The dog as a possible animal model for human non-Hodgkin lymphoma: a review by Francisco Alvarez

Hematological Oncology Hematol Oncol 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/hon.2017

Invited Review

The dog as a possible animal model for human nonHodgkin lymphoma: a review Laura Marconato1, Maria Elena Gelain2 and Stefano Comazzi3* 1 2 3

Centro Oncologico Veterinario, Sasso Marconi, Italy Department of Comparative Biomedicine and Food Science Faculty of Veterinary Medicine, University of Padua, Padua, Italy Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milan, Italy

*Correspondence to: Stefano Comazzi, DVM, PhD, Dipl ECVCP, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133, Milan, Italy. E-mail: stefano.comazzi@unimi.it Received 18 January 2012 Accepted 7 May 2012

Abstract Lymphoma represents the most frequent hematopoietic cancer in dogs, and it shows signiﬁcant overlap with the human disease. Several environmental factors have been associated with canine lymphoma, suggesting that they may contribute to lymphomagenesis. Canine lymphoma often presents in advanced stage (III–V) at diagnosis and, most commonly, has an aggressive clinical course requiring prompt treatment, which relies on the use of polychemotherapy. In this review, we will summarize the state-of-the-art of canine lymphoma epidemiology, pathobiology, diagnostic work-up and therapy, and will highlight the links to the corresponding human disease, providing evidence for the use of dog as an animal model of spontaneous disease. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: lymphoma; dog; animal model

Introduction

Clinical presentation

An animal model is a non-human animal used for research of speciﬁc human diseases. To be valid, it must share with humans some biological analogies in terms of pathophysiology. It also needs to be of small size, genetically manipulable and have a short lifespan. Animal models for lymphoid cancers are generally immune-compromised mice in which xenografts of human tumour cells can be studied or engineered knockout mice in which a missing gene allows to study oncogenetic pathways [1,2]. Nevertheless, these models miss the vast gene network and interaction between cancer and immune system. Additionally, knockout mice typically investigate monogenic diseases, whereas lymphoma is polygenic. Furthermore, tumours are difﬁcult to be reproduced in immune-competent laboratory animals, and the small size of most laboratory animals does not allow clinical translation, thereby limiting trials. The canine model may overcome some of these disadvantages because lymphoma occurs spontaneously in immune-competent hosts, whose size is larger, thereby allowing to reproduce several approaches used in humans.

The clinical presentation of cNHL varies, depending upon the type, grade and site of involvement. Commonly, dogs develop aggressive high-grade multicentric lymphoma, being comparable with human non-Hodgkin lymphoma (hNHL). Conversely, Hodgkin-like lymphomas are rare. Diffuse large B-cell lymphoma (DLBCL) is by far the most diffuse subtype (Figure 1). Dogs with high-grade multicentric lymphoma usually show painless peripheral lymphadenopathy (Figure 2) or, less commonly, clinical signs related to the effects of tumour inﬁltration. T-cell lymphoma with a mediastinal mass can cause respiratory distress, cranial vena cava syndrome and hypercalcemiarelated polyuria and polidypsia. Conversely, indolent lymphomas are often insidious. Although rare, this indolent presentation is common in splenic marginal zone, follicular and mantle cell lymphoma [8,9]. The features of the six most frequent cNHL subtypes are summarized in Table 1.

Incidence

Classification and grading schemes of cNHL tend to reflect the analogous in people, by grouping lymphomas according to their cytological characteristics (Kiel-updated classification) [10] or clinical and morphological aspects (working formulation) [11]. The revised WHO classification based on the Revised European American Lymphoma (REAL) system attempts to group lymphomas by cell type, phenotypic, genetic and molecular aspects, thereby correlating the different classification schemes [12,13]. The high reproducibility of this classification confirms its usefulness when used by experienced pathologists [14].

Lymphohematopoietic disorders are common in dogs, with canine non-Hodgkin lymphoma (cNHL) having the highest incidence, making up 83% of all hematopoietic cancer [3]. The age-adjusted overall incidence of cNHL is 1.5 per 100 000 for dogs younger than 1 year and 84 per 100 000 for dogs being 10 years old [4]. The incidence of cNHL of 15–30/100 000 is similar to human incidence [5,6], with an estimated population of more than 75 million dogs living in USA only [7]. Copyright © 2012 John Wiley & Sons, Ltd.

Classiﬁcation

L Marconato et al.

Figure 1. Cytological (A: May Grünwald-Giemsa stain) and histopathological (B: Hematoxilin-Eosin stain) aspects of a diffuse large B-cell lymphoma, the most diffuse canine lymphoma subtype. Histological image was kindly provided by Dr L. Aresu, University of Padua, Italy

Figure 2. Classical clinical presentation of a systemic canine lymphoma with a peripheral lymphoadenomegaly

Staging The diagnosis of cNHL involves cytology and histopathology on an enlarged lymph node, aided further by invasive or non-invasive procedures to confirm the extent of cancer and to formulate a proper prognosis and therapeutic plan. The advantages of cytology include accuracy, minimal invasiveness, rapidity and low costs. Because in most cNHL the growth pattern is diffuse, cytologic smear is usually representative of neoplastic population. Accuracy and sensitivity are increased by adding flow cytometry to cytology [15]. Although not routinely performed, molecular biology (PCR for antigen receptors rearrangements, PARR) may help in differentiating clonal from reactive proliferations [16]. Although morphologic diagnosis of cNHL has traditionally relied on cytological details, histology is useful to define those entities requiring a structural evaluation of the nodal architecture by excisional biopsy. This is particularly true for small cell low-grade lymphoma, including follicular lymphoma, T-cell rich B-cell lymphoma, marginal lymphoma and mantle cell lymphoma [8,9]. Copyright © 2012 John Wiley & Sons, Ltd.

