Circulating tumoral cells (CTCs) detection by size-based isolation and reverse-transcription real-time polymerase chain reaction (RT-qPCR) in dogs with oral melanoma

Author(s)

Victor Nowosh

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2022-08-04

Examining board members

Cristina de Oliveira Massoco Salles Gomes; Elizabeth Bohland; Julia Maria Matera; Samantha Ive Miyashiro; Márcia de Souza Xavier

• Advisor: Cristina de Oliveira Massoco Salles Gomes

Abstract

Cancer is a relevant cause of death among humans and companion animals. In both cases, melanoma is a challenging diagnosis, with highly aggressive behavior and a high risk of metastasis. Similarities between malignant neoplasms in these species motivate the use of dogs as models for medical oncology studies. Nowadays, there is a focus on personalized medicine and, consequently, on liquid biopsy. This novel tool detects tumoral biomarkers, including circulating tumor cells (CTCs). It has the potential for patients prognostic, diagnostic, and therapeutic assessment. Besides its considerable interest in human oncology, liquid biopsy studies are still lacking in veterinary medicine and tend to focus on other biomarkers. Based on this, this thesis aims to apply two CTC detection methods in dogs with oral melanoma. In the first chapter, a bibliographic review was conducted on the subjects “canine melanoma” and “ liquid biopsy”. In the second chapter, we tested the enrichment of cells by density- gradient centrifugation and characterization by real-time reverse-transcriptase PCR for melan-A and tyrosinase transcripts. There were 31 dogs enrolled in this study. A pilot study has shown good sensitivity to this technique, and 29/31 patients (93%) had positive results. However, controls have shown a 43% false-positive rate. Agarose gel electrophoresis results suggested unspecific amplification. Many challenges inherent to the technique and particularities inherent to veterinary reality may have contributed to these results, and this experience is further discussed. In the third chapter, we tested the enrichment of cells by microfiltration, specifically Isolation by Size of Tumor Cells (ISET), followed by characterization with immunocytochemistry for melan-A, PNL2 and S100 markers. There were 10 dogs enrolled in this study. CTCs were detected and stained positive in 9/10 samples. The mean count of CTCs was 1,1 cells/mL of blood. 3/10 samples were positive for melan-A, 3/10 for PNL2, and 8/10 for S100, showing how heterogeneous these cells are. However, samples from non-melanocytic neoplasms were also stained for S100, showing this markers low specificity. A patient with fast tumoral progression had 18 total individual cells and tumoral microemboli. However, a patient with no detected CTCs had a similar progression. Thus, the absence of CTCs can not always be correlated with a better prognosis. Statistical analysis has shown a significant correlation between CTC count and lymph node metastasis, suggesting a potential capacity in prognosis assessment. We conclude that, with its adaptations, well-established methods in human medicine allow the detection of CTC in dogs with oral melanoma. ISET demonstrated a good performance in detecting CTCs and it also has shown a potential application in patient prognosis assessment. Large-scale clinical trials are needed to validate its clinical utilities, and dogs and humans may benefit more from these techniques. (AU)

• FAPESP's process: 17/22374-0 - Circulating tumour cell detection using isolation by size (ISET) and RT-qPCR in dogs with Melanoma
• Grantee: Victor Nowosh
• Support Opportunities: Scholarships in Brazil - Doctorate