description: B cell non-Hodgkin’s lymphoma (B-NHL) consists of different pathological entities that are frequently characterized by distinct genetic alterations. However, the knowledge on these genetic lesions in B-NHL is still limited. In order to obtain a more comprehensive view of genetic lesions in B-NHL, we performed genome-wide analysis of copy number (CN) alterations as well as allelic imbalances using Affymetrix SNP arrays in 190 B-NHL cases, including 64 samples of diffuse large B-cell lymphoma (DLBCL), 62 of follicular lymphoma (FL), 64 of mucosa-associated lymphoid tissue lymphoma (MALT-L). SNP array data were analyzed with CNAG/AsCNAR software, which enabled sensitive detection of CN alterations in allele-specific manner, and thus allelic imbalances, without depending on availability of paired normal controls. Most frequent numerical abnormalities in B-NHL were gains of chromosomes 3 and 18, although gains of chromosome 3 were less prominent in FL. Chromosomal deletions that lead to loss of heterozygosity (LOH) were commonly found in 1p, 6q and 10q. However, the more chracteristic feature of B-NHL genomes was high frequency of CN netural LOH or uniparental disomy (UPD), which was found in 35 cases of DLBCL (55%), 32 cases of FL (52%) and 18 cases of MALT-L (28%). It is widely distributed in the genome, but more frequently found in 1p, 1q, 6p, 6q and 12q. High-grade amplifications and homozygous deletions frequently provide a clue to identify relevant gene targets. In our series, 12 loci of high-grade amplifications and 14 loci of homozygous deletions were identified, and helped to specify the candidate genes. These regions included, FCGR2B amplified in 5 cases of DLBCL, RERE amplified in 2 cases of FL and CDKN2A/CDKN2B deleted in 9 cases of DLBCL. The most notable finding in the current study was, however, the identification of common genomic alterations in genes that regulate activation of NFkB pathway in more than 50% of B-NHL cases. Eight lymphoma cases harbored high-grade amplification of cREL gene, and gain including cREL was detected in 28 samples (14.7%). Fourteen cases had gains or amplification of TRAF6, and another 16 cases had deletion at 10q including PTEN. These abnormalities were supposed to cause dysregulation of NFkB. Aberrant NFkB activity has long been implicated in the pathogenesis of B-NHL, and our study confirmed that dysregulation of NFkB pathway was main mechanism of lymphomagenesis, providing further rationale that he treatment against malignant lymphoma with inhibitor of NF kappa B pathway.

Description: Majority of malignant lymphoma arising from the ocular adnexae are ocular adnexal MALT lymphomas (OAL). Several genetic abnormalities, including t(14;18)(q32;q21), trisomy 18 and trisomy 3 have been reported in OAL. However, none of them are found in more than half of cases with OAL by conventional methods. High density Single Nucleotide Polymorphism (SNP) array analysis with CNAG/AsCNAR algorithm allows high-resolution and genome-wide detection of both loss of heterozygosity (LOH) and copy number abnormality, especially uniparental disomy (UPD), without depending on the availability of paired normal DNA (Yamamoto et al, Am J Hum Genet. 2007; 81:114–26). UPD is acquired by somatic recombination and therefore not detected by conventional cytogenetic analysis or array CGH. In this study we analyzed DNA from OAL for the presence of LOH with or without copy number changes. Tissue samples from patients with OAL at our institute between 1995 and 2003 (N=32) were subject to SNP-array (250K NspI) analysis with CNAG/AsCNAR algorithm. The patients included 21 males and 11 females, and the median age of 56.5 years at the time of diagnosis (range, 15–90 years). Clinical stage was I (29 cases), II (1 case), and IV (2 cases). Trisomy of 3, 18, and 21 were found in 9, 7 and 1 cases, respectively. Overall, LOH due to UPD more than one chromosomal band were found in 16 cases (50%). Recurrent one allele deletion was detected at 6q23-24, 12p13 and 17q21 (N=15, 15 and 21, respectively). In total, 28 (82%) among 32 cases showed one or more of the above changes. Characterization of these recurrent genetic abnormalities might reveal the pathogenesis of OAL.

