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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

At AHIV-1 infection, the binding of the viral envelopeproteins to CD4þ is essential for viral transmission, andthis process is facilitated by interaction with the highlyconserved host lectin, galectin-1 (Gal-1) [1?3]. Withinthe tumor microenvironment, Gal-1 is expressed by bothtumor and stromal cells where it promotes tumorimmune escape and favors hypoxia-driven angiogenesis[4?6]. In sporadically occurring Hodgkin lymphoma,high Gal-1 expression at diagnosis is associated withpoorer treatment response [7], and high soluble Gal-1(sGal-1) correlates with adverse disease characteristics [8].Previous studies have shown that targeted inhibition ofGal-1 prevents tumor-induced immunosuppression[9,10] as well as inhibits tumor growth and metastasisin various tumor models [6,11?13].In conclusion, the results of our study indicate that Gal-1 is significantly associated with risk of lymphoma in HIV-infected individuals and may represent an attractive futuretarget for the management of HIV-associated lymphoma. Fil: Vase, MajaØlholm. University Aarhus; Dinamarca Fil: Ludvigsen, Maja. University Aarhus; Dinamarca Fil: Bendix, Knud. University Aarhus; Dinamarca Fil: Dutoit, Stephen H.. University Aarhus; Dinamarca Fil: Hjortebjerg, Rikke. University Aarhus; Dinamarca Fil: Petruskevicius, Irma. University Aarhus; Dinamarca Fil: Møller, Michael B.. Odense Universitetshospital; Dinamarca Fil: Pedersen, Gitte. Aalborg Universitet; Dinamarca Fil: Denton, Paul W.. University Aarhus; Dinamarca. Arhus Universitetshospital; Dinamarca Fil: Honoré, Bent. University Aarhus; Dinamarca Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Larsen, Carsten S.. Arhus Universitetshospital; Dinamarca Fil: D'Amore, Francesco. Arhus Universitetshospital; Dinamarca

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

Background: A potential link between breast implants and anaplastic large-cell lymphoma (ALCL) has been suggested. Methods: We examined lymphoma occurrence in a nationwide cohort of 19,885 Danish women who underwent breast implant surgery during 1973–2010. Standardized incidence ratios (SIR), with 95% confidence intervals (CI), for ALCL and lymphoma overall associated with breast implantation were calculated. Results: During 179,246 person-years of follow-up, we observed 31 cases of lymphoma among cohort members. No cases of ALCL were identified. SIRs for ALCL and lymphoma overall were zero (95% CI, 0–10.3) and 1.20 (95% CI, 0.82–1.70), respectively. Conclusions: In our nationwide cohort study, we did not find an increased risk of lymphoma in general, or ALCL in particular, among Danish women who underwent breast implantation. However, our evaluation of ALCL risk was limited by the rarity of the disease. Impact: Our results do not support an association between breast implants and ALCL and are consistent with other studies on cancer risk and breast implants. Cancer Epidemiol Biomarkers Prev; 22(11); 2126–9. ©2013 AACR.

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 15 patients with hairy cell leukaemia splenic epithelioid granulomas were demonstrated in 4 out of 13 investigated cases (31%) and liver granulomas in 2 out of 10 cases (20%). Granulomas were never found in bone marrow specimens. Histological stains for mycobacteria, fungi and bacteria failed to demonstrate an etiological agents and culture (sputum) for mycobacteria were only performed in 3 cases, 1 showing Mycobacterium Tuberculosis. Attention to the possible role of atypical mycobacterial infections as an explanation to the often reported unresponsive fever of unknown origin in hairy cell leukaemia and the use of lymph node and/or liver biopsies for culture as well as histology is recommended.

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.

