Circulating cell-free DNA does not harbour a diagnostic benefit in cats with feline diffuse iris melanomas

Abstract

Objectives: Feline diffuse iris melanoma (FDIM) is the most common malignant primary intraocular tumour in cats, with reported metastases rates between 19% and 63%. Currently, the only available diagnostic tool for a tentative diagnosis is histopathological examination of the enucleated eye. Therefore, the veterinary ophthalmologist is often faced with the dilemma of whether to enucleate an oftentimes visual eye or to continue monitoring, with the risk of metastases developing. In the past, cell-free DNA (cfDNA) gained more attention in human medicine, especially in the field of oncology. Prior studies have shown the use of cfDNA as diagnostic or prognostic markers in canine and human cancer patients. Therefore, the aim of this study was to investigate cfDNA concentration and integrity in cats with FDIMs compared with cats with benign iris naevi and without ocular abnormalities.

Methods: cfDNA from plasma of cats with iris melanoma (n = 34), iris naevus (n = 30) and without ocular abnormalities (n = 32) were extracted. Primer and probes for feline amyloid beta precursor protein ( APP) and beta actin ( ACTB) were designed for amplicons of various lengths and quantitative PCRs of extracted cfDNA were performed to measure cfDNA concentration and integrity of the plasma samples. Differences of cfDNA concentrations and integrity levels between the three groups (iris melanoma, iris naevi and controls) were analysed using the Mann-Whitney U-test.

Results: cfDNA concentration and integrity analysis revealed no significant differences between the cats with iris melanoma, iris naevus or the control group ( P >0.01). Cats with metastases showed similar cfDNA concentration and integrity to cats without metastases.

Conclusions and relevance: cfDNA concentration and integrity seem to be insufficient as a diagnostic or prognostic marker in cats with FDIMs.

Figure 1

Cell-free DNA (cfDNA) (a,b) concentration and (c,d) integrity of cats with iris melanoma (n = 33), iris naevus (n = 30) and cats without ocular abnormalities (n = 31; ‘control’). Results were achieved using qPCRs for (a,c) ACTB and (b,d) APP with increasing amplicon lengths (ACTB1–4, APP1–4); cfDNA integrity was determined as the ratio of longer fragments (amplicon 2, 3 or 4) to the shortest fragment (amplicon 1). Statistical analysis of differences between the three groups was performed using the Mann–Whitney U-test and revealed P values >0.01. Extreme values are represented with an asterisk and outliers as an open circle