ALBERT

Description: Abstract 331 Background and aims: Systemic peripheral T-cell lymphomas (PTCL) are malignancies responding poorly to conventional therapy. To evaluate the efficacy of a dose-dense approach consolidated by upfront high-dose chemotherapy supported by autologous stem-cell transplantation (HDT/ASCT) in PTCL, the Nordic Lymphoma Group conducted the, so far, largest PTCL-restricted prospective phase II study in previously untreated systemic PTCL. This is the final report of the NLG-T-01 study with a 5-years median follow up. Methods: Patients with previously untreated systemic PTCL aged 18–67 years were included. ALK-positive anaplastic large cell lymphoma (ALCL) cases were excluded. An induction regimen of six cycles of bi-weekly cyclophosphamide, doxorubicin, etoposide, vincristin and prednisone (CHOEP) was given. Age-based (〉60 yrs) omission of etoposide was recommended. If in complete or partial remission, patients received high-dose chemotherapy with carmustine, etoposide, cytarabine and melphalan/cyclophosphamide (BEAM/BEAC) followed by HDT/ASCT. Results: A total of 166 patients with previously untreated PTCL were enrolled. Of these, 160 were histopathologically confirmed and included the following subtypes: PTCL-not otherwise specified (PTCL-NOS) (n=62; 39%), ALK-negative ALCL (n=31; 19%), angioimmunoblastic lymphoma (AIL) (n=30; 19%), enteropathy-associated T-cell lymphoma (n=21; 13%), panniculitis-like (n=6; 4%), T/NK nasal-type (n=5; 3%), and hepatosplenic (n=5; 3%). The M/F ratio was 2.0 and the median age 57 yrs (range 22–67 yrs). The majority of the cases presented with advanced-stage disease (81%), B-symptoms (59%) and elevated s-LDH (62%). Nevertheless, 71% of all patients had a good performance score (PS) (WHO 0–1) at inclusion. With regard to the International Prognostic Index (IPI), risk factor distribution was as follows: 1 factor n=45 (28%), 2 factors n=52 (32%), 3 factors n=30 (19%), 4–5 factors n=33 (21%). Of the 160 patients, a total of 114 (71%) underwent HDT/ASCT with 90 in complete remission at 3 months post-transplant. Early failures occurred in 26% of the patients. The treatment related mortality was low (4%). At a median follow-up of 60 months, 83 patients were alive. The median follow-up for deceased patients (N=77) was 9 months. The consolidated 5-yr overall (OS) and progression-free survival (PFS) values for the entire cohort were 51% and 44%, respectively. Best results were obtained in ALK-negative ALCL with 5-yr OS and PFS of 70% and 61%, respectively. IPI was a useful overall prognostic discriminator for the low/low-intermediate vs. intermediate-high/high groups with regard to 5-yr OS (p=0,047) and 5-yr PFS (p=0,029). If applied separately to each of the four major subtypes, IPI had a predictive value for OS in AIL (p=0,02) and for PFS in both AIL (p=0,02) and PTCL-NOS (p=0,03). The clinicopathological parameters that showed a significant impact on OS and PFS were: female gender (correlated with a better outcome), age (analyzed as continuous variable), PS≥2 (correlated with adverse outcome), and cytotoxic phenotype (correlated with adverse outcome in AIL). All these parameters retained their prognostic value at multivariate level, except for cytotoxic phenotype, where multivariate analysis could not be performed because of too small numbers. Conclusions: Dose-dense induction followed by HDT/ASCT is well tolerated and leads to long-term PFS in 44% of patients with systemic PTCL. This represents an encouraging outcome, particularly considering the high median age and adverse risk profile of the present study population. Therefore, based on these results, dose-dense induction and HDT/ASCT should be considered in transplant-eligible PTCL patients. Disclosures: Jantunen: Genzyme: Honoraria.