INTRODUCTION: Post-transplant lymphoproliferative disorder (PTLD) incidence is difficult to determine, mainly because both early and other lesions may go unrecognised and unregistered. Few studies have included systematic pathology review to maximize case identification and decide more accurately PTLD frequency after long term post-transplantation follow-up.METHODS: A retrospective population-based cohort study including all kidney transplant recipients at two Danish centres (1990 - 2011; population covered 3.1 million; 2175 transplantations in 1906 patients). Pathology reports were reviewed for all patient biopsies to identify possible PTLDs. Candidate PTLDs underwent histopathological review and classification.RESULTS: Seventy PTLD cases were identified in 2175 transplantations (3.2%). The incidence rate (IR) after first transplantation was 5.4 cases per 1000 patient-years (95% CI: 4.0-7.3). Most PTLDs were monomorphic (58.5%), or early lesions (21.5%). Excluding early lesions and patients < 18 years, IR was 3.7 (95% Cl: 2.9-5.5). Ten PTLD patients were retransplanted, 2 developing further PTLDs. Post-transplant patient survival was inferior in PTLD patients, while death censored graft survival was not.CONCLUSIONS: Using registry data together with extensive pathological review and long follow-up, a rather high incidence of PTLD was found. This article is protected by copyright. All rights reserved.

AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods.METHODS: The DNA for PCR was extracted from frozen and FFPE tissue, cell lines and blood. PCR primers Vgamma1-8, Vgamma9, Vgamma10 or Vgamma11 (5' end labelled) combined with a mixture of JgammaP1/JgammaP/JgammaP2/Jgamma2 (unlabelled) were used. Monoclonal cases were sequenced and clonality, reproducibility, sensitivity and specificity analyses were carried out.RESULTS: In all cases the molecular test was found to be in agreement with the histological diagnosis. Discrepancies were found between frozen and FFPE tissue in 18 of 56 (32%) tests. The method was highly reproducible. The sensitivity was found to be 0.5% for cell lines and 1% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient.CONCLUSIONS: Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however, to correlate the results with conventional morphological and immunohistochemical studies.

The influence of wear debris on bone healing around orthopedic implants is debated. Hydroxyapatite (HA) particles and polyethylene (PE) particles have been shown to have a negative effect on osteoblast cultures in vitro. The present study investigated the in vivo effects of HA and PE particles on the mechanical fixation and gap healing around experimental HA implants. Nonloaded implants (n = 30) were inserted bilaterally into the proximal tibia of 15 dogs with a 2-mm gap to the bone. The peri-implant gap was either (1) empty (n = 6) or filled with (2) hyaluronic acid (n = 8), (3) hyaluronic acid and HA particles (n = 8), or (4) hyaluronic acid and PE particles (n = 8). After 4 weeks, the animals were killed. The implant interface was evaluated by pushout testing until failure and by histomorphometry. Both HA and PE particles were found to be phagocytosed by macrophage-like cells in the interfacial tissue. HA particles were also integrated in newly formed bone. We found no negative effect of the particulate material on mechanical fixation of the implants or on bone formation around the implants.

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.

Laser microbeam microdissection (LMM) is an increasingly important method for obtaining pure cell samples for genetic and proteomic analysis. Immunohistochemistry (IHC) and in situhybridization (ISH) are useful techniques for targeting specific cell populations for microdissection but are difficult to apply with the tissue support membranes often used during LMM. Using detection of cytokeratins and Epstein-Barr virus gene products in head and neck carcinoma as a model, we describe optimized protocols for membrane and section preparation and for low temperature antigen retrieval that allow IHC and ISH to be used reliably on membrane mounted paraffin tissue sections. Visualization of cellular targets was markedly improved by staining and this could be further improved using a variety of optical media before microdissection. Tissue fragments thus stained were suitable for subsequent polymerase chain reaction analysis of extracted DNA using standard techniques. These IHC and ISH procedures are generally applicable and will be useful for detecting a wide range of antigens and nucleic acids in paraffin sections in conjunction with LMM.