A proper staging also includes a complete blood count with examination of the peripheral smear for the presence of atypical cells and biochemical tests including lactate dehydrogenase [17]. Documenting bone marrow (BM) involvement is necessary for accurate staging and management [18]. Histological examination of BM via core biopsy is rarely performed in veterinary medicine and lacks standardization; thus, aspiration cytology is generally considered suitable to define BM infiltration in spite of different sensitivity between these two methods. Rarely, BM infiltration is not correlated with hematologic abnormalities; vice versa, circulating blast cells do not necessarily imply BM infiltration, with their presence being attributable to the ‘overspill’ phenomenon. Similarly to human medicine, flow cytometry is of use also in identifying small amounts of involvement and, by CD34 labelling, in differentiating between lymphoma and acute lymphoblastic leukaemia, which harbours a different prognosis [19]. However, CD34 aberrant positivity has been occasionally reported in cNHL without BM involvement, the biological meaning of which is still to be elucidated [20,21]. Because of scarce cross-reactivity between human-specific and canine-specific antibodies, the panel of antibodies generally used for diagnosis and staging cNHL is more limited (Table 2) Conventional radiology, ultrasound and computed tomography remain the standard imaging modality for initial staging, aided by fine-needle aspiration of altered organs [22]. However, these techniques lack functional information, which impedes identification of disease in normal-sized organs [23]. 18F-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) may be an alternative, providing the advantage of detecting metabolic changes in areas involved before structural changes become visible. In dogs, PET is not part of cNHL staging primarily because of its expense, scarce scanner availability and limited radioisotope access. Recently, the proliferation marker 30 -deoxy-30 [18F] fluorothymidine has proven useful to detect early recurrence before clinical relapse [24]. Although preliminary, this finding is of great potential value, providing a basis for response-adapted therapy. The typical diagnostic algorithm is summarized in Figure 3.

Treatment It is commonly accepted that a ‘one-glove-ﬁts-all’ approach to treat cNHL has become obsolete. The ongoing dissection of subtypes, the expansion of multiagent chemotherapy and development of targeted drugs, the elaboration of stageadapted therapies and the assessment of minimal residual disease have been generated new aspects in the treatment of cNHL, contributing to the improvement of prognosis. Identifying reliable tools for proper patient selection is becoming crucial. Evaluating a speciﬁc constellation of clinical, clinico-pathological and pathological variables affecting prognosis is developing into one of these tools [25,26]. In spite of numerous therapeutic options, therapy has rarely been shown to be curative; thus, the main goal is Hematol Oncol 2012 DOI: 10.1002/hon

Centroblastic, polymorphic or immunoblastic

Pleomorphic mixed

Medium-sized macronucleolated cells (MMC)

Cutaneous T-cell lymphoma

Small clear cell

Peripheral T-cell not otherwise speciﬁed lymphoma

Marginal zone lymphoma (MZL)

Mycosis fungoides

T-zone lymphoma

Updated Kiel classiﬁcation

Diffuse large B-cell lymphoma

Lymphoma subtype

Freq. (%)

Low

High

Grade

Regional or generalized; indolent; variable lymphadenopathy (1 or more lnn or generalized)

Erythremic exfoliative dermatitis, nodule, plaques, ulcers; blood involvement (Sezary syndrome)

Multicentric: generalized painless lymphadenopathy; +/ hepatosplenomegaly; +/ bone marrow involvement; cutaneous; intestinal Splenic +/ bone marrow involvement; nodal; rarely extranodal (intestinal)

Multicentric: generalized painless lymphadenopathy; +/ hepatosplenomegaly; +/ bone marrow involvement

Most common clinical features

Small cells round to irregular nuclei; extended pale cytoplasma (hand mirror morphology)

Small cerebriform cells; convoluted nuclei; single, inconspicuous nucleoli; clear cytoplasm

Prevalence of medium-sized macronucleolated cells; rare immunoblasts and centroblasts

Small-sized to large-sized cells; irregular nuclei; pale cytoplasm

Prevalence of centroblasts or immunoblasts + MMCs and small blastic cells

Cell criteria

Histopathology

Coalescing marginal zone proliferation around fading germinal centres represented by residual benign mantel cells; intermediate mitotic index Epitheliotropic pattern; focal cluster of neoplastic cells in hyperplastic epidermis; low to intermediate mitotic index Interfollicular growth with follicle retention; the intervening areas of paracortex are ﬁlled with uniform population of smallintermediated sized lymphocytes; low mitotic index

Diffuse pattern; thinning of lymph node capsule; compression of peripheral sinus; large cells; multiple prominent nucleoli; frequent tingible bodies; high mitotic index Diffuse paracortical expansion in lymph node; thin capsule and compressed sinus; high mitotic index

Table 1. Main frequent canine lymphoma subtypes: frequency (Freq) [13], immunophenotype (IP), clinical and diagnostic aspects

Chlorambucil and prednisone

Median survival > 12 months (splenic MZL)

Splenectomy in case of splenic MZL; adjuvant chemotherapy questionable; CHOP-based protocols in case of nodal MZL Lomustine and prednisone; radiation therapy in case of localized lesions

(Continues)

