Abstract
Lymphoma and leukemia affect many species and may be attributed to several etiologies. Viruses including herpesvirus and retrovirus have been associated with lymphoma in cattle, cats, and nonhuman primates, humans and other species. Lymphoma or leukemia in sea otters have been rarely diagnosed in the wild or in animals living under human care. However, since 2006, 3 of 4 sea otter mortalities at the Vancouver Aquarium were due to complications related to leukemia and lymphoma. One 21-year-old female sea otter was diagnosed with chronic lymphocytic leukemia while two males (aged 12 and 16) were diagnosed with lymphoma. All three individuals were rescued as very young animals in Alaska and lived at the aquarium for the vast majority of their lives. Both male sea otters were treated aggressively with chemotherapy after initial diagnosis, living 148 and 404 days before being humanely euthanized due to declining quality of life. Due to the seemingly high incidence of blood-related cancer in this population of animals, sophisticated molecular techniques were employed to characterize the most recent neoplasia and investigate a potential etiology.
Whole genome sequencing was performed on peripheral blood and a lymphos needle biopsy. The tumor biopsy was sequenced on the Illumina HiSeq X Ten and Oxford Nanopore MinION, generating 264.5 and 4.63 passed filtered Gbp, respectively. De novo assembly was performed on the DNA and RNA data using ABySS 1.3.4. RNA-Seq reads were assembled using alternate kmers for k38–k74 and then merged to create a working contig set. The genome was assembled using a single end k56 and k76 assembly, followed by a paired end k96 assembly to produce the working contig set. These data sets were analyzed to produce structural variants with trans-abyss 1.4.10 using Enhydra lutris kenyoni as the reference. These results were then compared to the genome bam files and filtered to produce putative somatic events. Somatic single-nucleotide variants and indels called by Strelka1 (v1.0.15, default parameters) using a matched tumor-normal sample against the Enhydra lutris kenyoni reference genome2. Calls made with an internal Strelka quality score of under 15 were dropped. A custom gene annotation database was made in-house (following http://snpeff.sourceforge.net/SnpEff_manual.html), allowing for variant annotation by snpEFF (v4.3p).3 The mutations observed in the sea otter tumor were subsequently compared to human and canine lymphomas.
The sea otter genome contains 24,129 genes, in which 17,421 were mapped to the human genome. We identified several mutated genes with a known involvement in human lymphomas. We observed a stop-gain mutation in EP400, a paralog of human EP300. EP300 is a tumor suppressor which has been previously implicated in human diffuse large B-cell and follicular lymphomas.4 We also observed an indel in SPEN, a NOTCH pathway regulator, the deregulation of which is associated with human hematopoietic neoplasms.5 These observations suggest gene and pathway deregulation are a shared genetic commonality between sea otter and human lymphomas. Future comparative analyses regarding the mutational spectrum seen in human, canine, and sea otter lymphoid cancers may elucidate etiology and treatment options and facilitate a One Health approach to neoplasia.
Acknowledgements
A great many people lent their expertise to our efforts to ensure that our otters had the highest level of care possible. The authors would like to thank our animal care specialists including Brian Sheehan, Phil Wong, Indrajit Canagaratnam, Kristy Heffron, Nicole Garza, Troy Neil and Kristen Plancarte and our amazing veterinary technicians Sion Cahoon, Chelsea DeColle and Gwyneth Nordstrom. A great number of experts around the globe who helped in various ways includes (but not limited to): Joerg Mayer, Kathi Ellis, Mike Murray, Dr. G. Ogilvie, Dr. Charney, Marina Ivancic, Allison Peterson, Daniel Lewer, Tanya Wright, Caroline Marschner, Todd Schmitt, Miriam Kleiter, Barbara Kitchell, Valerie MacDonald, Leah Mitchell, Steven Dow, Bruno Grande, Ryan Morin, Jasleen Grewal, Martin Jones, Simon Chan, Steven Bilobram, Patrick Plettner, Heather Kirk, Pawan Pandoh, Adrian Ally, Noreen Dhalla, Angela Tam, Armelle Troussard, Rene Warren, S. Austin Hammond and Inanc Birol.
* Presenting author
Literature Cited
1. Saunders CT, Wong WSW, Swamy S, Becq J, Murray LJ, Cheetham RK. 2012. Strelka: accurate somatic small-variant calling from sequenced tumor - normal sample pairs. Bioinformatics. 28:1811–1817.
2. Jones SJ, Haulena M, Taylor GA, Chan S, Bilobram S, Warren RL, et al. 2017. The genome of the Northern sea otter (Enhydra lutris kenyoni). Genes. 8.
3. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, et al. 2012. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEFF. Fly. 6:80–92.
4. Pasqualucci L, Dominguez-sola D, Chiarenza A, Fabbri G, Grunn A, Trifonov V, et al. 2011. Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature. 471:189–195.
5. Arcaini L, Rossi D, Lucioni M, Nicola M, Bruscaggin A, Fiaccadori V, et al. 2015. The NOTCH pathway is recurrently mutated in diffuse large B-cell lymphoma associated with hepatitis C virus infection. Haematologica. 100:246–52.