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 a study of 71 autopsies in patients with malignant haematological disorders (55 leukaemias and 16 multiple myelomas) we found an increased number of megakaryocytes in the lung capillaries in only one of 55 cases of leukaemia (43 acute and 12 chronic leukaemias) with a mean value of 3 megakaryocytes per cm2. The incidence of pulmonary megakaryocytes in 16 cases of multiple myeloma was identical to that in an unselected, consecutive series of hospital autopsies. The discrepancy between the increased megakaryocytopoiesis and previously reported high number of circulating megakaryocytes in chronic myeloid leukaemia, and the few megakaryocytes in the pulmonary blood vessels of histological sections of autopsy specimens is discussed.

The distribution of intrapulmonary megakaryocytes is equal in healthy persons, but altered in diseased persons, the highest counts being found in superior lobes (p = 0.0027). This may be explained by the altered blood supply to the lungs of diseased persons lying in bed. We recommend that lung sections should be taken from central areas of the superior lobes in order to standardize the sections and to compaire results of future investigations on intrapulmonary megakaryocytes.

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.

A retrospective investigation of 75 EDTA-decalcified Jamshidi biopsies collected over a 2-year period at Aarhus University Hospital was performed. The biopsies originated from 75 patients suffering from idiopathic myelofibrosis, other chronic myeloproliferative disorders, or other conditions with known associations with bone marrow fibrosis. The relative volumes of trabecular and woven bone, as well as haematopoietic and non-haematopoietic tissue, were estimated histomorphometrically. The degree of fibrosis was estimated semiquantitatively. Finally, the thickness of trabecular osteons was estimated from the number of lamellae by counting. Patients with idiopathic myelofibrosis had statistically significantly more bone tissue than the other groups of patients. The osteosclerosis was primarily due to woven bone. Larger cancellous osteons also suggested a positive balance in bone remodelling. The amount of bone tissue showed furthermore a statistically significant increase through the groups of polycythaemia vera, essential thrombocythaemia, chronic myelogenous leukaemia and idiopathic myelofibrosis. Parallel to the increase in the amount of bone, an increase in the degree of marrow fibrosis was detected. The positive correlation between the amount of bone and the degree of marrow fibrosis was statistically highly significant (p=0.0008).

We used the molecular cytogenetic in situ techniques chromosome painting and PRimed IN Situ labeling (PRINS) to elaborate the cytogenetic observations in two cases of the rare aberration der(16)6(1;16), which occurs in a wide variation of hematologic and nonhematologic malignancies [1–13]. Review of the literature showed that, in contrast to the chromosome 1 breakpoint, the breakpoint on chromosome 16 is associated with diagnosis as well as patient age.

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.

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

Supplemental digital content is available in the text., Background Posttransplant lymphoproliferative disorder (PTLD) is a feared complication to organ transplantation, associated with substantial morbidity and inferior survival. Risk factors for PTLD include T cell–depleting induction therapy and primary infection or reactivation of Epstein-Barr virus. Possible associations between certain HLA types and the risk of developing PTLD have been reported by other investigators; however, results are conflicting. Methods We conducted a retrospective, population-based study on 4295 Danish solid organ transplant patients from the Scandiatransplant database. Having identified 93 PTLD patients in the cohort, we investigated the association of HLA types with PTLD, Epstein-Barr virus status and time to PTLD onset. The outcomes survival and PTLD were evaluated using Cox regression; mismatching, and the PTLD-specific mortality were evaluated in a competing risk analysis. Results Risk of PTLD was associated with male sex (odds ratio, 1.70; 95% confidence interval, 1.07-2.71), and, in women, HLA-DR13 conferred an increased risk (odds ratio, 3.22; 95% confidence interval, 1.41-7.31). In multivariate analysis, HLA-B45 and HLA-DR13 remained independent predictive factors of PTLD. Mismatching in the B locus was associated with a reduced risk of PTLD (P < 0.001). Overall survival was poor after a PTLD diagnosis and was significantly worse than that in the remaining transplant cohort (P < 0.001). Conclusions Our data indicate risk-modifying HLA associations, which can be clinically useful after transplantation in personalized monitoring schemes. Given the strong linkage disequilibrium in the HLA region, the associations must be interpreted carefully. The large size, virtually complete ascertainment of cases and no loss to follow-up remain important strengths of the study.

