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In patients with parenchymal brain masses of uncertain origin responsive to corticosteroids, primary CNS lymphoma (PCNSL) should be considered. PCNSL is a rare but aggressive brain tumor that is highly sensitive to high-dose methotrexate (HDMTX)–based chemotherapy. We report a series of six patients with brain masses without histologic confirmation suspicious for PCNSL based on clinical and radiomorphologic criteria after exclusion of some infectious conditions. All patients were treated with HDMTX. We observed two complete responses, two partial responses, and one stable disease. One patient had progressive disease and received rescue whole-brain irradiation. All patients were alive without disease progression 12–48 months after HDMTX start. No symptoms of late neurotoxicity have occurred so far. The response and survival data in this small series of patients are encouraging and suggest a benefit for patients with suspected PCNSL after initial treatment with HDMTX.

WT1 is a transcription factor involved in differentiation and proliferation of acute myeloid leukemia (AML) blasts and is expressed in 90% of cases, as determined by nested reverse transcription polymerase chain reaction (RT-PCR). It is proposed to be a key molecule in leukemia promotion. To assess the relevance of WT1 expression, we analyzed blood and bone marrow samples from 58 AML patients (37 at diagnosis, 8 in hematological remission, and 13 at relapse) for the level of WT1 expression, using quantitative real-time RT-PCR. In addition, 21 randomly chosen samples were also analyzed for the quantitative expression of the main WT1 splice variants. As expected, samples from patients at the time of diagnosis or relapse showed significantly higher WT1 expression compared to samples from patients in remission or control samples. No striking difference in expression levels was found between various French-American-British (FAB) subtypes. The level of WT1 expression observed in patients at the time of initial diagnosis was similarly high in patients at relapse. Expression of the four main isoforms (E5+/KTS+, E5−/KTS+, E5+/KTS−, and E5−/KTS−) was found in all samples with significantly higher expression levels of the E5+ variants. Together, these findings support the potential of WT1 as a target for novel treatment approaches in AML.

Dear Editor, Cystic fibrosis (CF) is the most common life-limiting autosomal recessive hereditary disorder in Europe; the mean expected lifetime is approximately 35 years and has significantly increased over the last decades because of major advances in supportive treatment, especially treatment of infectious complications [1, 2]. In patients with CF, digestive tract cancers remain the majority of malignancies, whereas the risk for hematological neoplasias is low [5]. We hereby present a case of a patient with CF and newly diagnosed Hodgkin's Lymphoma (HL). Due to infectious issues and pulmonary comorbidity, the curative treatment of HL in this particular clinical setting is a challenge. A 21-year-old male patient with a history of CF was newly diagnosed with HL, Ann Arbor stage IIIb. Sputum samples repeatedly showed colonization with Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus fumigatus, and Mycobacterium abscessus that had already been present prior to the diagnosis of HL. The previous prophylactic antibiotic regimen at that time consisted of inhaled colistin (polymyxin E). To prevent infectious complications caused by A. fumigatus, Mycobacterium abscessus, gram-negative bacteria, or Pneumocystis jirovecii during chemotherapy-induced immunosuppression, the anti-infective prophylaxis was expanded to include voriconazole, clarithromycin, levofloxacinand trimethoprim/sulfamethoxazole, as well as regular prophylactic applications of G-CSF after chemotherapy to prevent neutropenia. Chemotherapy was based on a modified BEACOPPbasis regime without bleomycine due to the patient's impaired lung