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Approximately 5% to 10% of diffuse large B-cell lymphomas (DLBCLs) harbor an MYC oncogene rearrangement (MYC+). The prognostic significance of MYC+ DLBCL was determined in an unselected population of patients with newly diagnosed DLBCL treated with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (R-CHOP). Using a Vysis break-apart fluorescence in situ hybridization probe, 12 of 135 (8.8%) cases of MYC+ DLBCL were identified that had no defining high-risk features. MYC+ DLBCL was associated with an inferior 5-year progression-free survival (66% vs 31%, P = .006) and overall survival (72% vs 33%, P = .016). Multivariate analysis confirmed the prognostic importance of MYC for both progression-free survival (hazard ratio = 3.28; 95% confidence interval, 1.49-7.21, P = .003) and overall survival (hazard ratio = 2.98; 95% confidence interval, 1.28-6.95, P = .011). Cases of MYC+ DLBCL also had a higher risk of central nervous system relapse (P = .023), independent of other risk factors. The diagnosis of MYC+ DLBCL is likely underappreciated; and given the lack of defining risk factors, fluorescence in situ hybridization for MYC rearrangements should be performed in all patients with DLBCL. In the R-CHOP treatment era, MYC+ DLBCLs have an inferior prognosis. Treatment regimens similar to those used in Burkitt lymphoma may be more appropriate in this patient population and need to be prospectively tested.

Background BCL6 gene rearrangement is the most frequent chromosomal abnormality in diffuse large B-cell lymphoma, a malignancy characterized by genetic heterogeneity and wide variability in clinical outcome. The prognostic significance of BCL6 rearrangement has not been evaluated in the context of rituximab therapy for diffuse large B-cell lymphoma. We analyzed the effect of the BCL6 rearrangement on survival in patients with diffuse large B-cell lymphoma treated with CHOP and CHOP plus rituximab (R-CHOP). Design and Methods BCL6 rearrangement status was analyzed by fluorescence in situ hybridization with break-apart probes in 164 patients with diffuse large B-cell lymphoma treated with CHOP (n=65) or R-CHOP (n=99). Cell-of-origin immunophenotype including BCL6 protein expression were determined by immunohistochemistry on a tissue microarray. Results BCL6 rearrangement was detected in 19.5% of cases. The presence of the gene rearrangement was associated with a non-germinal center B-cell immunophenotype ( P =0.006), and showed no correlation with BCL6 protein expression. A trend toward inferior overall survival was observed in association with the BCL6 rearrangement among patients treated with R-CHOP ( P =0.08), but not among patients treated with CHOP ( P =0.64). However, BCL6 rearrangement also correlated with a high International Prognostic Index score ( P =0.02), and did not demonstrate independent prognostic value by multivariate analysis. Conclusions The introduction of rituximab may have altered the prognostic impact of BCL6 gene rearrangement in patients with diffuse large B-cell lymphoma. However, prospective analysis within large randomized clinical trials will be needed to clarify the prognostic significance of this biomarker in the rituximab era.

Transcription factors play a central role in malignant transformation by activating or repressing waves of downstream target genes. Therapeutic targeting of transcription factors can reprogram cancer cells to lose their advantages in growth and survival. The BCL6 transcriptional repressor plays a central role in the pathogenesis of diffuse large B-cell lymphomas (DLBCL) and controls downstream checkpoints, including the p53 tumor suppressor gene. We report that a specific inhibitor of BCL6 called BPI can trigger a p53 response in DLBCL cells. This was partially due to induction of p53 activity and partially due to relief of direct repression by BCL6 of p53 target genes. BPI could thus induce a p53-like response even in the presence of mutant p53. Moreover, sequential BCL6 peptide inhibitors followed by p53 peptide or small-molecule activators provided a more powerful antilymphoma effect than either treatment alone by maximally restoring p53 target gene expression. Therefore, tandem targeting of the overlapping BCL6 and p53 transcriptional programs can correct aberrant survival pathways in DLBCL and might provide an effective therapeutic approach to lymphoma therapy. [Cancer Res 2008;68(9):3361–9]

Loss of major histocompatibility class II (MHC class II) molecules on diffuse large B-cell lymphoma (DLBCL) has been associated with poor survival; however, none of these reports analysed a uniformly treated patient cohort. This study was designed to validate one MHC class II antigen, HLA-DR, as a prognostic marker in patients uniformly treated with the MACOP-B regimen. Immunostaining results were correlated with the international prognostic index (IPI) score and overall survival (OS). Of the 97 cases, 82 had interpretable staining. Of these, 52 expressed HLA-DR (median OS, 16.2 years) while 30 were negative (median OS, 4.2 years, P = 0.037). The IPI was also predictive of OS in the study group (P = 0.023). A Cox multivariate model established both IPI (P = 0.031) and HLA-DR (P = 0.04) as independent predictors of OS. This is the first demonstration of the prognostic relevance of HLA-DR in a uniformly treated DLBCL patient group.

Purpose: We aimed to assess the prognostic significance of follicular lymphoma–associated macrophages in the era of rituximab treatment and maintenance. Experimental Design: We applied immunohistochemistry for CD68 and CD163 to two large tissue microarrays (TMA). The first TMA included samples from 186 patients from the BC Cancer Agency (BCCA) who had been treated with first-line systemic treatment including rituximab, cyclophosphamide, vincristine, and prednisone. The second contained 395 samples from PRIMA trial patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, and randomized to rituximab maintenance or observation. Macrophage infiltration was assessed using Aperio image analysis. Each of the two cohorts was randomly split into training/validation sets. Results: An increased CD163-positive pixel count was predictive of adverse outcome in the BCCA dataset [5-year progression-free survival (PFS) 38% vs. 72%, respectively, P = 0.004 in the training cohort and 5-year PFS 29% vs. 61%, respectively, P = 0.004 in the validation cohort]. In the PRIMA trial, an increased CD163 pixel count was associated with favorable outcome (5-year PFS 60% vs. 44%, respectively, P = 0.011 in the training cohort and 5-year PFS 55% vs. 37%, respectively, P = 0.030 in the validation cohort). Conclusions: CD163-positive macrophages predict outcome in follicular lymphoma, but their prognostic impact is highly dependent on treatment received. Clin Cancer Res; 21(15); 3428–35. ©2015 AACR.

We studied the role of multiple biomarkers in determining outcome in follicular lymphoma (FL), concentrating in particular on the role of benign macrophages. The study group consisted of uniformly staged and treated patients with FL enrolled in a phase 2 trial between 1987 and 1993. All patients were younger than 61 years of age, had advanced-stage FL, and were treated with a multiagent chemotherapy regimen, BP-VACOP (bleomycin, cisplatin, etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisone), followed by involved region radiation. The median follow-up of living patients was 12.5 years, and the median survival was 16.3 years. The International Prognostic Index (IPI) was predictive of overall survival (OS) (P = .003). Biopsy specimens from all cases were stained with an anti-CD68 antibody. Of the 99 evaluable patients with FL, 87 had less than 15 CD68+ macrophages/high-power field (hpf) (median, 7; range, 1-14) and 12 had more than 15 CD68+ macrophages/hpf (median, 20; range, 16-25) with a median OS of 16.3 vs 5.0 years, respectively (P < .001). A multivariate Cox model that included the IPI score, the histologic grade, and the lymphoma-associated macrophage (LAM) score, showed IPI and LAM to be independent predictors of OS (P = .009 and P = .004, respectively). The LAM content of FL predicts survival, and these data support a prominent role for nonneoplastic immune cells in the biology of FL. (Blood. 2005;106:2169-2174)

Extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) type occur in a number of anatomic sites, but share overlapping morphologic and immunophenotypic features. Helicobacter pylori infection has been identified as an etiologic factor in gastric MALT lymphoma, and a growing list of other infectious organisms have recently been shown to be associated with MALT lymphomas at other anatomic sites. Although cause and effect has not been established for most of these infectious agents, our understanding of the biology has significantly improved, in part through the application of standard cytogenetic analyses. The common karyotypic alterations that characterize MALT lymphomas include the trisomies 3 and 18, the translocations t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), t(3;14)(q27;q32), and the recently described t(3;14)(p14.1;q32). This apparent complexity of cytogenetic alterations that have now been implicated in the pathogenesis of extranodal MALT lymphoma serves as a paradigm for molecular cross talk in neoplastic disease. Recent data have shown that at least three of the disparate translocations affect a common signaling mechanism, and thus unify all three under a common pathogenesis, resulting in the constitutive activation of the nuclear factor kappa B (NF-κB) pathway. It may be that the new MALT-related translocation involving the FOXP1 gene and other as yet undiscovered translocations may all have in common increased NF-κB signaling.

Helicobacter pylori are gram-negative spiral microaerophilic organisms that belong to the Campylobacterales order, Helicobacteracea family. They are capable of colonizing the harsh environment of the human stomach. Over 50% of the world’s population carries this infection. 1 H pylori have colonized the human stomach since time immemorial. 2 In virtually all infected individuals, H pylori causes inflammation in the form of chronic active gastritis, which progresses in 10%–20% of affected persons to peptic ulcer disease, gastric adenocarcinoma, and/or mucosa-associated lymphoid tissue (MALT) lymphoma. However, only a very small minority (1%–2%) of infected individuals will develop a malignant disease. 3 One of the most distinctive features of H pylori is the genetic diversity it displays between clinical isolates. 4 H pylori isolates are highly diverse, with evidence of a constantly changing genome, primarily caused by point mutations, substitutions, insertions, and/or deletions. 5,6 Moreover, mixed infections are frequent and may lead to exchange of DNA fragments between H pylori strains in a single host. This suggests co-evolution of H pylori with its human host and that disease may be caused both by strain-specific properties that increase virulence and by host susceptibility. Such strains possibly evolve through exercising significant flexibility in gene content and gene regulation. Among many of the host–pathogen interactions that potentially could occur, some could prove beneficial in which the co-evolved bacteria and the host reach an almost symbiotic relationship. H pylori may not be just a bad bug in all instances. A large body of data have implicated H pylori in the pathogenesis of gastric MALT lymphoma, including epidemiologic, biological, and molecular genetic studies. Interestingly, both H pylori and MALT lymphoma were born almost simultaneously, when H pylori was described by Marshall and Warren 7 and MALT lymphoma was recognized as a distinct entity by Isaacson and Wright. 8 These latter investigators noted morphologic similarities between the histology of the condition known as immunoproliferative small intestinal disease, a subtype of primary intestinal B-cell lymphoma, and primary low-grade gastric B-cell lymphoma. Moreover, the histologic features differed from that of comparable nodal low-grade B-cell lymphomas in that the overall architecture and cytology of these lymphomas bore a resemblance to normal MALT tissue rather than recapitulating the structure of lymph nodes. Roughly one third of all non-Hodgkin’s lymphomas in adults are of extranodal origin. MALT lymphomas, by definition arising at mucosal/epithelial sites, collectively account for approximately 8% of all non-Hodgkin’s lymphomas, with gastric MALT lymphoma being the most common extranodal site. This review summarizes the published data that support a causative role for H pylori infection in the pathogenesis of gastric MALT lymphomas. The epidemiology of H pylori is discussed and its role in the pathogenesis, including factors affecting the infectious organism and the host. A major focus of this review addresses the relationship between the immune response, H pylori, and MALT lymphomas. This is followed by a description of the morphology, phenotype, and unifying concepts of the molecular genetics of MALT lymphomas. Treatment strategies of gastric MALT lymphoma are not discussed because these are beyond the scope of this review. Finally, we conclude with a brief discussion of H pylori–negative MALT lymphomas, preventative measures for reducing infection, and the direction of future research into MALT lymphomas.

We report a case of a man presenting with an unexplained fever, pancytopenia and hepatosplenomegaly without lymphadenopathy. Bone marrow flow cytometry strongly suggested hepatosplenic γδ T-cell lymphoma and infiltration of bone marrow samples by pathological T-lymphocytes confirmed the diagnosis. Despite chemotherapy the patient died 1 year after diagnosis. This is a rare disease that should be considered in the differential diagnosis of hepatosplenomegaly especially when it presents with B-symptoms and no lymph node enlargement. There is no standard therapy and the prognosis is poor.

