State of the Art and Comparative Aspects in Canine Lymphoma!

s of invited lectures Workshop Proceedings Page 5 Abstracts of invited lectures SESSION ON PATHOGENESIS Chairs: Hugo Murua Escobar, Franco Guscetti 01 Comparative Transcriptional Analysis of Canine and Human Diffuse Large B Cell Lymphoma (DLBCL). Molecular Signatures of NF-κB Activation and Sub-Classification of DLBCL Ted Hupp Edinburgh Cancer Research UK Centre, Edinburgh, United Kingdom E-mail: [email protected] Hupp T.1, Mudaliar, M.A.V.2, Haggart, R.D.2, Miele, G.3, Sellar, G.3, Tan, K.A.L.4, Goodlad, J.R.5, Milne, E.4, Vail, D.M.6, Kurzman, I.6, Crowther, D.3, and Argyle D.J.4 1Edinburgh Cancer Research UK Centre, Edinburgh EH4 2XR, UK; 2Translational Medicine Research Collaboration, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK; 3Pfizer Inc, Translational Medicine Research Collaboration, Ninewells Hospital, Dundee DD1 9SY, UK; 4Royal (Dick) School of Veterinary Studies and Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK; 4Western General Hospital, Department of Pathology, University of Edinburgh, UK; 6University of WisconsinMadison School of Veterinary Medicine, University of Wisconsin-Madison, Madison, USA We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-κB) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph nodes. The comparisons at gene level were performed by mapping the probesets in canine microarrays to orthologous genes in humans and vice versa. A considerable number of differentially expressed genes between canine lymphoma and healthy lymph node samples were also found differentially expressed between human DLBCL and healthy lymph node samples. Principal component analysis using a literature derived NF-κB target gene set mapped to orthologous canine array probesets and human array probesets clearly separated the healthy and cancer samples in both datasets. The analysis demonstrated that for both human and canine DLBCL there is activation of the NF-κB/p65 canonical pathway, indicating that canine lymphoma could be used as a model to study NF-κB-targeted therapeutics for human lymphoma. The model was further validated by identification of molecular signatures sub-classifying canine DLBCL into activated Bcell-like (ABC) or germinal centre B-cell-like (GCB) types. Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 6 02 Apoptosis in human and canine lymphoma: the Bcl-2 family of proteins Franco Guscetti Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland E-mail: [email protected] Deregulated apoptosis contributes to malignant transformation and progression and to the development of resistance to therapy. Intrinsic apoptosis has a main protective role against the establishment of neoplastic cell clones. The Bcl-2 family exerts a central function in this pathway by regulating the integrity of the outer mitochondrial membrane and the efflux of pro-apoptotic factors such as cytochrome c from the mitochondria into the cytosol. This results from the mainly stoichiometric interplay of three subgroups, i.e. the anti-apoptotic members (prototype: Bcl-2), the multidomain pro-apoptotic members (Bax, Bak) and the BH3-only proteins, which can act either as sensitizers or as activators. Since the initial discovery of the oncogenic properties of Bcl-2 in human follicular lymphoma an ample body of basic research studies involving lymphoid cell lines and murine tumor models has implicated the involvement of members of the Bcl-2 family of proteins in lymphomagenesis. Three potential blocks have been postulated, including absence or loss of function of activator BH3-only proteins, or of Bax/Bak, and overexpression of anti-apoptotic members. Further, the status of mitochondrial priming, which can be functionally assessed using BH3-peptides, has been shown to predict response to therapy as a result of the relative abundance of Bcl-2 family members in the cell. Recently, the potential of this protein family as a therapeutic target has emerged with the development and current testing in clinical phase I/II trails of small molecule inhibitors of antiapoptotic Bcl-2 proteins. They partly showed efficacy as single agents, e.g. for the treatment of CLL. At the sequence level, canine Bcl-2 family proteins display high levels of homology with their human counterparts especially at the BH-domains where they show almost 100% identity. Rare preliminary reports of use of such inhibitors in dogs with tumors are available, albeit this has not been reported yet with lymphoma. In canine lymphoma, the Bcl-2 family of proteins has received little attention so far. Our approach is to develop immunohistochemical methods for Bcl-2 proteins in biopsies collected for histopathology with the intent of establishing, in the mid-term, predictive markers. Previous