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Galectin-1 (Gal-1) has been associated with adverse prognosis in several cancers including lymphoma entities with CD30 expression. However, Gal-1 expression has not been systematically assessed in peripheral T-cell lymphomas (PTCL). Specimens from 169 nodal PTCL were assessed for intratumoural Gal-1 expression by immunohistochemistry. Overall survival (OS) in groups exhibiting high and low Gal-1 expression was compared in the cohort and in a subset analysis of CD30-positive PTCL only. Gal-1 expression was also correlated with biomarkers of the tumour microenvironment. No significant difference in OS based on Gal-1 expression was observed in the entire PTCL cohort. However, in the CD30-positive cohort, patients with high Gal-1 levels had significantly poorer outcome (5 years OS 10%, 95% confidence interval CI, 1-36) than their low Gal-1 counterparts (5 years OS 48%, 95% CI, 30-64, P =.021). In univariate analyses age 60 or younger, non-elevated lactate dehydrogenase (LDH), and performance score less than 2 correlated with superior survival but high Gal-1 expression significantly predicted adverse outcome at both univariate (HR 2.5, 95% CI, 1.1-5.7, P =.026) and multivariate levels (HR 3.2, 95% CI, 1.2-8.5, P =.017). Tumours with high Gal-1 had few cytotoxic T cells in the tumour microenvironment. High intratumoural Gal-1 expression before therapeutic intervention correlates with adverse outcome in nodal CD30+, ALK− PTCL patients.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) constitutes a heterogeneous category of lymphomas, which do not fit into any of the specifically defined T-cell lymphoma entities. Both the pathogenesis and tumor biology in PTCL-NOS are poorly understood. Protein expression in pretherapeutic PTCL-NOS tumors was analyzed by proteomics. Differentially expressed proteins were compared in 3 distinct scenarios: (A) PTCL-NOS tumor tissue (n = 18) vs benign lymphoid tissue (n = 8), (B) clusters defined by principal component analysis (PCA), and (C) tumors from patients with chemosensitive vs refractory PTCL-NOS. Selected differentially expressed proteins identified by proteomics were correlated with clinico-pathological features and outcome in a larger cohort of patients with PTCL-NOS (n = 87) by immunohistochemistry (IHC). Most proteins with altered expression were identified comparing PTCL-NOS vs benign lymphoid tissue. PCA of the protein profile defined 3 distinct clusters. All benign samples clustered together, whereas PTCL-NOS tumors separated into 2 clusters with different patient overall survival rates (P = .001). Differentially expressed proteins reflected large biological diversity among PTCL-NOS, particularly associated with alterations of “immunological” pathways. The 2 PTCL-NOS subclusters defined by PCA showed disturbance of “stress-related” and “protein metabolic” pathways. α-Enolase 1 (ENO1) was found differentially expressed in all 3 analyses, and high intratumoral ENO1 expression evaluated by IHC correlated with poor outcome (hazard ratio, 2.09; 95% confidence interval, 1.17-3.73; P = .013). High expression of triosephosphate isomerase (TPI1) also showed a tendency to correlate with poor survival (P = .057). In conclusion, proteomic profiling of PTCL-NOS provided evidence of markedly altered protein expression and identified ENO1 as a novel potential prognostic marker.

To the editor: Peripheral T-cell lymphomas (PTCLs) represent a group of rare hematological cancers of mature T-cell or natural killer cell origin accounting for 10% to 15% of all lymphomas.[1][1] Although many patients have poor outcomes, some achieve long-term survival.[2][2],[3][3] Thus

Post-transplant lymphoproliferative disorders (PTLDs) are potentially fatal, often Epstein-Barr virus (EBV)-driven neoplasias developing in immunocompromised hosts. Initial treatment usually consists of a reduction in immunosuppressive therapy and/or rituximab with or without chemotherapy. However, patients who relapse do poorly, and new treatment options are warranted. With the introduction of the immunoconjugate brentuximab vedotin, the CD30 antigen has become an effectively targetable molecule. Therefore, we investigated the frequency and level of CD30 expression in PTLDs. We identified 108 patients with PTLDs diagnosed during 1994-2011, of whom 62 had adequate paraffin-embedded tissue for tissue microarray construction. Immunohistochemical expression of CD30 was consistently detected in all types of PTLD (overall 85.25%), including the monomorphic subtypes, and was correlated with a more favorable outcome. For diffuse large B-cell lymphoma (DLBCL)-type PTLD this was regardless of EBV status, and remained significant in multivariate analysis. Cell-of-origin had no independent prognostic value in our series of DLBCL PTLD.

Clinical trials (CTs) are needed to improve the outcome for peripheral T-cell lymphomas (PTCL), and accrual into CTs is one of the main recommendations in international treatment guidelines. The use of risk-adapted strategies has been suggested as a way to optimize treatment outcome in PTCL. The aim of the present study was to evaluate CT eligibility and selected prognostic indices in a population-based PTCL cohort of 481 PTCL patients identified from the Danish Lymphoma Registry in the period 2000–2010. According to five predefined parameters (age, performance status, P-creatinine, P-ALAT and measurable tumour lesion), patients were subdivided into four groups: ‘younger fit’, ‘elderly fit’, ‘frail’ and ‘not CT eligible’. International prognostic index (IPI), prognostic index for T-cell lymphoma (PIT) and anaplastic lymphoma kinase (ALK) protein expression were tested at subtype-specific level. Overall, 41% of the patients were considered eligible for interventional CTs implicating curatively intended multiagent chemotherapy, including, if considered appropriate, consolidating stem cell transplantation (SCT), as part of the upfront management strategy. Moreover, 28% was elderly fit and eligible for interventional CT, including those with SCT as part of the trial design. Approximately 7% were defined as ‘too frail’ for aggressive treatment schedules, whereas 24% were deemed not to be eligible for any CT. Both overall and progression-free survivals were effectively predicted by IPI and PIT (p

OBJECTIVE: HIV-infected individuals have an increased risk of developing lymphoma. We sought to identify markers predictive of lymphoma development by comparing protein expression patterns in serum obtained at the time of HIV diagnosis from patients who later developed malignant lymphoma or benign lymphadenopathy, with samples from patients with no subsequent history of neoplasia.DESIGN: All patients were identified retrospectively from the Danish HIV cohort.METHODS: Serum samples (N = 21), obtained at time of HIV diagnosis, were subjected to high-resolution two-dimensional gel electrophoresis. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. A tissue microarray, containing diagnostic HIV-lymphoma tissue samples (N = 40), was used to investigate immunohistochemical expression of markers in tumoural lesions.RESULTS: Fourteen differentially expressed protein spots were detected. Using principal components analysis, spots containing immunoglobulin J chain, apolipoprotein A-I, procollagen C-endopeptidase enhancer-1 and complement C4-A were associated with lymphoma development (P CONCLUSION: We identified several differentially expressed protein spots present already at the time of HIV diagnosis. Analysis of biological differences correlating to lymphoma development at this early stage of a possible malignant transformation may lead to the identification of predictive markers. Further investigation of the potential clinical application of differentially expressed proteins as risk stratification markers for monitoring HIV-positive individuals is warranted.

A 10-year-old boy presented with headache and visual disturbance. During work-up in hospital he developed acute renal failure with a maximum creatinine level of 534 μmol/l. Complete blood count was normal. Kidney and bone marrow biopsy both showed massive infiltration of lymphoblasts of T-cell linage. Renal function normalized rapidly on prednisolone therapy. Kidney involvement in acute lymphoblastic leukemia is uncommon and renal failure due to leukemic infiltration is only sporadically reported. This case emphasizes the importance of kidney and bone marrow biopsy in cases of unexplained acute renal failure even with normal hematology.

In a Danish bi-regional registry-based study, we conducted an analysis of the incidence and clinical importance of secondary acute myeloid leukaemia (AML). In a total of 630 cases of AML, we found 157 (25%) cases of secondary AML. The secondary leukaemia arose from MDS (myelodysplastic syndrome) in 77 cases (49%), CMPD (chronic myeloproliferative disorder) in 43 cases (27%) and was therapy-related AML (t-AML) in 37 cases (24%). Median age at diagnosis of AML was 69 yr in secondary cases when compared to 66 yr in de novo cases (P = 0.006). In univariate analyses, secondary AML was associated with an inferior complete remission (CR) rate (P = 0.008) and poorer overall survival (OS, P = 0.003) whereas in complete remitters, disease-free survival (DFS) of secondary cases was equal to that of de novo cases. Interestingly, in all further analyses of CR-rates, OS and DFS, when correcting for the influence of age, cytogenetic abnormalities, performance status and leucocyte count (WBC), presence of secondary AML completely lost prognostic significance. We conclude that the presence of secondary AML does not per se convey an unfavourable prognosis and that patients with secondary AML should be offered the chance of benefiting from treatment according to current frontline AML protocols.

