ALBERT

Description: Autologous Stem Cell Transplantation (ASCT) is standard of care in relapsed diffuse large B-cell lymphoma(DLBCL) and other lymphoproliferative disorders (relapsed Hodgkin´s disease, mantle cell lymphoma (MCL) or T-cell lymphoma). BCNU, Etoposide, Ara-C, Melphalan(BEAM) is a standard conditioning regimen, but BCNU is known to be associated with interstitial pneumonia (range 2 to 20%) and an increased risk of death compared with other regimens. Therefore a less toxic conditioning protocol might improve the results in lymphoma patients. Bendamustineshowed promising results in B- and T-cell lymphoma and dose escalation is safe and feasible. Here we report promising results with bendamustinereplacing BCNU in the BEAM regimen described as Benda-EAM, previously published in a phase two dose finding study (Visani, Blood 2011). Fourty-one patients with Hodgkin´s (HL)(n=9) or Non-Hodgkin (n=32) lymphoma were consecutively treated with Benda-EAM (bendamustineon two consecutive days at a dose of 200 mg/m2 per day). Eleven patients were diagnosed with DLBCL, ten patients with MCL, six patients with follicular lymphoma (FL), three patients with T-cell lymphoma (TCL) and two patients with greyzone lymphoma (GZL). Twenty-seven patients were male and fourteen female with a median age of 52 years (range 22-71) and 25% were above the age of sixty. The median lines of previous therapies were 2 (range: 1-4). All patients had chemosensitive disease and before transplantation 34 patients (83%) were in complete (CR) and 7 (17%) in partial remission (PR). A median number of 4,20*106 CD34+ cells/kg (range: 1,60-13,30) were infused. All patients showed engraftment with a median time to achieve an absolute neutrophil count 〉 1*109/L of 10 days (range 8-13) and to platelets 〉20*109/L of 12 days (range 7-110). The median time of fever was 5 days (range: 0-15). The most common grade 3 and 4 toxicity during the whole treatment period were diarrhea (n=10), mucositis (n=7), infections (n=9) and febrile neutropenia (n=6), followedby nausea (n=4) and cardiologic toxicities (n=3). There were no pulmonary toxicities observed and no transplant related mortality occurred. After a median follow-up of 37 months 22 patients (56%) are still in CR, while 19 patients (44%) showed progression after a median time of 7 months after transplantation (range 2-29 months). Until today nine patients received an additional allogeneic transplantation. Eleven patients (27%) have died (3 DLBCL, 3 HL, 2 MCL, 1 GZL, 1 TCL and 1 FL), all due to lymphoma progression. Thus the 1- and 2-year PFS are 73.2% and 57.9% and the 1- and 2-year OVS 85.4% and 79.4%, respectively. In conclusion Benda-EAM is feasible with a quite promising outcome. Currently an international randomized phase II trial comparing Benda-EAM with BEAM is recruiting. So far thirty-five of 110 planned patients are randomized and first results are expected for 2018. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: In Myelodysplastic Syndromes (MDS) the International Prognostic Scoring System (IPSS) is the golden standard to assess the patients risk profile. Although only three parameters were included in the score, namely the medullary blast cell count, the karyotype and the amount of cytopenias, multiple risk factors, among others age and gender, were discussed in its original publication (Greenberg et al., Blood89,6,1997:p2079–88). Their additional predictive importance was stated there, but not formally quantified. In a retrospective multicenter analysis (for sample details see below) the modulating impact of age, gender and FAB classification on the IPSS was studied and individualized score values of the IPSS for each age*gender*FAB group were estimated (see table below). While without consideration of age, gender and FAB classification the risk of a patient at the four IPSS levels is best represented by the values 0 to 3, these values vary considerably between age*gender*FAB subgroups, as e.g. an IPSS ‘low’ scoring female patient