Median survival > 12 months

Median survival of 6 months

Median survival < 12 months

Median survival of 12 months; complete remission rates of 65%–90%

Outcome

CHOP-based protocols with a maintenance phase; usually incorporation of lomustine

Discontinuous CHOP-based protocols without a maintenance phase; incorporation of cytosine arabinoside in case of bone marrow involvement

First-line treatment

The dog as model for human non-Hodgkin lymphoma

Hematol Oncol 2012 DOI: 10.1002/hon

Radiation therapy and Median survival CHOP-based protocols in of 6–8 months case of mediastinal involvement; CHOP-based protocols with incorporation of lomustine in case of nodal involvement Thin capsule; focal perinodal colonization; diffuse cortical and medullary ﬁlling; lack of tingible bodies; high mitotic index Medium-sized cells; round to convoluted nuclei with dusty chromatin; inconspicuous nucleoli Mediastinal; generalized painless lymphadenopathy; +/ hepatosplenomegaly; often bone marrow involvement; often hypercalcemia High T/ B 3 Lymphoblastic Precursor Tcell lymphoma

Lymphoma subtype

Table 1. (Continued)

Updated Kiel classiﬁcation

Freq. (%)

Grade

Most common clinical features

Cell criteria

Histopathology

First-line treatment

Outcome

L Marconato et al.

to induce long-term remission without further compromising dog’s quality of life. With complete remission rates approaching 80%–90%, polychemotherapy, based on principles of induction therapy and prolonged maintenance phase, had its greatest impact on remission duration and survival when compared with single agent strategies. Stage-adapted and immunophenotypeadapted subtype-specific therapy designs have become major objectives of more recent clinical trials [9,18,27,28]. In dogs, corticosteroids, vincristine, cyclophosphamide and doxorubicin (‘CHOP’-based protocols) with or without L-asparaginase remain the backbone of induction therapy. Cytarabine can be added to the protocol in a myeloablative regimen in case of BM involvement [18]. Conventional chemotherapy yields complete responses in over 80% of dogs; nevertheless, the duration of this remission is short, only lasting 6–11 months [3]. Post-remission therapy includes maintenance chemotherapy or consolidation with total-body radiation therapy (RT). However, the optimal type and duration of post-remission therapy, the value of further intensifications and the optimal selection and timing of RT still are debated. In the case of T-cell lymphomas, there is a tendency in favour of maintenance therapy. Conversely, several trials have raised doubts about the need of prolonged treatments in dogs with B-cell lymphomas because there are no obvious survival benefits in the face of increased toxicity and higher costs [27]. Although CHOP is a standard protocol for B-cell lymphomas, it has been unsuccessful in a substantial number of T-cell cases. A significantly lower complete response rate has been observed among dogs with T-cell lymphoma treated with a CHOP regimen than among dogs with B-cell lymphoma administered with the same treatment [27,29,30]. Phenotype itself seems to represent an independent prognostic factor. Therefore, standard CHOP chemotherapy may be inadequate for T-cell lymphoma, and other chemotherapy regimens, such as MOPP and DMAC, have been developed [31,32] using alternative anticancer drugs, aiming at increasing dose intensity. Nevertheless, these alternative treatment modalities did not dramatically improve the outcome, with an overall survival time of 120–239 days. Although RT plays a major role in the management of hNHL, its place, modalities and indications require further research in cNHL. Best results were obtained when treating minimal residual disease with low-dose RT delivered in a consolidation setting to the whole body [33]. These results were encouraged by reports that half and total body irradiation was safe when used in conjunction with systemic chemotherapy [34,35]. Dose-intense chemotherapy combined with autologous hematopoietic stem cell transplantation has been recently explored, but advantages over standard chemotherapy are still unclear [36].

The dog as a possible epidemiologic model Because of improved vaccination programmes, better nutrition, more advanced diagnostic techniques and treatment options nowadays, dogs live longer, allowing them to Hematol Oncol 2012 DOI: 10.1002/hon

The dog as model for human non-Hodgkin lymphoma

Table 2. Most widely used antibodies in diagnosis of canine lymphoma Antigen

Source

CD45

AbD Serotec

CD18

AbD Serotec

CD3 CD3 CD3 CD5 CD5 CD4 CD8 CD20 CD22 CD21 CD79a CD34 CD90 MHC II

AbD Serotec Santa Cruz DAKO AbD Serotec DAKO AbD Serotec AbD Serotec Lab Vision Abcam AbD Serotec DAKO Pharmingen AbD Serotec, P.F. Moore AbD Serotec

CD11d

AbD Serotec

TCR ab

P.F. Moore UC Davis, CA P.F. Moore UC Davis, CA

TCR gd

Clone

Target species

Prevalent use

Expected positivity

YKIX716.13 CA12.10C12 CA1.4E9 YFC118.3 CA17.2A12 CD3-12 A0452 policlonal YKIX322.3 CD5-54-F6 YKIX302.9 YCATE55.9 LV-CD20 RFB-4 CA21D6 HM57 1H6 YKIX337.217 CA1.4G8 CA2.1C12 YKIX334.2 CA11.8H2 CA16.3D3 CA15.8G7

Canine

FC, IHC

Panleukocyte

Canine

FC, IHC

Panleukocyte

Canine Mouse cross-reactive Human cross-reactive Canine Canine Canine Canine Rabbit cross-reactive Rabbit cross-reactive Canine Human cross-reactive Human cross-reactive Canine

FC, IHC FC IHC FC IHC FC FC IHC IHC FC FC, IHC FC FC

T cells T cells (intracellular) T cells (intracellular) T cells T cells T helper, neutrophils T cytotoxic B cells (intracellular) B cells (intracellular) B cells B cells (intracellular) Precursor cells

Canine

FC, IHC

Canine

Dendritic, monocytes, macrophages, lymphocytes Macrophages, histiocytes, granular lympphocytes T-cell subsets

CA20.8H1

Canine

T-cell subsets

FC, flow cytometry, IHC, immunohistochemistry on formalin fixed and paraffin embedded tissue samples.