Introduction: Follicular lymphoma (FL) remains a significant clinical burden as it is an incurable disease and most patients will eventually suffer from disease progression. Two clinical events are associated with poor outcomes for patients with FL: (1) histological transformation (TFL) of their original FL into a high-grade, aggressive lymphoma subtype (2-3% of patients per year) and (2) early disease progression (PFL) where patients experience treatment failure within 2 years of receiving therapy (20% of patients). Despite recent high-throughput sequencing studies, the nature of tumor clonal dynamics leading to TFL or PFL is poorly understood and it is unknown if similar, or contrasting, modes of selection underpin these FL clinical events. Materials & Methods:We assembled a study cohort consisting of 21 patients: 15 experiencing TFL and 6 PFL. For each TFL and PFL patient, we obtained primary biopsies (T1; taken at the time of the initial FL diagnosis), biopsies at transformation/progression (T2) and matched normal samples. We performed whole genome sequencing on each specimen and identified single point mutations and copy number alterations using MutationSeq and TITAN, respectively. We compared T1 to T2 somatic mutation profiles and identified mutations associated with extinction of T1 clones and expansion of T2 clones. To validate these patterns, we selected 192 positions from each patient for deep-targeted sequencing validation (~10733X) in their T1, T2, and normal samples. We applied a statistical model (PyClone) to estimate cancer cell fraction (CCF) of each validated mutation. These CCF estimates were used to construct clonal phylogenies (Citup) and infer clonal dynamic patterns during their evolutionary histories. The Wright-Fisher model of genetic drift was used to model tumor evolution. Results: Temporal analysis of mutational burden revealed that mutational burden was significantly higher in T2 (8162 mutations ± 2146) than in T1 (6373 ± 2630) tumors for both TFL and PFL patients (Wilcox P < 0.001). This was independent of time interval between sampling (Spearman R2 = 0.029, P = 0.456). Mutation variant allelic fraction (VAF) distributions revealed that all distributions showed evidence of shared clonal ancestry between T1 and T2 tumors accompanied by substantial numbers of T1 and T2-specific mutations. We selected ≥ 192 mutations per patient from these distributions and performed deep-targeted amplicon sequencing, validating 96.3% of mutations and acquiring precise VAFs to infer clonal dynamics. In 13 of 15 TFL patients (87%), we observed dramatic clonal dynamics, characteristic of T2 tumors dominated by clones (or phylogenetic lineages) that were absent or extremely rare in T1 tumors (< 1% CCF). Digital droplet PCR was used to confirm the existence of both scenarios (confirming a clone as rare as 2 out of approximately 105 cells). Tumor evolution modeling demonstrated that this mode of evolution was driven through positive selection for mutations that confer fitness advantages and not by genetic drift. In contrast, PFL patients exhibited markedly different patterns of clonal dynamics compared to TFL patients. 4 of 6 PFL patients (67%) harbored readily detectable clones at T1, which expanded to full clonal prevalence during treatment with immuno-chemotherapy. Tumor evolution modeling demonstrated that this mode of evolution could be explained under neutral evolutionary dynamics (drift). Conclusions: We have shown that histological transformation and early progression manifest through divergent modes of tumor evolution. As the transformation phenotype may arise after diagnosis, more frequent monitoring of these patients will be required to determine the exact timing of the evolutionary inflection point that elicits transformation. In comparison, prediction of early treatment resistance should be achievable through comprehensive characterization of the genetic and clonal composition at diagnosis; this would ultimately identify patients who may benefit from upfront alternative therapies without the need to first endure predictable early treatment failure. Disclosures Sehn: roche/genentech: Consultancy, Honoraria; amgen: Consultancy, Honoraria; seattle genetics: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; TG therapeutics: Consultancy, Honoraria; celgene: Consultancy, Honoraria; lundbeck: Consultancy, Honoraria; janssen: Consultancy, Honoraria. Connors:Millennium Takeda: Research Funding; Seattle Genetics: Research Funding; F Hoffmann-La Roche: Research Funding; Bristol Myers Squib: Research Funding; NanoString Technologies: Research Funding. Scott:Janssen: Consultancy; Celgene: Consultancy; Roche: Honoraria; BC Cancer Agency: Patents & Royalties: Inventor on a patent licensed to NanoString Technologies.

Introduction : Follicular lymphoma (FL) remains an incurable malignancy as patients eventually experience progressive disease. A subset of patients is at risk of early lymphoma-related mortality due to histological transformation (TFL) to aggressive lymphoma (2-3% of patients per year) or early progression after immuno-chemotherapy, each of which leads to shortened survival. Mutations associated with transformation and/or early progression have been reported in the literature (e.g. CDKN2A, TP53, B2M) and the m7-FLIPI clinico-genetic risk model defines a high-risk patient group with poor failure-free survival after first-line treatment. However, the current knowledge of genetic alterations underlying transformation and/or early progression is inadequate to explain the majority of transformed cases and accurately predict early progressive disease. Materials and Methods: We performed targeted capture-based sequencing of 94 genes in a large cohort of fresh-frozen and formalin-fixed and paraffin-embedded patient specimens (402 samples in total). This cohort comprised 277 samples from 159 patients that experienced transformation (including 128 samples at T1 (the time of FL) and 149 samples at T2 (the time of transformation) with 118 paired biopsies) and 125 samples from 125 patients (pre-treatment samples only) presenting with either early progression within 2.5 years after starting immunochemotherapy (n=41) or late or never progression for at least 5 years after starting immunochemotherapy or observation (n=84). Mutations were called using MutationSeq and putative single nucleotide polymorphisms were filtered out using either matching germline samples (n=84) or dbSNP (v147). Bayesian proportion tests were used to compare the prevalence of gene mutations between groups. Results : We first compared T1 (n=128) and T2 (n=149) samples from 159 patients experiencing transformation. Eleven genes were more commonly altered in transformed lymphoma. These included genes that had previously been associated with transformation, such as TP53, B2M, MYC and EBF1, as well as genes that have not yet been implicated as contributing to this process, for example EZH2, CCND3, PIM1 and ITPKB. Moreover, mutations in GNA13, S1PR2 and P2RY8, that have been implicated in dissemination of germinal centre B cells, were enriched in T2 samples. In addition, cell-of-origin classification was available for 108 of the TFL cases with DLBCL histology, 18 and 90 of which were of the ABC and GCB subtype, respectively. Although the number of ABC-TFL cases was small, we observed higher percentages of BCL10 (16% versus 1%, Fisher P=0.004), CD79B (22% versus 4%, Fisher P=0.005) and MYD88 mutations (28% versus 9%, P=0.006) in ABC-TFL than in GCB-TFL, suggesting that B-cell receptor signaling and NF-κB signaling are important contributors to the ABC phenotype in TFL. Next, we assessed the association of gene mutations with patient outcomes contrasting early progressers (n=41) and late or non-progressers (n=84). Eleven genes were mutated more commonly in early progressers than in late progressers, including KMT2C, TP53, BTG1, MKI67, XBP1 and SOCS1. Overall, 32 out of 41 early progressers (78%) had mutations in at least one of the 11 early progression-associated genes, but none of the individual early progression-associated genes were mutated at a frequency > 22%. Thus, early progression appears to be related to relatively infrequent genetic alterations. None of the early progression-associated gene mutations form part of the m7-FLIPI outcome predictor. Furthermore, in our cohort that was enriched for clinical extremes, the m7-FLIPI was similarly associated with early progression when compared to the FLIPI but it was not superior, having higher specificity (89% versus 76%), but lower sensitivity (36% versus 63%). Conclusions: We found novel associations of gene mutations with transformation and showed that TFL is molecularly heterogeneous, with the ABC subtype being characterized by differential gene mutation when compared to the GCB subtype. With regards to progressive disease after immuno-chemotherapy, our approach identified early progression as a distinct clinico-genetic disease category that is imperfectly captured by traditional prognostic tools. Disclosures Connors: Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; F Hoffmann-La Roche: Research Funding; NanoString Technologies: Research Funding; Millennium Takeda: Research Funding. Scott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Trabalho final de mestrado integrado área científica de Ortopedia, apresentado á Faculdade de Medicina da Universidade de Coimbra Introdução: A fratura do escafoide é a fratura mais frequente dos ossos do carpo e a segunda com maior incidência ao nível do membro superior. Pela sintomatologia discreta, diagnóstico difícil e pelo seu suprimento sanguíneo precário e terminal, as fraturas localizadas na porção proximal do escafoide estão mais propensas à pseudartrose e à necrose avascular do fragmento ósseo proximal, quando não tratadas adequadamente e em tempo útil. A evolução natural da pseudartrose do escafoide é para uma artrose do punho, situação conhecida por SNAC (“scaphoid nonunion advanced collapse”). Atualmente, o tratamento da pseudartrose proximal do escafoide é cirúrgico, com fixação interna e sempre com a aplicação de enxerto ósseo, sendo este vascularizado ou não vascularizado, com indicações que são ainda motivo de controvérsia científica. O objectivo deste trabalho é tentar esclarecer as indicações de cada uma destas técnicas. Materiais e métodos: Foi efectuada uma pesquisa na PUBMED e na Base de artigos da Biblioteca Central dos Hospitais da Universidade de Coimbra, de onde foram selecionados estudos que utilizassem a técnica de enxerto ósseo vascularizado ou não vascularizado, no tratamento da pseudartrose proximal do escafoide cárpico, tendo sido seleccionados 6 artigos que correspondiam aos nossos critérios. Pela inexistência de estudos comparativos específicos para as pseudartroses proximais, foram também seleccionados dois estudos comparativos entre as duas técnicas de enxerto, mas relativamente a todas as localizações de pseudartrose do escafoide. Resultados: As taxas de consolidação nos diversos estudos foram variáveis, entre 67,8 e 100 %, sendo que foi atingido pleno de consolidação em alguns estudos, com ambas as técnicas de enxerto. Por outro lado, as taxas mais baixas de consolidação foram verificadas em estudos com a técnica de enxerto ósseo não vascularizado. A necrose avascular do fragmento ósseo proximal foi verificada em diversos pacientes, e nestes casos foram atingidas altas taxas de consolidação com ambas as técnicas. Todavia, num dos estudos comparativos a taxa de consolidação foi significativamente inferior quando se aplicou enxerto ósseo não vascularizado, sendo a presença de necrose avascular o fator considerado influenciador. Em termos clínicos e funcionais, verificaram-se melhorias significativas relativamente ao pré-operatório, não existindo diferenças significativas entre as duas técnicas quando a consolidação da pseudartrose foi alcançada. Nos casos em que se registou consolidação da pseudartrose, não houve evolução para artrose do punho. Discussão: A técnica de enxerto ósseo não vascularizado é cirurgicamente menos complexa, não implica a dissecação de pedículo vascular, nem técnicas microcirúrgicas para realização de anastomoses. Os enxertos ósseos do dorso do rádio distal, vascularizados ou não, pela sua proximidade ao escafoide e usando uma única incisão cirúrgica dorsal, tornam-se vantajosos em relação a outras localizações. A falta de consenso científico na escolha dos métodos de avaliação clinica e funcional dos diversos estudos e de trabalhos com um número significativo de casos, tornam difícil tirar conclusões acerca do valor de cada uma das técnicas de enxerto ósseo com um nível de evidência consistente. De igual forma, o tempo de seguimento pós-operatório não permite conhecer a evolução do processo degenerativo do punho. Conclusão: A aplicação de enxerto ósseo não vascularizado é o método de eleição no tratamento da pseudartrose proximal do escafoide cárpico. O enxerto ósseo vascularizado deve ser reservado para casos seleccionados, nomeadamente na presença de necrose avascular do fragmento proximal, apesar de continuar a ser uma matéria de controvérsia científica Introduction: The scaphoid is the most commonly fractured carpal bone and is the second in terms of incidence in fractures of the superior member. Through its mild symptoms, difficult diagnosis, and its precarious and terminal blood supply, fractures located in the proximal part of the scaphoid are more prone to nonunion and avascular necrosis of the proximal bone fragment, if not treated adequately and in a timely manner. When the nonunion of scaphoid is left untreated, osteoarthritis of the wrist develops in a situation known as SNAC (“scaphoid non-union advanced collapse”). Currently, treatment of proximal scaphoid nonunion is surgery, with internal fixation, and always with the application of bone graft, which is vascularized or non-vascularized, with indications that are still a matter of scientific controversy. The objective of this study is to clarify the indications of each of these techniques. Material and Methods: A search was conducted on PUBMED and on the Article base of the Central Library of the Hospitais da Universidade de Coimbra, from which, studies that utilized vascularized or non-vascularized bone grafting techniques in the treatment of proximal nonunion of the carpal scaphoid were selected, being that six articles corresponded to our criteria. Because of the inexistence of comparative studies for this specific scaphoid location, were also selected two studies comparing the two techniques of graft, but for all locations of scaphoid nonunions. Results: Union rates presented in the diverse studies varied between 67.8 and 100 %, being that complete union was achieved in some studies with both grafting techniques. Moreover, the low rates of union were verified in studies of the non-vascularized bone graft. Avascular necrosis of the proximal bone fragment was found in several patients, and in these cases, high union ratios were achieved with both techniques. However, in one of the comparative studies, the union rate was significantly inferior when non-vascularized bone grafts was applied, and the presence of avascular necrosis was considered the influencing factor. In clinical and functional terms, there were significant improvements relative to the preoperative and no significant differences exist between the two techniques when union was achieved. In those cases where union was achieved, no osteoarthritis changes were verified. Discussion: The non-vascularized bone grafting technique is surgically less complex and implicates neither the dissection of the vascular pedicle nor microsurgical techniques for the realization of anastomoses. Vascularized or non-vascularized bone grafts of the dorsum of the distal radius require a single surgical dorsal incision due to its proximity to the scaphoid, therefore becoming advantageous in comparison to grafts of other areas. The lack of scientific consensus in the selection of clinical and functional evaluation methods in the diverse studies and studies with a larger number of patients, makes it difficult to reach conclusions about the value of each bone grafting techniques with a level of consistent evidence. Similarly, the length of postoperative follow-up, is not provides information about the evolution of the degenerative process of the wrist. Conclusion: The use of non-vascularized bone graft is the method of choice in the treatment of proximal nonunion of the carpal scaphoid. The vascularized bone graft should be reserved for selected cases, particularly in the presence of avascular necrosis of the proximal fragment, although it remains a matter of scientific controversy