Description: Despite the success story of tyrosine kinase inhibitors (TKIs) for the treatment of Chronic Myeloid Leukemia (CML), patients can develop resistances against the drugs. The main known causes for resistance are mutations or over-expression of the BCR/ABL fusion protein, reduced bioavailability of the drugs and activation of compensatory molecular pathways. It is hypothesized that during disease progression, genomic instability of CML cells increases, which may lead to new genomic lesions harboring additional mechanisms of resistance. In this context, we studied genomic DNA profiles of 32 Imatinib resistant CML patients with high density 250K SNP arrays (Affymetrix). Molecular allelokaryotyping for allele specific copy number and loss of heterozygosity analysis was performed with the CNAG software. Single DNA samples from 27 patients were extracted after they had acquired resistance to Imatinib or alternative TKIs such as Nilotinib or Dasatinib. DNA from 12 patients could be analyzed in sequential samples from the initial diagnosis timepoint and a second timepoint upon the emergence of TKI resistance. All patients were positive for BCR/ABL by PCR and FISH. 10 relapse patient samples had known BCR/ABL mutations of which two were T315I mutations. High density allelokaryotyping confirmed pre-existent data on unbalanced translocations, amplifications and deletions from routine cytogenetics: 5 samples displayed a genomic duplication of the BCR/ABL fusion gene, 4 samples had trisomy 8, 1 sample showed deletion of chromosome 17p, 1 sample had heterozygous deletion of chromosome 9. Apart from this, SNP array analysis revealed numerous new submicroscopic genomic lesions. After exclusion of genomic copy number polymorphisms (CNPs) by comparison to recorded CNPs in the UCSC Genome Browser (http://genome.ucsc.edu/) the following results were obtained: Two patients displayed common heterozygous microdeletions of the reciprocal ABL/BCR fusion product. Furthermore, single samples displayed heterozygous micro-deletions on chromosomes 1, 2, 10, 12, 15, 17, and 22 or microduplications on chromosomes 2,3,6, 8, 9, 11, 12, 14, 15, 22. The affected regions contained potentially interesting genes in respect to resistance to therapy such as tumor suppressor candidate MBP-1, apoptosis related protein RERE, metastasis associated gene MTA3, nuclear body associated gene SP100, alpha-T-catenin (CTNNA3), Cbl-interacting protein Sts-1 and the DNA repair associated gene RAD51. As a new genomic alteration in CML, we detected acquired uniparental disomy (UPD) in 5 samples with a common site of UPD on chromosome 19q in 2 patients. In conclusion, in 14 out of 39 TKI resistant cases, high density SNP arrays enabled us to identify submicroscopic copy number lesions and regions of UPD containing promising candidate genes, which merit further research as sites conferring TKI resistance.

Description: Abstract 276 LEF1 is a member of the LEF/TCF family of DNA-binding transcription factors, which interact with beta-catenin in the WNT signaling pathway. N-terminal LEF1 mutations that impair beta-catenin binding are commonly found inhuman sebaceous skin tumors, and expression of an N-terminal–deleted Lef1 mutant that lacks the beta-catenin binding domain leads to sebaceous skin tumors. The intracellular domain of NOTCH1 has also been shown to function as a co-activator of LEF1, leading to the up-regulation of target genes distinct from those activated by beta-catenin binding. Recently, inactivating mutations of LEF gene have been reported in T-cell acute lymphoblastic leukemia (T-ALL) cases. In this study, we analyzed the frequencies and clinical significance of LEF1 mutations in pediatric T-ALL and T-cell non-Hodgkin's lymphoma (T-NHL). Mutation analysis was performed on 138 of the primary T-ALL and T-NHL patient samples. The clinical data were available 55 newly diagnosed T-ALL and 14 T-NHL patients. These children, aged under 15 years were enrolled into the Japan Association of Childhood Leukemia Study (JACLS) protocol ALL-97 between 1997–2001 and JACLS trial NHL-T98 between 1998–2002. At the time of diagnosis, bone marrow and/or peripheral blood cells were obtained from T-ALL patients and lymph nodes and/or pleural effusions were obtained from T-NHL patients. A total of 69 patients were included in the present study; 49 were male and 20 female; 55 were children diagnosed with T-ALL (median age of 9.5 years; range: 2.0 – 15.0 years) and 14 with T-NHL (median age of 11.0 years; range: 3.7 – 15.0 years). We performed high-resolution array comparative genomic hybridization (array CGH) and sequencing was performed on the entire coding region of LEF1. High molecular weight genomic DNA was used for microarray analysis using Affymetrix GeneChip 50K XbaI, HindIII or 250K NspI, according to the manufacturer's instructions. Genome-wide detection of allelic imbalances was performed using CNAG/AsCNAR software. We identified mono or biallelic LEF1 deletions in 7.5% (5 of 67) of these primary samples. An additional 11.6% (16 of 138) of the cases harbored sequence alterations of LEF1 gene. Twelve of the single nucleotide alterations were found in exons 1, 2 and 3, and four of the frame-shift mutations were found in exons 3, 7, and 8. Frame-shift mutations were novel LEF1 mutations, and mutations in exons 3 and 8 were positioned outside catalytic