Tasmanian devils have spawned two transmissible cancer clones, known as devil facial tumour 1 (DFT1) and devil facial tumour 2 (DFT2). DFT1 and DFT2 are transmitted between animals by the transfer of allogeneic contagious cancer cells by biting, and both cause facial tumours. DFT1 and DFT2 tumours are grossly indistinguishable, but can be differentiated using histopathology, cytogenetics or genotyping of polymorphic markers. However, standard diagnostic methods require specialist skills and equipment and entail long processing times. Here, we describe Tasman-PCR: a simple PCR-based diagnostic assay that distinguishes DFT1 and DFT2 by amplification of DNA spanning tumour-specific interchromosomal translocations. We demonstrate the high sensitivity and specificity of this assay by testing DNA from 557 tumours and 818 normal devils. A temporal-spatial screen confirmed the reported geographic ranges of DFT1 and DFT2 and did not provide evidence of additional DFT clones. DFT2 affects disproportionately more males than females, and devils can be co-infected with DFT1 and DFT2.Overall, we present a PCR-based assay that delivers rapid, accurate and high-throughput diagnosis . CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor 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 tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples 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). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. 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 imperiled Tasmanian devil population.

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