New Developments in Canine Lymphoma

s of invited lectures Workshop Proceedings Page 5 Abstracts of invited lectures SESSION ON PATHOGENESIS Gene Expression Profiles in Canine Lymphoma Kristy L. Richards Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 E-mail: [email protected] Canine B-cell lymphoma mimics the human disease in several fundamental ways, including histology, B-cell specific markers (e.g. CD20, PAX5, and CD79a), responsiveness to CHOP-based chemotherapy, and gene expression profiles that mirror activated B-cell (ABC) and germinal center B-cell (GCB) cellof-origin subtypes in human DLBCL. However, in other important ways, canine B-cell lymphoma differs from human DLBCL. These include mutation spectra from exome sequencing studies, prognostically important immunohistochemistry markers like BCL2 and c-MYC (“double expressor phenotype“), and less frequent cure rates after CHOP-based chemotherapy. These differences highlight the importance of credentialing each pathway that is targeted by new therapeutics, to be sure that it accurately mirrors the human disease. We have developed a program for experimental therapeutic development involving more accurate animal models, including canine clinical trials with pet dogs under veterinary oncology care. We call this program PATh (Progressive Assessment of Therapeutics) and it engages researchers from across the Cornell campus. As an example of our program, we are developing a new therapeutic agent with activity against fascin, an actin bundling protein. Fascin overexpression has been shown to be a poor prognostic factor both in human and in canine cancers. Cancer cells often upregulate fascin, and in many cancer types, this is associated with poor prognosis, due to increased invasive and metastatic potential. We have developed fascin inhibitors that prevent actin bundling and block cancer metastasis in mice. We have also shown that fascin is upregulated in approximately half of canine Bcell and T-cell lymphoma cases. We have further shown that a fascin inhibitor, NP-G2-044, kills human and canine lymphoma cells in vitro, and in vivo in mice bearing canine diffuse large B-cell lymphoma (DLBCL) xenografts. We are currently working on a canine clinical pilot trial to treat 10 dogs with lymphoma to measure clinical benefit. We are also using RNAseq data from canine B-cell lymphomas to understand what genes are differentially expressed in high-fascin vs. low-fascin individuals. Together, our studies will further validate the pet dog as a useful addition to the therapeutic development pipeline. We intend to advance human and canine health by using clinical trials, in areas where dogs and human overlap in their molecular characteristics. This strategy should advance both canine and human health. Selected References 1. Richards KL, Motsinger-Reif AA, Chen HW, Fedoriw Y, Fan C, Nielsen DM, Small GW, Thomas R, Smith C, Dave SS, Perou CM, Breen M, Borst LB, Suter SE. Gene profiling of canine B-cell Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 6 lymphoma reveals germinal center and postgerminal center subtypes with different survival times, modeling human DLBCL. Cancer Res. 2013;73:5029-5039. 2. Elvers I, Turner-Maier J, Swofford R, Koltookian M, Johnson J, Stewart C, Zhang CZ, Schumacher SE, Beroukhim R, Rosenberg M, Thomas R, Mauceli E, Getz G, Palma FD, Modiano JF, Breen M, Lindblad-Toh K, Alfoldi J. Exome sequencing of lymphomas from three dog breeds reveals somatic mutation patterns reflecting genetic background. Genome Res. 2015;25:1634-1645. 3. Curran KM, Schaffer PA, Frank CB, Lana SE, Hamil LE, Burton JH, Labadie J, Ehrhart EJ, Avery PR. BCL2 and MYC are expressed at high levels in canine diffuse large B-cell lymphoma but are not predictive for outcome in dogs treated with CHOP chemotherapy. Vet Comp Oncol. 2016. 4. Wilson-Robles H, Budke CM, Miller T, Dervisis N, Novosad A, Wright Z, Thamm DH, Vickery K, Burgess K, Childress M, Lori J, Saba C, Rau S, Silver M, Post G, Reeds K, Gillings S, Schleis S, Stein T, Brugmann B, DeRegis C, Smrkovski O, Lawrence J, Laver T. Geographical differences in survival of dogs with non-Hodgkin lymphoma treated with a CHOP based chemotherapy protocol. Vet Comp Oncol. 2017. 5. Chen L, Yang S, Jakoncic J, Zhang JJ, Huang XY. Migrastatin analogues target fascin to block tumour metastasis. Nature. 2010;464:1062-1066. 6. Yamada N, Mori T, Murakami M, Noguchi S, Sakai H, Akao Y, Maruo K. Fascin-1 expression in canine cutaneous and oral melanocytic tumours. Vet Comp Oncol. 2012;10:303-311. 7. Huang FK, Han S, Xing B, Huang J, Liu B, Bordeleau F, Reinhart-King CA, Zhang JJ, Huang XY. Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization. Nat Commun. 