Figure 3. Diagnostic algorithm generally used to diagnose canine lymphoma

experience diseases occurring in older animals, including cancer [37]. Additionally, because of their shorter lifespan when compared with humans, the latency period between exposure to a potential carcinogenic substance and cancer development is shorter. Dogs share the same environment with humans, being chronically and sequentially exposed to outdoor pollutants or other possible carcinogens (including Copyright © 2012 John Wiley & Sons, Ltd.

passive tobacco smoking), yet they do not indulge in occupational activities or lifestyles that may confound interpretation of epidemiological studies [38,39]. Pets, therefore, play a useful tool as sentinel hosts for cancer, possibly leading to early identiﬁcation of carcinogenic hazards in the general environment, predicting human risk and assessing health effects [38,39]. Given the fact that pets and people may Hematol Oncol 2012 DOI: 10.1002/hon

L Marconato et al.

develop the same type of cancer when subjected to the same risk factors, monitoring dogs that share the same environment may help identify a link between the carcinogen exposure and the occurrence of malignancy [40,41]. Epidemiological studies on cNHL are limited, but some authors identified an association with living in industrial areas or contact with chemicals, whereas the association with exposure to 2,4diCl-phenoxiacetic acid herbicides is controversial between different studies, as well as exposure to electromagnetic fields [42–45]. An epidemiological study in France showed a correlation between the incidence of cNHL and hNHL and reported a strong association between cNHL and the distribution of waste incinerators, radioactive waste or other polluted sites [46]. Recently, a 2.39-fold increase in the odds ratio for cNHL was found associated to the waste management crisis in the area of Naples, Italy, similarly to what was reported in people residing in the same area [47,48]. Another important issue refers to the influence of breed. Similarly to humans, and differently from rodents, dogs have the most phenotypical diversity among all other animals. Breeds can be considered genetic clusters with a specific predisposition to different lymphoma subtypes. DLBCL is prevalent in Cavalier King Charles Spaniel and Bassethound, and peripheral T-cell lymphoma in Irish wolfhound and Shih-tzu [49], whereas Boxer tends to develop T-lymphoblastic lymphoma [50].

The dog as a possible pathogenetic model In addition to the clinical, clinical–pathological and histological correspondences, also genomic instability is similar between cNHL and hNHL [51,52]. The correlation between genetic factors and development and progression of cNHL is more signiﬁcant because the confounding effects of human genetic heterogeneity are not present in purebred dogs. Few studies have investigated cNHL by using conventional cytogenetic techniques because of the high diploid chromosome number (2n = 78), similar size, morphology and banding pattern of canine autosome [53,54]. However, Hahn et al. [53] demonstrated genomic imbalance as a consequence of aneuploidy in 43 out of 61 cases of cNHL with trisomy of CFA 13 as primary aberration, being present in 75% of cases, and this correlated with longer ﬁrst remission and survival. The introduction of comparative genomic hybridization in veterinary medicine to investigate chromosomal copy number aberration (CNA) [51], the availability of new cytogenetic resources [55,56] and the complete canine genome map [57] lead to the detection of genomic imbalance as a common feature in cNHL. However, the global level of genomic instability in cNHL is lower than in hNHL with a limited proportion of genome involved in CNA [52,58]. Main aneuploidies include gains of CFA 13 and 31, analogous to partial gain of HSA 4 and 8 and gain of HSA21 in hNHL, respectively. In particular, the subchromosomal regions CFA13q-11q21.1/HSA8q22-qtel and CFA31q11-qtel/HSA21q11-qtel harbour genes involved in tumour pathogenesis such as c-myc. In people, Copyright © 2012 John Wiley & Sons, Ltd.

