RESUMO
Changes in the copy numbers of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) have shown promising diagnostic utilities among patients with head and neck squamous cell carcinoma (HNSCC). Considering the absence of objective prognostic tools for HNSCC surveillance, this study aimed to assess the utility of saliva-based cf-nDNA and cf-mtDNA in predicting the overall survival of patients with HNSCC. The study included ninety-four patients with a confirmed HNSCC diagnosis with a mean follow-up time of 32.04 months (±19.1). A saliva-based liquid biopsy was collected from each patient. A multiplex quantitative PCR was used to determine the absolute number of cf-nDNA and cf-mtDNA. The Kaplan-Meier estimator and Cox proportional hazards regression models were used to assess overall survival. The absolute copy numbers of cf-nDNA and cf-mtDNA were statistically significantly higher among the deceased patients than among the censored ones (p < 0.05). Individuals with elevated levels of cf-nDNA or cf-mtDNA were associated with a significantly poorer overall survival (p ≤ 0.05). A univariate analysis showed that only the absolute copy number of cf-mtDNA was the sole predictor of overall survival. However, the multivariate analysis showed that all the absolute copy numbers of cf-nDNA, the absolute copy numbers of cf-mtDNA, and the stage of HNSCC were predictors of overall survival. Our study confirms that saliva is a reliable and non-invasive source of data that can be used to predict the overall survival of patients with HNSCC, where cf-mtDNA levels act as the sole predictor.
RESUMO
Annexin V (ANXV), mostly characterized by its ability to interact with biological membranes in a calcium-dependent manner. ANXV interacts mainly with phosphatidylserine (PS), for that fluorescent ANXV widely produced and used as a sensitive and specific probe to mark apoptotic cells or any PS-containing bilayers membranes. Many reports described the prokaryotic expression of recombinant human ANXV. To overcome some of E. coli expression limitations, we aimed in this work to investigate unconventional alternative expression system in mammalian cells for producing secreted human ANXV in fusion with the super folder green fluorescent protein (sfGFP). HEK239T cells were transfected using polyethylenimine (PEI) and pcDNA-sfGFP-ANXV plasmid. Forty-eight hours post transfection, direct fluorescence measurement, immunoblotting and ELISA confirmed the presence of secreted sfGFP-ANXV in cells supernatant. The yield of secreted 6 × His-tagged sfGFP-ANXV after affinity purification was estimated to be around 2 µg per 1 ml of cells supernatant. The secretion system was proper to produce a fully functional sfGFP-ANXV fusion protein in quantities enough to recognize and bind PS-containing surfaces or liposomes. Besides, biological assays such as flow cytometry and fluorescent microscopy confirmed the capacity of the secreted sfGFP-ANXV to detect PS exposure on apoptotic cells. Taken together, we present mammalian expression as a quick, affordable and endotoxin-free system to produce sfGFP-ANXV fusion protein. The secreted sfGFP-ANXV in eukaryotic system is a promising biotechnological tool, it opens up new horizons for additional applications in the detection of PS bearing surfaces and apoptosis in vitro and in vivo assays.