2015;6:7465. Methylation Profile in Canine Lymphoma Luca Aresu Department of Comparative Biomedicine and Food Science, University of Padova (IT) / Institute of Oncology Research (IOR), Bellinzona, CH E-mail: [email protected] Canine B-cell lymphoma (BCL) is the most common hematopoietic cancer in dog, being considered a curable disease in less than 10% of dogs. In the remaining 90% of dogs the disease is rapidly fatal if left untreated. Dogs diagnosed with BCL may respond differently to traditional chemotherapeutic and chemo-immunotherapeutic protocols, but responsiveness remains scarce, with a significant number of dogs succumbing to lymphoma early in the disease course. Studies on gene expression profiling and chromosomal aberrations in cBCL have significantly extended the understanding of pathogenesis and clinical outcomes. Recently, mechanisms controlling gene expression in DLBCL have been described. However, the biology of this tumor is still not entirely explained by genomic events and transcriptional programs, and much less is known about epigenetic changes. Epigenetic mechanisms are fundamental for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. In this contest, DNA methylation is perhaps the most extensively studied epigenetic mechanism in mammals and evidence shows its role in the development and progression of various human cancers. By definition, DNA methylation is a heritable epigenetic modification characterized by the transfer of a methyl group to the cytosine residue of CpG dinucleotides by DNA-methyltransferases (DNMTs). In mammals, plants and other organism, most CpG dinucleotides are methylated on cytosine residues; conversely CpG Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 7 dinucleotides within gene promoters tend to be protected from methylation. Physiologically, methylation is extremely important in numerous cellular processes, including embryonic development, genomic imprinting, X-chromosome inactivation and chromosomal stability. In contrast, defects in DNA methylation are closely associated with cancer and two forms of aberrant DNA methylation are found in cancer: 1) the overall loss of 5-methyl-cytosine (global hypomethylation) and 2) gene promoter-associated (CpG island-specific) hypermethylation. While the precise consequences of genome-wide hypomethylation are still debated (activation of cellular proto-oncogenes, induction of chromosome instability), hypermethylation of gene promoters is associated with gene inactivation. The biological consequence of DNA methylation is a double-edged sword: the neoplastic process is promoted by local hypermethylation resulting in silencing of tumour suppressor genes and in parallel by global hypomethylation triggering reactivation of cellular protooncogenes. A plethora of studies reported that silencing of tumour suppressor genes and other cancer-related genes may occur through hypermethylation in the absence of obvious genetic change. So far, different techniques for methylation analysis have now been published. The bisulfite conversion of DNA results probably one of the most efficient to study methylation and is based on the selective chemical conversion of the unmethylated cytosines into uracil by treatment with sodium bisulfite, which is then amplified as thymine during PCR. In contrast, the methylated cytosines are not converted, such that, in the final sequencing result, the 5-methylcytosine will be still detected as cytosine. A limited number of reports in canine lymphoma are available and only investigating DNA methylation aberrations at single gene level. Methylation-specific PCR and pyrosequencing have shown significative results in these studies but it’s now evident that perturbation of methylation should be analysed at the whole genome level, also to identify gene-interactions that orchestrate tumor transcriptome and phenotype. In the recent years, the research in our laboratory has aimed at providing a better understanding of the epigenetic mechanisms underlying the pathogenesis of B-cell lymphoma in dog. Given the previous considerations for the first time we analysed the genome-wide DNA methylome by means of a canine DNA CpG microarray. The platform was developed and probe design was carried out by the Agilent bioinformatic support team using proprietary prediction algorithms to locate CpG Islands on the C. familiaris draft genome as deposited on Ensembl database (CanFam 3.1) and to design high quality oligo-probes. Microarray probes were selected in order to provide the highest possible coverage of dog genome. Coding DNA sequence (CDS) regions and CpG islands were given top priority. The X chromosome was excluded from analysis and probe design. A total of 170,000 probes (60mers, sense orientation) were designed on both CpG and CDS regions. In details, 102,000 probes were designed targeting a total of 36,807 CpG regions while 68,000 probes were directed against 672 CDS; average base pair tiling was 90 bp. The array was used for the first time in veterinary medicine and a bioinformatic pipeline was constructed ad hoc. In the experimental design we compared tissues obtained from 40 dogs affected by DLBCL with lymph nodal tissue obtained by 8 germ-free dogs used as control. Results showed that DLBCLs are characterized by a widespread aberrant methylation affecting 1,194 