Purpose Our aim was to reliably identify patients with advanced-stage classical Hodgkin lymphoma (cHL) at increased risk of death by developing a robust predictor of overall survival (OS) using gene expression measured in routinely available formalin-fixed paraffin-embedded tissue (FFPET). Methods Expression levels of 259 genes, including those previously reported to be associated with outcome in cHL, were determined by digital expression profiling of pretreatment FFPET biopsies from 290 patients enrolled onto the E2496 Intergroup trial comparing doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and Stanford V regimens in locally extensive and advanced-stage cHL. A model for OS separating patients into low- and high-risk groups was produced using penalized Cox regression. The model was tested in an independent cohort of 78 patients enriched for treatment failure but otherwise similar to patients in a population-based registry of patients treated with ABVD. Weighted analysis methods generated unbiased estimates of predictor performance in the population-based registry. Results A 23-gene outcome predictor was generated. The model identified a population at increased risk of death in the validation cohort. There was a 29% absolute difference in 5-year OS between the high- and low-risk groups (63% v 92%, respectively; log-rank P < .001; hazard ratio, 6.7; 95% CI, 2.6 to 17.4). The predictor was superior to the International Prognostic Score and CD68 immunohistochemistry in multivariate analyses. Conclusion A gene expression–based predictor, developed in and applicable to routinely available FFPET biopsies, identifies patients with advanced-stage cHL at increased risk of death when treated with standard-intensity up-front regimens.

In classical Hodgkin lymphoma (CHL), 20%-30% of patients experience relapse or progressive disease after initial treatment. The pathogenesis and biology of treatment failure are still poorly understood, in part because the molecular phenotype of the rare malignant Hodgkin Reed-Sternberg (HRS) cells is difficult to study. Here we examined microdissected HRS cells from 29 CHL patients and 5 CHL-derived cell lines by gene expression profiling. We found significant overlap of HL-specific gene expression in primary HRS cells and HL cell lines, but also differences, including surface receptor signaling pathways. Using integrative analysis tools, we identified target genes with expression levels that significantly correlated with genomic copy-number changes in primary HRS cells. Furthermore, we found a macrophage-like signature in HRS cells that significantly correlated with treatment failure. CSF1R is a representative of this signature, and its expression was significantly associated with progression-free and overall survival in an independent set of 132 patients assessed by mRNA in situ hybridization. A combined score of CSF1R in situ hybridization and CD68 immunohistochemistry was an independent predictor for progression-free survival in multivariate analysis. In summary, our data reveal novel insights into the pathobiology of treatment failure and suggest CSF1R as a drug target of at-risk CHL.

The Web Assisted Laboratory for Control (WALC) engineering on- line education is described. This on-line platform was developed by a multidisciplinary team. WALC enables the access to several virtual and remote labora- tories experiences on automation, process control and numerical methods. The virtual environment enables the registered user to test different control systems (hydraulic, mechanical, electrical) working in open and close loop. This environment is simulated based on the numerical solutions of the differential equations that describe the real world systems. An industrial automation network is available allowing the user to test different programs running on PLCs. The re- mote access to the platform is confined to pre-registered users and under a booking system ensuring that only the registered user is on-line. Other functionality are depicted. Based on user’ feedback analysis, and since this platform was developed as a dynamic learning tool, their needs are fulfilled., Fundação para a Ciência e a Tecnologia (FCT)

Background: MYC and BCL2 are critical driver genes for non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). However, the clinical impact of MYC and BCL2 genetic alterations, apart from translocations, has not been comprehensively investigated using high-resolution genetic assays, such as next generation sequencing and high-resolution SNP arrays. Moreover, correlations with cell of origin (COO) subtype, determined by gene expression profiling, have not been widely studied in a large homogeneously treated cohort. We determined the frequency and clinical impact of MYC and BCL2 genetic aberrations in DLBCL in a large population-based cohort uniformly treated with R-CHOP. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were treated with R-CHOP in BC. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of MYC and BCL2 was performed using a Truseq Custom Amplicon assay (Illumina) on the Miseq platform. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Dual positivity for MYC (cut off; 40%) and BCL2 (cut off; 50%) proteins identified a dual protein expresser (DPE) phenotype. COO classification was achieved using the Lymph2Cx assay based on NanoString technology in 299 patients and the Hans algorithm in 32 cases with low tumor content ( Results: COO determination revealed 193 cases to be GCB subtype, 107 cases ABC/non-GCB and 30 were unclassifiable. Using next generation sequencing, 310 SNVs were detected in MYC (29/347; 8% of cases) and BCL2 (88/347; 25% of cases), with mean redundant coverage depths of 633-fold. All MYC and 98% of BCL2 mutations were misssense mutations. Analysis of copy number alterations using GISTIC 2.0 revealed significant focal gain/amplification (gain/amp) peaks affecting 8q24, including the MYC locus (68/341; 20% of cases) and 18q21, including the BCL2 locus (82/341; 24% of cases). MYC and BCL2 translocations were detected in 38/283 (13%) and 90/300 (30%) of tumors, respectively. MYC gain/amp, BCL2 mutation, BCL2 translocations and double MYC/BCL2 translocation (DHIT) were seen significantly more often in GCB-DLBCL (26% vs 10%, p=0.001; 35% vs 11%, p With a median follow up of 6.5 years for living patients, the presence of MYC translocation, BCL2 gain/amp and BCL2 mutation were associated with an inferior 5y-time to progression (TTP, 53% vs 30%, p=0.019; 60% vs 32%, p=0.009 and 52% vs 17%, p=0.026, respectively) in ABC subtype. In GCB subtype, BCL2 translocation, MYC gain/amp and MYC translocation were associated with an inferior 5y-TTP (85% vs 62%, p=0.001; 80% vs 63%, p=0.011; and 79% vs 61%, p=0.013, respectively). In a multivariate Cox model of TTP including IPI and DPE, BCL2 gain/amp remained prognostic (HR=2.4 [1.3-4.5], p=0.008) independent of IPI and DPE in ABC-DLBCL. In GCB-DLBCL, BCL2 translocation and/or MYC gain/amp showed strong prognostic value (HR=3.0 [1.4-6.4], p=0.006) independent of IPI and DPE. In the IPI high risk group (IPI=3-5), the presence of a BCL2 translocation and/or MYC gain/amp defined a remarkably poor outcome group in GCB-DLBCL (5y-TTP; 29%). Similar poor outcome was observed in ABC-DLBCL cases that harbored BCL2 gain/amp (5y-TTP; 22%). Conclusions: The DHIT genotype was only seen in GCB-DLBCL. High-resolution genomic assays identified extremely poor prognostic groups within each COO subtype on the basis of MYC and BCL2 genetic status in a large uniformly R-CHOP-treated population-based cohort of DLBCL. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Background: Diffuse large B cell lymphoma (DLBCL) is divided into two distinct molecular subtypes, germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. Genetic landscape studies of DLBCL have revealed several GCB-DLBCL specific mutations, including CREBBP, GNA13, EZH2, TNFRSF14, BCL2 and MEF2B. Functional studies have recently shown that the inactivation of Gα13 signaling pathway genes, including GNA13, together with BCL2 over-expression, allows GC B-cells to escape the germinal center niche and widely disseminate. Although these findings revealed a critical role of genetic alterations of Gα13 signaling pathway in GC-driven mouse models of lymphomagenesis, clinical correlation is lacking. Here we analyzed the clinical impact of genetic alterations of Gα13 signaling pathway in a large population-based DLBCL cohort. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of GNA13, P2RY8, ARHGEF1, S1PR2 and RHOA was performed using a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Cell-of-origin classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014; 123) in 299 patients and the Hans algorithm (Hans, Blood 2004; 103) in 32 cases with low tumor content ( Results: Using next generation sequencing, 225 SNVs and 5 Indels were detected in GNA13 (16%), P2RY8 (18%), ARHGEF1 (6%), S1PR2 (3%) and RHOA (6%). SNP 6.0 microarrays revealed heterozygous deletions in GNA13 (2%), ARHGEF1 (1%), S1PR2 (4%) and RHOA (8%), but homozygous deletion was not found in any of these five loci. GNA13, P2RY8 and ARHGEF1 mutations were significantly more frequent in the GCB subtype than ABC subtype (26% vs. 6%; p In the cases with mutations of any of the five Gα13 signaling pathway genes, BCL2 over-expression (cut off; 50%) and translocation was associated with increasing stage (p=.018 and p=.005, respectively), but not in wt cases (p=.53 and p=.63, respectively). Specifically, in the cases with GNA13 and P2RY8 mutations individually, BCL2 over-expression was associated with advanced stage (stage III/IV, p=.018 and p=.037, respectively), but not in wild type (wt) cases. Importantly, BCL2 over-expression in the cases harboring Gα13 pathway mutations was not significantly associated with other poor risk features, including any other IPI factors or bone marrow involvement, indicating that genetic alterations in Gα13 signaling pathway accompanied by BCL2 over-expression might promote lymphoma dissemination into lymph nodes but not extranodal sites. With a median follow up of 6.5 years for living patients, there was no prognostic impact of harboring any isolated Gα13 pathway mutation in GCB-DLBCL patients. However, in cases with any Gα13 pathway mutations, BCL2 over-expression was significantly associated with an inferior 5y-time to progression (TTP; 90% vs 62%, p=.003) and disease-specific survival (DSS; 90% vs 71%, p=.042), but not in wt cases (Fig 1). In a Cox model of TTP including the IPI, BCL2 over-expression remained prognostic in the cases harboring any Gα13 pathway mutations (HR=4.13 [1.42-12.01], p=.009), but not in wt cases (HR=1.70 [0.62-4.68], p=.31). In cases with any Gα13 pathway alterations including copy number loss, BCL2 over-expression was also significantly associated with an inferior TTP (HR=3.64 [1.39-9.57], p=.009) independent of IPI, but not in the cases without genetic alterations (HR=1.75 [0.57-5.34], p=.33). Conclusions: Genetic alterations in Gα13 signaling pathway genes cooperate with BCL2 over-expression to promotes lymphoma dissemination to nodal sites and is associated with the poor outcome in GCB-DLBCL Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

STP-53912) and by the Fundacao para a Ciencia e Tecnologia (FCT BD13230/2003), Portugal. Follicular lymphoma patients display heterogeneous overall survival and variable risk of transformation. Recent studies have highlighted the role of the microenvironment. The contribution of microvessel density to follicular lymphoma survival remains controversial. We used a quantitative tumor mapping approach to determine whether the degree of vascularization correlated with outcome in a uniformly treated cohort. Whole-tissue sections of diagnostic biopsies from 84 cases were stained for CD34 and tumor-to-vessel-distance that encompassed 90% of the tumor (TVD90) was determined using image analysis. Twenty-one cases with lower TVD90 showed inferior overall survival (P=0.0001) and high risk of transformation (P=0.01). These cases significantly correlated with increased Lymphoma-Associated Macrophages (x(2)=0.025). In multivariate analysis macrophages content, IPI and TVD90 were independent predictors of overall survival (P=0.05, P=0.001 and P=0.01, respectively) and IPI and TVD90 predicted risk of transformation (P=0.008 and P=0.08, respectively). Increased angiogenesis is an independent marker of inferior survival and may promote transformation. publishersversion published

Background During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of μ heavy chain complexed with VpreB and λ5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma. Design and Methods Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas. Results We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%). Conclusions We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.