In 74 cases of acute myeloid leukaemia (AML) the relation between pretreatment myeloperoxidase (MPO) activity in polymorphonuclear leucocytes (PMN) and the incidence of infection in the preremission phase of the disease was investigated retrospectively. 36 patients had abnormal numbers (greater than 4%) of MPO-deficient PMN and 38 had normal numbers. In the first group more patients experienced fever attacks, more showed an infectious focus and an aetiological cause was demonstrated more frequently than among patients in the second group. This difference was statistically significant (P less than 0.01). Furthermore, the patients in the first group experienced more fever attacks, showed more infectious focus and had infectious microorganisms demonstrated in more febrile episodes than patients in the second group (P less than 0.01). The differences were not explained by differences in the incidences of neutropenia or other parameters investigated. It is concluded that decreased MPO activity in PMN from AML patients may contribute to the increased susceptibility to infections and that in the preremission phase of the disease it may account for approximately 15% of the infections.

In 45 cases of primary myelodysplastic syndrome; 16 refractory anaemia (RA), 11 RA with ring sideroblasts (RA+), 13 RA with excess of blasts (RAEB), 5 chronic myelomonocytic leukaemia (CMML), the relations between myeloperoxidase (MPO) activity in polymorphonuclear leucocytes (PMN), neutrophil alkaline phosphatase (NAP) activity, absolute number of PMN and thrombocytopenia were investigated. 11 patients (26%) showed abnormal numbers (greater than 4%) of MPO-deficient PMN and 27 (75%) showed abnormal NAP activity (NAP score; greater than 134.0, less than 15.0), mostly decreased. No significant correlations between MPO activity and NAP activity were demonstrated, nor were any significant correlations found with the other parameters investigated. The FAB-subtypes, RAEB and CMML, showed a significant correlation to thrombocytopenia (p = 0.028) and to pancytopenia (p = 0.024). The findings may support the view that at least some of the myelodysplastic syndromes may be fundamentally the same disease as acute myeloid leukaemia.

Myeloperoxidase (MPO) activity of blood granulocytes was estimated in 96 cases of acute myeloid leukaemia (AML), in 35 patients obtaining complete remission and in 14 of these patients during later relapse. As the pretreatment value of MPO activity was the same in patients who died before obtaining remission as in patients obtaining complete remission, determination of MPO-deficient PMN has no prognostic value with respect to the probability of obtaining complete remission. While half of the untreated patients had increased numbers of MPO-deficient PMN in the blood, all patients in complete remission had normal MPO activity (P = 0.0002). A normalization of the MPO activity after induction therapy, therefore suggests remission. Positive correlations could be demonstrated between an initially abnormal MPO activity and abnormal activity at relapse as well as between an initially normal MPO activity and normal activity at relapse (P = 0.0004). It is concluded that determination of the % of MPO-deficient PMN in the blood, may be a useful indicator of complete remission in AML, and in serial determinations during the remission phase also an indicator of threatening relapse.

In western countries, the age distribution of Hodgkin lymphoma (HL) follows a characteristic bimodal curve showing an early and a late peak at approximately 35 and 70 years, respectively. Furthermore, the presence of latent Epstein-Barr Virus (EBV) genome in the Hodgkin Reed-Sternberg cells, the tumour cell population of classical HL (cHL), has been found to have adverse prognostic impact in elderly, but not in younger cHL patients. We have characterized the protein expression in tumour tissue samples from younger (≤55yrs) and elderly (>55yrs) cHL patients and correlated the findings with EBV status. Differentially expressed proteins according to patients' age as well as tumoral EBV status belonged to different biological functional domains, such as apoptosis, cytoskeletal organization, response to oxygen levels and regulation of catabolic/metabolic processes. The differential expression of selected proteins, cytosolic aminopeptidase, heterogeneous nuclear ribonucleoprotein K, serotransferrin, and alpha-1-antitrypsin, was further validated by western blot analysis. Discovery based proteomics characterising biological features distinctive for subsets of cHL patients may be useful for the identification of novel biomarkers with potential therapeutic relevance. An evaluation of the prognostic impact of protein expression pattern in general and individually expressed proteins in particular is warranted. This article is protected by copyright. All rights reserved.

In 145 adult patients diagnosed with non-M3 acute myeloid leukaemia (AML) the relevance of FAB-subtype and immunophenotype to in vitro cellular drug resistance towards the anthracyclines aclarubicin (Acla) and daunorubicin (Dau), and the nucleoside analogue cytarabine (Ara-C), as well as other antileukaemic drugs, was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. We demonstrate that high CD14 expression is highly significantly associated with high cellular Ara-C and Dau resistance in univariate as well as multivariate analyses. FAB subtypes with highest and lowest cellular Ara-C resistance were M4 and M5, respectively (P < 0.01, one-way anova), whereas FAB subtypes with highest and lowest cellular Dau resistance were M4 and M1, respectively (P < 0.01, one-way anova). By contrast, no significant differences in cellular drug resistance towards Acla could be demonstrated among FAB subtypes. Furthermore, in two cohorts of AML patients treated by two different regimens for remission induction over a period of 15 yr (1985-94, n = 159 and 1995-99, n = 76, respectively) we demonstrate in univariate analyses a significance of CD14 expression with respect to clinical outcome. With the exception of significance to probability of obtaining complete remission in the first cohort (P = 0.03, logistic regression), this significance was, however, lost in multivariate analyses. It was demonstrated that FAB-M4 patients were older than M5 patients and that high CD14 expression was associated with the presence of secondary AML and older age. We conclude that although cases with high blast cell CD14 expression (and FAB-M4 cases) were more resistant to Ara-C as well as Dau in vitro, the clinical and biological significance of this may be debatable because of interactions with major prognostic factors in AML.

Introduction. HIV infected individuals have an increased risk of developing lymphoma even in the era of combined antiretroviral therapy. Galectin-1 (Gal-1) is known to promote various immunomodulatory functions, including Treg expansion (Dalotto-Moreno et al, Cancer Res 2013), promotion of tolerogenic dendritic cells (Ilarregui et al, Nat Immunol 2009) and apoptosis of fully-differentiated effector T-cells (Toscano et al, Nat Immunol 2007). In the context of cancer, Gal-1 is expressed on both tumor cells and cells in the tumor microenvironment, and is usually associated with immune privilege, tumor escape and hypoxia-driven angiogenesis (Juszczynski et al, Proc Natl Acad Sci 2007; Cedeno-Laurent et al, Blood 2012). Previously, high intratumoral Gal-1 levels have been suggested as an unfavorable outcome predictor in patients with classical Hodgkin lymphoma (cHL) (Kamper et al, Blood 2011). Furthermore, several in vitro studies revealed the benefit of Gal-1 inhibition with regards to overcoming treatment resistance e.g., after anti-VEGF and anti-CD20 therapy (Croci et al, Cell 2014; Lykken et al, Blood 2016). Thus, Gal-1 inhibition may prospectively be an important tool in lymphoma treatment. In this study, we have investigated the Gal-1 expression in pre-therapeutic tumoral tissue samples from patients with HIV-associated lymphoma and its correlation to clinicopathological features at lymphoma diagnosis. Methods. Adequate pre-treatment formalin-fixed paraffin embedded samples from 40 HIV-positive lymphoma patients were included in a tissue micro array. The study samples were immunohistochemically characterized by a panel of monoclonal antibodies including CD3, CD4, CD8, CD10, CD20, CD79a (MRQ-48), CD30 (Ber-H2) and MUM1, granzyme B, CD68 and CD163. Epstein-Barr virus (EBV) proteins were visualized by latent membrane protein 1 and Epstein-Barr nuclear protein 2. Expression of EBV-encoded smallRNAs was analyzed by in-situ hybridization. Immunohistochemical cell-of-origin (COO) evaluation was carried out according to the Hans classifier. Gal-1 expression was digitally quantified as an area fraction (AF) of the total core area. Estimates of differences between groups were evaluated using Students t test, Pearsons χ2, Fisher's exact test or Spearman correlation where appropriate. Optimal cut-off values of the AF were established by a ROC analysis and calculated using Youden's index for diffuse large B-cell lymphoma (DLBCL) (n=22). Outcome was estimated by Kaplan-Meier time-to-event analyses and compared using the log-rank test. Independent prognostic values were tested by Cox-regression analysis in a multivariate model for factors showing a crude association with p Results. High intratumoral Gal-1 expression in HIV-associated DLBCL tissue correlated with improved outcome (p=0.041), Figure 1. Patients with high Gal-1 expression had a higher occurrence of nodal disease and B-symptoms (p=0.006). Gal-1 expression did not vary according to tumoral EBV status, latency type, International prognostic index (IPI), clinical stage or COO signature. In multivariate analysis adjusted for rituximab treatment, both Gal-1 expression and IPI retained independent prognostic value. Furthermore, intratumoral Gal-1 expression correlated positively with a Th1-signature of the tumor microenvironment including the macrophage marker CD68 (p Conclusion. In HIV-associated DLBCL, intratumoral Gal-1 expression positively correlated with a Th1-signature of the tumor microenvironment. In addition, high pre-therapeutic intratumoral Gal-1 expression was found to be an independent predictor of improved outcome in HIV-associated DLBCL. Interestingly, this is the reverse of our previous findings in non-overtly immunocompromised patients with cHL (Kamper et al, Blood 2011). Further investigations on the potential clinical application of intratumoral Gal-1 expression as a predictive marker in different lymphoma types of the immunocompromised vs immunocompetent host are warranted. Figure. Figure. Disclosures d'Amore: CTI LIfe Sciences: Honoraria, Other: Advisory Boards; Servier: Honoraria, Other: Advisory Boards.