c-myc is mainly involved in B-cell lymphomas because of its frequent aberrant fusion with immunoglobulin genes and, consequently, transcriptional dysregulation [59]. Recently, the same rearrangement was detected in three cases of high-grade cNHL morphologically identified as sporadic Burkitt lymphoma [60]. A more extensive study, however, identified gain of CFA 13 as a common feature of both B-cell and T-cell lymphomas as well as carcinomas and sarcomas, suggesting that this recurrent CNA is probably linked to cancer progression rather than initiation [52,61,62]. Conversely, other CNAs seem to be associated with different lymphoma subtypes, such as the deletion within CFA 26 encompassing the IGLl gene, a hallmark of cDLBCL that is not conserved in humans, being instead characterized by the deletion of IGLk|. These findings are correlated with the predominance of IGLl usage in dogs compared with people [63]. T-cell lymphoma shows a higher frequency and wider distribution of chromosomal aberration and a correlation with deletion of CFA 11q16, orthologous to two regions located on HSA 5 and 9, leading to loss of CDKN2A/B and p16/Rb pathway inactivation in high-grade cPTLC [64]. As an evidence of conservation of this aberration, also hPTCL is associated with deletion of HSA5 5q, and this is correlated with longer survival in the European population but not in Japanese patients [65,66]. Different subtypes of T-cell and B-cell lymphomas are differently distributed in the human and canine population: T-zone and nodal marginal zone lymphomas are common in dogs and infrequent in people, whereas other subtypes are infrequent in both species, such as the mantle cell lymphoma. Conversely, follicular and Burkitt-like lymphomas are rare in dogs and common in people. The availability of canine and human datasets would be useful to understand the heritable and sporadic genetic defects underlying the lymphoma subtypes in both species. The epigenetics modification of DNA is considered nowadays a crucial step in tumorigenesis; the methylation of CpG islands is a mechanism of gene silencing identified in hNHL [67]. Promoter methylation is generally inversely correlated with gene expression, and aberrant hypometilation can lead to genomic instability in malignant cells. This cutting-edge lymphoma biomarker is under investigation also in cNHL. Pelham et al. found genomic hypometilation in cNHL, and the analysis of DLC1 gene, a tumour suppressor gene with high sequence similarities to the human orthologue, revealed an abnormal hypermethylation of promoter region in 21 cNHL [68,69]. As a matter of fact, gene expression profiling revealed the presence of two different subtypes of hDLBCL showing different biological behaviour; the activated B-cell type is more aggressive and related to a higher NF-B activity, which promotes proliferation and inhibits apoptosis [70]. Even if data regarding gene expression profiling in cNHL are not available yet, constitutive and increased NF-kB activities targeting gene expression were detected recently in primary cDLBCL tissue. This suggests the possibility to use this as a spontaneous model for activated B-cell DLBCL in people [71]. Finally, since the discovery of microRNA (miRNA) and their role in post-transcriptional regulation of gene Hematol Oncol 2012 DOI: 10.1002/hon

The dog as model for human non-Hodgkin lymphoma

expression, several studies have demonstrated their crucial involvement in tumorigenesis [72], and the evidence of a downregulation of Mir-15a and Mir-16-1 in chronic lymphocytic leukaemia was the ﬁrst clue of miRNA involvement in pathogenesis of hematopoietic malignancies [73]. Mortarino et al. [74] demonstrated the dysregulation of miRNA also in cNHL with the evidence of an upregulation of miR-17-5p in B-cell lymphoma and miR-181a in T-cell lymphoma. Also, Uhl and co-workers investigated miRNA expression in lymphoma cell lines; all tumours had an increased expression of mir19a + b, an oncogene acting primarily as inhibitor of apoptosis, and a decrease of tumour suppressor miR-218, whereas T-cell lymphomas showed an increased expression of miR-17-5p and B-cell lymphomas a decrease of miR181a [75]. Although preliminary, these data demonstrated the role of speciﬁc miRNA cluster dysregulation also in cNHL.

combination therapies under clinical investigation to explore their potential beneﬁts as integrated treatment, thereby as a prelude investigating these approaches for people with [36,79–81].

Conﬂict of Interest The authors have no competing interest.

Ethics This is a review article dealing with animal pathology. All the procedures described fulﬁl requirements of internal or national ethic committee of the authors of the manuscript or the cited references.

REFERENCES The dog as a possible therapeutic model Anticancer drug development is a multimillion dollar and time-consuming process, involving the design of carefully constructed steps eventually leading to the testing of new molecules in phase I and II clinical trials. A critical part of the preclinical stage of this process is to demonstrate antitumor efficacy in a relevant tumour model in vivo. Over other laboratory species, clinical trials recruiting pets with cancer for research purposes offer several potential advantages. Because of lack of gold standard treatments, pet owners may have the opportunity to enroll their pets in new clinical trials being potentially of benefit in terms of improved quality of life and prolonged survival. Because the regulation for the development of new drugs is not so strict as for humans, informed owner’s consent is often enough for recruiting dogs. Also, pets are typically enrolled earlier in the disease course as compared with humans, who are typically initially treated with conventional therapies and, only after failure, may be asked to participate to experimental trials. Additionally, in dogs, cancer progresses more rapidly, and clinical trials are completed earlier, allowing to assess toxicity data and efficacy in a more restricted time. To complete a randomized clinical trial in pet dogs, 1–3 years is required, whereas in people, it may require 15 years [76]. This rapid schedule provides the opportunity to integrate the findings on animals within human trials, including toxicity, response, pharmacokinetics, pharmacodynamics, dose, regimen, schedule, biomarkers and responding histologies, thereby acting as a bridge to the clinical application. The short remission interval offers the opportunity to test novel antitumoral drugs and new strategies. Many other interesting agents currently in clinical trials have already demonstrated efficacy and safety in cNHL. The list includes GS-9219 (a prodrug of the nucleotide analogue 9-(2phosphonylmethoxyethyl) guanine) [77], ABT526 (a modified thrombospondin-I peptide) [78] and I-kappa kinase inhibitors [71]. In the next future, a canine model should be useful for clinical trials on novel kinase inhibitors or histone deacetylase inhibitors. In addition, there are several Copyright © 2012 John Wiley & Sons, Ltd.

