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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

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.

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 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.

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 as well as in predicting long-term outcome. Purpose: To quantify subtypes of immune-competent cells in biopsy specimens from nonruptured chronic tendinopathic Achilles tendons and healthy control tendons. In addition, to examine whether findings in baseline cell biopsy specimens can predict the long-term presence of Achilles tendon symptoms. Study Design: Cross-sectional and case-control study; Level of evidence, 3. Methods: Fifty patients with nonruptured chronic Achilles tendinopathy and 15 healthy participants were included. At time of inclusion, an ultrasound examination was performed immediately before an ultrasound-guided Achilles tendon biopsy specimen was obtained. Tissue samples were evaluated immunohistochemically by quantifying the presence of macrophages (CD68-PGM1+, CD68-KP1+), hemosiderophages (Perls blue), T lymphocytes (CD2+, CD3+, CD4+, CD7+, CD8+), B lymphocytes (CD20+), natural killer cells (CD56+), mast cells (NaSDCl+), Schwann cells (S100+), and endothelial cells (CD34+) using a stereological technique. A follow-up examination was conducted more than 4 years (range, 4-9 years) after the biopsy procedure to evaluate the long-term presence of Achilles tendon symptoms. Results: Macrophages, T lymphocytes, mast cells, and natural killer cells were observed in the majority (range, 52%-96%) of biopsy specimens from nonruptured chronic tendinopathic Achilles tendons. CD68-KP1+ macrophages (0.29% vs 0; P = .005) and CD34+ endothelial cells (3% vs 0.97%; P = .04) were significantly more numerous in tendinopathic tendons compared with healthy tendons. The presence of iron+ hemosiderophages was more frequently observed in biopsy specimens obtained from the group who was asymptomatic at follow-up compared with the symptomatic group (42% vs 5%; P = .02). Conclusion: This study provides evidence for the presence of immune-competent cells in the majority of biopsy specimens from nonruptured chronic tendinopathic Achilles tendons. Macrophages and endothelial cells were significantly more numerous in tendinopathic tendons than in healthy tendons. The presence of iron+ hemosiderophages in baseline biopsy specimens was associated with a good prognosis. Clinical Relevance: New insight into the role of immune-competent cells in chronic Achilles tendinopathy.

AIMS: Macrophage infiltration has been associated with prognosis in several cancers, including lymphoma, but has not been assessed systematically in anaplastic large cell lymphoma (ALCL). The aim of the study was to correlate expression of the macrophage-associated antigens CD68 and CD163 with pre-therapeutic parameters and outcome in a cohort of treatment-naive ALCL patients.METHODS AND RESULTS: Pre-therapeutic tumour specimens from 52 patients with ALCL were included in a tissue microarray. The intratumoral macrophage content was assessed by immunohistochemical staining for CD68 and CD163, and quantified using digital image analysis. Anaplastic lymphoma kinase (ALK)-positive patients were significantly younger and had a favourable outcome compared with ALK-negative ALCL patients (median age: 42 versus 59 years; P = 0.008). However, ALK expression was not a significant predictor when adjusting for age. Although classical risk factors were distributed evenly between the compared groups, high intratumoral content of CD68 and/or CD163 correlated with poor outcome, in both univariate and multivariate analyses. High intratumoral CD163 content showed the strongest adverse association with both overall and progression-free survival in ALK-negative patients (P < 0.001).CONCLUSIONS: A high content of intratumoral CD68- and/or CD163-positive macrophages correlates with an adverse outcome in ALK-negative ALCL.

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 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).

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.

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.

Migration of wear debris to the periprosthetic bone is a major cause of osteolysis and implant failure. Both closed-pore porous coatings and hydroxyapatite (HA) coatings have been claimed to prevent the migration of wear debris. We investigated whether HA could augment the sealing effect of a porous coating under both stable and unstable conditions.We inserted porous-surfaced knee implants, with and without HA coating, in 16 dogs, according to a paired, randomized study design. 8 dogs had 2 implants inserted into each knee using a stable implant device and 8 dogs received 1 implant in each knee using a micromotion (500 microm) implant device. Implants had a peri-implant gap of 0.75 mm. We then injected polyethylene (PE) particles or a control solution into the knee joints on a weekly basis.After 16 weeks, the rating of particles around stable implants was reduced by the HA coating froma median value of 2 (1-4) to 1 (0-1) (p = 0.01) and during micromotion from 3 (2-4) to 1 (0-3) (p = 0.002). HA-coated implants had superior bone ongrowth during stable and unstable conditions. We found no difference in bone ongrowth between PE-exposed and vehicle-exposed implants.Compared to a pure plasma-sprayed porous coating, a layer of HA coating provides bettter bone ongrowth and protects the bone-implant interface against the migration of wear debris under both stable and unstable conditions.

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