Challenges of an Emerging Disease: The Evolving Approach to Diagnosing Devil Facial Tumour Disease

Devil Facial Tumour Disease (DFTD) is an emerging infectious disease that provides an excellent example of how diagnostic techniques improve as disease-specific knowledge is generated. DFTD manifests as tumour masses on the faces of Tasmanian devils, first noticed in 1996. As DFTD became more prevalent among devils, karyotyping of the lesions and their devil hosts demonstrated that DFTD was a transmissible cancer. The subsequent routine diagnosis relied on microscopy and histology to characterise the facial lesions as cancer cells. Combined with immunohistochemistry, these techniques characterised the devil facial tumours as sarcomas of neuroectodermal origin. More sophisticated molecular methods identified the origin of DFTD as a Schwann cell, leading to the Schwann cell-specific protein periaxin to discriminate DFTD from other facial lesions. After the discovery of a second facial cancer (DFT2), cytogenetics and the absence of periaxin expression confirmed the independence of the new cancer from DFT1 (the original DFTD). Molecular studies of the two DFTDs led to the development of a PCR assay to differentially diagnose the cancers. Proteomics and transcriptomic studies identified different cell phenotypes among the two DFTD cell lines. Phenotypic differences were also reflected in proteomics studies of extracellular vesicles (EVs), which yielded an early diagnostic marker that could detect DFTD in its latent stage from serum samples. A mesenchymal marker was also identified that could serve as a serum-based differential diagnostic. The emergence of two transmissible cancers in one species has provided an ideal opportunity to better understand transmissible cancers, demonstrating how fundamental research can be translated into applicable and routine diagnostic techniques.

The Genome of the Zebra Mussel, Dreissena polymorpha: A Resource for Comparative Genomics, Invasion Genetics, and Biocontrol

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate a high-quality chromosome-scale genome assembly. Through comparative analysis and transcriptomics experiments we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact Steamer-Like Elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

Cathelicidin-3 associated with serum extracellular vesicles enables early diagnosis of a transmissible cancer

The identification of practical early diagnosis biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumour disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (Sarcophilus harrisii). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumour lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analysed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples. The antimicrobial peptide cathelicidin-3 (CATH3) was enriched in serum EVs of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis; 93.8% sensitivity and 94.1% specificity). As antimicrobial peptides can play a variety of roles in the cancer process, our results suggest that the specific elevation of serum EV-associated CATH3 may be mechanistically involved in DFTD pathogenesis. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperilled Tasmanian devil population.

Traits of a mussel transmissible cancer are reminiscent of a parasitic life style

Some cancers have evolved the ability to spread from host to host by transmission of cancerous cells. These rare biological entities can be considered parasites with a host-related genome. Still, we know little about their specific adaptation to a parasitic lifestyle. MtrBTN2 is one of the few lineages of transmissible cancers known in the animal kingdom. Reported worldwide, MtrBTN2 infects marine mussels. We isolated MtrBTN2 cells circulating in the hemolymph of cancerous mussels and investigated their phenotypic traits. We found that MtrBTN2 cells had remarkable survival capacities in seawater, much higher than normal hemocytes. With almost 100% cell survival over three days, they increase significantly their chances to infect neighboring hosts. MtrBTN2 also triggered an aggressive cancerous process: proliferation in mussels was ~ 17 times higher than normal hemocytes (mean doubling time of ~ 3 days), thereby favoring a rapid increase of intra-host population size. MtrBTN2 appears to induce host castration, thereby favoring resources re-allocation to the parasites and increasing the host carrying capacity. Altogether, our results highlight a series of traits of MtrBTN2 consistent with a marine parasitic lifestyle that may have contributed to the success of its persistence and dissemination in different mussel populations across the globe.

Contemporary and historical selection in Tasmanian devils ( Sarcophilus harrisii ) support novel, polygenic response to transmissible cancer

Tasmanian devils ( Sarcophilus harrisii ) are evolving in response to a unique transmissible cancer, devil facial tumour disease (DFTD), first described in 1996. Persistence of wild populations and the recent emergence of a second independently evolved transmissible cancer suggest that transmissible cancers may be a recurrent feature in devils. Here, we compared signatures of selection across temporal scales to determine whether genes or gene pathways under contemporary selection (six to eight generations) have also been subject to historical selection (65–85 Myr). First, we used targeted sequencing, RAD-capture, in approximately 2500 devils in six populations to identify genomic regions subject to rapid evolution. We documented genome-wide contemporary evolution, including 186 candidate genes related to cell cycling and immune response. Then we used a molecular evolution approach to identify historical positive selection in devils compared to other marsupials and found evidence of selection in 1773 genes. However, we found limited overlap across time scales, with only 16 shared candidate genes, and no overlap in enriched functional gene sets. Our results are consistent with a novel, multi-locus evolutionary response of devils to DFTD. Our results can inform conservation by identifying high priority targets for genetic monitoring and guiding maintenance of adaptive potential in managed populations.