In 98 patients with chronic myeloproliferative disorders (45 chr. myeloid leukaemia (CML), 19 myelofibrosis primaria (MP), 28 polycythaemia vera (PV) and 6 idiopathic thrombocythaemia (IT)) the incidences of increased numbers of MPO-deficient polymorphonuclear (PMN) were 60% in CML, 32% in MP, 7% in PV and 0% in IT patients. The CML figure differed significantly from the others (p less than 0.001). This study confirms the finding of low NAP scores in CML compared to normal or high NAP scores in the other groups of the myeloproliferative syndrome. The incidences of increased numbers of MPO-deficient PMN in this study are comparable to those found in the primary myelodysplastic syndromes and in acute myeloid leukaemia. The finding supports the view that some of the CML cases and may be other cases of the chronic myeloproliferative disorders may be fundamentally the same disease as in primary myelodysplastic syndromes and in acute myeloid leukaemias.

Abstract 1584 Background: Tissue microarray (TMA) is a well established histopathological technique for high-throughput analysis of gene product expression, widely used in studies of malignant diseases. However, systematic validation of its use and performance in rare and heterogeneous entities such as peripheral T-cell lymphomas (PTCL) has not yet been reported. We report on the use of virtual TMAs to validate the adequacy of proposed TMA construction protocols for use in nodal PTCL and to determine the number of cores needed to represent whole slide (WS) results. In addition, we systematically applied digitalized histopathological tools to compare automated quantification protocols for immunohistochemical stains with traditional manual assessment techniques. Methods: Immunohistochemically stained WS were digitalized using a Nanozoomer (Hamamatsu Photonics KK, Japan). Subsequently, a novel virtual TMA with six 1 mm-diameter cores per case (Fig. 1) was designed to analyze 30 PTCL cases (n=10 PTCL-NOS, n=10 AITL, n=10 ALCL). We compared agreements scoring immunohistochemical stains for CD2, CD30 and Ki67 in WS versus TMA cores, using different quantification approaches i.e. 1) digital automated quantification; 2) manual stereological counting. In addition, we assessed the minimum number of TMA cores required to reflect adequately the results derived from WS analysis. Associations were analyzed using Bland-Altman plots and correlation plots. Results: The number of digitally simulated TMA cores required to represent adequately matched WS was found to be 3 or 4, depending on the biomarker under study. Agreement, evaluated by Bland-Altman plots using 4 TMA cores, was good between both manually and digitally quantified WS, and corresponding results comparing digital WS with digital TMA cores. Comparing manually and digitally quantified WS by correlation plots yielded correlation coefficients of 0.93 (CD2), 0.89 (Ki67), and 0.57 (CD30), respectively. Correlation coefficients obtained from comparison of a virtual TMA with 4 cores to digital WS were 0.95 (CD2), 0.89 (Ki67), and 0.99 (CD30). The lower agreement between CD30 stained WS is consistent with the known high degree of heterogeneity in staining for this antigen found in 2 of the 3 PTCL subtypes analyzed (PTCL-NOS and AITL), especially since the manual stereological counting was done in randomly sampled counting frames as opposed to whole slide evaluation by digital image analysis. Conclusions: We show that use of a digitally simulated virtual TMA is a cost-effective and tissue-saving way to validate the effectiveness of different planned TMA construction protocols, to provide adequately representative results when analyzing biomarker expression compared with WS stains. Thus, this approach is applicable in determining the number of TMA cores needed to adequately represent the tumor heterogeneity found in PTCL. Furthermore, automated digital quantification matches traditional stereological counting technique for assessing biomarker expression, and is a useful analysis tool for TMA-based studies in PTCL and, by implication, other tumors. Disclosures: No relevant conflicts of interest to declare.

Considerable effort has been spent identifying prognostic biomarkers in classic Hodgkin lymphoma (cHL). The aim of our study was to search for possible prognostic parameters in advanced-stage cHL using a proteomics-based strategy. A total of 14 cHL pretreatment tissue samples from younger, advanced-stage patients were included. Patients were grouped according to treatment response. Proteins that were differentially expressed between the groups were analyzed using 2D-PAGE and identified by liquid chromatography mass spectrometry. Selected proteins were validated using Western blot analysis. One of the differentially expressed proteins, the carbohydrate-binding protein galectin-1 (Gal-1), was further analyzed using immunohistochemistry HC and its expression was correlated with clinicopathologic and outcome parameters in 143 advanced-stage cHL cases. At the univariate level, high Gal-1 expression in the tumor microenvironment was correlated with poor event-free survival (P = .02). Among younger (≤ 61 years) patients, high Gal-1 was correlated with poorer overall and event-free survival (both P = .007). In this patient group and at the multivariate level, high Gal-1 expression retained a significant predictive impact on event-free survival. Therefore, in addition to its functional role in cHL-induced immunosuppression, Gal-1 is also associated with an adverse clinical outcome in this disease. Fil: Kamper, Peter. Aarhus University Hospital. Department of Hematology; Dinamarca Fil: Lugvigsen, Maja. University Aarhus; Dinamarca Fil: Bendix, Knud. Aarhus University Hospital. Institute of Pathology; Dinamarca Fil: Hamilton Dutoit, Stephen. Aarhus University Hospital. Institute of Pathology; Dinamarca Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Boe Møller, Michael. Odense University Hospital. Department of Pathology; Dinamarca Fil: Nyengaard, Jens. Aarhus University Hospital. Center for Stochastic Geometry and Advanced Bioimaging. Stereology & Electron Microscopy Laboratory; Dinamarca Fil: Honoré, Bent. University Aarhus; Dinamarca Fil: d'Amore, Franceso. Aarhus University Hospital. Department of Hematology; Dinamarca

Abstract 5074 Background: PTLD is a lymphoid proliferation that develops as a consequence of immunosuppression. It represents monoclonal B-cell, or rarely T-cell, proliferations, occurring in a setting of decreased T-cell immune surveillance. It has been shown that HLA-B mismatching is associated with increased risk of skin cancer in recipients of renal transplants. An association between skin cancer related to human papillomavirus and HLA-A11 has also been established in this group of patients. Bakker et al has shown that HLA-B mismatching is a risk marker of PTLD. More specifically, Subklewe et al found that HLA-B18 and HLA-B21 were associated with increased risk of PTLD, whereas mismatch at HLA-A03 and HLA-DR7 level reduced the risk of PTLD. We therefore hypothesized that mismatch of certain HLA alleles may be risk predictors of PTLD after renal transplantation. Methods: According to the national renal transplantation database of the Danish Society of Nephrology, 872 renal transplantations in 793 patients were performed at Aarhus University Hospital between 1990 and 2005. A total of 11 cases of PTLD were retrospectively identified through the National Danish Pathology database and individually reviewed by an experienced hematopathologist (KB). PTLD patients were investigated according to transplantation procedure, clinicopathological patient characteristics, type of lymphoma, outcome and HLA haplotype of both donor and recipient. Results: Univariate Cox regression analysis showed no positive correlation between risk of PTLD and number of HLA-B mismatches, or with HLA-A or HLA-DR mismatches. Conversely, we found a decreased risk with one/two HLA-B mismatches compared to no mismatches (p Conclusions: Our data suggest not only that the risk of developing PTLD is unaffected by an increasing number of HLA-B mismatches, but that a low number of such mismatches may even protect against PTLD developement. In our analysis, we found a moderate correlation between PTLD and HLA-B37, and a stronger one with HLA-DR6. The latter observation has never been previously reported. Further analyses with larger cohorts of renal transplant patients are needed to clarify the role of HLA antigens as risk markers for PTLD in order to establish whether a pre-emptive PTLD monitoring of renal transplant patients expressing certain haplotypes/haplotype mismatches is justified. Disclosures: No relevant conflicts of interest to declare.

Udgivelsesdato: 2009-Aug-13 The aim of the study was to investigate the expression of angio- and lymphangiogenic molecules (vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4) in non-Hodgkin lymphomas (NHL) treated in the pre-rituximab era. Pre-therapeutic lymph-node biopsies from 155 patients with NHL (64 follicular lymphomas (FLs), 47 de novo diffuse large B-cell lymphomas (DLBCL) and 44 peripheral T-cell lymphomas (PTCL)) were stained by in situ hybridization and immunohistochemistry. Tumor cell expression of VEGF, VEGF-C and their receptors was detected in most of the analyzed biopsies. In FL, diffuse intratumoral VEGF staining correlated with shorter overall survival (OS) (p = 0.008) and diffuse KDR staining was associated with a higher risk of histologic transformation (p = 0.05). In DLBCL, high KDR expression predicted poor treatment response (p = 0.03) and had a significant adverse impact on OS (p < 0.001). In PTCL, diffuse tissue distribution of VEGF mRNA correlated with an unfavorable 5-year OS (p = 0.004).