Chromosomal translocations are critically involved in the molecular pathogenesis of B-cell lymphomas, and highly recurrent and specific rearrangements have defined distinct molecular subtypes linked to unique clinicopathological features(1,2). In contrast, several well-characterized lymphoma entities still lack disease-defining translocation events. To identify novel fusion transcripts resulting from translocations, we investigated two Hodgkin lymphoma cell lines by whole-transcriptome paired-end sequencing (RNA-seq). Here we show a highly expressed gene fusion involving the major histocompatibility complex (MHC) class II transactivator CIITA (MHC2TA) in KM-H2 cells. In a subsequent evaluation of 263 B-cell lymphomas, we also demonstrate that genomic CIITA breaks are highly recurrent in primary mediastinal B-cell lymphoma (38%) and classical Hodgkin lymphoma (cHL) (15%). Furthermore, we find that CIITA is a promiscuous partner of various in-frame gene fusions, and we report that CIITA gene alterations impact survival in primary mediastinal B-cell lymphoma (PMBCL). As functional consequences of CIITA gene fusions, we identify downregulation of surface HLA class II expression and overexpression of ligands of the receptor molecule programmed cell death 1 (CD274/PDL1 and CD273/PDL2). These receptor-ligand interactions have been shown to impact anti-tumour immune responses in several cancers(3), whereas decreased MHC class II expression has been linked to reduced tumour cell immunogenicity(4). Thus, our findings suggest that recurrent rearrangements of CIITA may represent a novel genetic mechanism underlying tumour-microenvironment interactions across a spectrum of lymphoid cancers.

Despite advances in treatments for Hodgkin's lymphoma, about 20% of patients still die from progressive disease. Current prognostic models predict the outcome of treatment with imperfect accuracy, and clinically relevant biomarkers have not been established to improve on the International Prognostic Score.Using gene-expression profiling, we analyzed 130 frozen samples obtained from patients with classic Hodgkin's lymphoma during diagnostic lymph-node biopsy to determine which cellular signatures were correlated with treatment outcome. We confirmed our findings in an independent cohort of 166 patients, using immunohistochemical analysis.Gene-expression profiling identified a gene signature of tumor-associated macrophages that was significantly associated with primary treatment failure (P=0.02). In an independent cohort of patients, we found that an increased number of CD68+ macrophages was correlated with a shortened progression-free survival (P=0.03) and with an increased likelihood of relapse after autologous hematopoietic stem-cell transplantation (P=0.008), resulting in shortened disease-specific survival (P=0.003). In multivariate analysis, this adverse prognostic factor outperformed the International Prognostic Score for disease-specific survival (P=0.003 vs. P=0.03). The absence of an elevated number of CD68+ cells in patients with limited-stage disease defined a subgroup of patients with a long-term disease-specific survival of 100% with the use of current treatment strategies.An increased number of tumor-associated macrophages was strongly associated with shortened survival in patients with classic Hodgkin's lymphoma and provides a new biomarker for risk stratification.

PurposeHypoxia-inducible factor (HIF) controls the expression of genes in response to hypoxia, as well as a wide range of other cellular processes. We previously showed constitutive stabilization of HIF-1α in the majority of patients with diffuse large B-cell lymphoma (DLBCL). To our knowledge, the prognostic significance of HIF in lymphoma has never been investigated.Patients and MethodsWe studied the immunohistochemical protein expression of HIF-1α on tissue microarrays from 153 patients with DLBCL treated in sequential cohorts with cyclophosphamide, doxorubicin, oncovin, and prednisone (CHOP) or rituximab-CHOP (R-CHOP) from 1999 to 2002. Results were correlated with patient outcome.ResultsMedian follow-up for all patients was 80 months. Among all patients, HIF-1α was expressed in 62% of germinal center and 59% of non–germinal center patients. With HIF-1α analyzed as a dependent variable, there were no survival differences in CHOP-treated patients. In the R-CHOP group, however, HIF-1α protein expression correlated with significantly improved progression-free survival (PFS) and overall survival (OS). Five-year PFS for HIF-1α–positive patients was 71% v 43% for HIF-1α–negative patients (P = .0187), whereas 5-year OS was 75% and 54%, respectively (P = .025). In multivariate analysis with International Prognostic Index criteria, HIF-1α remained a significant predictor for PFS (P = .026) and OS (P = .043). Compared with other biomarkers, HIF-1α correlated only with BCL6 (P = .004). In terms of gene expression, we found several common gene associations of HIF-1α and the stromal-1 signature with genes predominantly involved in regulation of the extracellular matrix (eg, BGN, COL1A2, COL5A1, and PLOD2).ConclusionThe expression of HIF-1α protein is an important independent favorable prognostic factor for survival in patients with DLBCL treated with R-CHOP.

Primary cutaneous follicle center lymphoma (PCFCL) is characterized by a proliferation of follicle center cells in the skin. A definitive diagnosis is frequently delayed because of difficulties in interpretation of the histopathologic findings. It has an excellent prognosis with a 5-year survival over 95% and its risk of transformation has not been established. We describe a case report of man with a gastric diffuse large B-cell lymphoma (DLBCL) referred to our clinic because of nodules in the back that had gradually developed over a period of 10 years. A biopsy performed 3 years before was interpreted as reactive follicular hyperplasia. A new skin biopsy revealed a diffuse large B-cell lymphoma and immunoglobulin heavy chain gene rearrangements from the initial skin biopsy (PCBCL) and the DLBCL gastric biopsy were studied by polymerase chain reaction and an identical clonal rearrangement was detected which was highly suggestive of a transformation lymphoma. publishersversion published

P>Diffuse large B cell lymphoma (DLBCL) is clinically and biologically heterogeneous. In most cases of DLBCL, lymphoma cells co-express vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, suggesting autocrine in addition to angiogenic effects. We enumerated microvessel density and scored lymphoma cell expression of VEGF, VEGFR1, VEGFR2 and phosphorylated VEGFR2 in 162 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone)-like regimens. VEGFR2 expression correlated with shorter overall survival (OS) independent of International Prognostic Index (IPI) (P = 0 center dot 0028). Phosphorylated VEGFR2 (detected in 13% of cases) correlated with shorter progression-free survival (PFS, P = 0 center dot 044) and trended toward shorter OS on univariate analysis. VEGFR1 was not predictive of survival on univariate analysis, but it did correlate with better OS on multivariate analysis with VEGF, VEGFR2 and IPI (P = 0 center dot 036); in patients with weak VEGFR2, lack of VEGFR1 coexpression was significantly correlated with poor OS independent of IPI (P = 0 center dot 01). These results are concordant with our prior finding of an association of VEGFR1 with longer OS in DLBCL treated with chemotherapy alone. We postulate that VEGFR1 may oppose autocrine VEGFR2 signalling in DLBCL by competing for VEGF binding. In contrast to our prior results with chemotherapy alone, microvessel density was not prognostic of PFS or OS with R-CHOP-like therapy.

Access control models describe frameworks that dictate how subjects (e.g. users) access resources. In the Role-Based Access Control (RBAC) model access to resources is based on the role the user holds within the organization. Although flexible and easier to manage within large-scale authorization frameworks, RBAC is usually a static model where access control decisions have only two output options: Grant or Deny. Break The Glass (BTG) policies can be provided in order to break or override the access controls within an access control policy but in a controlled and justifiable manner. The main objective of this paper is to integrate BTG within the NIST/ANSI RBAC model in a transparent and secure way so that it can be adopted generically in any domain where unanticipated or emergency situations may occur. The new proposed model, called BTG-RBAC, provides a third decision option BTG. This allows break the glass policies to be implemented in any application without any major changes to either the application or the RBAC authorization infrastructure, apart from the decision engine. Finally, in order to validate the model, we discuss how the BTG-RBAC model is being introduced within a Portuguese healthcare institution where the legislation requires that genetic information must be accessed by a restricted group of healthcare professionals. These professionals, advised by the ethical committee, have required and asked for the implementation of the BTG concept in order to comply with the said legislation.

Previous studies of follicular lymphoma (FL) patients treated heterogeneously have suggested that decreased numbers of regulatory T cells correlates with improved survival. We studied advanced-stage FL patients from a single institution phase 2 trial. All patients were treated uniformly with multiagent chemotherapy and radiation. Tissue microarrays were constructed using diagnostic biopsies available in 105 patients and stained with CD4, CD8, CD25, and forkhead/winged helix transcription factor 3 (FOXP3) antibodies. Both cell content and cell distribution were evaluated. For all antibodies, there were cases with a predominant intrafollicular or perifollicular localization of cells (follicular pattern) while others displayed a diffuse pattern. The median follow-up of living patients was 17.1 years. The International Prognostic Index score predicted overall survival (OS; P = .004) but not risk of transformation (RT). Cell content did not impact survival, while immunoarchitectural patterns of CD4/CD8 were significant for progression-free survival (PFS; P = .056), CD25 for both PFS and OS (P = .002 and P = .024, respectively), and FOXP3+ predicted PFS, OS, and RT (P = .001, P < .001 and p = .002, respectively). A Cox multivariate model showed both International Prognostic Index score and FOXP3+ pattern were independent predictors of OS (P = .008 and P < .001, respectively), while only FOXP3+ pattern predicted RT (P = .004). We conclude that FOXP3+ cell distribution significantly predicts survival and RT in FL.

Dissertação de Mestrado apresentada no Instituto Superior de Psicologia Aplicada para obtenção de grau de Mestre na especialidade de Psicologia Social e das Organizações O presente estudo pretendeu relacionar Competência Psicológicas, Formulação de Objectivos e Rendimento em contexto desportivo, recorrendo a uma amostra de 12 atletas de Basquetebol em escalão de formação Júnior (sub-19). Assim, procurou-se verificar num estudo pré-experimental se existiam alterações nas competências psicológicas e no Rendimento desportivo após a formulação de objectivos individualmente com as atletas. Para a recolha dos valores de Competências Psicológicas em Pré e Pós teste utilizou-se o Inventário das Competências Psicológicas do Desporto que é uma versão traduzida e adaptada do Psychological Skills Inventory for Sport - P.S.I.S. (Form R-5) (Mahoney, 1987; Mahoney, Gabriel & Perkins, 1987, cit. por Cruz, 1993). As medidas de rendimento desportivo (número de Turnover´s de cada atleta durante a competição) foram recolhidas através da recolha dos valores em estatística competitiva através da visualização os jogos. No que diz respeito à Formulação de Objectivos esta foi efectuada através da Formulação de Objectivos Intervalares (F.O.I.) criado em 1984 por O'Block e Evans. Após a intervenção individual com as atletas e comparando os resultados de pré e pós intervenção, verificou-se que nas medidas psicológicas existiram alterações significativas na “autoconfiança” resultando uma melhoria dos valores; em relação ao “Controlo de Ansiedade” também existiram diferenças significativas mas neste caso existindo um decréscimo nos valores médios; em relação à escala de “Espírito de equipa” não existiram diferenças entre os valores médios, sendo praticamente idênticos após a intervenção; em relação à “Concentração” não existem diferenças significativas nos valores, no entanto existiu alguma melhoria nos valores médios. Por último não existindo diferenças significativas para a “Motivação”, neste caso também verificou-se um decréscimo dos valores. Referenciando agora as medidas de Rendimento Desportivo verificou-se que após a intervenção existiram diferenças significativas nos valores, tanto para a contabilização de “após 1 minuto jogado” como para “após 10 minutos jogados”, diferenças estas para valores superiores da medida “Turnover”, ou seja, para um decréscimo no rendimento desportivo. This study sought to relate the psychological, Formulation of Objectives and Performance in the athletic context, using a sample of 12 basketball players in training junior-level (sub- 19). Thus, we have studied a pre-experimental study whether there were changes in psychological skills and sports performance after the formulation of objectives with individual athletes. To collect the values of Psychological Skills in Pre and Post test we used the Psychological Skills Inventory for Sport which is a translated version of the Psychological Skills Inventory for Sport - PSIS (Form R-5) (Mahoney, 1987, Mahoney, Gabriel & Perkins, 1987, cit. By Cruz, 1993). The measures of athletic performance (number of Turnover's of each athlete during the competition) were collected through the collection of statistical values in competitive by viewing the games. With regard to the formulation of objectives this was achieved through the formulation of range object (FOI) created in 1984 by O'Block and Evans. After intervention with the individual athletes and comparing the results of pre and post intervention, it was found that the psychological measures there were significant changes in "confidence" resulting in an improvement in values, in relation to the Control of Anxiety "there were also significant differences but in this case there is a decrease in the average values related to the scale of “team spirit” there were no differences between the mean values are virtually identical after the intervention, in relation to “Concentrations” no significant differences in values, however there some improvement in the average values. Finally no significant difference for the "Motivation", in this case also there was a decrease of values. Referencing now the measures of income Sports found that after the intervention there were significant differences in values for both the accounting "after 1 minute played" as to "after 10 minutes played," these differences to greater measure of "turnover", result a decrease in sports-performance.