domain and DNA binding region. Missense mutations in exon 1 were located highly conservative lesion of beta-catenin binding domain of LEF1. Of the 8 LEF1 alterations detected in 16 cases also had NOTCH1 mutations. Analysis of the available clinical data showed that LEF1 gene alteration was not a significant predictor of event-free survival in children with T-ALL treated in JACLS ALL-97 and NHL-T98 protocol. However, LEF1 inactivation was associated with a younger age at the time of diagnosis, but not with sex, white blood cell count, central nervous system involvement, or the presence of an anterior mediastinal mass at the time of diagnosis. Furthermore, analysis of the T-ALL cell surface immunophenotype obtained at the time of diagnosis showed that all LEF1-alterd cases were characterized by developmental arrest at a cortical stage of T-cell differentiation. The clinical significance of these gene alterations and detailed data is discussed. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 295 Myelodysplastic syndromes (MDS) are a hetreogenous groups of myeloid neoplasms characterized by cytopenia of varying degrees and transition to acute myeloid leukemia (AML). MDS is one of the most frequent hematopoietic malignancies, particularly in the elderly. At present, allogeneic hematopoietic stem-cell transplantation is the only treatment that can induce long-term remission in MDS, but it is not applicable to most patients because of their advanced age and is associated with a high rate of treatment-related death and many complications such as chronic graft-versus-host disease. International Prognostic Scoring System (IPSS) is commonly used as a prognostic tool, but it is unsatisfactory from the point of view of genetic changes in MDS. Identification of the underlying genetic aberrations in MDS and the development of proper classification and targeted therapy are anticipated. To date, a number of gene mutations have been identified and implicated in the pathogenesis of MDS, including NRAS, TP53, RUNX1, cFMS, c-CBL, TET2, ASXL1, and more recently, IDH1, IDH2 and EZH2. However, only a part of MDS cases are able to be associated with these genetic changes. There are some remaining areas where copy number alterations and aUPDs are commonly observed and target genes have not been identified, and our knowledge about the genetic basis of MDS is thought to be still incomplete. Recently, next-generation resequencing technologies have been shown to be effective for the identification of disease-related gene and been successfully used to determine the genetic basis of some neoplastic disorders, such as AML and diffuse large B-cell lymphoma. More recently, the resequencing technology targeted for all protein-coding subsequences (i.e., whole exome analysis) has enabled cost-effective comprehensive mutation analysis of coding sequences, and has been successfully applied to identifying some Mendelian disorders. In this study, we performed a whole exome analysis of ten MDS patients in order to obtain a comprehensive registry of genetic lesions in MDS. Entire exon sequences were enriched by using SureSelect Human All Exon kit (Agilent Technologies) and were subjected to resequencing analysis using Illumina Genome Analizer IIx. On average, 12 gigabases (Gb) of sequence were generated per one tumor sample, in which more than 60% of mapped reads contained exon sequences. 〉 80% of exons were sequenced at the depth of 〉20 and average fold-coverage was 〉50 times. Because remission samples were difficult to obtain in MDS patients, paired CD3-positive T cells were used as a normal control. By comparing sequences in tumors and paired T cells, a number of candidate gene mutations and insertions-deletions, including those in IDH2, CKAP, TMEM146, CLEC1A, and other genes, which were validated by Sanger sequencing. Now, we are performing Sanger sequencing for some candidate genes, which were commonly mutated in more than one resequenced patients and were located within the regions of recurrent aUPDs in a cohort of 170 MDS subjects, assessing their prevalence in MDS. Our results suggested that target-capture resequencing technology is a powerful method to identify new gene mutations that are implicated in the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2149 Recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing, because these CBL mutations were shown to results in aberrant tyrosine kinase signaling, which would lead also to activation of RAS signaling pathways. We and others reported that CBL mutations occurred in a variety of myeloid neoplasms, including de novo acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and MDS/myeloproliferative neoplasm, especially in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The importance of CBL mutations concerning leukemogenesis is substantially increased. We investigated CBL mutations in 20 therapy-related leukemia/MDS (t-Leuk/MDS) cases and 26 infant leukemia cases. Homozygous mutation of theCBL gene (P417R), which was located in the RING finger domain, was identified in one out of 20 (5%) t-Leuk/MDS cases. This patient was a 5 year-old boy, whose biopsied specimen of the buccal lymph node showed malignant lymphoma (diffuse large T cell type, MT1(+), MB1(-), UCHL1(+)). No pathogenic nucleotide changes were identified in the CBL gene in the initial sample. Subsequently, the