Background: KRT16 codifies for acidic-intermedium cytokeratin constitutively expressed in squamous epithelia. Its role in carcinogenesis may be related to its expression in the hyperproliferative phase on epidermal cultures, but yet need to be clarified. In a previous cDNA microarray analysis, the KRT16 gene was differentially expressed between healthy cervical tissue and a high grade squamous intraepithelial lesion (HSIL) with confirmed HPV-18 transforming infection. Gene expression profile differences between uterine cervix healthy tissue, low grade, and high grade squamous intraepithelial lesion (LSIL, HSIL) could contribute to identify potential biomarkers for cervical premalignant lesions progression. Design: A chromogenic in situ hybridization (CISH) assay, (RNAscope), was performed to evaluate the KRT16 mRNA in situ expression in a tissue microarray (TMA) constructed from excision specimens (conization and hysterotomy) from 169 patients, with a mean age of 35.37 years (SD ±10.4), with representative areas from normal cervix tissue, LSIL and HSIL. The RNAscope 2.5HD Red KRT16 assay, and the RNAscope 2.5HD Red E6/E7 HPV-HR18 assay, designed to detect E6/E7 mRNA for eighteen different high-risk (HR) HPV genotypes, were performed. Stainings were visually scored from 0 to 4 by two pathologist based on the average number of dots per cell according to a semi-quantitative analysis; score values were compared using GraphPad Prism 7.0 software. A p< 0.05 was considered statistically significant. Results: The KRT16 mRNA in situ expression in healthy cervical tissue, LSIL and HSIL was determined by CISH in a cervical TMA including, normal cervix: 84, LSIL: 77, and HSIL: 157, tissue cores. The mean KRT16 mRNA expression scores in squamous epithelium lining normal cervix (0.95, 95% CI: 0.70-1.20), LSIL (0.88, 95% CI:0.60-1.15), and HSIL (1.11, 95% CI:0.89-1.33), were not significantly different (p=0.573). Mean HPV E6/E7 mRNA expression score in HSIL was significantly higher than mean HPV E6/E7 mRNA expression in LSIL (p<0.0001). There was a positive-low correlation between KRT16 mRNA expression and E6/E7 HR-HPV mRNA expression (Spearman r =0.27). Conclusions: Although previous cDNA microarray analysis identified differential expression of KRT16 between healthy cervical tissue and a HSIL with confirmed HPV-18 transforming infection, this was not confirmed when comparing its in situ mRNA expression between squamous epithelium lining normal cervix, LSIL and HSIL. Also, there is not a strong correlation with the expression of E6/E7 mRNA for eighteen different HR-HPV. The fact that KRT16 is constitutively expressed in basal cells may help to elucidate this phenomenon. The role of KRT16 in proliferative phase of basal cells and cervical carcinogenesis remains unclear, further studies are required to define it. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. KRT16 and E6-E7 HR-HPV mRNA in situ expression in normal cervical tissue, and low-grade and high-grade squamous intraepithelial lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2153.

Background: Cervical cancer is the fourth most common cancer in women worldwide, with around 85% of cases occurring in underdeveloped countries. The diagnosis of premalignant lesions is ​​the objective of cervical cancer prevention programs, aimed at increasing the chances of cure and survival time. In a previous study of our group cDNA of TMEM45A gene was detected as differentially expressed between healthy cervical tissue and a high grade squamous intraepithelial lesion (HSIL) with confirmed HPV-18 transforming infection. TMEM45A gene family is highly expressed in squamous epithelium, playing roles in differentiation, senescence and chemoresistance in some sort of epithelial carcinomas. The overexpression of this product may confer antiapoptotic pathways, contributing to the development of cervical squamous cell carcinoma. We compared the TMEM45A in situ mRNA expression in healthy cervical tissue, low grade squamous intraepithelial lesion (LSIL) and HSIL, using RNAscope chromogenic in situ hybridization (CISH). Design: TMEM45A expression was evaluated by CISH using a tissue microarray (TMA) constructed from cervical excision samples (conization and histerectomy) from 169 patients, with a mean age of 35.37 years (SD ±10.4), from a reference center for cervical pathology in the Colombian Caribbean. Stained sections from the donor blocks were reviewed by two pathologist, verifying the original sign-out diagnosis, and representative areas from normal cervix, LSIL and HSIL were selected. RNAscope 2.5HD Red TMEM45A assay was performed as a technical service in Advanced Cell Diagnostics´s laboratories (Newark, CA). The staining was visually scored by two pathologist from 0 to 4 based on the average number of dots per cell, according to a semi-quantitative analysis. The mean score values for each lesion group were compared using GraphPad Prism 7.0 software. A p< 0.05 was considered statistically significant. Results: The TMEM45A mRNA in situ expression was determined in a cervical TMA including, normal cervix: 76, LSIL: 69, and HSIL: 138, tissue cores. There was a higher mean staining score for TMEM45A mRNA in HSIL (1.51, 95% CI:1.26-1.76), compared to normal cervix (1.10, 95% CI:0.80-1.40). Mean score of TMEM45A in situ mRNA expression in HSIL was significantly higher than mean TMEM45A in situ mRNA expression in the normal cervix lining squamous epithelium (p=0.046). Conclusions: TMEM45A expression analysis identified differential expression between normal cervix lining squamous epithelium and HSIL. This finding supports the possible role of TMEM45A in carcinogenesis of well differentiated epithelial lesions. Additional studies to detect the TMEM45A expression at protein with samples of women from the Colombian Caribbean are in progress to validate these results. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. Differences in mRNA expression of a candidate gene, between normal cervical tissue and cervical premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2530.