We have designed a multiplex PCR, which allows for fast and high throughput demonstration of the BCL-1/IGH and BCL-2/IGH fusion DNA observed primarily in mantle cell- and follicular non-Hodgkin's lymphoma (NHL). Blood (PB) and/or bone marrow (BM) from 258 patients suspected of NHL have prospectively been evaluated. Eleven patients (4%) were found t(11;14)+ and 37 patients (14%) t(14;18)+. Comparing these results to standard diagnostic methods of PB and/or BM identified PCR+ samples that were normal by morphology (BCL-1/IGH: 1/11; BCL-2/IGH: 17/37). Equally important, patients who were not clonal in PB and/or BM by flow cytometry were identified as PCR+ (BCL-1/IGH: 3/11; BCL-2/IGH: 23/37). We conclude that this multiplex approach allows for easy and sensitive molecular determination of molecular lesions in NHL, which have diagnostic and prognostic importance. Udgivelsesdato: 2007-null

Udgivelsesdato: 2007-Mar The aim of the study was to evaluate angiogenesis in different subtypes of non-Hodgkin's lymphoma (NHL) and to correlate angiogenic scores to clinical endpoints. Pre-therapeutic lymph node biopsies from 308 patients with NHL [107 follicular B-cell lymphoma (FL), 94 diffuse large B-cell lymphoma (DLBCL), 107 peripheral T-cell lymphoma (PTCL)] were studied. Microvessels were scored according to the Chalkley and microvessel density method (MVD) methods. Vascular endothelial growth factor (VEGF) protein expression was evaluated by immunohistochemistry. Both Chalkley and MVD methods showed, that the lymphoma subtypes differed significantly in angiogenic scores (P < 0.001). Angiogenic scores in tumor area were highest in PTCL, and lowest in FL. However, a remarkable high microvessel density was found in interfollicular areas of FL. In FL, high interfollicular MVD scores predicted progressive disease and poorer overall and event-free survival (P = 0.024 and 0.013). High interfollicular Chalkley scores correlated with transformation to DLBCL (P = 0.01). VEGF expression was detected in all NHL subtype, and the strongest expression was found in PTCL. In FL, patients with diffuse VEGF expression in lymphoma cells had poorer overall survival than those with focal expression.

Hodgkin lymphoma (HL) is characterized by a minority of neoplastic, Hodgkin, Reed-Sternberg (HRS) cells surrounded by a heterogeneous background of non-neoplastic cells. Previous studies have described a prognostic role of the cellular composition in HL. The aim of the present study was to correlate parameters related to the intratumoral cellular microenvironment with clinicopathological features and prognosis in a cohort of previously untreated HL patients. For this purpose a tissue microarray Array (TMA) consisting of pairs of representative cores (2mm) from each tissue specimen and from positive and negative controls was constructed. Immunohistochemical stains of cellular antigens were assessed and graded. Tissue samples and clinical record data from a total of 87 HL patients were analysed. Histological subtypes were distributed as follows: nodular sclerosis (NS)61 cases (71%), mixed cellularity (MC) 15 (17%), nodular lymphocyte- predominant (NLP) 9 (10%) and lymphocyte depleted 2 (2%). The median age was 36.7 yrs (range 5–83 yrs and the male to female ratio 1.6. 56 had localised (67%) 27 had advanced stage disease (32%). Anaemia and leukocytosis were present in 4 (5%) and 15 (17%) cases, respectively. All patients were treated according to standard guidelines (chemo- radiotherapy for localised disease and ABVD/COPP- based chemotherapy for advanced stage disease). Immunohistichemical stains were used to characterise and quantify the neoplastic and non-neoplastic cells within tumor lesions. Following antigens were studied: CD3, CD20, CD30, CD15, CD10, STAT-6, MUM-2, FAS, p53 and MIB-1. Among histological subtypes NLP HL was, as expected, the one most frequently displaying CD20 positivity, while CD15 and CD30 were more common in classical HL (cHL). A strong CD3 expression in the non-neoplastic by-stander population was inversely correlated with the presence of leukocytosis in the peripheral blood. An opposite pattern was found in cases, mostly cHL, with high expression of STAT-6 in the tumor cell population. In addition to leukocytosis STAT-6 expression also correlated to younger age (60 yrs or less) and presence of B-symptoms. Time-related endpoint analysis was restricted to NS, representing the largest subgroup (n=61). Interestingly, a significant adverse impact on overall survival (OS) was detected in cases with a strong tumor cell expression of STAT-6. Even more marked was the unfavorable prognostic value of low or lacking tumor cell expression of CD30 (p=0.0012) or by-stander cell expression of CD3 (p=0,0133). All these findings were far more evident in younger (≤ 60 yrs) NS-HL patients than in their elderly counterparts. These three parameters where not, or only minimally, influenced by classical clinical prognosticators such as clinical stage, B-symptoms or anemia. Moreover, the adverse prognostic influence of these factors was mutually increased in those cases, where they occurred in combination (high STAT6 + low CD30: p=0.0001; low CD30 + low CD3: p=0.0004; high STAT6 + low CD3: p=0.0017). The role of the STAT family of proteins and that of T-regulatory cells in the pathophysiology of HL should be further elucidated.

Background: PTLD is a lymphoid proliferation that develops as a consequence of immunosuppression and represents monoclonal B-cell, or rarely T-cell proliferations, occurring in a setting of decreased T-cell immune surveillance. PTLD is a major complication after solid organ transplantation. The disease is often unpredictable and devastating of nature resulting in a high rate of morbidity and patient mortality. Methods: Between 1990 and 2005 872 renal transplantations in 793 patients were performed at Skejby University Hospital in Aarhus and 11 cases of PTLD were identified retrospectively. PTLD Patients were investigated according to transplantation procedure, patient characteristics, type of lymphoma, treatment and outcome. Results: 11 of 793 renal transplant recipients (1,4%) developed PTLD. Patients (7 male, 4 female) ranged from 19 to 73 years of age at the time of diagnosis (mean age: 42 years). 8 had a cadaveric renal transplant while 3 received a transplant from related donor. Lymphomas were classified as diffuse large B-cell lymphoma in 10 cases and as Burkitt lymphoma in 1 case. No cases were of T-cell origin or Hodgkins lymphomas. Six cases were EBV positive (54%). Out of 11 cases, 5 were diagnosed within 1 year after transplantation, among which 4 were EBV positive, whereas 6 had a latency period of more than 1 year, among which only 2 were EBV positive. The mean latency time between grafting and diagnosis of PTLD among the early lesions was 7 months and among late lesions 6 years and 2 months (ranging from 1 month to 10 years and 8 months). Sites involved in PTLD were renal graft in 2 cases (both early lesions), lungs in 1 case, bowel system in 4 cases, CNS in 1 case, isolated lymph nodes in 1 case, widespread disease in 1 case and uncertain location in 1 case. All patients were treated by tapering of the immunosuppressive regimen. Two were treated with additional surgery and 3 with chemotherapy - all 5 of them are alive and in complete remission. Among those 5 patients 3 have preserved graft function (60%) and 2 returned to dialysis. One patient was treated with anti-CD20 monoclonal antibody, 3 had both conventional chemotherapy and anti-CD20 immunotherapy and 2 did not receive additional treatment. All 6 were dead at the end of the study period. With a median follow-up period of 5 years and 2 months (ranging from 6 months to 11½ years) 5 patients of 11 PTLD cases (45%) were alive with no sign of lymphoma relapse. Conclusions: PTLD is a severe complication, usually running an aggressive course and the outcome remains poor. The incidence in our population-based regional study material is 1,4%. Overall survival after a median follow-up of 62 months was 45%, with 60% of survivors maintaining graft function.

Background: PTLD comprises a diverse spectrum of hematological conditions, ranging from early lesions, characterized by reactive-like proliferations, to monomorphic lesions, resembling overt lymphoma. Â In most cases, the lesions are believed to arise as the result of reduced immune surveillance secondary to the use of immunosuppressive drugs post-transplant. This view is supported by the observation that PTLDs, particularly those characterized by early or polymorphic lesions, may regress spontaneously upon reduction of the immunosuppressive treatment. Studies in sporadic lymphomas have identified distinct microenvironmental characteristics, predictive of the clinical behavior, but data is scarce in the immunocompromised setting. Therefore, the aim of this study was to investigate the tumor microenvironment in a population-based cohort of PTLD. Methods: We identified 108 PTLD patients diagnosed in the period 1994-2011. Of these, 62 cases had adequate tissue for tissue microarray construction. All biopsies were reviewed and classified according to the WHO 2008 criteria. Immunohistochemically stained sections were digitally quantified using Tissuemorph (Visiopharm Integrator System 4.0.3.0, Visiopharm, Denmark). Determination of optimal cut-off values of the area fraction (AF) was established by a ROC-curve. ROC analyses were performed in every disease entity and used for endpoint analyses. Results: Themedian age was 45 yrs (range 2-77 yrs) with a M/F ratio of 3:1. The majority presented in stage I-II (61%), and B symptoms and extranodal disease were common features (40% and 42%, respectively). Most tissue samples were EBV-positive (85%). The EBV latency pattern was predominantly latency II (41%) or III (43%). The AF of galectin-1 (gal-1) positive cells was clearly correlated to latency type (p=0.0001), whereas FOXP3 and programmed death-1 (PD-1) did not show such correlation (fig 1). Overall survival (OS) was significantly higher in cases with high levels of PD-1 expression, with 5-yrs OS of 39% (23-55%) and 69% (47-83%) for low and high levels, respectively (p=0.01). Expression levels of FOXP3 and gal-1 had no impact on OS in the total cohort (fig 1). In the monomorphic setting, a low AF of FOXP3 positive cells was associated with an increased risk of progression (OR 6.1; 95% CI: 1.4-26.0). This did not, however, translate into an inferior OS (p=0.163). To specifically characterize the tumor microenvironmental features of DLBCL-type PTLD with respect to cell of origin (COO), 30 evaluable cases were analyzed according to the Hans classifier; 10 (33%) were germinal center (GC) type and 20 (67%) of non-GC type. Cases of non-GC subtype had a significantly shorter time to PTLD of 1.16 yrs (CI: 0.64- 2.11), than those of GC-subtype (3.66 yrs; CI: 1.64- 8.16) (p=0.023) and a lower AF of gal-1 positive cells (p=0.042). The 5-yrs OS was 58% (32-77%) and 0% for non-GC versus GC-tumors, respectively (p=0.075). High levels of FOXP3 expression were associated with superior OS in the non-GC sub-group (p=0.04); gal-1 and PD-1 had no influence in the COO setting. Conclusion: The present study is one of the few attempts to describe tumor microenvironmental features in PTLD and relate them to outcome. In a population-based PTLD cohort, we found that specific immune cell subsets were variably expressed in different PTLD subtypes, and that high expression of PD-1 (whole cohort) and FOXP3 (non-GC DLBCL only) correlated with significantly better outcome. Â | | N(%) pts in TMA | | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------- | | Organ tx(%) | | | Kidney Heart Lung | 47(75.8) 9(14.5) 6(9.7) | | Type PTLD(%) | | | Early/polymorphic lesions Monomorphic PTLD Diffuse large B-cell lymphoma Peripheral T-cel lymphomal, NOS* T-Anaplastic large cell lymphoma Burkitt lymphoma Hodgkin lymphoma-type PTLD Marginal zone lymphoma | 18(29.0) 38(61.2) 32(51.6) 1(1.6) 3(4.8) 2(3.2) 4(6.5) 2(3.2) | ![Figure 1][1] Figure 1 Disclosures No relevant conflicts of interest to declare. [1]: pending:yes