Description: [§ share last authorship] Background: In 2000-2010, the first large prospective trials in peripheral T-cell lymphoma (PTCL) showed outcomes burdened by high failure rates during induction. Concurrently, trials with the anti-CD52 monoclonal antibody alemtuzumab (ALZ) yielded promising responses in PTCL while demonstrating the feasibility of combining ALZ with CHOP. Hence, the Nordic Lymphoma Group initiated the randomized ACT-1 trial to test, in younger patients (pts) (18-65yrs), the addition of ALZ to CHOP + autologous stem cell transplant (ASCT). Primary endpoint was the 3 years event-free survival (EFS). Here, we present the final analysis of the ACT-1 trial (ClinicalTrials.gov: NCT00646854). Patients and Methods: Overall, 136 pts were randomized (43% of planned sample size due to slow accrual), five did not receive study treatment, and 131 were analyzed (ALZ-CHOP: 65; CHOP: 66). Due to lack of tumoral CD52 expression, anaplastic large cell lymphomas (ALCL) were not included in the ACT-1 trial. An amendment tapering ALZ dose from 360 mg (30 mg on days 1+2 of each CHOP course) to 120 mg (30 mg on day 1 of CHOP courses 1-4) was introduced early on due to systemic fungal infections in 2 pts. Of the 65 pts treated with ALZ-CHOP, 4 received the pre- and 61 (94%) the post-amendment dose. Monitoring for CMV- and EBV-DNA and antimicrobial prophylaxis were mandatory. Results: The median observation time for the Full Analysis Set was 66 months and the median age 51 yrs. The ALZ-CHOP and CHOP cohorts were well balanced with regard to classical prognostic factors and histological subtypes (PTCL-NOS 58% vs 54%, AILT 21% vs 25%, other 21% vs 21%). Feasibility: Neither CHOP nor ALZ-CHOP pts experienced substantial treatment delay. ALZ exposure did not affect stem cell harvest nor hematopoietic recovery. Grade 4 leucopenia was more frequent in ALZ-CHOP pts (73% vs 35%; p=0.001), whereas the occurrence of grade 3-4 anemia and thrombocytopenia did not differ significantly. After ALZ dose amendment, the frequency of bacterial and fungal infections of grade ≥3 was similar in both treatment arms. ALZ treated pts had more viral events (22/57=42% vs 4/23=17%), mainly due to asymptomatic CMV reactivations. The ratio of serious adverse events per ALZ-CHOP treated patient dropped markedly (from 3.25 to 0.86, comparable with 0.46 for CHOP) after dose amendment. Additional toxicity was mild and similar in both arms. Treatment related mortality was 4% (5% vs 3%). Efficacy: Complete remission (CR) was 52% in ALZ-CHOP vs 42% in CHOP. Primary refractory disease occurred for ALZ-CHOP and CHOP in 23% and 38% of pts, respectively. Overall, females had a significantly better outcome than males (p=0.004), also after adjustment for classical prognostic factors. Analyzing time-related endpoints without knowledge of CD52 expression, 3-years EFS, progression-free, and overall survival (PFS, OS) did not differ significantly between ALZ-CHOP and CHOP (EFS 35% vs 26%, PFS 37% vs 26%, OS 52% vs 50%). Fig.1A shows EFS by treatment arm, by gender, and by gender and treatment arm. Although not significantly different, EFS, PFS and OS values of ALZ-CHOP treated females in the ACT-1 trial were consistently higher than those of non-ALZ treated females or of males regardless of treatment group. RNA sequencing from evaluable pre-therapeutic tumor biopsies defined a signature of differentially expressed genes to be predictive of clinical outcome in ALZ-CHOP but not CHOP treated pts (n=33). Tumor microenvironment genes were prominent in determining response to ALZ. Tumors rich in B-cell milieu showed good responses, while the opposite was observed in tumors with signatures enriched with high endothelial cell genes (p

Description: Introduction: Several recent publications have advanced our knowledge of the prognostic significance of clonal cytogenetic abnormalities in MDS, yet the genetic risk assessment of the rare karyotypic aberrations in MDS patients (pts) remains unknown. Using the German-Austrian (G-A) Cytogenetics Database, we previously defined 24 cytogenetic prognostic subgroups; however, 12 subgroups characterized by non-complex (isolated or one additional abnormality only) karyotypes with del(9q), del(15q), t(15q), del(12p), −X, t(1q), t(7q), t(17q), −21, t(11q23), +19, t(5q) were observed infrequently (