The activated B cell–like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by constitutive activation of the nuclear factor-κB (NF-κB) pathway. In this study, we showed that the NF-κB pathway induced the expression of the cytokines interleukin (IL)-6 and IL-10 in ABC DLBCL cell lines, which also have high levels of total and phosphorylated signal transducer and activator of transcription (STAT) 3 protein, suggesting autocrine signaling. Using RNA interference for STAT3, we defined a gene expression signature of IL-6 and IL-10 signaling through STAT3. Based on this signature, we constructed a molecular predictor of STAT3 signaling that defined a subset of ABC DLBCL tumors with high expression of STAT3, IL-6, and/or IL-10 and their downstream targets. Although the STAT3-high and STAT3-low subsets had equivalent expression of genes that distinguish ABC DLBCL from germinal center B cell–like DLBCL, STAT3-high ABC DLBCLs had higher expression of signatures that reflected NF-κB activity, proliferation, and glycolysis. A small-molecule inhibitor of Janus kinase signaling, which blocked STAT3 signature expression, was toxic only for ABC DLBCL lines and synergized with an inhibitor of NF-κB signaling. These findings suggest that the biological interplay between the STAT3 and NF-κB pathways may be exploited for the treatments of a subset of ABC DLBCLs.

Purpose The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab. Patients and Methods DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome. Results In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis. Conclusion We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

A tissue microarray (TMA) containing diagnostic biopsies was used to develop predictors of outcome in a group of 105 patients having advanced-stage follicular lymphoma (FL). The patients were staged and uniformly treated, and the usable cases had been randomly divided into a subgroup of 50 patients with outcomes identified, and a reserved subgroup of 43 patients whose outcomes were masked for blind testing of the predictors. Using training-input data from some patients with known outcomes, parallel cascade identification developed two predictors of overall survival based on a number of biomarkers. Both predictors had statistically significant performance over the remaining patients with known outcomes. The first predictor had been identified with model architectural settings and encoding scheme chosen, for the particular training input used, to enhance classification accuracy over remaining patients in the known subgroup. The second predictor was obtained without changing the settings and encoding scheme, but from an entirely different training input corresponding to novel cases from the TMA. Not surprisingly, the first predictor showed much higher accuracy over the known subgroup, but when tested over the reserved subgroup of 43 patients, averaged about 58% correct and did not reach statistical significance. The other predictor performed very similarly over the known and the reserved subgroups, with prediction on the reserved subgroup highly significant at p = 0.0056 in Kaplan-Meier survival analysis. We conclude that a predictor based on a number of biomarkers obtainable at diagnosis has the potential to improve prediction of overall survival in FL.

Aims: To analyse the possible activation of distinctmolecular pathways in mucosa-associated lymphoidtissue (MALT) lymphoma, we determined the preval-ence of trisomies 3, 12, 18 in MALT lymphomas fromdifferent organs, as well as the prevalence of trans-locations of the MALT1 gene in a subset of primarybreast MALT lymphomas. We compared the numericalcytogenetic alterations in lymphomas, precursorlesions and in normal non-haematolymphoid tissuefrom the same organs.Methods and results: Forty-two samples of paraffin-embedded tissue (29 MALT lymphomas from stomach,breast, parotid and thyroid; two Sjo¨gren’s syndrome;two Hashimoto’s thyroiditis and nine reactive samples)were studied by fluorescence in situ hybridization(FISH). Analysed together, the cases of gastric, parotidand thyroid MALT lymphomas presented trisomy 3 in46%, trisomy 12 in 28% and trisomy 18 in 21% of thecases. In contrast to other locations, trisomy 3 was notpresent in the majority of the cases of primary breastMALT lymphomas. None of the nine breast casespresented MALT1 gene rearrangements. Half of thecases of preneoplastic lesions exhibited trisomy 3 andtrisomy 12; none exhibited trisomy 18.Conclusions: Trisomy 3 is the most frequent numericalabnormality in gastric, parotid and thyroid but not inprimary breast MALT lymphomas. MALT1 gene re-arrangements are also rare in this location, suggestingthat distinct molecular pathways may be activated inbreast cases.Keywords: cytogenetics, FISH, MALT, non-Hodgkin’s lymphoma, pathogenesisAbbreviations: FISH, fluorescence in situ hybridization; MALT, mucosa-associated lymphoid tissue; MZL, marginalzone lymphoma

A tissue microarray (TMA) containing diagnostic biopsies was used to develop predictors of outcome in a group of 105 patients having advanced-stage follicular lymphoma (FL). The patients were staged and uniformly treated, and the usable cases had been randomly divided into a subgroup of 50 patients with outcomes identified, and a reserved subgroup of 43 patients whose outcomes were masked for blind testing of the predictors. Using training-input data from some patients with known outcomes, parallel cascade identification developed two predictors of overall survival based on a number of biomarkers. Both predictors had statistically significant performance over the remaining patients with known outcomes. The first predictor had been identified with model architectural settings and encoding scheme chosen, for the particular training input used, to enhance classification accuracy over remaining patients in the known subgroup. The second predictor was obtained without changing the settings and encoding scheme, but from an entirely different training input corresponding to novel cases from the TMA. Not surprisingly, the first predictor showed much higher accuracy over the known subgroup, but when tested over the reserved subgroup of 43 patients, averaged about 58% correct and did not reach statistical significance. The other predictor performed very similarly over the known and the reserved subgroups, with prediction on the reserved subgroup highly significant at p = 0.0056 in Kaplan-Meier survival analysis. We conclude that a predictor based on a number of biomarkers obtainable at diagnosis has the potential to improve prediction of overall survival in FL.

Background: Although R-CHOP has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL), 40% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Recent studies using next-generation sequencing strategies have described the landscape of recurrent mutations in DLBCL. However, with the exception of TP53 and FOXO1, little is known about the clinical relevance of recurrent mutations and importantly, the interactions of these genetic alterations in DLBCL. Moreover, an integrated analysis of copy number alterations and recurrent mutations annotated across cell-of-origin (COO) distinctions for a large cohort of DLBCL cases who have received uniform therapy is lacking. The present study examined the frequency and clinical impact of recurrent genetic aberrations of DLBCL using high-resolution technologies in a large population-based DLBCL cohort. Methods: We analyzed 348 newly diagnosed DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency (Vancouver) with available DNA extracted from fresh frozen biopsy material (tumor content >30%). Matched germ line DNA was available for 67 patients. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Targeted re-sequencing of the coding exons of 56 genes was performed using a Truseq Custom Amplicon assay. Gene selection was based on mutational frequencies that have been previously described in DLBCL mutational landscape publications. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Tissue microarrays were constructed using duplicate 0.6mm cores from 332 cases, and breakapart FISH assays for MYC, BCL2 and BCL6 and IHC staining for MYC, BCL2 and cell of origin proteins were performed. COO classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014;123) in 299 patients as well as Hans algorithm (Hans, Blood 2004;103) in 32 cases with low tumor content. 194 cases were assigned to GCB subtype, 107 cases, ABC/non-GCB and 30 were unclassifiable. Results: In the mutation analysis, we identified 2,757 SNVs and 245 small indels. The mean depth of coverage was 634. Recurrent mutation frequencies varied between 0 and 58, with a mean of 8.25 per case. 98% of cases harbored at least one mutation and 95% of cases multiple mutations. 10 mutated genes were detected significantly more frequently in the GCB subtype including CREBBP, GNA13, EZH2, TNFRSF14, IRF8,STAT3, BCL2, SGK1, MEF2B and CD83, and 4 mutated genes, MYD88, CD79B, PRDM1 and PIM1, in the ABC subtype. In the copy number analysis, 45 significant amplification peaks and 57 deletion peaks were revealed by the GISTIC algorithm. As previously reported, 9p21.3, including CDKN2A,were more frequently detected in the ABC subtypes. With a median follow up of 6.5 years for living patients, the 5 y disease specific survival (DSS) and time to progression (TTP) of all patients were 72% and 64%, respectively. The clinical cohort was representative of registry data from BC based on a comparison of patient characteristics and survival outcomes with 1,194 control DLBCL R-CHOP patients. The ABC subtype was significantly associated with an inferior DSS and TTP (both p Conclusions: Our approach using next generation sequencing and high resolution SNP array provides an accurate estimation of frequency and clinical significance of recurrent genetic alterations of DLBCL in a uniformly R-CHOP-treated large population-based cohort of patients. Disclosures No relevant conflicts of interest to declare.

Introduction: Recent studies have established that concurrent MYC and BCL2 protein expression by immunohistochemistry (IHC) identifies a subgroup of patients with diffuse large B-cell lymphoma (DLBCL) with a poor outcome. Classic dual translocation MYC/ BCL2, so called ‘double hit' disease, is associated with a high risk of central nervous system (CNS) relapse; however the impact of concurrent MYC and BCL2 protein expression on the risk of CNS relapse remains unknown. Further, robust biological markers that accurately predict the risk of CNS relapse in DLBCL would also be of value in clinical practice. Methods: Cases of pre-treatment formalin fixed paraffin embedded DLBCL in two tissue microarrays were independently scored by two expert hematopathologist (GWS and KLT or PF and AM) for expression of MYC (Epitomics Y69), BCL2 (Dako 124), CD10, BCL6 and MUM1 by IHC. MYC and BCL2 positivity were defined as ≥ 40% and ≥ 50% cells with staining, respectively, in accordance with previously established cutoffs (Johnson, JCO 2012; 30). Cases with discordant scores were reviewed by a third hematopathologist (RDG) to reach a consensus. Cell of origin (COO) was assigned according to the Hans IHC algorithm (Hans, Blood 103: 2004) as well as by the recently described gene expression profiling Lymph2Cx 20 gene assay based on NanoString technology (Scott, Blood 2014; 123) in the subset of patients with ≥ 40% tumor content. Patients treated with at least one cycle of R-CHOP chemotherapy with curative intent were included and those with established CNS disease at diagnosis were excluded. Results: 447 patients were identified with the following baseline clinical characteristics: Median age 65 y (16-92y); males n=280, 63%; performance status ≥ 2, n= 147, 33%; stage 3 or 4 disease n=242, 54%; elevated LDH n=219, 47%; EN > 1 n= 80, 17%. With a median follow-up of 6.75 years for living patients, the 3 year time to progression, progression-free and overall survival for all patients were 68%, 66%, and 73%, respectively. In total, 131 (29%) were MYC+BCL2+ and 316 (71%) were non-MYC+BCL2+. By COO assignment using the Hans algorithm (n=444), 192 were non-GCB (43%) and 252 were GCB (57%) and by the Lymph2Cx (n=308); 103 were ABC (33%), 172 were GCB (56%) and 33 (11%) were unclassifiable. The 2 year cumulative risk of CNS relapse for the whole cohort was 4.3%. The cumulative risk of CNS relapse was higher in cases that were MYC+BCL2+ (2 year risk 9.4% vs 2.4%, P=0.001) with similar results obtained if classic MYC+BCL2+ double hit cases are excluded. There were no cases of CNS relapse in cases MYC+ alone by IHC. By COO, patients with a non-GCB phenotype by the Hans algorithm had an increased risk of CNS relapse (2 year risk 6.9% vs 2.6%, P=0.03) and similarly, cases assigned as ABC DLBCL by the Lymph2Cx assay also identified a group with a higher risk of CNS relapse compared to GCB cases (9.5% vs 2.5%, P=0.03) (Figure 1). In Cox regression multivariate analysis including the COO (Hans), IPI group (0/1 vs 2/3 vs 4/5) and MYC/BCL2 IHC, only the IPI (HR 2.18, P=0.02) and MYC+BCL2+ IHC (HR=3.76, P=0.007) were associated with an increased risk of CNS relapse. Similar results were obtained using the Lymph2Cx COO designation. Within the IPI risk groups, MYC+BCL2+ status further stratified patients in the intermediate risk group (IPI 2 or 3, n=206) into a higher risk group (2 year CNS relapse 12.6%) and a low risk group (2 year CNS relapse 2.9%) (P=0.01). A similar trend was observed in the high IPI risk group (IPI 4 or 5, n=86, 2 year CNS relapse MYC+BCL2+ 17.2% vs 4.7%, P=.0.18) but it was not useful in the low IPI risk group (IP1 0 or 1 (n=155), 2 year CNS relapse 4% vs 1%, P=0.39) where the overall risk was low. Within the COO subgroups, MYC+BCL2+ status also defined a group at high cumulative risk of CNS relapse within the non-GCB subtype (12.9% vs 3%, P=0.001) and by the Lymph2Cx defined ABC subtype (16.9% vs 2.2%, P= 0.03) and a trend was observed for GCB defined by Lymph2Cx (6.6% vs 1.5%. P=.08) but not by Hans criteria (P=0.40). Conclusion: Concurrent expression of MYC and BCL2 protein in DLBCL defines a group of patients at high risk of CNS relapse, independent of the IPI and COO. MYC+BCL2+ status may help to further risk stratify patients in the intermediate and high IPI risk groups and within the ABC subtype to identify patients who should undergo additional diagnostic testing and in whom to explore the effectiveness of prophylactic CNS strategies. Figure 1 Figure 1. Disclosures Savage: F Hoffmann-La Roche: Other. Sehn:Roche: Research Funding. Connors:Seattle Genetics, Inc.: Research Funding; Roche: Research Funding. Gascoyne:Hoffman La-Roche: Research Funding.

