Preclinical diagnostics

Preclinical markers (biological markers) that allow the detection of latently infected animals were seen as impor­tant for controlling the spread of DFTD through free- ranging populations of Tasmanian devils, particularly those animals selected for zoo-based insurance popula­tions.

4.1 Haematology and biochemistry

Haematological and biochemical reference intervals have been published for free-ranging DFTD-free popu­lations of Tasmanian devils (Peck et al. 2015) (see Appendix 1). Subsequent analysis of blood from animals with DFTD demonstrated haematological and biochem­ical changes consistent with chronic debilitation and inflammation, which are not specific for DFTD (Peck et al. 2016) and therefore not specific for the diagnosis of DFTD.

4.2 Metabolomics

Biomarkers are chemicals of a range of types (e.g. pro­teins, mRNA and other metabolites) found in a variety of body fluids and two approaches are used for chemical analysis: targeted (known metabolite) and non-targeted (select all metabolites and study differences between normal and affected groups). The latter approach was initially taken by researchers working on DFTD (Gather­cole 2012). In metabolomics, samples are most commonly analysed using nuclear magnetic resonance (NMR) or mass spectrometry (MS). These two methods can also be preceded with a separation technique such as liquid chro­matography (LC), gas chromatography (GC) or capillary electrophoresis (CE). The majority of techniques that involve separation utilise MS detection rather than NMR.

Karu et al. (2016) published the first metabolomics study of Tasmanian devils suffering from DFTD. Using the non-targeted approach and LC high-resolution mass spectrometry they found that a range of peptide segments of the N-terminal end of fibrinogen were the strongest markers for DFTD, consistent with the marked inflam­matory effect of the tumour. Other identified potential markers included amino acid and lipid metabolites, and cortisol and urea were the most significant health predic­tors.

ERBB3, a member of the epithelial growth factor (EGF) family, is expressed in early embryonal development and plays an important role in the neural crest development and Schwann cell regulation pathways. ERBB3 is upregu- lated in several human cancers (e.g. breast, colon, gastric, ovarian and prostate), where excess extracellular domain transcripts have been detected by ELISA. DFT cells were found to express ERBB3 immunohistochemically (Hayes et al. 2017). A survey of serum samples from 15 healthy Tasmanian devils, 8 with DFTD and 12 affected with another commonly diagnosed cancer (cutaneous lym­phoma) for the presence of ERBB3 moieties by ELISA, was undertaken. Those with DFT or cutaneous lymphoma had elevated levels of ERBB3 compared with clinically normal animals. ERBB3 appears to be a useful metabolite for the early diagnosis of DFTD, but has not yet been adapted for field application. ERBB3 overexpression and cooperation in neoplastic transformation and potential as an oncogene may also make it a therapeutic target (Hayes et al. 2017). Another possible early detection candidate for DFT1 is the elevated expression of cathelicidin-3 (CATH3) in serum-derived extracellular vesicles. While

CATH3 has a role in antimicrobial immune responses in Tasmanian devils their elevated presence in the serum of free-ranging Tasmanian devils affected with DFTD, allowed a high predictive power of pre-clinical DFTD diagnosis (Espejo et al. 2022). The assay is yet to be vali­dated as a fit for purpose test.

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