Background: T-cell malignancies originating from regulatory T (Treg) cells are almost exclusively confined to human T-cell leukemia virus 1 (HTLV-1) associated adult T-cell leukemia/lymphoma (ATLL), although sporadic cases of other peripheral T-cell lymphomas (PTCLs) with hypothesized Treg derivation have been reported. Patients and methods: We investigated a series of 169 well characterized PTCLs for the expression of Treg-cell phenotypic markers FOXP3, CD25 and CD4 by immunohistochemistry (IHC) using tissue microarray on formalin-fixed paraffin embedded tissue. Clinico-pathological data for patients enrolled were retrieved from the Danish Lymphoma Registry and medical records. Treg tumors were further invetsigated by Affymetrix OncoScan analysis and Illumina Infinium HumanMethylation450 BeadChips. Results: Variable amounts of FOXP3-positive Treg cells were often (99% of the cases) found as part of the non-neoplastic cellular infiltrate. However, in five cases (3%) classified as PTCL, not otherwise specified (PTCL-NOS) the vast majority of what morphologically appeared to be the neoplastic cell population displayed a strong positivity for FOXP3, CD4, CD25 and CD3 suggesting a probable Treg origin of these cells. Cases were HTLV-1 negative and showed monoclonal rearrangements of the T-cell receptor genes. All cases were male older than 60 years and showed an aggressive clinical course with overall survival less than two years, despite all patients had low IPI-risk profile at the time of diagnosis. The PTCL with Treg phenotype showed a complex pattern of chromosomal imbalances. Moreover, as compared to normal T-cell subsets their DNA methylation profiles were mostly related to that of normal Treg cells but significantly differed from that. Conclusions: We suggest that FOXP3-positve PTCL, in the absence of HTLV-1 infection, constitute a distinct entity separated from PTCL-NOS, occurring predominantly in elderly male patients and characterized by a highly aggressive clinical behavior. Further genomic analyses are ongoing. The identification and characterization of these cases may be useful to guide upfront therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Introduction: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of mature T-cell lymphomas, probably composed by different biologically related subsets that have not yet been conclusively identified. In the WHO classification, PTCL-NOS accounts for 25-30% of all mature T-/NK-cell malignancies. The clinical outcome is generally poor with a 5-yr overall survival of 30-35% after conventional treatment strategies. The aim of the study was to apply proteomic analysis in PTCL-NOS and to use the protein expression profiles to characterize clinically relevant subsets within this heterogeneous entity by means of unsupervised cluster analysis. Methods: Archival frozen tumor tissue samples from 20 patients diagnosed with PTCL-NOS from 1991 to 2010 were analyzed for protein expression by high-resolution two-dimensional gel electrophoresis. Individual protein spots were visualized with fluorescence staining and the expression profiles were identified. All patients were homogeneously treated with curatively intended anthracycline-containing combination regimens. Clinico-pathological features were obtained from the Danish Lymphoma Registry (LYFO) and from patient records. Hyperplastic tonsils from healthy adults were included as reference tissue (n=8). Principal component analysis and unsupervised hierarchical cluster analysis were performed on the basis of the protein expression profiles. Differentially expressed (two-fold or higher, Mann-Whitney U-test) proteins between the detected clusters were identified by liquid chromatography - tandem mass spectrometry. Results: Unsupervised cluster analysis defined three distinct clusters: one containing all reference samples and two additional ones further subdividing the PTCL-NOS cases in two separate subsets. Patients from these two PTCL-NOS subsets had significantly different responses to treatment and survival (p = 0.001). The differentially expressed proteins were primarily involved in (i) promotion of tumor growth, (ii) regulation of cellular metabolism, and (iii) immune responses. Conclusion : Proteomic analysis identified shared protein expression patterns and potential prognostic markers in subsets of PTCL-NOS. Disclosures No relevant conflicts of interest to declare.

Introduction: When lymphomas originally diagnosed as indolent (IL) transform to a more aggressive histology (TIL) the result is often an accelerated clinical course and a shortened survival. The purpose of this study was (i) to investigate whether autologous stem-cell transplantation (ASCT) performed at the time of transformation improved the outcome compared to rituximab-containing chemotherapy alone, (ii) to identify clinical pre-transformation factors influencing post-transformation outcome. Methods: Patients aged 18-68 yrs with histologically verified sequential TIL diagnosed between 1999 and 2012 at 3 Danish tertiary lymphoma referral centers were identified using the National Danish Pathology Registry. Pre-therapeutic clinico-pathological features as well as treatment- and outcome data at baseline and follow-up were collected through the Danish lymphoma registry (LYFO) and patient records. The patient cohort was subdivided into 2 major subsets according to treatment strategy at transformation i.e. ASCT or no ASCT. Selected characteristics of potential clinical relevance, treatment background and response rates were compared using χ2 test, Fisher's exact test or t test and tested in a multivariate analysis using a Cox proportional Hazards model. Treatment outcomes were estimated as 5-year overall (OS) and progression-free survival (PFS) and compared using the log-rank test. Results: Fifty-seven patients with sequential TIL were included of which 39 patients received ASCT after initial immunochemotherapy and 18 did not. The two treatment cohorts (non-ASCT vs. ASCT) were comparable in all features including time to transformation (3.4 yrs vs. 6.5 yrs; p=0.10) and treatment with rituximab at IL-stage (n=5, 28% vs. n=7, 18%; p=0.49). Outcome determinants were tested at a median follow-up of 3.1 yrs (0.1-13.4 yrs) from the time of TIL diagnosis. The 5-yr OS was 57% if ASCT was performed and 36% if it was not (p=0.10). In terms of 5-yr PFS, ASCT treated patients had significantly higher values as compared to not transplanted ones (47% vs. 6%; p=0.003). This associated superior outcome was irrespective of prior rituximab treatment (Prior rituximab: 21% vs. 0%; p=0.02. No prior Rituximab 52% vs. 9%; p=0.03). However, the favorable impact of ASCT was greatest in patients who were rituximab-naïve at transformation (5-yr OS: 21% vs. 64%; 5-yr PFS: 21% vs. 52%). When comparing the outcome of sequential TIL patients based on the number of prior chemotherapy regimens, a higher number of prior regimens (>2) correlated with an inferior outcome (5-yr OS: 70% vs. 22%, p=0.05; 5-yr PFS: 64% vs. 23%, p=0.04) as did a high FLIPI-score. No influence on outcome was observed with regard to type of treatment at IL-stage, induction at TIL-stage (CHOP-like vs. platinum based), age or WHO-performance score at TIL diagnosis. Conclusions: ASCT improved the outcome in sequential TIL. The beneficial impact of ASCT was greater in patients, who were rituximab-naïve at transformation. Factors with negative influence on outcome were a high FLIPI-score and the number of treatment regimens prior to transformation. Disclosures No relevant conflicts of interest to declare.