Gene expression profiling studies have reported up-regulated mRNA expression of the FOXP1 forkhead transcription factor in response to normal B-cell activation and high expression in a poor prognosis subtype of diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to investigate the prognostic importance of FOXP1 protein expression in an independent series of DLBCL.First, the specificity of our FOXP1 monoclonal antibody was verified by confirming that it did not recognize the closely related FOXP2, FOXP3, or FOXP4 proteins. FOXP1 protein expression was then analyzed by immunohistochemistry using a DLBCL tissue microarray constructed from 101 previously untreated de novo cases from the British Columbia Cancer Agency. FOXP1 expression was scored as either positive (30% positive nuclei) or negative (30% positive nuclei). The overall survival curves clearly showed that patients grouped as FOXP1-positive (40%) had a significantly decreased overall survival (P = 0.0001). FOXP1-positive patients had a median overall survival of 1.6 years compared with 12.2 years in FOXP1-negative cases. In addition, FOXP1-positive patients showed a clear trend to earlier progression in comparison to the FOXP1-negative patients. The analysis of FOXP1 expression within low, medium, and high International Prognostic Index groupings found that FOXP1-negative patients had better overall survival within each group indicating that FOXP1 expression has predictive value independent of the International Prognostic Index subgrouping, a finding that was confirmed in multivariate analysis. These initial results suggest that FOXP1 expression may be important in DLBCL pathogenesis.

Diffuse large B-cell lymphoma (DLBCL) can be divided into prognostically important subgroups with germinal center B-cell-like (GCB), activated B-cell-like (ABC), and type 3 gene expression profiles using a cDNA microarray. Tissue microarray (TMA) blocks were created from 152 cases of DLBCL, 142 of which had been successfully evaluated by cDNA microarray (75 GCB, 41 ABC, and 26 type 3). Sections were stained with antibodies to CD10, bcl-6, MUM1, FOXP1, cyclin D2, and bcl-2. Expression of bcl-6 (P

Objectivo: Descrever os aspectos clínico-radiológicos da Histiocitose de Células de Langerhans (HCL) numa série de doentes. Material e Métodos: Reviram-se 20 casos de HCL, nove com diagnóstico segundo os actuais critérios anátomo-patológicos (positividade para CD1a e/ou presença de grânulos de Birbeck em microscopia electrónica), outros nove apenas com critérios morfológicos, e dois casos com diagnóstico patológico estabelecido fora da instituição, não susceptível de aferição anátomo- patológica. Resultados: A razão sexo masculino:feminino foi 4:1 e a média de idades de apresentação da doença 8 anos (variação: 2 meses - 44 anos). Os quatro órgãos mais frequentemente envolvidos foram, por ordem decrescente: osso, gânglios linfáticos, pele e hipófise. Dois doentes tinham doença unifocal óssea, oito apresentavam doença multifocal-unissistémica, em sede óssea ou ganglionar, e dez doença multissistémica. As lesões líticas foram o achado radiológico mais frequente, distribuindo-se predominantemente na calote craniana, ossos ilíacos e fémures. As alterações pulmonares constituiram achado minoritário. Conclusão: A série em estudo incluiu uma proporção grande de casos com gravidade clínica (10 doentes com HCL multissistémica), tendo-se observado em número relativamente restrito a forma menos grave da doença, correspondente ao clássico granuloma eosinófilo. A aparência das alterações radiológicas foi contudo similar à descrita na literatura. Objective: To describe the clinical and radiological features of Langerhans Cell Histiocytosis (LCH) in a series of patients. Material And Methods: We reviewed 20 cases of LCH, nine with diagnosis by the current pathological criteria (positivity for CD1a and/or presence of Birbeck granules identified at ultrastructural examination), and nine only with morphological criteria. In the other two cases, the pathological diagnosis was made outside our institution and could not be reviewed. Results: Male:female ratio was 4:1 and the average age of presentation of the disease was 8 years (range: 2 months - 44 years). The four most affected organs were, in decreasing order: bone, lymph nodes, skin and hypophysis. Two patients had unifocal bone disease, eight multifocal unisystem involvement in the bone or lymph nodes, and ten presented multisystem LCH. Lytic lesions were the most common radiological finding, affecting mainly the skull, iliac bones and femurs. Pulmonary lesions were rather infrequent. Conclusion: This series included a large proportion of severe cases (10 patients with multisystem LCH), while the mildest form of the disease, classically designated as eosinophilic granuloma, was observed only in a few patients. The appearance of the radiological findings was however similar to the described in the literature.

Primary effusion lymphoma (PEL) is one of the least common of the AIDS-related lymphomas, accounting for less than 1–4% of cases. Clinical manifestations depend on the extent and distribution of disease and, as in the majority of patients no detectable mass lesion is found, symptoms are related to fluid accumulation, dyspnoea (pleural or pericardial effusions), abdominal distension (ascites) or joint swelling. The median survival after diagnosis, even with aggressive chemotherapy, remains poor and remissions are often of short duration. We present the case of a 31-year-old man with AIDS and diagnosis of PEL, in whom sustained and complete remission of the tumour was achieved with adjunctive ganciclovir therapy. Since the disease is so uncommon, there is a paucity of data to guide the treatment of these patients; ganciclovir might be a potential antiviral therapeutic option, as demonstrated by the 2-year remission achieved in our patient.

Abstract 1586 Background Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by the t(11;14) translocation and concomitant over-expression of cyclin D1. MCL has a variable natural history; while some patients have prolonged survival similar to other indolent B-cell lymphomas, most follow an aggressive course with short survival. While the t(11;14) is pathognomonic of MCL, it is not necessary for disease pathogenesis, as a subset of MCL cases lack the translocation. Furthermore, in vivo models demonstrate that cyclin D1 over-expression alone is unable to bring about the disease, and that deregulation of additional cellular pathways is required for its pathogenesis. Assessment of microRNA (miR) expression in MCL may help determine mechanisms of gene deregulation and reveal pathways involved in disease pathogenesis. In this study we examined MCL in relation to both aggressive and indolent B-cell NHL to determine a miR signature that characterizes MCL. Design and Methods Total RNA from a training set of 36 B-cell NHL cases (19 indolent and 17 aggressive) and 32 MCL was applied to a high-throughput quantitative real-time PCR platform assessing the expression of 365 miRs [TaqMan Human MicroRNA Array v1.0 (Early Access) or TLDA]. miRs that were differentially expressed between MCL and aggressive NHL, and between MCL and indolent NHL were then validated using RNA from a second, independent, set of B-cell NHL cases (28 indolent and 28 aggressive) and 50 MCL cases. Validated miRs were determined and potential targets for each miR were examined. A map of targets common to the MCL miR signature was created, revealing important proteins involved in MCL pathogenesis. Results 66 miRs (11 over-expressed, 55 under-expressed) were differentially expressed between MCL and aggressive B-cell NHL and 8 miRs (7 over-expressed, 1 under-expressed) were differentially expressed between MCL and indolent B-cell NHL (false discovery rate = 0.2). 6 miRs from each group were chosen for validation in an independent set of MCL and NHL cases. Of these 12 miRs, 7 miRs validated (2 were under-expressed in MCL relative to aggressive B-cell NHL, and 5 were over-expressed in MCL relative to indolent B-cell NHL). Genes and pathways involved in disease pathogenesis are most likely targeted by multiple miRs. We thus determined a set of 123 genes predicted to be targets of this MCL miR signature, based on five miR target prediction databases from the mirDIP (microRNA data integration) portal. These genes were significantly enriched for focal adhesion and integrin signalling, proteasome-mediated degradation, and the PI3K signalling pathway. Conclusions Using the largest set of MCL cases evaluated to date, a 7-miR signature characteristic of MCL was discovered. The gene targets of these miRs are enriched for roles in proteasome-mediated protein degradation, consistent with the reported sensitivity of MCL to proteasome inhibitors. In addition, these miRs are predicted to be involved in regulation of PI3K/AKT signalling, confirming reports of the importance of this pathway in MCL pathogenesis. Enrichment of target genes involved in focal adhesion and integrin signalling indicate the importance of MCL-stromal interactions and motivates further study into the role of the tumor microenvironment in MCL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 430 Introduction: 25–30% of patients with advanced stage classical Hodgkin lymphoma (cHL) develop progressive disease despite treatment with ABVD. While more intensive upfront treatments, such as BEACOPP, result in a higher failure free survival, this comes at the expense of increased toxicity and morbidity. 10–15% of patients with advanced stage cHL will ultimately succumb to the disease, with similar outcomes using different upfront regimens likely reflecting the planned use of second-line high dose therapy. The International Prognostic Factors Project Score, a prognostic tool developed using freedom from progression of disease as the outcome, has been used in clinical practice and has guided clinical trials. We hypothesized that biological factors, as measured by gene expression profiling in pretreatment formalin fixed paraffin embedded tissue (FFPET) samples, might provide a robust predictor of overall survival (OS). Method and Patients: NanoString technology was used to quantitate 261 mRNA species in diagnostic FFPET samples from patients in the Intergroup E2496 trial, which compares ABVD with Stanford V chemotherapy in locally advanced and advanced stage cHL. The 261 genes included individual genes and genes that represent cell and cellular process signatures that have been associated with outcome in cHL. Total RNA was extracted from whole section scrolls from the 309 FFPET blocks available. A penalized binary logistic regression model with leave-one-out cross validation was used to produce a parsimonious predictive model for OS. A threshold was selected that maximized the Chi square of the Log Rank (Mantel-Cox) test between the identified low and high risk groups. An independent cohort of patients with advanced stage cHL enriched for treatment failure, consisting of 59 patients from British Columbia treated with ABVD and 71 patients from Portugal treated with Stanford V, was used to test the generated model and threshold. Results: Gene expression of adequate quality was produced in 293 of the 309 samples (95%). At a median follow up of 5.3 years, 36 (12%) of the 293 patients had died. 51 genes were differentially expressed (t-test p < 0.05) with respect to OS, including genes that are part of macrophage, cytotoxic/NK cell and apoptosis signatures. A predictive model for OS was produced with an area under the curve (AUC) of the receiver operating characteristic (ROC) curve of 0.73. The predictor identified a high-risk group, comprising 39% of the cohort, with a 77% estimated 5-year OS compared with 96% in the low risk group (Figure 1A, log-rank p < 0.0001). The predictor was validated in the independent cohort giving an AUC of the ROC curve of 0.73 and an estimated 5-year OS of 71% in the high-risk group compared with 91% in the low risk group (Figure 1B, log-rank p = 0.006). Discussion: We have produced a parsimonious gene expression-based predictor of OS in cHL, developed in, and applicable to, widely available FFPET using NanoString technology. The predictor identifies, at diagnosis, a clinically meaningful proportion of patients at significantly higher risk of death when treated with ABVD or Stanford V. Further evaluation of the gene expression signature with alternative treatments, ideally in the context of clinical trials, is needed to understand the impact of more intensive cytotoxic therapies versus novel targeted approaches. Disclosures: Horning: Genentech: Employment, Equity Ownership. Connors:Seattle Genetics: Consultancy, Research Funding.

