Advances in human papillomavirus detection and molecular understanding in head and neck cancers: Implications for clinical management.

Head and neck cancers (HNCs), primarily head and neck squamous cell carcinoma (HNSCC), are associated with high-risk human papillomavirus (HR HPV), notably HPV16 and HPV18. HPV status guides treatment and predicts outcomes, with distinct molecular pathways in HPV-driven HNSCC influencing survival rates. HNC incidence is rising globally, with regional variations reflecting diverse risk factors, including tobacco, alcohol, and HPV infection. Oropharyngeal cancers attributed to HPV have significantly increased, particularly in regions like the United States. The HPV16 genome, characterized by oncoproteins E6 and E7, disrupts crucial cell cycle regulators, including tumor protein p53 (TP53) and retinoblastoma (Rb), contributing to HNSCC pathogenesis. P16 immunohistochemistry (IHC) is a reliable surrogate marker for HPV16 positivity, while in situ hybridization and polymerase chain reaction (PCR) techniques, notably reverse transcription-quantitative PCR (RT-qPCR), offer sensitive HPV detection. Liquid-based RT-qPCR, especially in saliva, shows promise for noninvasive HPV detection, offering simplicity, cost-effectiveness, and patient compliance. These molecular advancements enhance diagnostic accuracy, guide treatment decisions, and improve patient outcomes in HNC management. In conclusion, advances in HPV detection and molecular understanding have significant clinical management implications. Integrating these advancements into routine practice could ultimately improve patient outcomes.

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.

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.

Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant.

We recently reported that a genetic transformation of the RNA-Binding-Protein (McRBP), an RNA chaperone gene derived from common ice plant (Mesembryanthemum crystallinum), alleviated injury and loss of biomass production by salt stress in Eucalyptus camaldulensis in a semi-confined screen house trial. In this study, we assessed the potential environmental impact of the transgenic Eucalyptus in a manner complying with Japanese biosafety regulatory framework required for getting permission for experimental confined field trials. Two kinds of bioassays for the effects of allelopathic activity on the growth of other plants, i.e., the sandwich assay and the succeeding crop assay, were performed for three transgenic lines and three non-transgenic lines. No significant differences were observed between transgenic and non-transgenic plants. No significant difference in the numbers of cultivable microorganisms analyzed by the spread plate method were observed among the six transgenic and non-transgenic lines. These results suggested that there is no significant difference in the potential impact on biodiversity between the transgenic McRBP-E. camaldulensis lines and their non-transgenic comparators.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.).

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA-Binding-Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened-house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants' acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high-stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non-transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate-stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non-transgenic status survived the 70 mM NaCl treatment, but after 6-month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non-transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non-transgenic plants after 15-week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.

Transcriptional enhancement of a bacterial choline oxidase A gene by an HSP terminator improves the glycine betaine production and salinity stress tolerance of Eucalyptus camaldulensis trees.

Novel transgenic Eucalyptus camaldulensis trees expressing the bacterial choline oxidase A (codA) gene by the Cauliflower mosaic virus (CaMV) 35S promoter and the Arabidopsis thaliana heat shock protein (HSP) terminator was developed. To evaluate the codA transcription level and the metabolic products and abiotic stress tolerance of the transgenic trees, a six-month semi-confined screen house cultivation trial was conducted under a moderate-stringency salt-stress condition. The transcription level of the CaMV 35S promoter driven-codA was more than fourfold higher, and the content of glycine betaine, the metabolic product of codA, was twofold higher, with the HSP terminator than with the nopaline synthase (NOS) terminator. Moreover, the screen house cultivation revealed that the growth of transgenic trees under the salt stress condition was alleviated in correlation with the glycine betaine concentration. These results suggest that the enhancement of codA transcription by the HSP terminator increased the abiotic stress tolerance of Eucalyptus plantation trees.

Environmental risk assessment of impacts of transgenic Eucalyptus camaldulensis events highly expressing bacterial Choline Oxidase A gene.

Under the Japanese biosafety regulatory framework for transgenic plants, data for assessing a transgenic plant's impact on biodiversity must be submitted in order to obtain approval for a confined field trial. We recently reported the development of four novel transgenic Eucalyptus camaldulensis clones expressing the bacterial choline oxidase A (codA) gene, i.e., codAH-1, codAH-2, codAN-1, and codAN-2, and evaluated their abiotic tolerance by semiconfined screen house trial cultivation. Here we evaluated the impacts of the transgenic E. camaldulensis clones on productivities of harmful substances from those clones to affect soil microorganisms and/or other plants in the environment. A comparison of the assessment data between the transgenic trees and non-transgenic comparators showed no significant difference in potential impacts on biodiversity. The results contribute to sound-science evidence ensuring substantial equivalence between transgenic and non-transgenic E. camaldulensis.