regions, corresponding to 823 genes. The hypermethylated sequences were enriched in neighbourhood (upstream ≤ 10kb) or promoter regions while the hypomethylated sequences were preferentially located in gene bodies and downstream regions. This was quite expected considering that CpGIs are highly susceptible to DNA methyltransferases in cancer determining gene silencing. Conversely, CpG-poor regions undergo a global decrease of genomic DNA methylation affecting genome stability, transcriptional elongation, and RNA splicing. Functional analysis of differentially methylated genes identified 22 Biological Process (BP), 5 Molecular Function (MF), 2 Cellular Component (CC) Gene Ontology (GO) terms and 3 KEGG pathways as significantly enriched. Overall, 19 out of 22 GO_BP enriched terms were Abstracts of invited lectures Workshop Proceedingss of invited lectures Workshop Proceedings Page 8involved in the development/morphogenesis of anatomical structures, including 10 terms directlylinked to embryogenesis and 9 related to specific tissues development. All these terms were mainlyrepresented by genes playing key roles in regulating organogenesis (SHH, BMPs, GREM1), bodypatterning (HOX gene family) and tissues differentiation (FGFR2, FGF18, SOX9). Clustering analysisbased on the methylation levels of a subset of methylated CpGs identified three different subgroupsof DLBCL that were significantly associated to overall survival.Next generation sequencing is rapidly becoming a more affordable option, and permits to overtakelimits of microarray analysis such as requirement of speciesor transcript-specific probes and legacytechnology. Also, in the last years the costs of sequencing have dramatically decreased. Consideringthis innovation, in 2016, in our lab we profiled 50 DLBCLs for whole genome methylation profiles bya Methyl Binding Protein (MBD)-seq approach. MBPs are able to capture methylated DNA regionsthat are then sequenced through high-throughput technology. On the same samples we performedRNA-seq for transcriptomic profiling and array comparative genomic hybridization (ACGH) foridentification of chromosomal aberrations. The opportunity to investigate the three omics on thesame tissue has no precedents in veterinary medicine and the results have delineated the correlationof gene-expression with methylation in canine DLBCL. More specifically, it’s evident how thereduction of expression of about 400 tumor-suppressor genes is significantly correlated to the increaseof methylation at the promoter level. Conversely, the up-regulation of genes that are involved inDLBCL pathogenesis seems to be driven by copy number aberrations, most likely gains.In the next future, the major challenge will be to identify hypomethylating agents acting as specificinhibitors of DNA methylation and targeting down-expressed genes (i.e. tumor suppressor genes) toreactivate their expression. Few studies have now validated these molecules in veterinary medicinebut more robust data are needed to consider their use in the clinic. Selected References1. Aresu L. Canine Lymphoma, More Than a Morphological Diagnosis: What We Have Learned aboutDiffuse Large B-Cell Lymphoma. Front Vet Sci. 2016 Aug 31;3:77.2. Jain S, Aresu L, Comazzi S, Shi J, Worrall E, Clayton J, Humphries W, Hemmington S, Davis P,Murray E, Limeneh AA, Ball K, Ruckova E, Muller P, Vojtesek B, Fahraeus R, Argyle D, Hupp TR.The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use inComparative Medicine. PLoS One. 2016 Feb 19;11(2).3. Gaurnier-Hausser A, Mason NJ. Assessment of canonical NF-κB activity in canine diffuse large B-celllymphoma. Methods Mol Biol. 2015;1280:469-504.4. Aricò A, Ferraresso S, Bresolin S, Marconato L, Comazzi S, Te Kronnie G, Aresu L. Array-basedcomparative genomic hybridization analysis reveals chromosomal copy number aberrations associatedwith clinical outcome in canine diffuse large B-cell lymphoma. PLoS One. 2014 Nov 5;9(11).5. Mudaliar MA, Haggart RD, Miele G, Sellar G, Tan KA, Goodlad JR, Milne E, Vail DM, Kurzman I,Crowther D, Argyle DJ. Comparative gene expression profiling identifies common molecularsignatures of NF-κB activation in canine and human diffuse large B cell lymphoma (DLBCL). PLoSOne. 2013 Sep 4;8(9).6. Kim M, Costello J. DNA methylation: an epigenetic mark of cellular memory. Exp Mol Med. 2017 Apr28;49(4):e322. doi: 10.1038/emm.2017.10.7. Hoffman RM. Is DNA methylation the new guardian of the genome? Mol Cytogenet. 2017 Apr4;10:11. doi: 10.1186/s13039-017-0314-8. eCollection 2017.8. Tanić M, Beck S. Epigenome-wide association studies for cancer biomarker discovery in circulatingcell-free DNA: technical advances and challenges. Curr Opin Genet Dev. 2017 Feb;42:48-55.9. Yokoi K, Yamashita K, Watanabe M. Analysis of DNA Methylation Status in Bodily Fluids for EarlyDetection of Cancer. Int J Mol Sci. 2017 Mar 30;18(4). Abstracts of invited lecturesWorkshop Proceedingss of invited lecturesWorkshop Proceedings Page 9SESSION ON DIAGNOSTICS Clonality Testing in the Diagnosis of Canine Lymphoma