Enrique Boccardo

Gastrin-releasing peptide receptor: a promising new biomarker to identify cervical precursor lesions and cancer

This study aimed to verify the relation between gastrin-releasing peptide receptor (GRPR), oncogenic Human Papillomavirus (HPV) and cervical lesions severity. GRPR mRNA levels were evaluated in cervical cancer-derived cell lines and in primary keratinocytes expressing HPV16 oncogenes by RT-PCR. GRPR protein expression was assessed by immunohistochemistry in organotypic cell cultures derived from keratinocytes transduced with HPV16 oncogenes and in 208 cervical samples, including 59 non-neoplastic tissue, 28 cervical intraepithelial neoplasia grade 3 (CIN3), 44 squamous cell carcinomas (SCC) and 77 adenocarcinomas (ADC). Generic primers (GP5+/GP6+) were used to identify HPV infection in tissue samples. Experiments involving cell lines were analyzed through non-parametric tests (Kruskal Wallis), and Fisher's Exact Test for human tissues samples. All statistical tests were considered significant at p <0.05. Immunohistochemical evaluation was conducted independently and blindly by two observers (AD- LO). Any discordant findings were resolved through discussion to reach a consensus score. GRPR mRNA levels were not increased in cells expressing HPV16 or HPV18 oncogenes. However, at the protein level, GRPR was upregulated in organotypic cell cultures containing HPV oncogenes. Besides, it was identified an association between GRPR expression and cervical lesion severity (p < 0.0001). The detection rate of high-risk HPV DNA was directly correlated with cervical disease. Nonetheless, HPV infection was not directly associated with GRPR in cervical samples. GRPR expression is highly predictive of cervical lesion severity, irrespective of HPV infection and might contribute to improving patient's therapeutic management as well as being used a marker of disease progression.

Association of NK6 homeobox 1 promoter methylation with HPV infection, histological sub-type, and patient outcomes in cervical lesions.

To evaluate NK6 homeobox 1 (NKX6.1) promoter methylation in cervical lesions and its association with human papillomavirus (HPV)16/18 infection, histological sub-type, and patient outcomes using clinical and bioinformatic data. A total of 207 cervical tissue samples, including cervicitis (n = 22), cervical intraepithelial neoplasia grade 3 (n = 20), adenocarcinoma in situ (n = 6), adenocarcinoma (n = 59), and squamous cell carcinoma (n = 100), were analyzed by methylation-specific polymerase chain reaction and bisulfite sequencing. HPV genotyping was performed with the INNO-LiPA assay. The Cancer Genome Atlas data (n = 309) were examined for methylation at 27 cytosine-phosphate-guanine sites and their association with overall survival. NKX6.1 promoter methylation was detected in 26.6% of cervical samples and was significantly associated with neoplastic lesions, particularly squamous sub-types (p = .002), with comparable frequencies in cervical intraepithelial neoplasia 3. Logistic regression confirmed that NKX6.1 methylation and HPV16/18 infection were independently associated with squamous cell carcinoma. The Cancer Genome Atlas analysis revealed 11 cytosine-phosphate-guanine sites within NKX6.1 significantly correlated with overall survival, with loci, such as cg12401926 and cg18297736 linked to poorer outcomes. These prognostic effects were locus-specific and not observed when global methylation was considered. NKX6.1 promoter methylation represents an early event in cervical carcinogenesis and is associated with squamous histology. Although global methylation showed no prognostic relevance, site-specific cytosine-phosphate-guanine methylation patterns demonstrated significant survival associations, supporting NKX6.1 as a potential locus-dependent prognostic biomarker in cervical cancer.