Description: Abstract 3565 Enteropathy-associated T-cell lymphoma (ETCL) is a rare lymphoma often, but not always, associated with celiac disease and characterized by poor prognosis when treated with conventional chemotherapy. In previous studies long-term survival has been achieved in only 10–20% of the patients. Limited data is available on the feasibility and efficacy of intensive induction chemotherapy followed by autologous stem transplantation (ASCT) in this rare lymphoma entity. We therefore specifically analysed the outcome of ETCL patients included in a large prospective phase II study (NLG-T-01) performed by the Nordic Lymphoma Group. The NLG-T-01 study included 160 patients with systemic alk-negative peripheral T-cell lymphoma over the period 2002–2007. The patients received CHOEP-14 × 6 followed by ASCT after BEAM or BEAC in responsive patients. The study included altogether 21 patients (13 %) with ETCL. There were 16 males and 5 females with a median age of 55 years (32-65) at diagnosis. Eighteen patients (86 %) had advanced disease, three patients (14 %) had a bulky tumour, nine patients (43 %) presented with B symptoms and four (19%) with elevated serum lactate dehydrogenase. Response status after three and six courses was CR or CRu in 67 % patients. Fourteen patients (67 %) received BEAM or BEAC supported by blood stem cell graft (median number of stem cells infused 5.4 × 106/kg). Of these, 6 patients relapsed with a median of 219 days from ASCT. Of the 7 patients (33%), who did not reach ASCT because of refractory/progressive disease, 5 died early due to lymphoma. At a median follow-up of 45 months, 10 patients (45 %) are alive. The progression-free survival is 40 %. One patient (5%) died due to early transplant-related cause (disseminated candidiasis). In this prospective study, intensive induction chemotherapy followed by ASCT was feasible in the majority of younger patients with EATL. In a subset of patients, who should clinically and biologically be further characterized, long-term outcome seems promising when compared to historical controls. Whether addition of other chemotherapeutic agents, antibodies such as alemtuzumab or other biologicals may further improve long-term outcome remains to be studied. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1548 Introduction: The introduction of Rituximab as supplement to chemotherapy has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL). Still, a fraction of patients are resistant or relapse shortly after treatment. Improved stratification of patients with DLBCL for standard immunochemotherapy or alternative treatment strategies is therefore urgently needed. Although DLBCL profiling based on mRNA expression may be helpful, this has not proven clinically efficient, and the prognostic value of immunohistochemical algorithms is controversial. In addition, novel therapeutic options are essential since the current alternative treatment modalities are often not curative. MicroRNAs (miRs) are particularly attractive molecules for clinical use since they are well conserved in formalin fixed paraffin embedded (FFPE) tissue, and novel data imply that they may be targeted directly in the patients. Materials and methods: RNA was extracted from diagnostic biopsies from DLBCL patients (n=97) treated uniformly with immunochemotherapy (R-CHOP n=80 or R-CHOEP n=17). GCB/non-GCB profiling was done by immunohistochemistry according to the Hans classification. MiR profiles were generated using Affymetrix microRNA version 1.0 arrays. Data analyses were performed using R/biocondutor and the webtool “SignS” that uses parallel computing for finding survival related genes and signatures from gene-expression datasets. Survival analysis was performed in R using the survival package. Univariate analysis was performed by comparing Kaplan-Meier survival estimates using Log-rank test. Cox proportional hazards regression model was used for multivariate analysis. Results and discussion: The median follow-up time for all patients was 3.4 years. The estimated 3-year over all survival probability was 82.8% (95% CI: 75.4%-90.9%). No