Description: INTRODUCTION: Myelodysplastic syndromes are dynamic diseases affecting the stem cell compartment in bone marrow presenting with different clinical courses ranging from stable, indolent disease to rapid progression to acute myeloid leukemias. So far, only 3 studies on karyotype analysis in MDS with a minimum of 30 patients have been published. Most knowledge about genetic evolution in MDS is based on the description of parallely existing subclones within one single examination. Thus, little is known about the real frequency, the time spans and the clinical impact of karyotype evolution. MATERIALS AND METHODS: So far, data from 322 patients with MDS or secondary AML and at least two successfully performed classical cytogenetic analyses are available from four centres of the Competence Network Acute and Chronic Leukemias. As yet, we retrospectively examined 268 patients out of this data set. Karyotype evolution (KE) was defined as acquisition of additional aberrations, expansion of an aberrant clone (〉20%) or development of a completely aberrant karyotype after an initial mosaic karyotype. RESULTS: In 44 cases (16%) KE was observed. In the mean 2.8 (range 2–9) cytogenetic examinations have been performed. In 27 cases additional aberrations occurred and in 17 cases the abnormal clone expanded in a subsequent analysis. Compared to stable courses, patients with KE had a tendency towards a shorter survival (p=0.15). In the group of patients with expansion of the aberrant clone the most frequent karyotypes were −7/7q- (4x), complex (3x), 5q- (3x) and +8 (3x). The most frequent karyotypes in which during the course of the disease additional aberrations occurred were complex (4x) and karyotypes with two miscellaneous aberrations (4x). The most frequent additional aberrations were 5q- (3x) and −17/17p- (3x). CONCLUSIONS: In sequential cytogenetic examinations KE is a frequent event. Patients with KE tend to have a shortened survival. In our collective no long-term survivor could be observed in the group displaying KE regardless of the therapy strategies (excluding allogeneic transplantation). In this multicentric study which encompasses the largest data base on sequential analyses in MDS to date, frequency, evolution patterns and prognostic relevance of karyotype changes have been studied allowing a better insight into the genetic dynamics of MDS.

Description: Multiple myeloma (MM) may be preceded by a monoclonal gammopathy of undetermined significance (MGUS), but it is at present unclear whether or not MM post-MGUS is biologically and clinically different from MM de-novo. To address this issue, we have performed a molecular cytogenetic analysis of 32 cases of MM post-MGUS (median time between recognition of MGUS and transition to MM, 7.6 years; range, 2.6 years to 19.5 years) and compared the findings with those of 256 patients with MM de-novo, in whom no previous history of MGUS had been documented. FISH studies of clonal plasma cells (cytoplasmic Ig positive) with probes for IgH translocations [t(14q32)], t(11;14)(q13;q32), t(4;14)(p16;q32), and deletion of 13q14 [del(13q14)] revealed results summarized in Table 1: Serial studies of MGUS plasma cells and MM post-MGUS plasma cells from 12 of these patients have thus far indicated that all chromosomal abnormalities observed at MM post-MGUS were already present in the MGUS plasma cells; most notably, there was one patient with t(4;14) plus del(13q) who had both abnormalities at the time of MGUS 94 months prior to transition to MM. Collectively, our data suggest that MM post-MGUS is characterized by a distinct chromosomal pattern, in particular a high frequency of t(14q32) plus del(13q14), frequent occurrence of a t(11;14), but low frequency of a t(4;14). We are currently studying the t(14q32) plus del(13q) chromosomal pattern in MGUS to investigate its potential value as a risk factor for transition from MGUS to MM. Table 1: FISH of MM post-MGUS versus MM de novo Abnormality MM post-MGUS MM de-novo P-value Any t(14q32) 24/32 (75%) 114/256 (44.5%) .05 t(11;14)(q13;q32) 9/32 (28.1%) 32/256 (12.5%) .05 t(4;14)(p16;q32) 2/32 (6.3%) 27/256 (10.6%) .37 del(13q14) 19/32 (59.4%) 102/256 (39.8%) .13 del(13q14) plus t(14q32) 18/19 (94.7%) 57/102 (55.8%) .11 del(13q14) plus t(11;14) 6/19 (31.6%) 9/102 (8.8%) .03