Background: Gene expression differences between cervical healthy tissue and premalignant lesions could contribute to identify potential biomarkers for each step of this tumor progression. In a previous cDNA microarray analysis, we identified the gene Retinol Binding Protein 1 (RBP1), differentially expressed between normal cervix and a high grade squamous intraepithelial lesion (HSIL) with HPV-18 transforming infection. RBP1 codifies a retinol-binding protein implied in differentiation of epithelia and may play a role on inhibition of E2F-dependent gene expression and suppress cell growth. Design: The expression of RBP1 mRNA was evaluated using RNAscope chromogenic in situ hybridization (CISH) in a tissue microarray constructed from specimens (conization and hysterotomy) obtained from 169 patients, with mean age of 35.37 years (SD ±10.4). Stained sections from donor blocks were reviewed by two pathologist, who chose representative areas of normal cervix, low grade squamous intraepithelial lesion (LSIL), and HSIL. RNAscope 2.5HD Red RBP1 assay, and RNAscope 2.5HD Red HPV-HR18 , that detect E6/E7 mRNA for eighteen different high-risk (HR) HPV genotypes, were performed, the stainings were visually scored by two pathologist from 0 to 4 in a semi-quantitative analysis, based on the average number of red dots per cell. The mean score values for each tissue group were compared using GraphPad Prism® 7.0. A p< 0.05 was considered statistically significant. Results: The in situ expression of RBP1 mRNA was determined in a cervical TMA including, normal cervix: 66, LSIL: 74, and HSIL: 130, tissue cores. There was not a statistically significant difference (p=0.965) between the RBP1 mRNA expression score in the squamous epithelium from normal cervix (0.42, 95% CI:0.23-0.61), and LSIL (0.51, 95% CI:0.28-0.74). But, its expression was higher in HSIL (1.30, 95% CI:1.09-1.50), with a very significant difference compared to squamous epithelium from normal cervix (p<0.0001). Mean HPV E6/E7 mRNA expression in HSIL was significantly higher than mean HPV E6/E7 mRNA expression in LSIL (p<0.0001). There was positive-moderate correlation between RBP1 mRNA expression and E6/E7 HR-HPV mRNA expression (Spearman r =0.55). Conclusions: The in situ mRNA expression of RBP1 is similar in LSIL and squamous epithelium lining normal cervix, but the significantly different expression between healthy cervical tissue and HSIL, considered as the real premalignant cervical lesion, confirms our previous findings. There is a moderate correlation with the expression of E6/E7 mRNA for eighteen different HR-HPV. We could hypothesized that this overexpression represent a failed mechanism of regulation induced by the HR-HPV infection in cervical carcinogenesis. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. Retinol binding protein-1 and E6-E7 HR-HPV mRNA expression in normal cervical tissue and high grade squamous intraepithelial lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2538.

Background Comparing the in situ differences in mRNA expression of candidate genes between normal cervical tissue and premalignant lesions, could contribute to detect progression risk biomarkers. Use of RNAscope chromogenic in situ hybridization (CISH) that amplifies specific mRNA signals and detects molecules as points that can be quantified, could help the detection of these biomarkers. Methods A tissue microarray was constructed from cervical samples (conization and histerectomy). Representative areas of normal cervix, low-grade (LSIL), and high-grade squamous intraepithelial lesions (HSIL) were chosen. RNAscope 2.5HD Red HPV-HR18 assay that detect E6/E7 mRNA for eighteen high-risk (HR) HPV genotypes, and RNAscope 2.5HD Red assays to detect mRNA from: KRT16, TMEM45A and RBP1, were performed. The stainings were scored by two pathologist from 0 to 4 in a semi-quantitative analysis based on the average number of dots per cell; mean score values were compared using GraphPad Prism 7.0. A p<0.05 was considered statistically significant. Results A total of 390 cervical tissues cores from 169 patients with mean age 36.08 years (SD ±10.48) were included. Mean mRNA expression of HR-HPV E6/E7, TMEM45A and RBP1 were significantly higher in HSIL compared to LSIL and normal cervix squamous epithelium. KRT16 mRNA expression scores in normal cervix squamous epithelium, LSIL, and HSIL, were not significantly different (Table 1). Conclusions CISH allowed to detect E6/E7 mRNA molecules for eighteen HR-HPV genotypes in routinely processed tissues, and to evaluate the expression of the described candidate genes in their context without destroying the tissue morphology. Identification of differential expression between normal cervical tissue and HSIL supports the possible role of TMEM45A and RBP1 in cervical cancer progression and its potential use as progression risk tissue biomarkers. Table 1.Biomarkers candidate genes expression profiles in normal cervix, LSIL and HSILCISH scoresMean (±SD)95% CINormal cervixLSILHSILp valueHR-HPV E6/E7 mRNAn=640.4098(±0.7611)0.2149-0.6048n=640.4098 (±0.7611)0.2149-0.6048n=1381.294 (±1.189)1.078-1.51<0.0001*TMEM45A mRNAn=761.105 (± 1.322)0.8031-1.407n=691.232 (±1.477)0.8771-1.587n=2381.514(± 1.491)1.264-1.7650.046*RBP1 mRNAn=660.4242(±0.7658)0.236-0.6125n=740.5135 (±0.9826)0.2859-1.159n=1301.3 (±1.186)1.094-1.506<0.0001*KRT16 mRNAn=840.9524(±1.15)0.7028-1.202n=770.8831 (±1.214)0.6076-1.159n=1571.115 (±1.41)0.8924-1.3370.573* Citation Format: Ines Benedetti, Felipe Ruiz, Reinhard Rodríguez. mRNA in situ expression of candidate genes for progression risk tissue biomarkers, in premalignant cervical lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 479.

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