immunohistochemical studies of human lymphoma were based on the combined assessment of Bcl-2 family members variably including Bcl-2, Bcl-x, Mcl-1, Bax and Bak; while it was early shown that overexpression of Bcl-2 was associated with worse prognosis, data on prognostic significance were inconsistent for the remaining proteins. This may be due to the heterogeneity of the cases studied. A recent work has found a significantly higher apoptotic activity and expression of pro-apoptotic Bcl-2 members in human tumors with a germinal center vs. non-germinal center B-like profile. We have carried out immunohistochemistry using tissue arrays containing over 80 classified, archival canine lymphoma samples. We put much emphasis on antibody validation using recombinant proteins, normal canine tissues and western blots. So far, we have analysed the expression of the Bcl-2 family members Mcl-1 and Bcl-x, Bak and Bax, and the BH3-only protein Bad in this material. These proteins were found to be frequently and variably expressed across lymphoma immunophenotypes and subtypes. B-cell neoplasms significantly expressed more Mcl-1 than T-cell tumors. Bak and Bax were frequently expressed. Interestingly, the expression of Bax was significantly positively correlated with the percentage of cleaved caspase-3 positive apoptotic cells, indicating an association with the apoptotic activity of the tumors. Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 7 Selected References Bai M, Skyrlas A, Agnantis NJ, Kamina S, et al. Diffuse large B-cell lymphomas with germinal center B-celllike differentiation immunophenotypic profile are associated with high apoptotic index, high expression of the proapoptotic proteins bax, bak and bid and low expression of the antiapoptotic protein bcl-xl. Mod Pathol. 2004 Jul;17(7):847-56. Dettwiler M, Croci M, Vaughan L, Guscetti F. Immunohistochemical Expression Study of Proapoptotic BH3-Only Protein Bad in Canine Nonneoplastic Tissues and Canine Lymphomas. Vet Pathol. 2013 Feb 15. [Epub ahead of print] Letai AG. Diagnosing and exploiting cancer's addiction to blocks in apoptosis. Nat Rev Cancer. 2008 Feb;8(2):121-32. Ni Chonghaile T, Sarosiek KA, Vo TT, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science. 2011 Nov 25;334(6059):1129-33. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008 Jan;9(1):47-59. 03 Proliferation index (Ki67 and s-phase) in human and canine lymphoma Fulvio Riondato Dept. of Veterinary Sciences, University of Torino, Italy E-mail: [email protected] Both s-phase fraction (SPF) and Ki67 index (Ki67) correlate with tumor grading of human nonHodgkin lymphoma (NHL) and cut-off values to discriminate between aggressive and indolent forms have been suggested. Most importantly, both indexes present independent prognostic significance. However, contradictory results have been reported especially concerning Ki67. This is due in part to the heterogeneity of Ki67 and SPF values between and among different NHL histotypes. Other factors accounting for the contrasting results are the differences in the cut-off values adopted and in the method of Ki67 evaluation. More recently both indexes have been studied in specific lymphoma entities demonstrating the need for a different interpretation in relation to the histotype of NHL. These studies showed that a low Ki67 was associated to a worse prognosis in DLBCL and to a better prognosis in indolent NHL. Similarly, a better prognosis has been linked to a lower SPF in indolent lymphomas and to a higher SPF in aggressive lymphomas using different specific threshold values. Moreover, there is evidence of improved efficacy of the IPI (International Prognostic Index) score when combined with Ki67 evaluation. In patients affected by DLBCL and presenting with low or intermediate IPI score, a higher overall survival has been highlighted for Ki67 <70% and <75%, respectively; in mantle cell lymphoma a combined biological index (MIPIb) adding Ki67 to the MIPI (Mantle Cell Lymphoma IPI) score was useful to stratify patients into different risk groups. Very few studies have been carried out on the assessment of SPF and Ki67 in canine lymphoma, and similarities with the human counterpart have been found. In a study by Teske, SPF varied among the different canine lymphoma entities but didn’t show diagnostic or prognostic significance. In dogs, like in humans, Ki67 resulted as a very good indicator of grade of malignancy: in a study by FournelFleury, 21% was identified as the cut-off value being able to discriminate between lowand highgrade NHL (the cut-off was reduced to 10% if mycosis fungoides cases were excluded). In this study Ki67 varied significantly among subtypes with marginal zone lymphoma (MZL) presenting the lowest index (3%). However, a certain degree of heterogeneity