difference in survivability was observed between the R-CHOP and the R-CHOEP treated cohort (P=0.145). High IPI (〉 2) was significantly associated with inferior overall survival (OS, P = 0.038), but not progression free survival (PFS, P = 0.083). Univariate analysis showed that in this cohort the Hans classification was not prognostic (P=0.73; (52 GBC and 37 non-GCB subtypes; 8 NA)). Seven miRs were differentially regulated between GCB and non-GCB using a cutoff of P〈 0.05. Five miRs were upregulated in non-GCB lymphomas: miR-625, miR-222, miR-221, miR-155 and miR-503, two were downregulated (miR-181a, miR-181b). For survival analysis, we initially applied a multivariate approach (Robust likelihood-based survival modeling, RBsurv), which identified a subset of miRs that significantly associates with poor survival. These include one upregulated miR, and four down regulated miRs. In order to obtain cross validated survival estimates, we applied three different algorithms; FCSM(SignS), TGD(SignS) and GLMboost(SignS) to the same sample set. These combined bioinformatic models identified a total of 17 deregulated miRs that significantly associate with survival. Among these, 9 are predicted by more that one algorithm, and interestingly, all 4 models identify a novel upregulated and potential oncogenic miR in patients treated by immunochemotherapy. When the cross-validated predictors were combined into a unified robust “miR-survival-predictor”, the performance is as good as, or even better, than the IPI. In addition, our model is a superior predictor of survival than the GCB/non-GCB classification according to Hans. Our data are currently being validated in a test set of 60 patients, and functional studies of the novel putative oncomiR are ongoing. Disclosures: No relevant conflicts of interest to declare.

Description: Systemic PTCL, with the exception of alk-positive anaplastic large cell lymphoma (ALCL), have a poor prognosis. ASCT has been shown to have a favourable impact on relapsed PTCL. Therefore, the NLG designed a prospective multicenter phase II study to evaluate the impact of a dose-intensified induction schedule (6 courses of two-weekly CHOEP) consolidated in 1st PR/CR with high-dose therapy (BEAM) followed by ASCT in previously untreated systemic PTCL. This is the largest prospective PTCL-specific trial published so far. Newly diagnosed non-primary cutaneous PTCL cases aged 18–67 yrs were eligible for enrollment. Cases of alk-positive ALCL were excluded. From Oct 2001 to Feb 2006, 99 histologically confirmed PTCL cases were included in the study: PTCL unspecified (n=41), alk-neg ALCL (n=24), AILT (n=15), enteropathy-type (n=12), panniculitis-like (n=3), T/NK nasal-type (n=2), hepatosplenic (n=2). The M/F ratio was 1.8 and the median age 55 yrs (range 20–67 yrs). Although almost 2/3 of the cases presented with advanced-stage disease (62%), B-symptoms (61%) and/or elevated s-LDH (63%), the majority of them (65%) had a good performance score (WHO 0–1) at diagnosis. Of the 77 patients, where information was available for all 6 induction courses, 68 (88%) were in CR (31) or PR (37) after the 3rd and 66 (86%) after the 6th course. A total of 58 patients (75%) went through ASCT indicating that at least a fourth of this younger patient cohort has a primary refractory disease and fails therapy before reaching the transplant. Treatment-related toxicity after both induction and high-dose treatment was manageable. Of the 58 transplanted patients, 50 (86%) were still in remission at re-evaluation short after transplant. In 39 patients follow-up data one year post-transplant were available: 30 are still in CR and 9 have relapsed, suggesting that post-transplant relapses probably account for another 25% of the original patient cohort. In conclusion, the present data indicate that a time- and dose-intensified schedule is feasible and effective in previously untreated systemic PTCL. Continuous remissions are not uncommon, but a longer follow-up is needed to further characterize long-term remission rates and evaluate their impact on time-to-treatment failure and overall survival.