patient was treated with chemotherapy including VP-16 (200 mg/m2) given twice weekly. Nineteen months after initial diagnosis, he was diagnosed as having therapy-related leukemia with t(5;21) and MLL gene rearrangement due to VP-16. Furthermore, CBL gene mutations were found in 3 of 26 (12%) infant leukemia cases with 11q23 translocation/MLL gene rearrangement. CBL gene mutations were located in splice site in intron 8 and the RING finger domain (Y371H, in 2 cases), SNP array analysis (Affymetrix, GeneChip) of these cases with mutated CBL gene disclosed 11q-acquired uniparental disomy in all cases, but not in cases with wild-type CBL. CBL mutation has not been reported in acute leukemia with 11q23 translocation/MLL gene rearrangement. To our knowledge, these are the first t-Leuk/MDS case and infant cases with 11q23 translocation/MLL rearrangement, suggesting that CBL is mutated in a unique subset of t-Leuk/MDS and infant leukemia which is considered to play a pathogenic role in the development of t-Leuk/MDS and infant leukemia. Further accumulation of cases with t-Leuk/MDS and infant leukemia having the CBL mutation is needed. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 198 Background: Idiopathic aplastic anemia (AA) is a syndrome characterized by pancytopenia and bone marrow (BM) hypoplasia, which are caused by the auto-immune destruction of hematopoietic stem cells (HSCs). However, little is known about the nature of HSCs that survive the occurrence of autoimmune insults and maintain hematopoiesis both during and after aplastic diathesis. While the severity of the autoimmunity is the major determinant of BM failure in patients with AA, some intrinsic genetic changes in HSCs could also be involved in the disease process, since the clonality of the residual, persistent hematopoiesis under the aplastic state has been well recognized. Objectives/Methods: To characterize the nature of the HSCs that support hematopoiesis in AA, peripheral blood (PB) specimens obtained from 317 patients with AA were subjected to the genome-wide analysis of genetic lesions using Affymetrix® 500K SNP arrays. For the normal controls, 1746 PB specimens from the Japan Marrow Donation Program (JMDP) were also analyzed. All specimens were genotyped for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles. In the eligible cases, PB leukocytes and CD34+ BM cells were also examined to determine their expression of HLA-A antigens using allele-specific monoclonal antibodies by flow cytometry (FCM). To identity the common HLA types associated with AA, a total of 6,629 registries from JMDP who had received allogeneic bone marrow transplantation between 1992 and 2008 were employed, where the HLA frequencies in AA (N=406) were compared with those among other hematopoietic disorders, in acute myeloid leukemia (N=1,822), acute lymphocytic leukemia (N=1,406), chronic myeloid leukemia (N=1,014), myelodysplastic syndromes (N=824), non-Hodgkin's lymphoma (N=565), and other neoplastic disorders (N=392). Results: A number of genetic alterations were detected in our AA case series, among which the most conspicuous was acquired uniparental disomy (UPD), or the copy number-neutral loss of heterozygosity, involving the 6p arms (6pUPD). The 6pUPD was identified in 38 patients (12%) but not in any JMDP donor specimens, and it commonly affected the HLA locus, which was expected to result in the loss of one HLA haplotype. In fact, loss of HLA-A expression from the missing haplotype was confirmed by FCM in all 13 6pUPD-positive cases thus far tested, whereas the HLA-A expression from both haplotypes was preserved in the 58 samples without 6pUPD. The loss of HLA-A expression in the 6pUPD-positive cases was found in multiple lineages of leukocytes, including granulocytes, monocytes, B cells, BM CD34+ cells, and to a lesser extent in T cells. Of particular interest was the fact that the missing HLA haplotypes that were predicted from the SNP array data were extremely biased to particular class I HLA alleles, including HLA-A*02:01, HLA-A*02:06, A*31:01, B*40:02, and B*40:06. Moreover, when the frequencies of these alleles were compared among the 6,629 JMDP registries, they were shown to be strongly associated with AA in comparison to other non-significant HLA alleles, where the odds ratios for these alleles with regard to non-significant alleles as for the risk of the development of AA were 2.00 (95%CI; 1.52 – 2.62) for A*02:01, 2.37 (95%CI; 1.80 – 3.12) for A*02:06, 1.46 (95%CI; 1.06 – 2.02) for A*31:01, 2.07 (1.56 - 2.77) for B*40:02, and 2.67 (1.95 - 3.66) for B*40:06. Conclusions: AA patients frequently show permissive hematopoiesis with 6pUPD, which is thought to develop due to the occurrence of auto-immune insult based on the Darwinian principle of “survival of the fittest” (Figure). The tight association of AA with particular class I antigens that are invariably missing in permissive hematopoiesis with 6pUPD strongly supports the hypothesis that the auto-immunity responsible for AA is primarily mediated by cytotoxic T cells which target a relatively limited species of auto-antigens presented on HSCs through these relevant HLAs. Our findings therefore also provide a solid basis for isolating the target auto-antigens responsible for the development of AA in the future studies. Disclosures: No relevant conflicts of interest to declare.