Search

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls ( n = 40) and neuroblastoma patients with localized ( n = 10) and metastatic disease ( n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel ( PHOX2B, TH, CHRNA3) and a cell cycle regulation panel ( E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non-invasive nature of these so-called liquid biopsies provides an attractive alternative to tissue biopsy-based cancer diagnostics. This study aimed to investigate if circulating cell cycle-related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real-time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non-invasive biomarker to diagnose canine tumours.

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.