Description: Introduction Disruption of miR34a, -b, and -c has been implicated in lymphomagenesis, and in CLL it has been suggested that miR34a expression is a surrogate marker for TP53 disruption associated with a poor prognosis. P53 is a transcription factor for the miR34s, and overexpression of miR34s in p53 deficient cells can reinstate p53 functions (Chang et al, Mol Cell 26, 2007, He et al, Nature 447, 2007). However, in cellular senescence miR34a may be activated independently of p53 (Christoffersen et al, Cell Death Differ17, 2010). A recent multicenter study shows that in diffuse large B-cell lymphoma (DLBCL), TP53 disruption is still a negative prognostic factor for survival after the implementation of Rituximab (Xu-Monette et al, Blood 19, 2012). Information on the role of the miR34s in normal CD19+ B-cells (PBL-B), activated B-cells, and de novo diffuse large B-cell lymphoma (DLBCL) is limited. Aims Given that MIR34A and MIR34B/C locate to regions of allelic loss in DLBCL (1p36.23 and 11q23.1, respectively), and the importance of the miR34 targets in DLBCL pathogenesis, we investigated a large panel of newly diagnosed cases of DLBCLs for MIR34A and MIR34B/C promoter methylation, TP53 mutational status, clinical presentation patterns, and outcome. Methods MIR34A/B/C promotor methylation was performed by MsMCA and bisulfite sequencing. Histone modifcations at the MIR34A/B/C promotor was examined by ChIP RT-qPCR. Expression of miR34s was performed using miRCURY LNA™ Universal RT-qPCR. The coding sequences and splice sites of exons 5-9 of the TP53gene were scanned for mutations by PCR and denaturing gradient gel electrophoresis (DGGE). Differences in clinical characteristics using the one-way Anova, the Pearson chi-square, or Fisher’s exact tests. Overall survival was estimated using the Kaplan-Meier method and log-rank test. For assessment of independent predictors of survival a multivariate Cox regression hazard model with backward stepwise (likelihood ratio) entry was applied. Effects not meeting a p-value 〈 0.05 were removed from the model. Results We show that only miR34a-5p is expressed in PBL-B, and significantly induced in activated B-cells (P=0.017) and reactive lymph nodes (P

Description: Abstract 4108 Background: In Philadelphia-negative chronic myeloproliferative neoplasms (MPN) increased microvascular density, bizarre vessel architecture and increased number of pericytes are distinct histopathological features; apart from the characteristic proliferation of myeloid cell lines and the progressive accumulation of connective tissue in the bone marrow. Pericytes express several markers such as CD146, CD271, Smooth Muscle Actin (SMA), Desmin, Platelet-derived growth factor receptor beta and Neuron-glial 2. However, these markers are also expressed in other cell types of which some are related to vascular structures. Immunofluorescence labelling is the golden standard for detection of co-expressed cellular antigens, but due to the crowded cellular environment in bone marrow and excessive autofluorescence, identification of cell types by light microscopy is preferred. Aim: This is a methodological study aiming to identify pericyte marker profiles by light microscopy in bone marrow biopsies, contributing to our understanding of the pathogenesis of MPN. Method and results: Formalin fixed, decalcified and paraffin-embedded blocks of bone marrow trephine specimens from normal donor (n=1) and patients with primary myelofibrosis (PMF) (n=3) were included. Specimens were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique), inspired by the work of Glass et al. 2009 (J Histochem Cytochem). Briefly, antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualised with Amino-Ethyl-Carbazole (AEC). After imaging, erasing of AEC with 96% alcohol and blocking of immunoreagents, the slides were stained with a traditional double immuno-labelling procedure. We successfully applied up to four layers of antibodies using CD146, SMA, CD34, CD271, and Ki67 in different combinations; either displayed as single or single followed by traditional double sequential staining runs (figure 1). In addition to the conventional light microscopy analysis we applied a Photoshop color palette, where the different immunohistochemical reactions in the staining sequence were assigned to the different color channels creating a single composite image. The SE-technique significantly improves morphological studies especially in bone marrow trephines with the cells of interest intermingled with other cells. Additionally, the SE-technique makes it possible to detect more than two antigens regardless of immunoglobulin type or animal host. Conclusion: To our knowledge, the SE-technique described in this study, is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow trephines at light microscopic level. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms. The SE-technique displayed as single staining images and Photoshop color palette combined image. Panel A-D shows an identical area in the different steps of the method. A) CD146. Positive pericytes (arrow). B) SMA. Positive pericytes (arrow). C) CD34. Negative pericytes (arrow). D) Combined image with CD146 (green channel), CD34 (red channel), and SMA (blue channel). Coexpression of CD146/CD34 is seen as yellow reaction deposit, and coexpression of CD146/SMA as cyan reaction deposit (arrow). Note, as a negative control, the CD146 positive fat cell in A (arrowhead) - negative in B and C, and SMA positive pericyte in B (arrow) – negative in C. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3566 Primary systemic T-cell lymphoma of anaplastic large cell type (ALCL) is an aggressive and predominantly nodal subtype of lymphoma, further subdivided based on expression of the ALK-protein, with ALK-pos ALCL occurring predominantly in younger patients and associated with a favourable prognosis. ALK-neg ALCL is believed to carry a prognosis similar to that of other nodal peripheral T-cell lymphomas and previous studies have shown long-term survival rates below 50%. There is retrospective data suggesting a benefit from ASCT in first-line treatment of this lymphoma subtype. We analyzed the outcome of ALK-neg ALCL patients included in a prospective phase II trial, NLG-T-01, conducted by the Nordic Lymphoma Group. The NLG T-01 trial enrolled 160 patients aged 18–67 years from the Nordic countries with systemic ALK-neg peripheral T-cell lymphoma within the period 2002–2007. The treatment schedule consisted of 6 courses of CHOEP-14 followed by ASCT (BEAM or BEAC) in responding patients. Patients 〉60 years received CHOP-14 as induction. Altogether, the trial included 31 patients with ALK-neg ALCL (19% of the study population). Median age was 56 years (22-65) with a male:female ratio of 2.4. Stage III-IV was found in 18 patients (58%), B-symptoms in 19 patients (61%) and 6 patients (19%) had a bulky lesion (〉10cm). Pre-therapeutic serum lactate dehydrogenase was elevated in 18 patients (58%) and performance score was 2–4 in 10 patients (32%). After 3 and 6 courses of chemotherapy, response status was CR or CRu in 29% and 58% of the patients, respectively. In total, 24 out of 31 patients (77%) underwent BEAM/BEAC therapy followed by ASCT. Four patients did not respond or had disease progression during induction chemotherapy. The remaining 3 patients did not undergo ASCT for other reasons (mobilization failure, lung insufficiency, patient decision, respectively). Overall response rate after ASCT was 74% for the entire initial population and 96% for those undergoing ASCT. Median follow-up was 45 months. Six patients relapsed after ASCT. There was a total of nine deaths (29%): six due to lymphoma, two due to toxicity and one from second malignancy (colon cancer). With a median follow-up of 45 months, 3-year overall and progression-free survival values were 73% and 64%, respectively. Intensive chemotherapy followed by ASCT was feasible in the majority of the patients included in this prospective trial. Long-term outcome appears promising when compared to previously published data, with the survival curve suggesting a plateau. In ASCT eligible patients, intensive induction chemotherapy consolidated by upfront ASCT is an effective treatment that yields outcome results at least as good as those obtained in age-comparable patients with diffuse large B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Fas (APO-1/CD95) is a cell-surface receptor involved in cell death signaling. Germline mutations in the Fas gene have been associated with autoimmune lymphoproliferative syndrome, and somaticFas mutations have been found in multiple myeloma. We have examined the entire coding region and all splice sites of theFas gene in 150 cases of non-Hodgkin’s lymphoma. Overall, mutations were identified in 16 of the tumors (11%). Missense mutations within the death domain of the receptor were associated with retention of the wild-type allele, indicating a dominant-negative mechanism, whereas missense mutations outside the death domain were associated with allelic loss. Fas mutations were identified in 3 (60%) MALT-type lymphomas, 9 (21%) diffuse large B-cell lymphomas, 2 (6%) follicle center cell lymphomas, 1 (50%) anaplastic large cell lymphoma, and 1 unusual case of B-cell chronic lymphocytic leukemia with a marked tropism for skin. Among the 16 patients with somaticFas mutations, 15 showed extranodal disease at presentation, and 6 relapsed in extranodal areas. Ten of 13 evaluable patients showed features suggestive of autoreactive disease. Our data indicate that somatic disruption of Fas may play a role in the pathogenesis of some lymphomas, and suggest a link between Fas mutation, cancer and autoimmunity. © 1998 by The American Society of Hematology.