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.

Trisomy 8 is a frequently acquired cytogenetic abnormality in myeloid malignancies, but may also represent a constitutional chromosome abnormality with a wide phenotypic variation. We report a case of myelodysplastic syndrome (MDS) that developed in a child with trisomy 8 mosaicism and normal phenotype. Bone marrow (BM) cells all showed trisomy 8 with additional clonal abnormalities in most cells. Based on the present case and a review of previously published cases of myeloid malignancies in patients with trisomy 8 mosaicism, it appears likely that the malignant cells developed from the trisomic cell population, suggesting that constitutional trisomy 8 may be a predisposing condition to myeloid malignancies. Trisomy 8 in malignant cells is usually considered an acquired abnormality, but this implies a risk of ignoring a constitutional trisomy 8 mosaicism. Examination for constitutional trisomy 8, despite a normal phenotype, may therefore be warranted in hematologic malignancies with trisomy 8 of BM cells to evaluate further the possible association and to preclude erroneous use of trisomy 8 as a tumor marker.

Background Limited data exist on the presence and function of immune-competent cells in chronic tendinopathic tendons, and their potential role in inflammation and tissue healing. Objectives To quantify subtypes of immune-competent cells in biopsies from non-ruptured chronic tendinopathic Achilles tendons and healthy control tendons. Methods Fifty patients with non-ruptured chronic Achilles tendinopathy and 15 healthy subjects were included. At time of inclusion, the Achilles tendons were examined clinically and evaluated with ultrasound with regard to tendon thickness and Doppler flow grade immediately before an ultrasound-guided tendon biopsy was obtained. Tissue samples were evaluated immunohistochemically by quantifying the presence of macrophages (CD68-KP1 + ), iron + hemosiderophages (Perls Blue), T-lymphocytes (CD3 + ), B-lymphocytes (CD20 + ), natural killer cells (CD56 + ), mast cells (NaSDCl + ), Schwann cells (S100 + ) and endothelial cells (CD34 + ) using a stereological technique. Cell counts were expressed as number of positive cell profiles per biopsy area (c/a), except for CD68-KP1 and CD34, which were quantified using a point counting grid to estimate number of grid hits of stained cells which were then calibrated to the total biopsy area (expressed as cell fractional area (%)). Descriptive data are reported as median and range if nothing else specified. Comparisons between groups were made with Mann-Whitney U tests or Fisher9s Exact Tests. The level of significance was set at P Results Macrophages, T-lymphocytes, mast cells, and natural killer cells were observed in the majority (range: 52–96%) of biopsies from non-ruptured chronic tendinopathic Achilles tendons. Conclusions This study provides evidence for the presence of immune-competent cells in the majority of biopsies from non-ruptured chronic tendinopathic Achilles tendons. Macrophages and endothelial cells were significantly more numerous in chronic tendinopathic tendons than in healthy tendons. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.1684

This study addresses the clinical problems regarding access of wear debris to the bone-implant interface and the possible dissemination of polyethylene (PE) particles to distant organs. We inserted two implants into each knee of 7 dogs allowing access of joint fluid to the bone-implant interface with a 0.75 mm initial gap around the implant. Hydroxyapatite (HA)-coated and non-coated (Ti) titanium alloy implants were randomly allocated to each distal femoral condyle. PE particles were repeatedly injected into the right knee joint 3 weeks after surgery for a period of 49 weeks, while only vehicle was injected into the left knee joint. We found huge amounts of PE particles mainly in the bone-implant interface around Ti implants. Infiltration of mononuclear inflammatory cells was present around 3 of 7 Ti implants in relation to PE particles. HA implants had approximately 70% bone ongrowth. In contrast, no bone ongrowth was seen on any Ti implants, all being surrounded by a fibrous membrane. The number of PE particles was evaluated semi-quantitatively. More PE particles were found around Ti implants than with HA implants (p < 0.002). Specimens from iliac lymph nodes, liver, spleen and lung were examined and showed dissemination of PE particles only in regional lymph nodes.