Abstract 800 Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) accounting for ~6% of all NHL. It is sensitive to combination chemotherapy, but remission durations are short without approaches such as stem cell transplantation (SCT). Most patients are incurable, but the clinical course is variable, with some patients succumbing quickly, while others survive >10 years. MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression by inhibiting mRNA translation. miRs are useful in the prognostic assessment of tumors, but work to date examining differences between MCL and normal lymphoid tissues, have only identified 2 miRs involved in MCL prognosis (Zhao JJ, Blood, 2010; Di Lisio L, Leukemia, 2010). We used a novel approach to identify a prognostic miR signature in MCL. We hypothesized that a miR signature defining aggressiveness can be obtained by comparing miR expression profiles of aggressive NHL with indolent NHL, and that this signature when applied to a set of MCL cases, may aid in MCL prognosis. Total RNA was extracted from 135 formalin-fixed paraffin-embedded samples obtained at primary diagnosis (Table 1). RNA from a training set of 19 indolent and 20 aggressive NHL cases was analyzed on a high-throughput quantitative real-time PCR (qRT-PCR) platform assessing the expression of 365 miRs and 3 endogenous controls (TaqMan Human MicroRNA Array v1.0: TLDA, ABI) using the DDCt method. A two-sample Wilcoxon Rank sum test corrected for false discovery rate was used to assess the significance of differential expression for each miR between aggressive and indolent NHL. The 14 most significantly differentially expressed miRs (p Fig. 1. Psrincipal component analysis demonstrating separation of MCL cases into a good prognosis group in red (median OS: 88 months, range: 41–131 months) and a poor prognosis group in blue (median OS: 15 months, range: 4–40 months) based on expression of a 9 miR aggressiveness signature. Fig. 1. Psrincipal component analysis demonstrating separation of MCL cases into a good prognosis group in red (median OS: 88 months, range: 41–131 months) and a poor prognosis group in blue (median OS: 15 months, range: 4–40 months) based on expression of a 9 miR aggressiveness signature. Table 1. Sample breakdown Training set Number Aggressive cases Diffuse large B-cell lymphoma 5 Primary mediastinal B-cell lymphoma 5 Burkitt lymphoma 5 Atypical Burkitt 5 Indolent cases Small lymphocytic lymphoma/CLL 5 Extranodal marginal zone lymphoma 5 Follicular lymphoma Grade 1 3 Grade 2 3 Grade 3a 3 Validation set Aggressive cases Diffuse large B-cell lymphoma 7 Primary mediastinal B-cell lymphoma 5 Burkitt lymphoma 3 Atypical Burkitt 4 Indolent cases Small lymphocytic lymphoma/CLL 5 Extranodal marginal zone lymphoma 5 Follicular lymphoma Grade 1 5 Grade 2 5 Grade 3a 5 MCL cases Conventional 19 Blastoid/pleomorphic 11 Prolymphocytoid 1 Multiple lymphomatoid polyposis 1 Normal benign lymph nodes 20 TOTAL 135 Table 2. MCL clinical data Features Total % Gender Male 23 72 Female 9 28 ECOG 0 12 38 1 17 53 2-3 3 9 Stage 1 2 6 2 8 25 3 7 22 4 15 47 B symptoms 9 28 Extranodal sites 5 16 Lines of therapy 0 2 6 1 14 44 2 3 9 3 6 19 4 3 9 >4 4 13 Types of therapy Observation alone 2 6 Anthracycline-based 18 56 Rituximab 14 44 SCT 3 9 Bortezomib 3 9 Radiation 14 44 Median Range Age (yrs) 69 37–90 M-IPI score 6.6 5.3–8.7 Ki-67 (%) 25 7.5–90 Time to 1st treatment (months) 0.8 0.1–99.1 Overall survival (months) 34 4–131 Disclosures: Kuruvilla: Hoffman LaRoche: Honoraria, Research Funding; Celgene: Research Funding; Amgen: Honoraria; Otsuka: Honoraria; Genzyme: Honoraria.

Abstract 3944 Poster Board III-880 Background MCL is a clinically aggressive lymphoma characterized by frequent relapses and short survival. Clinical behavior and response to therapy vary considerably and are often unpredictable based on clinical factors. Prior gene and protein expression studies have demonstrated proliferation and TP53 expression as biological mechanisms that strongly influence survival (Rosenwald Cancer Cell 2003 & Katzenberger Blood 2006 and Louie Blood 1995). Recent studies have highlighted the role of the microenvironment in follicular lymphoma (FL) including numbers of lymphoma-associated-macrophages (LAM), as important prognostic factors (Dave NEJM 2004 & Farinha Blood 2005). Within most tumors, macrophages display an M2 phenotype that promotes tumor growth and angiogenesis. In MCL, the clinical impact of LAM is unknown. Our aim was to assess the prognostic impact of LAM in MCL patients from a single institution experience using tissue microarrays (TMA), immunohistochemistry and clinical correlates. Methods A TMA block was built with duplicate 0.6mm cores of paraffin-embedded formalin-fixed diagnostic biopsies from 185 patients treated at the BC Cancer Agency (1983-2004). A subset of cases was also analyzed for the presence of the t(11;14) by FISH. Primary and subsequent therapy included multiple regimens: single or multi-agent chemotherapy with or without rituximab, stem cell transplant, radiation and therapeutic splenectomy. Standard immunohistochemistry was performed with CD20, CCND1, CD68, CD163 (an M2 marker), CD34, TP53 and Ki67. TP53 and Ki67 IHC were scored using image analysis (VIAS). Univariate and multivariate correlates of overall survival (OS) were determined using SPSS software. Results The median follow-up of living patients was 5.75 years. The IPI predicted OS (p=0.0013). Numbers of cases with interpretable staining varied for the different biomarkers (range 157 to 180). All 185 cases were either CCND1 positive and/or t(11;14) FISH positive. Both proliferation (quartiles of Ki67+ cells) and TP53 (dichotomized based on cut-off >40%) were significant predictors of OS independent of IPI (OS, p=0.0002). Cases showing more than 5% infiltrating CD68+ cells (18/185) had a 4-y OS of 5% vs. 35% (p=0.0081). These CD68+ cases significantly correlated with 12 cases showing more than 5% infiltrating CD163+ cells (x2=0.001), which also significantly influenced OS (p=0.008). 17 cases had increased microvessel density (MVD), defined by CD34+ cells, which correlated with inferior OS (p=0.02). Interestingly, cases with increased MVD often had >5% CD163+ cells (x2=0.05), but not increased CD68+ cells. Multivariate analysis including IPI, TP53, Ki67, CD68, CD163 and CD34, showed IPI (RR=1.8, 95%CI=1.0-3.1, p=0.047), TP53 (RR=2.3, 95%CI=1.3-4.3, p=0.006), Ki67 (RR=2.2, 95%CI=1.2-4.2, p=0.012) and CD68 (RR=2.1, 95%CI=1.1-3.8, p=0.022) to be independent predictors of OS. Finally, in the 27 patients who received rituximab at some time during their disease course, increased CD68+ cells did not significantly affect OS (p=0.1), suggesting that, similar to FL, the prognostic effect of LAM may be abolished by rituximab. Conclusions The number of LAMs is an important prognostic factor in MCL, independent of clinical parameters, proliferation and TP53 expression. Expression of CD163, described as an M2 polarized macrophages, also predicts survival and correlates with increased angiogenesis. These preliminary data suggest that the use of rituximab may abrogate the prognostic impact of LAM in MCL. In summary, similar to other lymphoma subtypes, the microenvironment is important in MCL biology and prognosis and may be an important new target for therapy in this aggressive disease. Disclosures: Connors: Roche Canada (F Hoffmann-La Roche): Research Funding. Gascoyne:Roche Canada, Genentech, Lilly, Millennium : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