Long-term follow-up data are presented on changes in peripheral blood counts and Hb requirements of 11 patients with myelodysplastic syndromes (MDS) during iron chelation treatment with desferrioxamine for up to 60 months. The erythroid marrow activity was indirectly evaluated by repeated determinations of the serum transferrin receptor concentration. The efficacy of iron chelation was evaluated by repeated quantitative determination of the liver iron concentration by magnetic resonance imaging. Reduction in the Hb requirement ( > or = 50%) was seen in 7/11 (64%) patients. Five patients (46%) became blood transfusion independent. Platelet counts increased in 7/11 (64%) patients and the neutrophil counts in 7/9 (78%) evaluable patients. All patients in whom iron chelation was highly effective showed improvement of erythropoietic output accompanied by an increase in the serum transferrin receptor concentration. It is concluded that reduction in cytopenia in MDS patients may be accomplished by treatment with desferrioxamine, if the iron chelation is efficient and the patients are treated for a sufficiently long period of time. Exactly how treatment with desferrioxamine works remains a challenge for further investigation.

Abstract 4427 Very rarely, Philadelphia positive (Ph+) chronic myeloid leukemia (CML) presents with isolated thrombocythemia (CML-T) with no or only slight elevation of neutrophil counts. Due to their rarity, such patients have not been studied in detail, especially whether they constitute a separate disease entity, where the initiating leukemic hit originates from a more lineage-committed progenitor cell type than the one affected in classical CML. To evaluate this, we studied a 68-year-old woman who during a follow-up for follicular lymphoma presented with a sudden thrombocythemia of 2062×109/L, a basophil count of 0.94×109/L but no increase in neutrophil count (3.9×109/L). A bone marrow (BM) biopsy revealed slight to moderate hyperplasia with an increased number of small, hypolobulated megakaryocytes, but no increase in the myelopoietic compartment. Apart from the Ph1 positivity, no molecular aberrations were seen by cytogenetic analysis, array CGH, or extensive molecular testing; noteworthy, the JAK2 V617F mutation was absent. The patient was treated with three different Tyrosine Kinase Inhibitors (TKI's), all of which had to be terminated after a total of 26 weeks of therapy due to severe side effects (dyspnea, acrocyanosis of the fingers and fatigue). At present, she is on pegylated interferon alpha-2a, which is well tolerated. In a longitudinal study of the stem cell biology in this patient we centered on three BM aspirates obtained at diagnosis as well as 3 and 9 months following diagnosis. Subsets of progenitor- and mature lineage committed cells were FACS-purified and analyzed for the presence of leukemic cells by FISH and qPCR technique and additionally subjected to growth in a 14-day methylcellulose assay. At diagnosis, the CD34+ compartment consisted of 10% immature (CD34+CD38-) and 90% mature (CD34+CD38+) progenitors, both of which were enriched for Ph+ cells (74% and 78%, respectively) (Figure 1A-B). Moreover, the colony-forming cells (CFC's) from sorted CD34+ cells were BCR-ABL+ with the erythroid lineage (BFU-E; 94%) dominating and only 4% myeloid lineage (CFU-G, CFU-M, and CFU-GM) and 2% multi-lineage (CFU-GEMM). By qPCR analysis of picked colonies we, moreover, observed that all types of colonies were BCR-ABL+ (Figure 1C). After three months of TKI therapy the patient responded with a 2.6 log decrease in BCR-ABL expression in BM mononuclear cells, the BCR-ABL+ CFC's disappeared and the distribution of erythroid and myeloid colonies normalized. However, specific analysis of the progenitor compartment showed reductions of only 0.5 log in CD34+CD38- cells and 1.6 log in CD34+CD38+ cells. At 9 months after the diagnosis, after pausing TKI treatment for 3 months, BCR-ABL expression in peripheral blood resurged and further analysis identified Ph+ cells by FISH in CD66+ neutrophils (10%) and CD19+CD10+ B-cell precursors (2%), and by expression of BCR-ABL in CD19+ B-cells (positive in one of three triplicate wells) and CD3+ T-cells (0.000068 K562 equivalents) (Figure 1D). These data show, despite the highly unusual presentation with isolated thrombocythemia, that the origin of this form of CML is very similar to classical CML with pan-lineage involvement and a resistance of progenitor cells to TKI therapy. The selective expansion of megakaryocytic lineage cells in this form of CML, however, remains enigmatic and will require further insight into the mechanics of differentiation in the malignant clone at the level of common myeloid progenitors or earlier. Figure 1. Delineation of stem cell biology in a CML patient presenting with isolated thrombocythemia. (A) FACS sorting of CD34+CD38− and CD34+CD38+ cells at diagnosis and 3 months after. (B) Initial treatment response in whole BM and FACS sorted CD34+CD38+ and CD34+CD38− cells. (C) Lineage involvement of the CML clone 9 months after diagnosis (after 3 months of pausing TKI treatment). (D) Growth of FACS sorted CD34+ cells in CFC-assay at diagnosis and 3 and 9 months after. : BCR-ABL expression measured by qPCR, and normalized to K562 equivalents (K562eq). : Fraction of Ph+ cells evaluated by FISH. Figure 1. Delineation of stem cell biology in a CML patient presenting with isolated thrombocythemia. (A) FACS sorting of CD34+CD38− and CD34+CD38+ cells at diagnosis and 3 months after. (B) Initial treatment response in whole BM and FACS sorted CD34+CD38+ and CD34+CD38− cells. (C) Lineage involvement of the CML clone 9 months after diagnosis (after 3 months of pausing TKI treatment). (D) Growth of FACS sorted CD34+ cells in CFC-assay at diagnosis and 3 and 9 months after. : BCR-ABL expression measured by qPCR, and normalized to K562 equivalents (K562eq). : Fraction of Ph+ cells evaluated by FISH. Disclosures: No relevant conflicts of interest to declare.

Abstract 5202 We here report the observation of the development of a splenic marginal zone lymphoma (SMZL) after years of active chronic visceral Leishmania (VL) infection, implying a possible pathogenic link between these two conditions, where the neoplastic process is preceded by a prolonged period of chronic antigen stimulation. In 2004, a 60-year old male developed attack-like episodes of septic fever with chills, rigor and subsequent profound fatigue. He had a 4 years previous medical history of severe asthma-like symptoms and hypereosinophilia. A bone marrow (BM) and liver biopsy were inconclusive. A whole-body computed tomography showed a moderately enlarged spleen. No evidence of bacterial, fungal or viral infection was found; the symptoms persisted, although mitigated, with occasional exacerbations over the following 3 years. By 2007 the patient was admitted again and a new BM biopsy was taken, still showing reactive changes. Due to increased splenomegaly, a diagnostic splenectomy was performed. Examination of the spleen revealed a primary splenic marginal zone lymphoma (SMZL). In 2010 the patient was admitted with septicaemia and constitutional symptoms. A BM biopsy showed the presence of numerous Leishmania amastigotes and a retrospective review of all previous biopsies (since 2004) identified Leishmania amastigotes in all of them. In fact, a thorough travel history covering the previous two decades revealed several visits, often twice a year, to the Mediterranean basin, e.g. Greece and Malta, areas endemic for Leishmania infantum (L. infantum). Visceral Leishmaniasis is a frequent opportunistic infection in HIV-infected immunodeficient individuals but uncommon in cancer patients, and only a few cases of VL in patients with already well-established malignant lymphoma have been reported. Chronic antigenic stimulation by microbial pathogens has been proposed as a pathogenetic factor in a variety of marginal zone lymphomas. Tissue specific immune responses toward Leishmania parasites have been shown in rodents, and the spleen is a ‘safe harbor’ for the long-term persistence of visceralizing Leishmania. Sustained antigenic stimulation with upregulation of critical signaling pathways may result in prolonged polyclonal B-cell proliferation, and a subsequent increased risk of malignant transformation. This case is the first to provide evidence for a possible link between chronic antigen stimulation by long-lasting L. infantum infection and the development of a SMZL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3586 The prognosis of leukemia patients suffering from secondary AML (sAML) compared to that of patients with de novo AML is dismal. The group of sAMLs is heterogeneous and includes AML arising from an antecedent myelodysplastic (MDS) or myeloproliferative neoplasm (MPN), and AML caused by cytotoxic therapy (tAML). In the present retrospective population- and national registry-based analysis we identified 612 (27%) patients as having some form of sAML. Cytogenetic risk group patterns and clinical outcomes among the different categories of sAML were compared to those of 1635 de novo AML cases identified in a total population of 2261 patients (data missing in 14 cases). The cohort represents >90% of all AML patients diagnosed and treated in Denmark during the eleven-year period January 2000 through December 2010. The following groups of sAMLs were identified: A. Patients with an antecedent MDS or chronic myelomonocytic leukemia (324 cases), B. Patients with antecedent MPN (excluding chronic myeloid leukemia, 108 cases), C. Patients previously treated with chemo- and/or radiotherapy for another hematological neoplasm (113 cases), and D. Patients previously treated with chemo- and/or radiotherapy for another non-hematological neoplasm or disease (67 cases). For all 1168 curatively treated patients in the total cohort, presenting cytogenetic abnormalities (categorized according to revised MRC-criteria, D. Grimwade et al. Blood, 2010), age, leukocyte count, and type of leukemia (secondary vs. de novo) were all prognostic parameters found to be highly statistically significant to probability of attainment of complete remission (CR) and to overall survival (OS) in univariate as well as multivariate analyses, data not shown. There were strikingly fewer patients showing favorable cytogenetic abnormalities among sAMLs. Focusing on the above defined 4 categories of sAML, patterns of cytogenetic risk group distribution were strikingly and statistically significantly different (nevaluable= 418, p-value, Chi-square Additionally, in the sAMLs we found age, cytogenetic abnormalities, and white blood cell count (WBC) to be highly statistically significant to probability of attainment of CR and to duration of OS. By contrast, we did not find the specific sAML category to be of significance to probability of attainment of CR or to duration of OS, Table 2, Fig. 1.Table 2.Factors of significance to probability of attainment of CR and to OS in 246 cases of secondary AMLProbability of CR (Logistic regression, nevaluable= 246)Probability of overall survival (Cox regression, nevaluable= 246)VariableOdds ratio (OR)95% CI of ORP valueHazard ratio95% CI of HRP valueAge1.071.03–1.11 In conclusion, from these analyses we confirm the prognostic significance of presence of sAML as well as other well established prognostic parameters in AML. We find cases of sAML-category D, i.e., patients previously treated with chemo- and/or radiotherapy for another non-hematological neoplasm or disease, to exhibit favorable cytogenetic abnormalities relatively frequently. Probability of attainment of CR and OS duration were similar in the four different specific categories of sAML. Well established prognostic parameters including age, cytogenetic abnormalities, and WBC are of significant prognostic value in sAML. Disclosures: No relevant conflicts of interest to declare.