1. Macor P, Secco E, Zorzet S, Tripodo C, Celeghini C, Tedesco F. An update on the xenograft and mouse models suitable for investigating new therapeutic compounds for the treatment of B-cell malignancies. Curr Pharm Des 2008; 14: 2023–2039. 2. Tarantul VZ. Transgenic mice as an in vivo model of lymphomagenesis. Int Rev Cytol 2004; 236: 123–180. 3. Vail DM, Young KM. Canine lymphoma and lymphoid leukemia. In Withrow and MacEwen’s Small Animal Clinical Oncology. (4th edn), Withrow SJ, Vail DM (eds.). WB Saunders: St. Louis, 2007; 699–712. 4. Dorn CR, Taylor DO, Schneider R. The epidemiology of canine leukemia and lymphoma. Bibl Haematol 1970; 36: 403–415. 5. Hahn KA, Bravo L, Adams WH, Frazier DL. Naturally occurring tumors in dogs as comparative models for cancer therapy research. In Vivo 1994; 8: 133–143. 6. Vail DM, MacEwen EG. Spontaneously occurring tumors of companion animals as models for human cancer. Cancer Invest 2000; 18: 781–792. 7. Rowell JL, McCarthy DO, Alvarez CE. Dog models of naturally occurring cancer. Trends Mol Med 2011; 17: 380–388. 8. Valli VE, Vernau W, de Lorimier LP, Graham PS, Moore PF. Canine indolent nodular lymphoma. Vet Pathol 2006; 43: 241–256. 9. Stefanello D, Valenti P, Zini E, et al. Splenic marginal zone lymphoma in 5 dogs (2001–2008). J Vet Intern Med 2011; 25: 90–93. 10. Fournel-Fleury C, Magnol JP, Bricaire P, et al. Cytohistological and immunological classification of canine malignant lymphomas: comparison with human non-Hodgkin’s lymphomas. J Comp Pathol 1997; 117: 35–59. 11. Parodi LA. Classification of malignant lymphoma in domestic animals: history and conceptual evolution. Eur J Vet Pathol 2001; 2: 43–49. 12. Valli VE, Jacobs RM, Parodi AL, Vernau W, Moore PF. Histological Classification of Haematopoietic Tumors of Domestic Animals. World Health Organization, Second Series, Vol. III. Armed Forces Institute of Pathology: Washington, DC, 2002. 13. Ponce F, Marchal T, Magnol JP, et al. A morphological study of 608 cases of canine malignant lymphoma in France with a focus on comparative similarities between canine and human lymphoma morphology. Vet Pathol 2010; 47: 414–433. 14. Valli VE, San Myint M, Barthel A, et al. Classification of canine malignant lymphomas according to the World Health Organization criteria. Vet Pathol 2011; 48: 198–211. 15. Comazzi S, Gelain ME. Use of flow cytometric immunophenotyping to refine the cytological diagnosis of canine lymphoma. Vet J 2011; 188: 149–155. 16. Burnett RC, Vernau W, Modiano JF, Olver CS, Moore PF, Avery AC. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003; 40: 32–41. Hematol Oncol 2012 DOI: 10.1002/hon

L Marconato et al.

17. Marconato L, Crispino G, Finotello R, Mazzotti S, Zini E. Clinical relevance of serial determinations of lactate dehydrogenase activity used to predict recurrence in dogs with lymphoma. J Am Vet Med Assoc 2010; 236: 969–974. 18. Marconato L, Bonfanti U, Stefanello D, et al. Cytosine arabinoside in addition to VCAA-based protocols for the treatment of canine lymphoma with bone marrow involvement: does it make the difference? Vet Comp Oncol 2008; 6: 80–89. 19. Vernau W, Moore PF. An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol 1999; 69: 145–164. 20. Wilkerson MJ, Dolce K, Koopman T, et al. Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules. Vet Immunol Immunopathol 2005; 15: 179–196. 21. Gelain ME, Mazzilli M, Riondato F, Marconato L, Comazzi S. Aberrant phenotypes and quantitative antigen expression in different subtypes of canine lymphoma by flow cytometry. Vet Immunol Immunopathol 2008; 15(121): 179–188. 22. Flory AB, Rassnick KM, Stokol T, Scrivani PV, Erb HN. Stage migration in dogs with lymphoma. J Vet Intern Med 2007; 21: 1041–1047. 23. LeBlanc AK, Jakoby BW, Townsend DW, Daniel GB. 18FDGPET imaging in canine lymphoma and cutaneous mast cell tumor. Vet Radiol Ultrasound 2009; 50: 215–223. 24. Lawrence J, Vanderhoek M, Barbee D, Jeraj R, Tumas DB, Vail DM. Use of 30 -deoxy-30 -[18F]fluorothymidine PET/CT for evaluating response to cytotoxic chemotherapy in dogs with non-Hodgkin’s lymphoma. Vet Radiol Ultrasound 2009; 50: 660–668. 25. Marconato L, Stefanello D, Valenti P, et al. Predictors of longterm survival in dogs with high-grade multicentric lymphoma. J Am Vet Med Assoc 2011; 238: 480–485. 26. Rao S, Lana S, Eickhoff J, et al. Class II major histocompatibility complex expression and cell size independently predict survival in canine B-cell lymphoma. J Vet Intern Med 2011; 25: 1097–1105. 27. Simon D, Nolte I, Eberle N, Abbrederis N, Killich M, Hirschberger J. Treatment of dogs with lymphoma using a 12-week, maintenance-free combination chemotherapy protocol. J Vet Intern Med 2006; 20: 948–954. 28. Rebhun RB, Kent MS, Borrofka SA, Frazier S, Skorupski K, Rodriguez CO. CHOP chemotherapy for the treatment of canine multicentric T-cell lymphoma. Vet Comp Oncol 2011; 9: 38–44. 29. Ruslander DA, Gebhard DH, Tompkins MB, Grindem CB, Page RL. Immunophenotypic characterization of canine lymphoproliferative disorders. In Vivo 1997; 11: 169–172. 30. Garrett LD, Thamm DH, Chun R, Dudley R, Vail DM. Evaluation of a 6-month chemotherapy protocol with no maintenance therapy for dogs with lymphoma. J Vet Intern Med 2002; 16: 704–709. 31. Siedlecki CT, Kass PH, Jakubiak MJ, Dank G, Lyons J, Kent MS. Evaluation of an actinomycin-D-containing combination chemotherapy protocol with extended maintenance therapy for canine lymphoma. Can Vet J 2006; 47: 52–59. 32. Brodsky EM, Maudlin GN, Lachowicz JL, Post GS. Asparaginase and MOPP treatment of dogs with lymphoma. J Vet Intern Med 2009; 23: 578–584. 33. Lurie DM, Gordon IK, Théon AP, Rodriguez CO, Suter SE, Kent MS. Sequential low-dose rate half-body irradiation and chemotherapy for the treatment of canine multicentric lymphoma. J Vet Intern Med 2009; 23: 1064–1070. 34. Williams LE, Johnson JL, Hauck ML, Ruslander DM, Price GS, Thrall DE. Chemotherapy followed by half-body radiation therapy for canine lymphoma. J Vet Intern Med 2004; 18: 703–709. 35. Gustafson NR, Lana SE, Mayer MN, Larue SM. A preliminary assessment of whole-body radiotherapy interposed within a chemotherapy protocol for canine lymphoma. Vet Comp Oncol 2004; 2: 125–131. 36. Frimberger AE, Moore AS, Rassnick KM, Cotter SM, O’Sullivan JL, Quesenberry PJ. A combination chemotherapy protocol with dose intensification and autologous bone marrow transplant (VELCAP-HDC) for canine lymphoma. J Vet Intern Med 2006; 20: 355–364.