Description: Abstract 1600 Introduction: Although rare, ocular adnexal lymphomas (i.e. lymphoma of the orbit, eyelids, conjunctiva, lacrimal gland and lacrimal sac), are among the most common malignant tumors involving the ocular adnexal regions. The main subtypes are low-grade mucosa associated lymphoid tissue (MALT) lymphoma and aggressive diffuse large B cell lymphoma (DLBCL). In rare cases low-grade MALT lymphoma are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal, disease. Material and methods: A total of 18 MALT lymphomas and 25 DLBCLs involving ocular adnexal sites were included in the study. All sections were analyzed immunohistochemically by two independent pathologists (ER, SH) using the following panel of antibodies: bcl-2, bcl-6, CD3, CD5, CD10, CD20, CD23, CD79α, cyclin D1, MUM-1 and Ki-67. Confirmed cases of DLBCL were categorized as either germinal centre B-cell-like (GCB) or non-GCB types according to the algorithm by Hans et al. Using LNA-based arrays from Exiqon, we performed global miRNA expression profiling of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The most differentially expressed microRNAs were confirmed by RT-qPCR analyses. Microarray processing was performed using the R environment. Results: Of the18 MALT patients (pts.) 15 pts. (83%) presented with Stage I lymphoma and 3 pts. (17%) had Stage IV. The 5-year overall survival for the entire population was 77%. In the DLBCL group 13 pts. (52%) presented with Stage I lymphoma, 3 pts. (12%) Stage II lymphoma, 1 pt. (4%) Stage III and 8 pts. (32%) presented with Stage IV lymphoma. Nine of the DLBCLs were classified as GCB and 16 as non-GCB type. The 5-year overall survival for the entire group was only 13%. Our miR arrays and confirmatory qPCR analysis revealed 43 miRNAs with significantly altered expression profiles (41 down- and 2 upregulated) in DLBCL compared to MALT lymphoma. Seven of the miRNAs down-regulated in DLBCL relative to MALT lymphoma showed enrichment for a direct transcriptional repression by the oncoprotein MYC. Supervised hieracical clustering analysis identified tree clusters: Cluster 1: MALT (high expression), cluster 2: DLBCL (intermediate expression), cluster 3 DLBCL (low expression). Thus, apparently the DLBCLs in cluster 2 seem to resemble MALT more than DLBCLs in cluster 3. We also report loss of miRNAs involved in the regulation of NFKB1 and DNA methyltransferases in DLBCL vs MALT. Conclusion: We conclude that fundamental differences in miRNA expression exist between ocular adnexal MALT lymphoma and DLBCL. Among the possible consequences of altered miRNA expression are increased NF-kB signaling and DNA hypermethylation. However, in line with the recent observations in gastric MALT/DLBCL transformation1, we suggest the differences may at least in part be caused by MYC transcriptional regulation of miRNAs in aggressive cases. The fraction of DLBCL that is reported to arise from MALT is exceedingly low. However, in the current study we find a group of DLBCLs (cluster 2), whose level of MYC regulated miRNA expression is intermediate between MALT (cluster 1) and DLBCL (cluster 3). Thus it could be speculated whether cluster 2 DLBCLs have developed secondary to a preceding MALT lesion, which, however was not detected by histology. Disclosures: No relevant conflicts of interest to declare.