function. To maximize curative potential, eight cycles of “EACOPPbasis” were initially planned. However, due to recurring infectious complications of increasing length and severity (fever of unknown origin after the first course of chemotherapy preceding primarily lower respiratory tract infections with distinctly increasing pulmonary infiltrates), the cytotoxic treatment had to be delayed for a total of 42 days after only six cycles. Recurrent fever of unknown origin and documented infections (increased lung infiltrates) were treated with adequate antibiotic agents (e.g., imipenem, ceftazidime, cefepime, and aztreonam, intermittently supplemented with clindamycin or vancomycin). Microbiological sampling (blood culture and respiratory specimens) did not yield any additional pathogens. Despite intensive antimycobacterial treatment with clarithromycin, amikacin, imipenem, cefoxitin, moxifloxacin, linezolid, or tigecyclin [4], M. abscessus was repeatedly cultured from respiratory samples [3]. Chest CT scans revealed a moderate increase of preexisting lung infiltrates (Fig. 1). Restaging procedures including positron emission tomography CT showed a persisting complete remission after 17 months of follow-up care. Infectious complications, Stefan Baumann and Friederike Kuchler contributed equally to this work. S. Baumann : F. Kuchler :M. Reinwald :N. Hartel : W.-K. Hofmann :D. Buchheidt (*) Department of Haematology and Oncology, Hospital Mannheim University, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany e-mail: dieter.buchheidt@umm.de

Limited penetration of posaconazole into cerebrospinal fluid in an allogeneic stem cell recipient with invasive pulmonary aspergillosis

Background The prophylactic use of systemic antifungal agents reduces morbidity and mortality after allogeneic hemopoietic stem cell transplantation (HSCT). However, the efficacy of this approach in pts receiving intensive chemotherapy or autologous HSCT is yet unproven. This trial was designed to evaluate the efficacy of L-AmB prophylaxis in high-risk neutropenic pts. Methods: 231 pts with hematological malignancies and expected neutropenia (N) of more than 10 days (d) following intensive chemotherapy or autologous HSCT were enrolled, 219 pts became neutropenic and were randomized to receive either 50 mg L-AmB i.v. every second d (arm A) or no systemic antifungal prophylaxis (arm B). Treatment with L-AmB started 1–3 d prior onset of N and was continued until neutrophil recovery, breakthrough IFI, intolerable toxicity or death. The level of significance was 0.05 (two-sided) for all statistical analyses. Calculations were performed using commercially (SPSSWIN 12.0) software, the calculations for GEE were performed using the software MAREG. Results Pt. characteristics: Eligible pts 219; arm A: 110; arm B: 109. Reasons for exclusion were: Absence of N (8), infection prior N (3) and pts decision (1). Baseline characteristics were balanced for age (mean 53.8 years), underlying disease (119 AML, 27 ALL, 64 NHL, 9 other), duration of N (mean 14.8 D) and treatment modality (primary 149, secondary 42, transplant 28). Primary endpoint: The incidence of proven and probable IFI was 5 of 110 pts (4.6%) in arm A and 22 of 109 pts (20.2%) in arm B (p = 0.001, RR = 2.9, CI 1.3 – 6.5). Key secondary endpoints: Pneumonia of unknown origin occurred in 6 pts (5.5%) vs. 28 pts (25.7%) (p < 0.001), the incidence of possible, probable and proven IFI was 11 pts (10.2%) vs. 42 pts (39.6%) (p < 0.001), systemic antifungals were used in 24 pts (22%) vs. 64 pts (59%) (p < 0.001), and death occurred in 4 pts (3.7%) vs. 9 pts (8.2%) (p = 0.16) in arm A vs. arm B. Toxicity: No grade 3 or 4 toxicity was noted. Laboratory abnormalities, including creatinine and liver function tests, were not different between the treatment groups. Conclusion: Intermittent application of low dose L-AmB is save. The significant lower incidence of IFI in pts treated with L-AmB prophylaxis supports its use in prolonged N.