Background The etiology of Achilles tendinopathy still remains unclear. Limited data exists on whether immune-competent cells are present in the tendon. Objectives To evaluate the presence of T and B lymphocytes, NK cells and macrophages in chronic Achilles tendinopathy using immunohistochemistry and unbiased stereological technique for quantification. Further, we wanted to correlate the amount of each cell type to disease duration as well as tendon thickness and vascularity evaluated by ultrasound immediately before biopsy. Methods 56 non-ruptured Achilles tendons from 25 women and 31 men with chronic tendinopathy were examined with Doppler ultrasound regarding tendon thickness and vascularity. 17 patients had received local steroid injection or other anti-inflammatory treatment within 6 months before the examination. From all tendons, a biopsy was obtained and stained with hematoxylin/eosin, Van Gieson, toluidin blue, Pearls Blue and NaSDCl in addition to the following immunohistochemical markers: CD2, CD3, CD4, CD7, CD8, CD20, CD34, CD56, CD68(KP1), CD68(PG-M1) and Granzyme-B. The area fraction count (AFC) of positive cells in each sample was determined by quantifying and calibrating the area fraction of positive cells to a standard area in 4 mm thick slides using unbiased stereological techniques. The Mann-Whitney rank sum test and the Spearman correlation test was used (p Results The median age of the patients was 50 years (range 33-69) and the median disease duration was 14 months (range 4-360). In 56 Achilles tendon biopsies, we found presence of CD2 + cells in 44 (79%), CD3 + cells in 49 (88%), CD4 + cells in 47 (84%), CD7 + cells in 38 (68%), CD8 + cells in 32 (57%), CD20 + cells in 10 (18%), CD34 + cells in 54 (96%), CD56 + cells in 31 (55%), CD68KP1 + cells in 54 (96%), CD68PG-M1 + cells in 49 (88%), Granzyme-B + cells in 4 (7%), iron positive cells in 13 (23%) and NaSDCl positive cells in 45 (80%). The T lymphocyte AFC correlated positively to A: B lymphocyte AFC (CD20∼CD2, CD4, CD7; p B: Macrophage AFC (CD68∼CD2, CD3, CD4, CD7, CD8; p C: NK cell AFC (CD56∼CD2, CD7, CD8; p Disease duration correlated to B lymphocyte (CD20) AFC (p=0.02, r-value=0.31) and NK cell (CD56) AFC (p=0.01, r-value=0.35). In addition, both B lymphocyte and NK cell AFC were significant higher in patients with a disease duration of more than 14 months compared to patients withh a shorter disease duration (p No correlation was found between tendon thickness, Doppler vascularity and any AFC. Conclusions In 56 biopsies from non-ruptured chronic tendinopathic Achilles tendons, we found a combined presence of macrophages, T and B lymphocytes and NK cells with no signs of granulocytic cellular infiltrate. Longer disease duration was associated with higher area fraction count of B lymphocytes and NK cells. These findings support the theory of chronic tendinopathy being driven by an immunologic process. Disclosure of Interest None Declared

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.

Invasive fungal infection is an increasing problem in severely immunocompromised patients. A 30-year-old man with profound pancytopenia due to acute myelogenous leukaemia acquired a severe invasive candida oesophagitis with total desquamation and expulsion of 90 mm of the distal oesophagus. The case report lends support to the concept of early recognition of fungal oesophagitis including species identification and possibly also antimycotic prophylaxis during immunosuppressive treatment.

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.