37. Paoloni M, Khanna C. Comparative oncology today. Vet Clin North Am Small Anim Pract 2007; 37: 1023–1032. 38. Van der Schalie HW, Gardner HS, Bantle JA, et al. Animals as sentinels of human health hazards of environmental chemicals. Environ Health Perspect 1999; 107: 309–315. 39. Backer LC, Grindem CB, Corbett WT, et al. Pet dogs as sentinels for environmental contamination. Sci Total Environ 2001; 274: 161–169. 40. Reif JS, Rhodes WH, Cohen D. Canine pulmonary disease and the urban environment. Arch Environ Health 1970; 20: 676–683. 41. Hayes HM Jr, Hoover R, Tarone RE. Bladder cancer in pet dogs: a sentinel for environmental cancer? Am J Epidemiol 1981; 114: 229–233. 42. Gavazza A, Presciuttini S, Barale R, Lubas G, Gugliucci B. Association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners. J Vet Intern Med 2001; 15: 190–195. 43. Hayes HM, Tarone RE, Cantor KP. On the association between canine malignan lymphoma and opportunity for exposure to 2,4-dichlorophenoxyacetic acid. Environ Res 1995; 70: 119–125. 44. Kaneene JB, Miller R. Re-analysis of 2,4-D use and the occurrence of canine malignant lymphoma. Vet Hum Toxicol 1999; 41: 164–170. 45. Reif JS, Lower KS, Ogilvie GK. Residential exposure to magnetic fields and risk of canine lymphoma. Am J Epidemiol 1995; 141: 352–359. 46. Pastor M, Chalvet-Monfray K, Marchal T, et al. Genetic and environmental risk indicators in canine non-Hodgkin’s lymphomas: breed associations and geographic distribution of 608 cases diagnosed throughout France over 1 year. J Vet Intern Med 2009; 23: 301–310. 47. Marconato L, Leo C, Girelli R, et al. Association between waste management and cancer in companion animals. J Vet Intern Med 2009; 23: 564–569. 48. Senior K, Mazza A. Italian "triangle of death" linked to waste crisis. Lancet Oncol 2004; 5: 525–527. 49. Modiano JF, Breen M, Burnett RC, et al. Distinct B-cell and T-cell lymphoproliferative disease prevalence among dog breeds indicates heritable risk. Cancer Res 2005; 65: 5654–5661. 50. Lurie DM, Milner RJ, Suter SE, Vernau W. Immunophenotypic and cytomorphologic subclassification of T-cell lymphoma in the boxer breed. Vet Immunol Immunopathol 2008; 125: 102–110. 51. Thomas R, Smith KC, Gould R, Gower SM, Binns MM, Breen M. Molecular cytogenetic analysis of a novel high-grade canine T-lymphoblastic lymphoma demonstrating co-expression of CD3 and CD79a cell markers. Chromosome Res 2001; 9: 649–657. 52. Thomas R, Seiser EL, Motsinger-Reif A, et al. Refining tumorassociated aneuploidy through ’genomic recoding’ of recurrent DNA copy number aberrations in 150 canine non-Hodgkin lymphomas. Leuk Lymphoma 2011; 52: 1321–1335. 53. Hahn KA, Richardson RC, Hahn EA, Chrisman CL. Diagnostic and prognostic importance of chromosomal aberrations identified in 61 dogs with lymphosarcoma. Vet Pathol 1994; 31: 528–540. 54. Winkler S, Murua Escobar H, Reimann-Berg N, Bullerdiek J, Nolte I. Cytogenetic investigations in four canine lymphomas. Anticancer Res 2005; 25: 3995–3998. 55. Langford CF, Fischer PE, Binns MM, Holmes NG, Carter NP. Chromosome-specific paints from a high-resolution flow karyotype of the dog. Chromosome Res 1996; 4: 115–123. 56. Breen M, Langford CF, Carter NP, et al. FISH mapping and identification of canine chromosomes. J Hered 1999; 90: 27–30. 57. Breen M, Jouquand S, Renier C, et al. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res 2001; 11: 1784–1795. 58. Thomas R, Smith KC, Ostrander EA, Galibert F, Breen M. Chromosome aberrations in canine multicentric lymphomas detected with comparative genomic hybridisation and a panel of single locus probes. Br J Cancer 2003; 89(8): 1530–1537. Hematol Oncol 2012 DOI: 10.1002/hon