Abstract 3521 Background Acute promyelocytic leukemia (APL) accounts for approximately 5% of all acute myeloid leukemias (AML). The characteristic molecular feature of APL is a fusion product named PML-RARA which acts as transcriptional repressor that affects gene expression patterns involved in differentiation, apoptosis, and self-renewal. The internal tandem duplication of the Fms-related Tyrosine-like Kinase 3 (FLT3-ITD) confers a poor prognosis in non-APL AML, however its effect in APL is still under discussion as several investigators found no prognostic influence for FLT3-ITD in APL. Aberrant DNA-promotor-methylation of tumor suppressor genes contributes significantly to leukemogenesis and oncogenic transformation. Deneberg et al. recently identified characteristic methylation profiles for cytogenetically normal AML, however no specific methylation profile was associated with FLT3-ITD in a study that excluded APL. To further elucidate the influence of aberrant methylation in FLT3-positive APL we carried out a genome wide DNA methylation analysis on APL samples with and without FLT3-ITD. Methods In total, genomic DNA from blasts of 54 APL patients at initial diagnosis (bone marrow n=32, peripheral blood n=22) were analyzed (median age 46 years, gender: 35 female, 19 male, blast count median 80%). The molecular analysis was carried out with written informed consent, with permission of the institutional review board and in accordance with the declaration of Helsinki. DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 25% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Pyrosequencing was performed to validate methylation changes as detected by the array-based analysis. Results The methylation pattern of FLT3-ITD-positive APL (n=18) patients was analyzed and compared to patients without FLT3-ITD (n=32) or D835 Mutation (n=4). We identified 133 CpGs that were significantly differentially methylated in FLT3-ITD-positive APL as compared to FLT3-ITD-negative APL. The most significant differential methylation was observed for 5 CpGs showing a strong hypomethylation of the chemokine (C-C motif) receptor 6 (CCR6) in FLT3-ITD-APL as compared to FLT3-negative APL (q-value < 6.9 *10−13). Other interesting target genes showing pronounced hypomethylation in FLT3-ITD positive APL samples belonged to the family of phosphatases such as the dual specificity phosphatase 5 (DUSP5), protein tyrosine phosphatase, receptor type, N polypeptide 2 (PTPRN2) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). The most prominent hypermethylation in FLT3-ITD APL was observed in CpGs within the coding region of suppressor of cytokine signaling 2 (SOCS2) and significantly discriminated between FLT3-ITD and FLT3-negative APL (q-value < 10−5). The results of the genome-wide analysis obtained with the Illumina 450K BeadChip were validated for 4 CPGs in 10 samples via pyrosequencing and showed a robust Pearson correlation of 0.92 suggesting a good and reliable performance of the Illumina 450 K Bead Chip Assay. Conclusions The current study represents a comprehensive genome wide methylation analysis of a clinically well-defined cohort of APL patients. We here demonstrate for the first time that in contrast to cytogenetically normal AML, APL patients with FLT3-ITDs display a highly specific and disease defining DNA methylation profile. Thereby key regulators of cellular growth signaling such as SOC2, PTPRN2 and DUSP5 are significantly differentially methylated in dependency of FLT3-ITD status. This suggests that a cooperative effect between PML-RARA and FLT3-ITD is mediated by dysregulation of DNA methylation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3801 Background Aberrant DNA methylation at promoter CpG islands is recognized as one of the hallmarks driving the pathogenesis of myeloid malignancies, especially myelodysplastic syndromes (MDS). Shen et al. recently characterized an algorithm of 10 aberrantly methylated gene loci which was predictive for overall survival and progression-free survival in a large cohort of MDS patients. A particular cytogenetic subgroup of MDS patients with a deletion on the long arm of chromosome 5 (5q-syndrome) has been shown to benefit from treatment with lenalidomide. However, the exact underlying molecular mechanism of MDS with isolated deletion (5q) is still not understood. To further elucidate on the role of deregulated DNA methylation we analyzed DNA-methylation profiles of bone marrow cells from patients with MDS with an isolated deletion (5q). Methods All patients were diagnosed and treated within a German multicenter trial investigating the safety of Lenalidomide in patients with low risk myelodysplastic syndromes and an isolated deletion (5q) after informed consent and according to the declaration of Helsinki. Bone marrow cells of 47 MDS patients with deletion (5q) at initial diagnosis were analyzed (median age 70 years, range 41 – 88 years, IPSS score: low n= 22; intermediate-1 n = 25). DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 15% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Gene ontology analysis was performed using GATHER (http://http://gather.genome.duke.edu/). Results Using a q-value of ≤ 0.05 for the beta-value of the array and excluding gender-specific chromosomal CpGs, 473,929 CpGs were evaluable for analysis. Gene Ontology analysis using the GATHER Tool showed a significant enrichment of genes mapped to 5q31 (p Highly significant differential methylation profiles between MDS patients with isolated (5q) were found between patients with low and intermediate-1 IPSS score. CpGs differentially hypermethylated in intermediate-1 risk versus lows risk patients affected the coding regions of interesting candidate genes such as platelet-derived growth factor receptor, beta polypeptide (PDGFRB), clathrin interactor 1 (CLINT1), both located at 5q33 and suspected to be involved in the pathogenesis of 5q deleted MDS. Furthermore, transcriptional regulators such as proline, glutamate and leucine rich protein 1 (PELP1), v-myb myeloblastosis viral oncogene homolog (avian) (MYB), genes known to be involved in cancer like trichorhinophalangeal 1(TRPS1), and tumor suppressors like forkhead box P1 (FOXP1) and genes thought to be involved in the pathogenesis of MDS like Minichromosome maintenance protein2(MCM2) did show differentially DNA-methylation according to our selection criteria. Conclusions We present a comprehensive genome wide methylation analysis of MDS patients with an isolated deletion (5q) with low and intermediate-1 risk according to IPSS. Thereby we detected sets of significantly differentially methylated CpGs between both risk groups. Correlation of these data to clinical parameters might help to further elucidate the contribution of aberrant methylation to the phenotype of MDS with isolated deletion (5q) and could possibly help establishing novel prognostic markers based on differential methylation. Moreover, unraveling the role of aberrant methylation patterns might result in new therapeutic treatment approaches at least in a subset of patients. Disclosures: Nowak: Celgene: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ottmann:Celgene: Clinical trial participation Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Schlenk:Celgene: Research Funding. Ganser:Celgene: Research Funding. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hofmann:Celgene: Honoraria, Research Funding. Nolte:Celgene: Research Funding.