within each type was reported. In a recent study on canine indolent lymphomas, Flood-Knapik described significantly different Ki67 Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 8 indexes in follicular lymphoma compared to marginal zone lymphoma, and hypothesized that Ki67 might be useful in detecting more aggressive MZL. Our preliminary flow cytometric data confirm that both SPF and Ki67 are very good markers to differentiate between low-grade and high-grade lymphomas (cut-off values of 3,9% and 12%, respectively); furthermore, Ki67 may have prognostic significance in DLBCL, with a better outcome associated to intermediate values (20-40%). However, a much larger series is necessary to get reliable results, considering the very low incidence of some NHL subtypes in dogs. Moreover, combining flow cytometric determination of cell size (FSC), immunophenotype, proliferative activity (Ki67) and apoptotic activity (Annexin-V) ,we developed a scheme to help in classifying different entities. Thus, flow cytometric analysis of SPF and Ki67 are useful in stratifying canine lymphoma cases according to biological behaviour. Further studies are warranted to confirm their utility as prognostic indicators in canine NHLs and to define their role in characterizing specific entities for comparative studies. Selected References: Duque et al. Cytometry 1993;14: 492-496 Lackowska et al. Pol J Pathol 2012; 1:18-24 Broyde et al. Am J Hematol 2009; 84:338-343 Teske et al. Exp Hematol 1993; 21:579-584 Fournel-Fleury et al. J Comp Pathol 1997; 117:61-72 Flood-Knapik et al. Vet Comp Oncol 2012 (epub ahead of print) 04 Role of canine lymphoma cell lines in research comparison with human lines Barbara Rütgen Clinical Pathology, Department of Pathobiology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria E-mail: [email protected] Since the 50ies of the last century cell lines serve as worldwide used tools for in vitro and in vivo studies. Stable well characterised cell lines ensure stable and reproducible experimental conditions throughout a research project and are used for preclinical drug testing, antigen production, development of animal models, basic research on tumorigenesis and for quality control in diagnostic testing. Establishment of leukemia/lymphoma cell lines is known to be difficult. Given the small amounts of sample material available and the lack of specific growth factors for optimal cell culture conditions the establishment of cell lines in veterinary medicine is particularly challenging. Nevertheless, also in humans success rates for the establishment of leukemia/lymphoma cell lines are poor. In humans, liquid sample material such as peripheral blood and pleural effusions show the highest success rate with 48% and 13%, respectively, whereas out of solid lymph node materials the success rate is only 3%. Approximately 1500 hematopoietic neoplastic cell lines have been described. Many of them were developed before the ‘genomics revolution’; thus their characterisation is often based solely on their clinical origin in combination with immunophenotyping analysis. In veterinary medicine only a few canine lymphoma/leukaemia cell lines are described and even a smaller number is well characterized. The first canine cell lines were established in the late 1980ies and early 1990ies and more recently in 2007, 2010 and 2013. Most of the well described cell lines, as in Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 9 humans, were established from effusion material or whole blood. One lymphoma cell line has been established from lymph node material and only two, OSW and CLBL-1, have been characterized in detail at the genomic level. The OSW cell line is characterized as T-cell line out of a peripheral T-cell lymphoma, whereas the CLBL-1 cell line is a B-cell line derived from a Diffuse Large Cell Lymphoma. As tools enabling genomic characterization are now available, previously established canine hematopoietic cell lines are revisited. Generation of a genotypic and phenotypic profile for each cell line is important to ensure its usefulness as in vitro and in vivo models of lymphoid neoplasia and for the development of adequate animal models for equivalent human disease entities. Canine haematopoietic malignancies are highly comparable with their human counterparts with regard to clinical presentation, tumour biology and response to therapy. Dogs share evolutionarily conserved chromosome aberrations and mutations within key oncogenes, indicative of common pathogenesis mechanisms. Moreover dogs have also been sharing their environment with humans for thousands of years and are outbred individuals, which makes canine lymphoma a suitable large animal model in comparative medicine. These facts make also canine lymphoma cell lines