Abstract 5103 An increased risk of alk-negative T-cell derived anaplastic large-cell lymphoma (T-ALCL) in patients with breast prostheses has been suggested, although unequivocal evidence of an association has not yet been provided. Several reports have also suggested that silicone, one of the major components of breast prostheses, activates the immune system, and induction of myeloma has been shown in a murine model. While association to breast prostheses has been described in as many as 48 patients with T-cell derived ALCL1, only one case of breast implant-associated B-cell derived lymphoma, displaying a follicular histology2, has been reported so far. We here present a case of DLBCL diagnosed in 2006 in a 66 year-old woman, who had undergone cosmetic implantation of bilateral breast prostheses 20 years previously. The disease initially involved right cervical and mediastinal nodes. She was treated with chemo-immunotherapy (rituximab + CHOP q 3 weeks for a total of 8 series) achieving a complete remission (CR) by summer 2006. Almost 1 year later, a nodal DLBCL relapse occurred at cervical level. The patient was again treated with chemoimmunotherapy (rituximab+ dexamethasone, high-dose cytosine arabinoside and cis-platin q 3 weeks for a total of 3 series). A new CR was obtained and consolidated with an autologous transplant with BEAM conditioning in March 2008. More than a year later, a new cervical node relapse occurred along with a small focus in the lung (not bioptically verified). From then on, the patient received multiple therapies, every time with initial chemosensitivity, but quickly followed by new progression as soon as therapy was discontinued. According to PET assessment, there has never been any tumour manifestation below the diaphragm, and no lymphoma infiltration was detected at any time in the bone marrow. As of June 2010, the patient developed multiple cutaneous and subcutaneous tumours corresponding to the anterior thoracic wall in close proximity to the upper quadrants of both breasts. These tumors were preceeded by an erythematous lesion clearly demarcating the cutaneous area of the anterior thoracic wall and breasts corresponding to the underlying implants. Cutaneous biopsies taken at this erythematous stage already revealed diffuse DLBCL infiltration of the skin and subcutis. Cutaneous and subcutaneous biopsies showed alk-negative, CD30-negative CD20-positive DLBCL. All previous lymph node biopsies are CD20 positive, bcl-2 and bcl-6 positive and negative for CD10. A fraction of tumour cells expressed MUM-1. By and large, no major changes in the immunohistochemical profile of the tumor have been observed since the original diagnosis in 2006. The striking anatomical localization of the latest relapse, but also the fact that the patient's disease over the years persistently manifested itself in lymph node drainage regions adjacent to or in the near proximity of the patient's breast implants, may be suggestive of a chronic antigenic stimulation eventually resulting in a malignant B-cell lymphoproliferation of DLBCL type. DLBCL histology has not previously been reported in possible association with breast implants. Disclosures: No relevant conflicts of interest to declare.

Abstract 4150 The immunotoxin gemtuzumab ozogamicin (Mylotarg‘, GO) comprises a potent cytotoxic agent, calicheamicin, and a recombinant humanized monoclonal antibody directed against CD33. GO is increasingly used for treatment of acute myeloid leukemia (AML) as CD33, a trans-membranous glycoprotein, is highly expressed on most leukemic blast cells, but, in normal cells confined to early multilineage hematopoietic progenitors and myelomonocytic precursors. Adverse effects of GO treatment are primarily related to myelosuppression (infections, fever, bleedings). However, severe sinusoidal obstructive syndrome (SOS) and hepatotoxicity have also been reported. Since the introduction of GO treatment into routine clinical practice, the incidence of SOS has been reported to span from 0% to 33%, but, the direct cause of the liver-associated effects remain unknown. Using immunohistochemistry we show that the CD33 receptor is widely distributed in the liver tissue and highly expressed on hepatocytes making them susceptible for targeted CD33 chemotherapy by GO. Moreover, utilizing CD163 as a specific marker for macrophages, double immunofluorescence staining revealed that Kupffer cells strongly express CD33. Therefore, the reported hepatoxicity associated with GO treatment may be due to accumulation of antibody-toxin conjugates in hepatocytes and Kupffer cells causing calicheamicin-induced damage. Disclosures: No relevant conflicts of interest to declare.

The vascular endothelial growth factor family (VEGF, VEGF-B, -C and -D) is the main regulator of angiogenesis and lymphangiogenesis via their receptors, Flt-1, KDR and Flt-4. The aim of the present study was to estimate the microvascular density and investigate the expression of VEGF, VEGF-C and its receptors Flt-1, KDR and Flt-4 in peripheral T-cell lymphomas (PTCL). Material and methods: Microvessel density (MVD) was determined after CD34 immunohistochemical (IH) staining of endothelial cells in pre-therapeutic lymph-node biopsies from 107 cases of PTCL (64 unspecified, 1 angiocentric, 10 angioimmunoblastic and 32 anaplastic large cell T/0). 44 of these cases (28 unspecified, 1 angiocentric, 4 angioimmunoblatic and 11 anaplastic large cell T/0) were investigated for expression of angiogenic molecules, VEGF, VEGF-C and their receptors by IH at protein level. Furthermore, VEGF and VEGF-C mRNA expression was detected by non-isotopic ′in situ′ hybridization. The angiogenic scores were correlated to pre-therapeutic clinical parameters, treatment response and survival. Results: Tumoral expression of VEGF, VEGF-C and of their receptors was detected in the majority of the PTCL biopsies investigated (VEGF mRNA: 73%, VEGF protein: 95%; VEGF-C mRNA: 79%; VEGF-C protein: 79%; Flt-1 and KDR: 100%; Flt-4: 26%). All biopsies contained VEGF, Flt-1 and KDR positive vessels and VEGF-C was also widely expressed by the endothelial cells (3 negative cases), while endothelial Flt-4 expression was detected only in 43% of cases. High MVD scores correlated with an advanced clinical stadium (Ann Arbor) (p=0.047). A strong diffuse VEGF mRNA expression significantly correlated with a poorer overall survival (p=0.0015) as compared to focal or negative ISH signal. The predictive value of this parameter persisted at multivariate level independently of the International Prognostic Index (HR: 3.95, p=0.012, Cox regression). Conclusion: VEGF, VEGF-C and their receptors are expressed by both lymphoma and endothelial cells in the majority of PTCL cases. VEGF expression seems to have an adverse impact on survival. Larger studies of angiogenesis/lymphangiogenesis in PTCL are needed to clarify the biological role of these molecules and identify possible therapeutic targets.

Hemoglobin liberated to plasma during intravascular hemolyses is rapidly bound to haptoglobin. The hemoglobin-haptoglobin complexes undergo endocytosis through the monocyte/macrophage specific scavenger receptor for hemoglobin (CD163). This mechanism protects against oxidative and NO-scavenging adverse effects of free hemoglobin. In this study, we describe a novel syndrome of severe intravascular hemolysis and serious hemolytic crisis due to impaired hemoglobin scavenging in three acute myeloid leukemia patients following CD33-directed therapy with the immunotoxin gemtuzumab ozogamicin (GO, Mylotarg™). A synchronous high free hemoglobin, haptoglobin, and low bilirubin after septicemia-induced intravascular hemolysis indicated abrogated clearance of haptoglobin-hemoglobin complexes. This was further supported by low levels of plasma soluble CD163 and a concordant low number of CD163-expressing monocytes. We show that CD163 positive monocytes and bone marrow macrophages coexpress CD33 thus suggesting that the GO-induced cellular cytotoxicity of CD33 positive cells eradicates a significant part of the CD163 positive monocytes and macrophages. The patients had severe inflammation and serious organ failure symptoms that may be a direct effect of the persistent high level of free hemoglobin. One of the patients had a fatal outcome whereas, the other two recovered from the hemolytic episode, and the peripheral blood CD163 expression returned to normal. The risk of a serious hemolytic crisis should be considered following CD33-targeted chemotherapy. Furthermore, the cases provide circumstantial evidence of a key role of CD163 plasma hemoglobin clearance in vivo.

