Biplex quantitative PCR to detect transcriptionally active human papillomavirus 16 from patient saliva.

Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.

The characterization of extracellular vesicles-derived microRNAs in Thai malaria patients.

BACKGROUND: Extracellular vesicles (EVs) have been broadly studied in malaria for nearly a decade. These vesicles carry various functional biomolecules including RNA families such as microRNAs (miRNA). These EVs-derived microRNAs have numerous roles in host-parasite interactions and are considered promising biomarkers for disease severity. However, this field lacks clinical studies of malaria-infected samples. In this study, EV specific miRNAs were isolated from the plasma of patients from Thailand infected with Plasmodium vivax and Plasmodium falciparum. In addition, it is postulated that these miRNAs were differentially expressed in these groups of patients and had a role in disease onset through the regulation of specific target genes. METHODS: EVs were purified from the plasma of Thai P. vivax-infected patients (n = 19), P. falciparum-infected patients (n = 18) and uninfected individuals (n = 20). EV-derived miRNAs were then prepared and abundance of hsa-miR-15b-5p, hsa-miR-16-5p, hsa-let-7a-5p and hsa-miR-150-5p was assessed in these samples. Quantitative polymerase chain reaction was performed, and relative expression of each miRNA was calculated using hsa-miR-451a as endogenous control. Then, the targets of up-regulated miRNAs and relevant pathways were predicted by using bioinformatics. Receiver Operating Characteristic with Area under the Curve (AUC) was then calculated to assess their diagnostic potential. RESULTS: The relative expression of hsa-miR-150-5p and hsa-miR-15b-5p was higher in P. vivax-infected patients compared to uninfected individuals, but hsa-let-7a-5p was up-regulated in both P. vivax-infected patients and P. falciparum-infected patients. Bioinformatic analysis revealed that these miRNAs might regulate genes involved in the malaria pathway including the adherens junction and the transforming growth factor-ß pathways. All up-regulated miRNAs could potentially be used as disease biomarkers as determined by AUC; however, the sensitivity and specificity require further investigation. CONCLUSION: An upregulation of hsa-miR-150-5p and hsa-miR-15b-5p was observed in P. vivax-infected patients while hsa-let-7a-5p was up-regulated in both P. vivax-infected and P. falciparum-infected patients. These findings will require further validation in larger cohort groups of malaria patients to fully understand the contribution of these EVs miRNAs to malaria detection and biology.

Current state of play for HPV-positive oropharyngeal cancers.

Clinically, HPV-positive oropharyngeal cancers (OPCs) have been shown to have a distinct prognosis, compared to HPV-negative tumours, particularly in survival rates and responses to treatment. These patients have better survival chances and improved prognosis, indicating that a more exhaustive knowledge of these distinctions would aid in the discovery of clinical approaches for both HPV-positive and negative tumours. Furthermore, there is increasing evidence that HPV-related oropharyngeal cancers constitute an epidemiological, molecular, and clinical distinct form as compared to non-HPV related ones therefore, the treatment of these specific subtype of oropharyngeal cancers should adopt a distinct clinical treatment pipeline. Our review will examine the current approaches for the diagnosis and treatment of OPC and discuss the relevance of de-escalation clinical trials in progress.

Application of salivary noncoding microRNAs for the diagnosis of oral cancers.

Oral cancer is on the rise globally and survival rates, despite improvements in clinical care, have not significantly improved. Early detection followed by immediate intervention is key to improving patient outcomes. The use of biomarkers has changed the diagnostic landscape for many cancers. For oral cancers, visual inspection followed by a tissue biopsy is standard practice. The discovery of microRNAs as potential biomarkers has attracted clinical interest but several challenges remain. These microRNAs can be found in bodily fluids such as blood and saliva which have been investigated as potential sources of biomarker discovery. As oral cancer is localized within the oral cavity, saliva may contain clinically relevant molecular markers for disease detection. Our review provides an outline of the current advances for the application of salivary microRNAs in oral cancer. We also provide a technical guide for the processing of salivary RNAs to ensure accurate clinical measurement and validation.

Human papillomavirus 16 E6 modulates the expression of miR-496 in oropharyngeal cancer.

Human papillomavirus (HPV), notably type 16, is a risk factor for up to 75% of oropharyngeal squamous cell carcinomas (SCC). It has been demonstrated that small non-coding RNAs known as microRNAs play a vital role in the cellular transformation process. In this study, we used an LNA array to further investigate the impact of HPV16 on the expression of microRNAs in oropharyngeal (tonsillar) cancer. A number of miRNAs were found to be deregulated, with miR-496 showing a four-fold decrease. Over-expression of the high risk E6 oncoprotein down-regulated miR-496, impacting upon the post-transcriptional control of the transcription factor E2F2. These HPV specific miRNAs were integrated with the HPV16 interactome to identify possible mechanistic pathways. These analyses provide insights into novel molecular interactions between HPV16 and miRNAs in oropharyngeal cancers.