valuable for canine and furthermore for comparative medicine. In humans, as well as in dogs, these cell lines represent important tools providing repeatable and reproducible results and, after sufficient characterization, these cell lines have the potential to be representative for several aspects of the respective diseases. Selected References: Seiser EL, Thomas R, Richards KL, Kathryn Kelley M, Moore P, Suter SE, Breen M. Reading between the lines: molecular characterization of five widely used canine lymphoid tumour cell lines. Vet. Comp. Onc. 2011;11,1:30–50. Kisseberth WC, Nadella MVP, Breen M, Thomas R, Duke SE, Murahari S, Carrie E. Kosarek CE, Vernau W, Avery AC, Burkhard MJ, Rosol TJ. A novel canine lymphoma cell line: a translational and comparative model for lymphoma research. Leuk. Res. 2007;31:1709–20. Rütgen BC, Hammer SE, Gerner W, Christian M, de Arespacochaga AG, Willmann M, Kleiter M, Schwendenwein I, Saalmüller A. Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse large cell lymphoma. Leuk. Res. 2010;34:932–938. Momoi Y, Okai Y, Watari T, Goitsuka R, Tsujimoto H, Hasegawa A. Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma. Vet. Immunol. Immunopathol. 1997;59:11–20. Nakaichi M, Taura Y, Kanki M, Mamba K, Momoi Y, Tsujimoto H, Nakama S. Establishment and characterization of a new canine B-cell leukemia cell line. J. Vet. Med. Sci. 1996;58:469–71. Rütgen BC, Willenbrock S, Reimann-Berg N, Walter I, Fuchs-Baumgartinger A, Wagner S, Kovacic B, Essler SE, Schwendenwein I, Nolte I, Saalmüller A, Murua Escobar H. Authentication of Primordial Characteristics of the CLBL-1 Cell Line Prove the Integrity of a Canine B-Cell Lymphoma in a Murine In Vivo Model. PLoS ONE 2012;7(6): e40078. doi:10.1371/journal.pone.0040078 Umeki S, Ema Y, Suzuki R, Kubo M, Hayashi T, Okamura Y, Yamazaki J, Tsujimoto H,Tani K, Hiraoka H, Okuda M, Mizuno T. Establishment of Five Canine Lymphoma Cell Lines and Tumor Formation in a Xenotransplantation Model. J. Vet. Med. Sci. 2013;75(4): 467–474. Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 10 05 The role of MMPs, VEGF and PDGF in canine lymphomas Luca Aresu Department of Comparative Biomedicine and Food Science, Legnaro, Padova CAP 35020, Italy E-mail: [email protected] Vascular Endothelial Growth Factor (VEGF) and enzymes involved in extracellular matrix (ECM) remodelling, including Matrix Metalloproteinases (MMPs), are recognized useful molecules for the early prognosis assessment of different human haematologic malignancies. Tumour cells in lymphoma and leukaemia can self-produce and secrete MMPs to promote invasion and increase their metastatic potential. These processes are controlled by tissue inhibitor metalloproteinases (TIMPs) that are co-secreted by MMP-producing cells. Among MMPs, MMP-2 and MMP-9 play a critical role in the modification of ECM and tumour invasion. In canine lymphoma VEGF and active-MMP-9 plasmatic levels are significantly higher when compared with healthy dogs and in T-cell lymphomas compared with B-cell lymphomas. Increased levels of VEGF in plasma are also correlated to the biological behaviour in T-cell lymphoma. Decreased VEGF and act-MMP-9 levels are observed in dogs with B-cell lymphomas during the chemotherapy protocol. This data is in agreement with human studies indicating that VEGF and MMP-9 might predict treatment response. Conversely, in T-cell lymphomas, VEGF and MMP-9 levels are unaffected. In lymph nodes affected by lymphoma, the most significant results are higher MMP-9 protein and mRNA expression levels in T-cell lymphomas compared with B-cell lymphomas and healthy control lymph nodes, indicating that MMP-9 may be associated with tumour phenotype. MMP-9 is also associated with prognosis since it is higher in HG T-cell lymphomas that are characterized by organ invasion and frequent bone marrow infiltration, and are considered highly aggressive. Biologically, Tcells are able to migrate across ECM barriers during the inflammatory process towards target tissues and the activation of MMP-9 can cause alteration of adjacent connective tissues and degradation of collagen. Interestingly, significantly higher levels of TIMP-1 mRNA are observed in T-cell lymphomas compared with B-cell lymphomas and controls. The in vitro model of T-cell lymphoma cell line (OSW) confirms this data. The most realistic hypothesis is that MMP-9 and TIMP-1 may act in concert in canine T-cell lymphoma: MMP-9 causes ECM degradation, whereas TIMP-1 shows an anti-apoptotic action. VEGF-A mRNA and protein expression are also correlated with prognosis and, moreover, the mRNA VEGF-A results are correlated with MMP-9 results in T-cell lymphoma. Future efforts should be directed to the functional VEGF polymorphisms, which have an effect on the regulation of gene