The dog as model for human non-Hodgkin lymphoma

59. Hartmann EM, Ott G, Rosenwald A. Molecular biology and genetics of lymphomas. Hematol Oncol Clin North Am 2008; 22: 807–823. 60. Breen M, Modiano JF. Evolutionarily conserved cytogenetic changes in hematological malignancies of dogs and humans – man and his best friend share more than companionship. Chromosome Res 2008; 16: 145–154. 61. Thomas R, Wang HJ, Tsai PC, et al. Influence of genetic background on tumor karyotypes: evidence for breed-associated cytogenetic aberrations in canine appendicular osteosarcoma. Chromosome Res 2009; 17: 365–377. 62. Winkler S, Reimann-Berg N, Murua Escobar H, et al. Polysomy 13 in a canine prostate carcinoma underlining its significance in the development of prostate cancer. Cancer Genet Cytogenet 2006; 169: 154–158. 63. Arun SS, Breuer W, Hermanns W. Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralbl Veterinarmed A 1996; 43: 573–576. 64. Fosmire SP, Thomas R, Jubala CM, et al. Inactivation of the p16 cyclin-dependent kinase inhibitor in high-grade canine nonHodgkin’s T-cell lymphoma. Vet Pathol 2007; 44: 467–478. 65. Zettl A, Rüdiger T, Konrad MA, et al. Genomic profiling of peripheral T-cell lymphoma, unspecified, and anaplastic large T-cell lymphoma delineates novel recurrent chromosomal alterations. Am J Pathol 2004; 164: 1837–1848. 66. Nakagawa M, Nakagawa-Oshiro A, Karnan S, et al. Array comparative genomic hybridization analysis of PTCL-U reveals a distinct subgroup with genetic alterations similar to lymphoma-type adult T-cell leukemia/lymphoma. Clin Cancer Res 2009; 15: 30–38. 67. Shaknovich R, Melnick A. Epigenetics and B-cell lymphoma. Curr Opin Hematol 2011; 18: 293–299. 68. Pelham JT, Irwin PJ, Kay PH. Genomic hypomethylation in neoplastic cells from dogs with malignant lymphoproliferative disorders. Res Vet Sci 2003; 74: 101–104. 69. Bryan JN, Jabbes M, Berent LM, et al. Hypermethylation of the DLC1 CpG island does not alter gene expression in canine lymphoma. BMC Genet 2009; 10: 73. 70. Bavi P, Uddin S, Bu R, et al. The biological and clinical impact of inhibition of NF-kB-initiated apoptosis in diffuse large B cell lymphoma (DLBCL). J Pathol 2011; 224: 355–366.

71. Gaurnier-Hausser A, Patel R, Baldwin AS, May MJ, Mason NJ. NEMO-binding domain peptide inhibits constitutive NF-kB activity and reduces tumor burden in a canine model of relapsed, refractory diffuse large B-cell lymphoma. Clin Cancer Res 2011; 17: 4661–4671. 72. Hall PA, Russell SH. New perspectives on neoplasia and the RNA world. Hematol Oncol 2005; 23: 49–53. 73. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99: 15524–15529. 74. Mortarino M, Gioia G, Gelain ME, et al. Identification of suitable endogenous controls and differentially expressed microRNAs in canine fresh-frozen and FFPE lymphoma samples. Leuk Res 2010; 34: 1070–1077. 75. Uhl E, Krimer P, Schliekelman P, Tompkins SM, Suter S. Identification of altered microRNA expression in canine lymphoid cell lines and cases of B- and T-cell lymphomas. Genes Chromosomes Cancer 2011; 50: 950–967. 76. Hansen K, Khanna C. Spontaneous and genetically engineered animal models; use in preclinical cancer drug development. Eur J Cancer 2004; 40: 858–880. 77. Vail DM, Thamm DH, Reiser H, et al. Assessment of GS-9219 in a pet dog model of non-Hodgkin’s lymphoma. Clin Cancer Res 2009; 15: 3503–3510. 78. Rusk A, Cozzi E, Stebbins M, et al. Cooperative activity of cytotoxic chemotherapy with antiangiogenic thrombospondin-I peptides, ABT-526 in pet dogs with relapsed lymphoma. Clin Cancer Res 2006; 12: 7456–7464. 79. Mason NJ, Coughlin CM, Overley B, et al. RNA-loaded CD40activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma. Gene Ther 2008; 15: 955–965. 80. Peruzzi D, Gavazza A, Mesiti G, et al. A vaccine targeting telomerase enhances survival of dogs affected by B-cell lymphoma. Mol Ther 2010; 18: 1559–1567. 81. Henson MS, Curtsinger JM, Larson VS, et al. Immunotherapy with autologous tumour antigen-coated microbeads (large multivalent immunogen), IL-2 and GM-CSF in dogs with spontaneous B-cell lymphoma. Vet Comp Oncol 2011; 9: 95–105.

Hematol Oncol 2012 DOI: 10.1002/hon