Prevalence and polymorphism of a mussel transmissible cancer in Europe

Transmissible cancers are parasitic malignant cell lineages that acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukemia-like disease. In Mytilus mussels, two lineages of Bivalve Transmissible Neoplasia (BTN), both emerged in a M. trossulus founder individual, have been described to date (MtrBTN1 and MtrBTN2). Here, we performed an extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping hundred SNPs of thousands of European Mytilus mussels. The genetic analysis allowed us to simultaneously obtain the genotype of hosts -M. edulis, M. galloprovincialis or hybrids- and the genotype of tumors of heavily infected individuals. In addition, a subset of individuals were systematically genotyped and analysed by histology in order to screen for possible non-transmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M. galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found but a few individuals with non-transmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2 genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M. trossulus individuals. The observed polymorphism is most likely due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two divergent sublineages, confirmed by mtCOI sequences, are co-spreading in the same geographic area, suggesting a complex diversification of MtrBTN2 since its emergence and host species shift.

First description of a widespread Mytilus trossulus-derived bivalve transmissible cancer lineage in M. trossulus itself

Two lineages of bivalve transmissible neoplasia (BTN), BTN1 and BTN2, are known in blue mussels Mytilus. Both lineages derive from the Pacific mussel M. trossulus and are identified primarily by their unique genotypes of the nuclear gene EF1α. BTN1 is found in populations of M. trossulus from the Northeast Pacific, while BTN2 has been detected in populations of other Mytilus species worldwide but not in M. trossulus itself. Here we examined M. trossulus from the Sea of Japan (Northwest Pacific) for the presence of BTN. Using hemocytology and flow cytometry of the hemolymph, we confirmed the presence of disseminated neoplasia in our specimens. Cancerous mussels possessed the BTN2 EF1α genotype and two mitochondrial haplotypes with different recombinant control regions, similar to that of common BTN2 lineages. This is the first report of BTN2 in its original host species M. trossulus. A comparison of all available BTN and M. trossulus COI sequences suggests a common and recent origin of BTN2 diversity in populations of M. trossulus outside the Northeast Pacific, possibly in the Northwest Pacific.

Gene expression of Tasmanian devil facial tumors differs among host populations

Background Transmissible cancers lie at the intersection of oncology and infectious disease, two traditionally divergent fields for which gene expression studies are particularly useful for identifying the molecular basis of phenotypic variation. In oncology, transcriptomics studies, which characterize the expression of thousands of genes, have identified processes leading to heterogeneity in cancer phenotypes and individual prognoses. More generally, transcriptomics studies of infectious diseases characterize interactions between host, pathogen, and environment to better predict population-level outcomes. Tasmanian devils have been impacted dramatically by a transmissible cancer (devil facial tumor disease; DFTD) that has led to widespread population declines. Despite initial predictions of extinction, populations have persisted at low levels, due in part to heterogeneity in host responses, particularly between sexes. However, the processes underlying this variation remain unknown. Results We sequenced transcriptomes from healthy and DFTD-infected devils, as well as DFTD tumors, to characterize host responses to DFTD infection, identify differing host-tumor molecular interactions between sexes, and investigate the extent to which tumor gene expression varies among host populations. We found minimal variation in gene expression of devil lip tissues, either with respect to DFTD infection status or sex. However, 4,088 genes were differentially expressed in tumors among our sampling localities. Pathways that were up- or downregulated in DFTD tumors relative to normal tissues exhibited the same patterns of expression with greater intensity in tumors from localities that experienced DFTD for longer. No RNA sequence variants were associated with expression variation. Conclusions Expression variation among localities may underly phenotypic variation in the tumor, potentially manifesting in morphological differences that alter ratios of normal-to-tumor cells within biopsies. Phenotypic variation in tumors may arise from environmental variation or differences in host immune response that were undetectable in lip biopsies, potentially reflecting variation in host-tumor coevolutionary relationships among sites that differ in the time since DFTD arrival.