expression. Platelet Derived Growth Factors (PDGFs) and receptors are known to induce tumour growth by directly stimulating growth, to stimulate angiogenesis and to recruit pericytes. Peripheral T-cell lymphomas and cutaneous T-cell lymphomas shows the most significant results with a high level of protein and mRNA expression of PDGF-B and PDGF-Rβ. Conversely, PDGF family seems not to be involved in the pathogenesis of Diffuse Large B-cell lymphomas and Marginal Lymphomas. The PDGF-B gene has been identified as the human homologue of the v-sis oncogene, and the strong expression of PDGF-B and PDGF-Rβ suggests that this autocrine signalling may be important in the malignant transformation of T-cell lymphomas. Conversely, this mechanism seems not to be associated with B-cell lymphomas. Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 11 SESSION ON DIAGNOSIS AND CLASSIFICATION Chairs: Frédérique Ponce, Stefano Comazzi 06 Cytology in classification and therapy of canine lymphomas Frédérique Ponce Vet Agro Sup, Veterinary School of Lyon, Lyon, France E-mail: [email protected] Ponce F., Belluco S., Marchal T., Fournel-Fleury C. Clinical Oncology Unit and Research Unit ICE 2011-03-101, Oncology. Vet Agro Sup, Veterinary School of Lyon, France. Following the Human Classifications of Non-Hodgkin’s Lymphomas (NHLs), the goal is to define canine disease entities, with their particular morphology, immunophenotype and clinical data. They must be recognized by pathologists and have clinical relevance. The first rationale for a precise morphological classification of NHLs appears obvious as the examination at the cellular level is now an essential step to make the diagnosis of the various subtypes of NHLs. In our experience, there appears to be a good correlation between morphology and phenotype allowing the pathologists to formulate a tentative diagnosis of B or T-phenotype by cytologic analysis in most cases. However, the immunophenotype always has to be precisely determined by immunolabelling. As in humans, the cytological diagnosis is based on: i. the architectural pattern (e.g. recognition of a follicular organization, aspect of the background) ; ii. the cell type ; iii. the differentiation block in the lymphomatous process (concept of transformation during the course of the disease) ; iv. the proliferating power (mitotic index, Ki67 index). The second rationale for a precise morphological classification is to allow oncologists to predict outcome and responses to chemotherapy. Hence, there are many distinct diseases each with their particular clinical presentation at the time of diagnosis, their own prognostic relevance and associated with distinctive responses to chemotherapy. This suggests that recognition and classification of the different subtypes of lymphomas is clinically justified in dogs, as in humans. The third rationale for a precise cytological classification is to allow, in the context of the daily diagnostic work, a precise diagnosis of the majority of subtypes and a regular cytological follow-up of the clinical course of indolent lymphomas until transformation. Cytological monitoring allows an earlier detection of transformation from a low to a high-grade lymphoma. In addition, fine needle aspirations are easily and quickly performed in case of infiltration of deeply located organs. In humans, there is still significant skepticism towards the role of cytology, and excisional biopsy is frequently regarded as essential for a precise classification of NHLs. However, cytology is an accurate method of diagnosing and typing common forms of human’s NHL. Awareness of the diagnostic limitations and pitfalls is of key importance in promoting the acceptance for a cytologic diagnosis. Excisional biopsy should be considered where the cytologic diagnosis is equivocal or lymphadenopathy persists without identified cause. Out of the new World Health Organization classification, two B-cell entities will be presented as examples and will be compared to their humans counterparts: i. the Diffuse Large B cell lymphoma ; Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 12 ii. The Marginal Zone lymphoma and the concept of transformation towards higher grade as in humans (indolent and aggressive). The definition of canine T-cell and NK cell neoplasms, based on morphological immunophenotypic features and postulated cell of origin is less well known than for B-cell tumors and still imprecise. However, two particularly striking clinico-morphological entities will be pointed out: i. the small clear cell/T-zone lymphoma with indolent clinical course, as a specific canine entity ; ii. the aggressive T-cell Lymphoma with Large Granular cells, compared to humans aggressive N/K cell lymphoma/leukemia, hepatosplenic T-cell lymphoma and enteropathy type.