Description: Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation of genes involved in cellular proliferation and differentiation and represents, in addition to genetic aberrations, an important mechanism of transcriptional silencing in the pathogenesis of age-associated diseases such as myelodysplastic syndromes (MDS). We have previously reported downregulation of enzymes associated with carnitine-metabolism (including the carnitine transporter OCTN2, which is responsible for carnitine uptake into cells) in healthy elderly persons, as well as a specific downregulation of microsomal carnitine palmitoyltransferase (mCPT; also known as glucose regulated protein GRP58 or endoplasmatic reticulum protein ERp61) a marker for proliferative potential of cells in MDS patients (J.Mol.Med, 81:435ff 2003). Aim of this study was to elucidate the mechanism of this downregulation by analysing the methylation status of the mCPT promoter in 14 MDS patients (2 RA, 5 RARS, 6 RAEB and one CMML). First, mRNA levels of mCPT, as well as a mitochondrial CPT (CPT1A) and OCTN2 were analysed from peripheral blood mononuclear cells using RTQ-PCR (reverse transcriptase quantitative real time PCR). Bisulfite modifications of genomic DNA were performed with commercially available kits (Epigentek, Chemicon, including methylated and unmethylated DNAs as controls) and subsequently analysed by real-time PCR with specific primers for methylated and unmethylated promoter sequences of the mCPT gene. Confirming earlier data, gene-expression of mCPT was reduced in all cases. Association of this downregulation with promoter hypermethylation was detected in 10/14 samples of MDS patients. 2 patients with unmethylated mCPT promoter had characteristic distinct MDS subtype-diagnoses: one RAEB (FAB) with single del(5)(q) and one CMML, thus suggesting different mechanisms of gene silencing in heterogeneous MDS. In two more patients with unmethylated mCPT (one RA and one RARS), relative OCTN2-mRNA-expression was elevated 10 fold as compared to hypermethylated samples, which were characterised by extremely low OCTN2-expression. Since L-carnitine has the potential to induce histone acetylation (J. Med. Genet.2003; 40; 76ff), which prevents hypermethylation of CpG islands, our data may provide an explanation for the concordant downregulation of mCPT and OCTN2 in these cases. In conclusion, our data confirm downregulation of mCPT in MDS. Besides methylation, other factors may also contribute to this phenomenon, because mCPT is located in the microsomal membrane where additionally multiple pre- and postranslational modifications such as prenylation, proteolysis and palmitoylation take place. The exploration of the epigenetic alterations mentioned above may help to develop novel strategies for therapy monitoring as clinical trials using epigenetically targeted therapies have yielded promising results for myelodysplastic syndromes.

Description: In myelodysplastic syndromes (MDS) cytogenetic analysis has a major role to assess the individual risk of patients. According to the International Prognostic Scoring System (IPSS) three cytogenetic subgroups can be distinguished: favorable [-y,del(5)(q),del(20)(q)], poor [chr. 7 abnormalities, complex] and intermediate [other abnormalities]. In a multicenter analysis the prognostic impact of karyotypic patterns in a series of 1159 primary MDS patients with cytogenetic data was investigated. Aim of the presented analysis was to study the influence of age and gender on this prognostic model. Median age was 66 years, median survival 37 months, 654 patients (56,4%) were male and 505 (43,6%) female. According to the three cytogenetic riskgroups defined by the IPSS the distribution and the median survival duration were as follows: favorable 739 pts. (63,8%), 53 months; intermediate 206 pts. (17,8%), 31 months; poor 214 pts. (18,4%), 11 months. These results are concordant with other published data, especially the original publication of the IPSS. Focussing on age and gender we divided the whole patient cohort in 4 subgroups:male, female, age at time of diagnosis