Abstract 1452 Background: Acute promyelocytic leukemia (APL) accounts for approximately 5 % of all acute myeloid leukemias. Recent experiments have shown that aberrant DNA-promotor-methylation of tumor suppressor genes may be an important mechanism contributing to leukemogenesis and oncogenic transformation. Figueroa et al. recently identified characteristic methylation profiles in a large cohort of acute myeloid leukemia patients using HpaII tiny fragment enrichment by ligation-mediated PCR and array-based methylation analysis. However, as APL is a rare entity, little is still known about the role of deregulated DNA methylation in APL. Methods: Bone marrow blast cells of 45 APL patients at initial diagnosis were analyzed (median age 47 years, gender: 28 female, 17 male). These samples have previously been characterized by high-density-SNP-array analysis (Nowak D et al., Abstract# 38779, ASH 2011), which allowed a segregation into defined molecular and clinical risk subgroups (samples with deletion of chromosome 7q: n = 6; samples with > 5 copy-number-alterations as detected by SNP array: n = 8; samples with white blood cell count at initial diagnosis > 10000/μl: n = 5; samples of patients who suffered relapse: n = 7; samples of patients with initial white blood cell count < 10000/μl: n=19). Furthermore, CD34+-cells from the bone marrow of 10 healthy donors (median age 32 years, gender: 5 female, 5 male) were analyzed as a healthy control group. DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs between subgroups was defined by a minimum mean methylation difference of 25% as expressed by the beta-value of the array data and statistical significance set at p ≤ 0.05 in a two-sided t-test. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), SAS 9.2./JMP-Genomics 4.2® (SAS Software, Cary, USA) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Results: Using a detection p-value of p ≤ 0.01 for the beta-value of the array and excluding X-chromosomal CpGs, 472.261 CpGs were evaluable for analysis. Highly significant differential methylation profiles between APL-blasts and CD34+-cells of healthy donors could be detected for 18163 CpGs (3.8 %). CpGs differentially hypermethylated in APL versus healthy individual affected the coding regions of interesting candidate genes such as transcriptional regulators microRNA 125b1 (MIR125B1), wilms tumor gene 1 (WT1), WT1 antisense RNA (WIT1), v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1), chromodomain helicase DNA binding protein 5 (CHD5), deleted in colon cancer (DCC) and homeobox genes like iroquois homeobox 1 (IRX5) and NK6 homeobox 2 (NKX6–2). CpGs in the vicinity of genes involved in regulation of the epigenetic landscape such as jumonji, AT rich interactive domain 2 (JARID2) and DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A) were significantly hypomethylated in APL as compared to the control group. The analysis of molecular and clinical subgroups revealed that methylation profiles frequently clustered according to the respective molecular lesions in the analyzed samples. Conclusions: We present a comprehensive genome wide methylation analysis of different APL patient subgroups defined by molecular aberrations and clinical risk profiles. Thereby we have detected sets of significantly differentially methylated CpGs in all subgroups. Integration of this data with results from high density SNP array analysis and detailed clinical outcome data may allow to elucidate the pathomechanistic contribution of aberrant methylation in APL and possibly establish novel prognostic markers based on differential methylation. Disclosures: No relevant conflicts of interest to declare.