133 Background: PMBCL represents a unique lymphoma type in the WHO classification and shares overlapping histological and gene expression features with both diffuse large B-cell (DLBCL) and classical Hodgkin lymphoma. Similar to DLBCL, PMBCL may show loss of expression of the major histocompatibility class II (MHC II) antigens, which correlates with inferior patient survival (Rimsza, Blood 2006). MHC II proteins are expressed on antigen-presenting cells and are important in eliciting immune responses. In DLBCL, decreased expression of HLA-DR is related to loss of immunosurveillence. Our aim was to validate HLA-DR as an important prognostic biomarker in PMBCL and correlate it with immune response in a cohort of patients treated with multi-agent chemotherapy. Methods: A tissue microarray block was built with duplicate 0.6mm cores of paraffin embedded diagnostic biopsies from 103 patients treated with CHOP/CHOP-like chemotherapy at the BC Cancer Agency (1980-2005). Standard immunohistochemistry was performed with CD20, HLA-DR (IgG2b), CD3, CD4, CD8, CD57, CD68 and cytotoxic markers (TIA1 & Granzyme B). HLA-DR expression was evaluated using a histoscore (intensity of staining and % of positive malignant cells) and correlated with the content (%) of different non-neoplastic infiltrating T-cell subsets and macrophages. Univariate & multivariate analyses were used to characterize overall survival (OS) and progression free survival (PFS). Results: The median follow-up of living patients was 10 years. The IPI predicted OS (p=0.042) but not PFS. Of the 92 cases with interpretable staining, 32 were positive for HLA-DR and 60 negative, with 10-year OS of 86% vs. 61% (p=0.006) and 2-year PFS of 78% vs. 53% (p=0.018), respectively. A Cox multivariate model established both HLA-DR status and IPI as independent predictors of OS (RR=0.3, 95%CI=0.12–0.75, p=0.01; RR=2.9, 95%CI=1.2–6.9, p=0.06, respectively). HLA-DR expression correlated significantly with increased content of all analyzed T cell markers, especially CD3, CD8 and TIA1 (x2, p Conclusions: We validated the negative prognostic importance of loss of HLA-DR expression by neoplastic B cells in PMBCL patients treated with multi-agent chemotherapy in a single institution experience. Loss of HLA-DR expression correlated with decreased numbers of infiltrating benign T cell populations, especially CD8+ and TIA1+ cells, where decreased cytotoxic T cell content correlated independently with inferior survival. This study shows loss of immunogenicity and immunosurveillance are key mechanisms in the response to treatment of PMBCL patients and suggests that specific therapies focused on this pathway may benefit patients. Disclosures: Connors: Roche Canada (F Hoffmann-La Roche): Research Funding. Gascoyne:Roche Canada, Genentech, Lilly, Millennium : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 268 INTRODUCTION: Classical Hodgkin lymphoma (cHL) is unique among lymphomas due to the scarcity of the malignant Hodgkin Reed Sternberg (HRS) cells, which are derived from clonal germinal center B cells. Investigations using laser capture microdissection permit more detailed analysis of these cells. However, most recent studies were limited by low case numbers and lack of available clinical data. PATIENTS AND METHODS: We studied 29 cases of cHL and the 5 HL lines KMH2, HDLM2, L428, L540, and L1236 by gene expression profiling. All patients were treated at the BC Cancer Agency between 1984 and 2006 and received at least 4 cycles of polychemotherapy and stage-dependent radiotherapy. The cohort also included 5 biopsies taken at relapse. Treatment failure was defined as disease progression or relapse at any time (n=14) after initiation of treatment; treatment success as absence of progression (n=15). We used laser microdissection (Molecular Machines & Industries Cellcut with Nikon Eclipse TE2000-S microscope) to study the enriched HRS cell compartment separately from the microenvironment. RNA extraction was performed on pools of 1000 microdissected HRS cells in each case. Gene expression profiles were generated using Affymetrix HG UA133 2.0 Plus arrays using two-cycle labeling reactions. HRS cell profiles were compared to microdissected germinal centers (GC), and HL cell line profiles compared to enriched tonsillar CD77+ centroblasts (MACS cell separation, Miltenyi). Furthermore, we compared gene expression profiles of treatment failures to those of treatment successes. RESULTS: We identified 1342 differentially expressed probesets (fold change >5, False Discovery Rate (FDR) adjusted p value 5, FDR-adjusted p value DISCUSSION: Using microdissection of HRS cells in a large number of cases we were able to further characterize the unique expression program of HL and refine the data inventory about dysregulated cellular functions and pathways in this disease. Overexpression of genes associated with NFκB, complement and hematopoietic progenitor cells proliferation correlate strongly with treatment failure. Further study using immunohistochemistry is currently ongoing to validate these findings and to develop clinically useful biomarkers. Disclosures: Gascoyne: Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 2923 Poster Board II-899 Background: MCL is characterized by the presence of the t(11;14) translocation that juxtaposes the cyclin D1 gene downstream of the immunoglobulin heavy-chain gene promoter resulting in enhanced G1μS phase transition leading to cellular proliferation. Despite the common genetic lesion, patients exhibit considerable heterogeneity in their clinical behavior and response to therapy. The clinical significance of the microenvironment in follicular lymphoma has been highlighted by gene expression studies demonstrating that a T-cell response signature correlates with a superior outcome compared to that seen with a macrophage signature. In contrast, the significance of the non-malignant cellular component in MCL biopsies remains unknown. We report the results of a retrospective study examining flow cytometry (FCM) of MCL diagnostic tissue biopsies, focusing on the non-malignant lymphoid cells. Method: Patients were included in the study if they had MCL diagnosed according to the 2008 WHO criteria, had FCM performed on their diagnostic nodal or tissue biopsy and had adequate clinical information that included baseline clinical characteristics, treatment regimens and clinical outcome. Patients were excluded if they were too frail to receive chemotherapy or if FCM was only performed on peripheral blood or bone marrow. 122 patients met the criteria for inclusion in the study; of which 56 were also assessable by tissue microarray (TMA). FCM data were re-analyzed for expression of CD3, CD4, CD8, CD19, CD20 and kappa and lambda light chains on cells gating on lymphoid populations. Estimates of the non-neoplastic B-cells were derived from total CD19/CD20 positive B cells excluding the light chain restricted population. ‘High' and ‘low' expressers for each marker were determined by examining frequency histograms for a trough. TMAs were prepared from diagnostic paraffin-embedded blocks according to established protocols. Sections were immunostained for CD3, CD4, CD8, CD68, CD34, TIA-1, CD163, FOXP3, PD-1, CD57, CD21, P53 and Ki67. Survival correlates were assessed by Cox regression using SPSS. Results: The median age was 67 y (range 22-94) with 69% being male. 88% had advanced-stage disease with 16% having a high IPI (4/5). 50 (41%) patients received rituximab as part of initial or subsequent therapy. Primary and secondary treatment regimens included: observation (19); single agent alkylators (33); CHOP-like with rituximab (39); CHOP-like without rituximab (32); CVP-like (11); higher intensity regimens (9); fludarabine-based (19); gemcitabine-based (9); bortezimib (4); flavopiridol (2); autologous stem cell transplant (12); allogeneic stem cell transplant (2); radiation (45); and therapeutic splenectomy (6). The median follow up of the living patients was 30 months. The 5-y OS for the group was 21%. FCM median tumor content was high at 84% (range 21-100%) with median CD3, CD4, CD8 and non-tumor CD20 populations of 12%, 7%, 4% and 1%, respectively. Univariate analysis revealed CD835% was also adversely associated with OS (p=0.028). Conclusion: While the non-neoplastic cellular infiltrate often constitutes only a minor fraction of the tumor mass in MCL, it appears to significantly influence response to rituximab therapy. Further studies are required to validate this observation prospectively and define the mechanism by which the cytotoxic T-cells exert their influence. Disclosures: Connors: Roche Canada: Research Funding. Gascoyne:Roche Canada, Genentech, Lilly, Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Follicular lymphoma (FL) is an indolent heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma is a dominant clinical event frequently associated with inferior survival. No consistent biological markers are predictive of survival or risk of transformation (RT). Recent studies have highlighted the role of the microenvironment, including regulatory T cells (Treg) as important prognostic factors in FL. Using tissue microarrays (TMA), we previously identified FOXP3+ T cell distribution but not cell density within the tumor is an independent adverse prognostic factor for both overall survival (OS), progression-free (PFS) survival and RT in advanced-stage FL pts treated uniformly with an aggressive treatment regimen (ASH 2007). Programmed death-1(PD1) belongs to the immunoglobulin CD28 receptor family and is characteristically expressed by germinal center associated T cells. They have imunoregulatory activity and are FOXP3-negative. Using the same cohort we assessed the role of PD1+ cells alone and in combination with FOXP3+ cells in FL prognosis. Methods: Between 1987 and 1993, 126 pts were enrolled on a phase II study of BP-VACOP chemotherapy with involved region radiotherapy. All patients were treatment naive, < 61y and had advanced-stage FL. Paraffin blocks were available for 105 pts. TMAs consisted of duplicate 1.0mm cores of diagnostic biopsies and were immunostained with both FOXP3 and PD1 (NAT105) specific antibodies. Both cell content and immuno-architectural patterns were determined and correlated with OS and RT including univariate and multivariate analysis. Results: There were 101 evaluable cases. The median follow-up of the living pts was 14 years. Histologic grade included 78 grade 1, 17 grade 2 and 6 grade 3a FLs. The IPI was predictive of OS (RR = 2.5, 95% CI =1.5–4.4, p = 0.002), but not RT (p = 0.14). Cases revealed zero to 731 FOXP3+ cells per core (median of 242 cells per core) and zero to 654 PD1+ cells per core (median of 102 cells per core). There was a predominantly intrafollicular or perifollicular localization of positive cells (“follicular pattern”) in 38 and 43 pts for FOXP3 and PD1, respectively. There was no significant correlation between cell density (χ2=0.1) or pattern (χ2=0.8) between markers. Separately, cell density did not impact prognosis. Yet, when quartiles were combined and dichotomized, cases with high cell content of FOXP3+/PD1+ cells correlated with inferior OS and increased RT in univariate (p = 0.002 and p = 0.01) and multivariate Cox models (RR = 2.18, 95% CI =1.2–3.9, p = 0.01 & RR = 3.4, 95% CI =1.3–8.8, p = 0.015). In univariate analysis, a PD1 follicular pattern correlated with inferior OS (p=0.027) but did not impact RT. When combined with follicular FOXP3+ cases, a subset of 15 “double follicular” FOXP3 & PD1 cases correlated with inferior OS in univariate (p=0.0016) and multivariate Cox models (RR = 2.1, 95% CI =1–4.3, p = 0.04). This subset of cases also showed a trend for increased RT (p = 0.09). Conclusions: Combined cell density and distribution within FL of immuno-regulatory T cell subsets are important predictors of OS & RT in advanced-stage FL patients treated uniformly with an aggressive treatment regimen. Our results reinforce the importance of the microenvironment in FL biology and further suggest an active role for immuno-modulatory T cells in pro-tumoral immunity.

HIF is a transcription factor that regulates gene expression in response to decreases in cellular oxygenation (hypoxia). Genes activated by HIF are involved in glycolysis, glucose transport, angiogenesis, cell proliferation, cell migration, cell metabolism, and cell survival. Many of these genes confer protection against the consequences of oxygen deprivation while others enhance resistance to chemotherapy or radiotherapy. Clinically, evidence of elevated HIF protein correlates with poor prognosis in lung, breast, colorectal, brain, pancreatic, ovarian, renal, and bladder cancers. Our preliminary tissue micro array (TMA) data suggested that HIF is frequently stabilized in lymphoma (BJH2008; 141:676), especially among patients with diffuse large B-cell lymphoma (DLBCL). We also found that constitutive expression of HIF-1α and/or HIF-2α is stabilized in a set of established lymphoma cell lines, underscoring the potential impact of HIF in lymphoma. To further investigate the importance of HIF, we examined TMAs for HIF-1α expression from 153 patients with DLBCL treated with CHOP or rituximab-CHOP (R-CHOP) at the BCCA between 1999–2002. Treatment was before (pre-rituximab) and after (post-rituximab) institution of a policy recommending the combination of rituximab with CHOP for all patients with newly diagnosed advanced-stage DLBCL (March 2001). Tissue sections were stained with monoclonal antibody to HIF-1α and expression (nuclear) was scored by computer (Automated Cellular Imaging System) and validated by pathology review. HIF was dichotomized as either negative (neg; no HIF staining) or positive (pos; score of 1 or greater). Of the 153 patients, 78 were consecutively treated with R-CHOP, while 75 had received CHOP. We found HIF pos expression in 63% (49/78) of patients treated with R-CHOP and 59% (44/75) treated with CHOP (p=NS). Further, based on the Hans algorithm, HIF was expressed in 62% of germinal center (GC) patients and 59% of non-GC patients (P=NS). In the R-CHOP group (see Figure 1A and 1B), the 5-year progression-free survival (PFS) was 45% for HIF neg patients vs 62% for HIF pos (log rank p=0.0187); while the 5-year overall survival (OS) was 48% for HIF neg vs 76% for HIF pos (log rank p=0.025). In patients treated with CHOP, there was no difference in outcome between HIF pos and HIF neg subsets. In multivariate analysis in the R-CHOP treated group, controlling for the International Prognostic Index (IPI), HIF pos expression remained a significant independent predictor for OS (p=0.03), while the IPI was of borderline significance (p=0.051). Comparison with other biomarkers showed that HIF did not correlate with expression of bcl-2, CD10, MUM-1, or FOXP1; while a high correlation was detected with bcl-6 and HIF (among HIF pos R-CHOP patients, 94% were bcl-6 pos and 6% were bcl-6 neg; p=0.004). We conclude that expression of HIF-1α is an important and heretofore undiscovered independent favorable prognostic factor for PFS and OS in DLBCL patients treated with R-CHOP, but not in patients treated with CHOP. These data suggest there may be an important biological interaction between CD20 monoclonal antibody-based therapy and HIF or downstream genes regulated by HIF. Further study of this observation is warranted. Figure 1. Survival according to HIF-1α status in patients treated with R-CHOP. Figure 1. Survival according to HIF-1α status in patients treated with R-CHOP.

Finding new prognostic markers in diffuse large B-cell lymphoma (DLBCL) is of importance because the disease is heterogeneous and current therapies fail to cure many affected patients. Previously, we showed that the expression of LMO2 mRNA was the strongest single predictor of superior outcome in patients with DLBCL in a multivariate model based on the expression of six genes (Lossos et al, NEJM 2004). Subsequently, we generated an anti-LMO2 monoclonal antibody and showed that LMO2 protein is expressed in a subset of germinal center-derived B-cell lymphomas including DLBCL, and that its expression predicted a superior outcome in DLBCL patients who were treated with anthracycline-based regimens (Natkunam et al, Blood 2006 & 2007). We have now expanded this cohort to include DLBCL patients treated at five international medical centers and have also tested the prognostic impact of LMO2 in patients treated with the addition of rituximab to CHOP (R-CHOP), a regimen which was recently demonstrated to improve the outcome of DLBCL patients (Coiffier et al, NEJM 2002; Sehn et al, JCO 2005; Habermann et al, JCO 2006; Pfreundschuh et al, Lancet Oncol 2006). Immunohistochemistry for LMO2 was performed on tissue microarrays containing cores of diagnostic biopsy specimens from patients with de novo DLBCL treated with anthracycline-based chemotherapy (280 patients) or with R-CHOP (80 patients) who had been followed for clinical outcome. The results show that in the pre-rituximab group staining for LMO2 was strongly correlated with overall survival (OS) and progression-free survival (PFS) (p=0.0018 and p=0.00071, respectively). Similarly, in the post-rituximab group LMO2 protein expression was associated with a significantly longer OS (p=0.0048) and PFS (p=0.0087). In multivariate Cox regression analyses, the expression of the LMO2 protein was found to be independent of the clinical IPI and most strikingly, was more robust than the conventional IPI in predicting OS and PFS, particularly in the post-rituximab DLBCL group of patients (OS p = 0.03, PFS p = 0.01). The capacity of LMO2 protein to identify DLBCL patients with improved outcome in an IPI-independent manner indicates its potential role in risk stratification of this disease. We conclude that the prognostic value of LMO2 protein expression remains significant in the era of R-CHOP treatment and recommend its assessment in all newly diagnosed DLBCL patients to confirm these results and eventually to optimize patient management.