Description: Background:Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy of the elderly with a heterogenous molecular pathophysiology. Whereas mutations in components of the RAS pathways are among the most common somatic mutations in CMML the JAK2 V617F mutation which is a typical finding in polycythemia vera and around 50% of patients with essential thrombocythemia and primary myelofibrosis, respectively, is by far less frequently detected in CMML but can be consistently found in a subgroup of patients in larger series. Due to the fact that JAK2 V617F-positive CMML is a rare disease the clinical, hematological and in vitro growth characteristics of this entity are poorly investigated. In the "Austrian Biodatabase for Chronic Myelomonocytic Leukemia (ABCMML)" we retrospectively and prospectively collect clinical, biologic, and molecular information of patients with CMML from different centers in a real life setting. Aims:Our aim was to characterize the clinical, hematological, molecular and biologic features of CMML patients harboring a JAK2 V617F mutation. Methods:The diagnosis of CMML was established according to diagnostic criteria of the World Health Organization (WHO) classification of 2008 (Vardiman et al, Blood 2009). Clinical and hematological data were obtained from patients records. For molecular characterization we used next-generation sequencing with amplicon-based target enrichment of 39 CMML associated genes. Only mutations with an allele burden of 〉10% were considered positive in this analysis. Autonomous colony-forming units granulocyte/macrophage (CFU-GM) growth in the absence of exogenous cytokines was assessed using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Results:Up to now targeted NGS data are available in 116 patients and in vitro culture data in 75 patients respectively. We identified 13 CMML patients who had a JAK2 V617F mutation with an allele frequency 〉10%. Clinical, hematological, and biologic characteristics in these patients were compared with 103 patients who had NGS sequencing and were negative for the JAK2 V617F mutation. As shown in Table 1 JAK2 V617F-positive CMML patients had significantly higher WBC counts, higher hemoglobin values, higher platelet counts and more pronounced splenomegaly as compared to JAK2 V617F-negative patients. On the other hand the percentage on monocytes in peripheral blood and the numbers of CFU-GM growing in vitro without addition of exogenous growth factors were lower in CMML patients with the JAK2 V617F mutation as compared to patients without this mutation. The majority of JAK2 V617F-positive patients had additional mutations that can be also found in JAK2 V617F-negative patients, in particular mutations in genes of epigenetic regulation and RNA-splicing, respectively. As shown in Figure 1 there was a trend towards a better survival of patients with the JAK2 V617F mutation as compared to JAK2 V617F-negative patients (p=0.05). In a JAK2 V617F-positive CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we were able to demonstrate the disappearance of constitutional symptoms and a durable spleen response lasting for over 56 months (Fig. 2). Conclusion:Out data show that CMML patients with the JAK2 V617F mutation have hematological, biologic and clinical characteristics different from JAK2 V617F-negative CMML patients. These findings suggest that JAK2 V617F-positive CMML patients should be regarded as a distinct subgroup which may benefit from specific targeted treatments. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celegene: Honoraria, Research Funding.

Description: We have generated a large, unique database that includes morphologic, clinical, cytogenetic, and follow-up data from 2124 patients with myelodysplastic syndromes (MDSs) at 4 institutions in Austria and 4 in Germany. Cytogenetic analyses were successfully performed in 2072 (97.6%) patients, revealing clonal abnormalities in 1084 (52.3%) patients. Numeric and structural chromosomal abnormalities were documented for each patient and subdivided further according to the number of additional abnormalities. Thus, 684 different cytogenetic categories were identified. The impact of the karyotype on the natural course of the disease was studied in 1286 patients treated with supportive care only. Median survival was 53.4 months for patients with normal karyotypes (n = 612) and 8.7 months for those with complex anomalies (n = 166). A total of 13 rare abnormalities were identified with good (+1/+1q, t(1q), t(7q), del(9q), del(12p), chromosome 15 anomalies, t(17q), monosomy 21, trisomy 21, and −X), intermediate (del(11q), chromosome 19 anomalies), or poor (t(5q)) prognostic impact, respectively. The prognostic relevance of additional abnormalities varied considerably depending on the chromosomes affected. For all World Health Organization (WHO) and French-American-British (FAB) classification system subtypes, the karyotype provided additional prognostic information. Our analyses offer new insights into the prognostic significance of rare chromosomal abnormalities and specific karyotypic combinations in MDS.