Aim: To develop a multiplex PCR method applicable in a clinical setting for the simultaneous detection of the chromosomal lesions t(11;14)(q13q32), t(14;18)(q32;q21), t(2;5)(p23;q35), t(11;18)(q21;q21), t(3q27;var), and t(8;14)(q24;q32) frequently found in non-Hodgkin lymphoma (NHL). Methods: DNA and RNA were prepared from 50μm lymph node (LN) sections by homogenization on a FastPrep instrument (Qbiogene, Cedex, France) followed by automated nucleic acid purification on a MagNa-Pure LC robot (Roche Diagnostics, Basel, Switzerland). The multiplex PCR was condensed in four PCR tubes. The first covered the MTC and mTCp94 region of BCL1/IGH fusion DNA, the MBR and MCR breakpoint regions of BCL2/IGH fusion DNA together with the control gene TCF20. The second included the API2/MLT and ALK/NPM breakpoints on cDNA along with β-ACTIN as a control gene. The third contained primers amplifying eight different fusions partners of BCL6 (IGH (14q32), IGL (22q11), HSP89α (14q32), HSP90β (6p12), PIM1 (6p21), TFR (3q26), TTF (4p13), and H4 (6p21)) on cDNA together with β-ACTIN as the control gene. The fourth tube harbored a long range PCR with primers detecting the CMYC/IGH breakpoints on genomic DNA (Cμ, Cγ, Cα, and joining region of the IGH (Basso et al., 1999, Am J Pathology)) together with ABL as a control gene. Patient samples and cell lines: One-hundred-and-twelve LN biopsies frozen in Tissue-Tek OCT Compound (Sakura, Vaerloese, Denmark) were randomly selected from consecutive patients referred with suspected hematological malignancy. The following cell lines were used as positive controls: B-CLL line JVM-2 (t(11;14)+), NHL lines DOHH-2 (t(14;18)+, t(8;14)+) and WSU-NHL (t(14;18)+), Burkitt’s lymphoma lines BL-41, BL-70 (t(8;14)+), and MD901 (t(3;22)+), T-NHL line Karpas 299 (t(2;5)+), and ALL line MD903 (t(3;14)+). Results: In pilot experiments employing cell lines and fresh LN material, this optimized multiplex PCR reaction proved to be simple and fast with a short turnover time, considering the large number of genetic aberrations detected. In a retrospective LN material encompassing 112 blinded samples, BCL1/IGH fusion DNA with breakpoint in the MTC region was detected once while BCL2/IGH was found in 20 samples (19 in MBR and one in MCR). BCL6/IGH fusion cDNA was found in three samples while the TTF gene was utilized twice as translocation partner to BCL6. Finally, CMYC/IGH fusion DNA was detected three times (1 IGHCα, 2 IGHCγ). All PCR products apart from CMYC/IGH were sequenced and verified the specific chromosomal lesions. Nineteen were excluded due to weak control bands in the first three PCR tubes, while 38 were excluded in the long range PCR detecting CMYC/IGH. Conclusion: We conclude that the NHL multiplex PCR described is an easy and timesaving method for identifying heterogeneous molecular disease markers in NHL. The standardized DNA- and RNA preparation together with the condensation into four PCR tubes, moreover, makes it convenient to the clinical setting. Application of this assay and identification of positive cases has the added advantage that quantitative real-time PCR monitoring residual disease can be applied.

Of eight patients with primary myelodysplastic syndrome (MDS) treated with Roacutane (13-cis retinoic acid, Roche, Basel, (13-RA)) 20 mg/m2 for 6 weeks and an additional 100 mg/m2 for 4 weeks (3 patients), 4 responded either with a slight increase in peripheral blood neutrophil count or a decrease in myeloperoxidase deficient neutrophils. In agar cultures 2 patients showed a concurrent increase in growth of day 11 colonies and clusters. In 2 of the patients a decrease in the number of immature bone marrow cells positive for the myeloid antibody anti-My7 was observed. Only minor alterations were seen in natural killer cell activity. In 4 patients showing clonal chromosomal abnormalities before treatment a disappearance of minor clonal abnormalities during treatment was observed, and in 3 chromosomal abnormalities reappeared after cessation of therapy. Even though the overall impact of 13-RA on the hematopoietic system was minor, the increase in myeloperoxidase normal granulocytes in the blood and the decrease in My7 positive cells and in clonal chromosomal abnormalities warrants further interest in this agent as a treatment modality in MDS. The side effects, especially experienced by the patients receiving 100 mg/m2, were, in spite of symptomatic treatment, of such a degree that only low dose treatment (10-20 mg/m2) administered for prolonged periods of time (3-6 months) would seem recommendable.

Relations between cytogenetic status, FAB-classification and an abnormal subpopulation of myeloperoxidase (MPO)-deficient polymorphonuclears (PMN) in 45 patients with myelodysplastic syndrome (MDS) are reported. Clonal abnormalities were demonstrated in 85% of the patients, with a lower incidence in the RA+ group (refractory anaemia with ring sideroblasts) compared to the others (p = 0.004). In 12 patients a spontaneous progression in cytogenetic aberrations occurred and in 7 of these (60%) a simultaneous progression in FAB-subtype was seen. The appearance of MPO-deficient PMNs was observed in 6 of these patients (55%). A progression in FAB-subtype was noted in further 4 patients and 2 additional patients developed MPO-deficient PMNs. Only one sufficient cytogenetic investigation was available in these patients. Thus 100% of the fully studied patients showed progression in cytogenetic abnormalities when a progression in FAB-subtype or a development of MPO-deficient PMNs was seen. 3 (49%) of the 8 patients developing MPO-deficient PMNs too showed a progression in FAB-subtype. Although no significant correlation to specific categories of structural aberrations or abnormalities in specific chromosome pairs could be demonstrated, clonal cytogenetic aberrations seem to be involved when the disease progresses and when MPO-deficient PMN develop.

In a prospective analysis of the diagnostic value of immunophenotyping in acute leukemias (ALs), all patients admitted to a pediatric and a haematological department suspected of AL were examined consecutively with a selected panel of monoclonal antibodies (Mabs) against leucocyte differentiation antigens during an 8-month period. A total of 189 samples obtained from blood, bone marrow, spinal fluid and lymph nodes in 120 cases were all analysed blindly. The results were correlated with a routine morphological/cytochemical evaluation. Differing results were obtained in seven out of 38 cases in which the immunologically defined diagnosis was acute myeloid leukemia (AML), and in one out of 21 cases with the primary diagnosis acute lymphoid leukemia (ALL). Immunological phenotyping disclosed two cases of hybrid leukemia, one case of biphenotypic and one case of bilineal leukemia. No evidence of malignancy was found in 36 cases, 30 cases of blood and bone marrow and six cases of spinal fluids, in every case in accordance with the pathological examination. These results demonstrate that a first-line immunological evaluation of bone marrow, blood and spinal fluid from patients suspected of AL is highly capable of discriminating between different malignant and nonmalignant haematological diseases and also between various types of leukemias. The immunological methods do, however, require a sufficient amount of material which was a limiting factor in 14 out of 120 examinations, mainly from patients treated with several cycles of cytostatics. It is concluded that immunophenotyping can be used as a first-line diagnostic tool in malignant haematological diseases.

Neutrophil alkaline phosphatase activity was estimated in 194 patients; 59 cases of chronic myeloid leukaemia (CML), 42 cases of polycythaemia vera (PV), 24 cases of primary myelofibrosis, 7 cases of idiopathic thrombocythaemia, 6 cases of leukaemoid reaction, 19 cases of secondary polycythaemia (PS) and 37 cases of the primary myelodysplastic syndrome (MDS). According to NAP activities the groups proved to be separate entities (p less than 0.00025). The incidence of decreased NAP score in the CML group was 85% and differed significantly from the other groups as a whole, as well as separately (p less than 0.001). The MDS group, the only group besides CML that showed decreased scores, also differed significantly from the others (p less than 0.001). The PS group, nearly always showing normal scores, differed significantly from the PV group (p less than 0.0052). A method evaluating single cell NAP activity proved superior to the score method in discriminating between the different groups. Thus, the incidence of decreased activity in the CML group was 93% compared with 85% by the score method and the incidences of increased activity in the PV, MP, IT, and LR groups were 79% to 100% compared with 25% to 67% by the score method. The latter difference was statistically significant (p = 0.029).

In 148 consecutive cases of untreated acute myeloid leukaemia (AML) the relation between an increased number of myeloperoxidase (MPO)-deficient polymorphonuclear leucocytes (PMN) and a classification modified after the proposals outlined by the French-American-British Co-operative Group, the FAB classification, was investigated. In 22 cases with minimal granulocytic component, 8 M5, 13 M6, 1 M7, no one (0%) showed an increased number of MPO-deficient PMN. In 3 (13%) of 23 cases with slight granulocytic component, 3 M0, 20 M1, and in 49 (57%) of 86 cases with granulocytic differentiation (53 M2, 1 M3, 32 M4), an increased number was demonstrated. This difference was statistically significant at a high level (P less than 0.0001). The number of MPO-deficient PMN could not be estimated in 15 cases of AML and 2 additional cases were mixed leukaemias. It is concluded that an increased number of MPO-deficient PMN in acute leukaemia speaks in favour not only of AML, but suggests the diagnosis of subtypes with some granulocytic component, most likely M1, M2, M3 or M4. Furthermore, the results support the concept that in AML at least some of the mature myeloid cells may be involved in the leukaemic process.