Background Myelodysplastic Syndromes (MDS) are heterogeneous groups of hematopoietic malignancies that are highly susceptible to transformation into acute myeloid leukemia and clinically manifest with signs of severe anemia. Numerous studies have revealed recurring molecular alterations in hematopoietic stem/progenitor cells from MDS patients as presumable cause for the persistent dysfunction of their hematopoiesis. However, how these events translate into disturbed regulation of erythropoiesis is poorly understood so far. Characterization of the transcriptional network and associated epigenetic changes in dysplastic erythroprogenitor cells might therefore aid in the elucidation of functional molecular consequences finally leading to impaired erythropoiesis. Methods CD71+ erythroprogenitor cells were isolated from purified bone marrow cells via magnetic cell separation for 15 MDS patients (IPSS low/int-1 risk n=11, int-2/high risk n=4) and 7 age-adjusted healthy donors. Extracted DNA and RNA were processed and hybridized to Affymetrix Exon 1.0 ST Array and Illumina Infinium HumanMethylation450 Beadchips according to manufacturers’ instructions. Data analysis was carried out using Qlucore Omics Explorer 2.3 and in-house scripts for correlation of the two datasets were developed using Python 2.7.5. Results Using a false discovery rate/q-value of ≤ 4% as a cutoff for differential gene expression and CpG DNA methylation, both datasets allowed highly distinct clustering into MDS IPSS low/int-1 risk, int-2/high risk and healthy donor groups. In our MDS low/int-1 risk cohort 63 genes have been identified as significantly up- and 13 as downregulated (fold change ≥1.5, p≤0.001) while 64 genes showed up- and 28 downregulation in the MDS int-2/high risk group (fold change ≥1.5 p≤0.01). Surprisingly, global DNA methylation profiling revealed that 741 CpGs were detected as hypo- but only 19 as hypermethylated in low/int-1 risk, whereas 231 CpGs demonstrated hypo- and 479 hypermethylation in int-2/high risk MDS CD71+ cells relative to healthy controls (mean CpG methylation difference ≥10%, p≤0.0001). In order to discover genes susceptible to DNA methylation associated regulation of transcription, individual CpG methylation values were correlated with corresponding exon probeset expression intensities for every single gene. Using this approach, starting with >23x106 possible CpG/probeset combinations, 4418 displayed significant positive correlation, whereas only 2726 were negatively correlated (R≥0.6 or R≤-0.6, mean methylation difference MDS vs. healthy ≥5%, p≤0.01). Consequently, in MDS low/int-1 risk we identified strong hypomethylation as putative cause for a 5.8-fold upregulation of GDF15, an important regulatory factor involved in iron homeostasis. Moreover, DNA hypermethylation associated 6.6-fold knockdown of the transcription factor GTSF1, which has been associated with increased apoptosis in gametogenesis, was demonstrated for MDS int-2/high-risk. In this cohort, we also observed a 3-fold upregulation of LY6E, which has been shown to result in a strong block of differentiation and maintenance of self-renewal in avian erythroprogenitor cells. Consistently, gene set enrichment analysis identified a stem cell like gene signature as highly enriched in MDS CD71+ BM cells but also gene sets involved in oxidative phosphorylation as well as regulation of cell cycle and apoptosis. Conclusion Our integrative approach reveals novel candidate genes implicated in disturbed erythropoiesis in MDS and allows distinctive separation between healthy donors and MDS risk groups by assessment of epigenomic and transcriptomic landscapes derived from CD71+ bone marrow cells. DNA hypomethylation induced gene upregulation surprisingly appears to be a common event in MDS erythropoiesis which co-occurs with DNA hypermethylation induced gene silencing. In addition, frequent detection of significant positive correlation between DNA methylation and gene expression might add an additional layer of complexity to the current dogma of epigenetic gene regulation. Finally, the distinctively hypermethylated geneset in MDS high-risk as compared to the unexpected global hypomethylation phenotype in MDS low-risk might suggest a mechanistic explanation for the selective efficacy of demethylating substances specifically in the higher risk patient groups. Disclosures: No relevant conflicts of interest to declare.