The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells.

To evaluate the histologic response rate of high-grade squamous intraepithelial lesions (HSIL) of the cervix after topical application of 5% imiquimod cream.

To investigate pathologic discrepancies between colposcopy-directed biopsy (CDB) of the cervix and loop electrosurgical excision procedure (LEEP) in women with cytologic high-grade squamous intraepithelial lesions (HSILs).

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.

This study aimed to evaluate the differences in cervical appearance among different human papillomavirus (HPV) genotypes in patients with high-grade squamous intraepithelial lesions (HSILs).

Over 10% of patients diagnosed with cervical intraepithelial neoplasia (CIN) have no lesions detected in their cervical conization specimens. The purpose of this study was to determine the factors related to the absence of such lesions. We particularly sought to investigate whether the expression of B7-H4 in precancerous lesions and cancer of the uterine cervix plays a role in the presence or absence of residual lesions in conization specimens and whether this protein is associated with T cells (i.e., Foxp3+ regulatory T cells, CD4+, and CD8+) and interferon-γ production. Of the 807 patients with CIN treated by conization, 104 (12.9%) had no lesions in their conization specimens. Seventy-five of these patients were deemed the study group and were matched with 75 patients who did have CIN detected in their conization specimens (the control group). Immunohistochemistry and immunofluorescence staining were used to detect B7-H4, Foxp3, CD4, CD8, and interferon-γ in the 75 pairs of specimens obtained via biopsy; 20 samples were found to have chronic cervicitis, and another 20 had squamous cell carcinoma of the cervix. Menopause, the absence of human papillomavirus, low-grade histological findings, and a diagnosis of CIN1 and CIN2 on biopsy correlated with a low probability of lesions on conization specimens. B7-H4 expression was detected in 11.1% of CIN2, 46.6% of CIN3, and 70% of cervical cancer samples, but not in tissues representing chronic cervicitis or CIN1. B7-H4 expression was associated with the presence of lesions on conization specimens, increased regulatory T cells, decreased CD8+ T cells, and lower interferon-γ production. These data suggest that close follow-up and thorough reevaluation should be considered for patients diagnosed with CIN2 who are negative for B7-H4 expression on biopsy before proceeding with cervical conization.

Human papillomavirus (HPV) infection is recognized as the most common sexually transmitted disease in the world, and there is a consensus on its role in the etiology of preneoplastic epithelial changes in the cervix. Through the process of lipid peroxidation, oxidative stress is found in the course of premalignant and malignant changes. Moreover, the level of physical activity can exert an influence on markers of oxidative stress, lowering the serum levels of these markers. Objective. To determine the relationship between levels of malondialdehyde (MDA) and the level of physical activity in women with squamous intraepithelial lesion (SIL) of the cervix. Methods. A cross-sectional study was conducted with 46 women participating in a cervical cancer control program. The women had been submitted to the cytopathological exam and were divided into two groups: 18 with SIL and 28 controls. MDA concentrations were determined, and the International Physical Activity Questionnaire (IPAQ) was administered on the same day as the gynecological appointment (prior to the Papanicolaou test). Results. The SIL group had higher MDA levels than the control group (mean: 47.63 ± 9.57 vs. 9.32 ± 4.79, respectively) and a lower IPAQ score (median: 713.5 vs. 1875, respectively). A weak correlation was found between the MDA level and IPAQ score (r 2 = -0.34, p = 0.018). Conclusion. The women with SIL had higher levels of oxidative stress and were less physically active than the women in the control group. These findings suggest that physical exercise exerts an influence on markers of oxidative stress in the development of intraepithelial squamous lesions.

Stratified mucin-producing intraepithelial lesion (SMILE) is a rare high-grade cervical precancerous lesion designated a variant of adenocarcinoma in situ (AIS) in the WHO classification. We aimed to determine HPV genotypes, immunohistochemical phenotype and mucin presence in SMILE. Between 2010 and 2018, SMILE was diagnosed in 34 out of 6958 (0.5%) cervical biopsies, in 23 patients. Twenty-six tissue samples from twenty-one patients were available for further analysis, including 13 with SMILE alone, 12 with SIL and/or AIS and one with HSIL, AIS and endocervical adenocarcinoma. HPV genotyping was performed using the Seegene Anyplex II HPV 28 assay. Of the 26 samples, a single HPV genotype was identified in the majority of cases (n = 22), including 12/13 SMILEs associated with SIL/AIS. All but one were high-risk HPV genotypes (23/24; 96.8%). We identified seven different HPV genotypes, the most common being HPV16 (n = 10; 43.5%), HPV18 (n = 8, 34.8%) and HPV 31 (n = 5, 21.7%). All SMILEs showed a strong positive reaction to p16, CK7, CK19 and high Ki67 expression comparable to adjacent HSIL and/or AIS if present. SMILE showed variable mucin presence and p40-positive squamous differentiation suggesting phenotypic diversity in cervical precancerous lesions infected by single HPV.

High-risk human papillomavirus (HR-HPV) has been demonstrated to be the necessary cause of cervical carcinoma. High-risk HPV detection has a prognostic significance for the women who are at increased risk of disease progression. HPV genotyping in cervical cancer precursor lesions is crucial for prevention and management of cervical cancer. This study was designed to investigate the distribution of HR-HPV genotypes among a group of patients with high-grade squamous intraepithelial lesions and higher, of the cervix, in Botswana.

DNA methylation is clinically relevant to important tumorigenic mechanisms. This study evaluated the methylation status of candidate genes in cervical neoplasia and determined their diagnostic performance in clinical practice. Cervical cancer and normal cervix tissue was used to select the top 5 discriminating loci among 27 loci in 4 genes (CCNA1, CADM1, DAPK1, JAM3), and one locus of JAM3 (region M4) was identified and confirmed with 267 and 224 cervical scrapings from 2 independent colposcopy referral studies. For patients with atypical squamous cells of unknown significance and those with low-grade squamous intraepithelial lesion, with JAM3-M4 compared to a triage marker of hrHPV testing, the specificity for cervical intraepithelial neoplasia 3 CIN3 and cancer cases (CIN3+) / no neoplasia and CIN1 (CIN1-) was significantly increased, from 21.88 to 81.82 and 15.38 to 85.18, respectively. The corresponding positive predictive value (PPV) was increased from 26.47 to 57.14 and 18.52 to 63.64, respectively. For hrHPV-positive patients, compared to a triage marker of cytology testing, JAM3-M4 showed increased specificity and PPV, from 30.67 to 87.65 and 38.82 to 82.14, respectively. We assessed whether JAM3-M4 could distinguish productive from transforming CIN2; the coincidence rate of JAM3-M4 and P16 was as high as 60.5%.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix -SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell® invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC; and explain why p16 overexpression fails to stop CC growth.

High oncogenic risk human papillomaviruses (HPVs) are closely associated with cancer of the cervix. However, HPV infection alone may not be sufficient to cause cervical cancer, and other factors or cofactors may have a cumulative effect on the risk of progression from cervical HPV infection to cancer. The present study investigates the cytosine‑adenine (CA) repeat polymorphism in the P1 promoter region of the insulin‑like growth factor‑1 (IGF‑1) gene among cervical precancerous and cancer patients and healthy control females. The association between these polymorphisms, tissue and blood serum levels of IGF‑1, and cervical cancer risk and progression is evaluated. The material for analysis consisted of blood cells and postoperative tissues from patients diagnosed with low‑grade squamous intraepithelial lesions (L‑SILs), high‑grade squamous intraepithelial lesions (H‑SILs) and invasive cervical cancer (ICC). A polymerase chain reaction amplification and the sequencing of DNA were used for the identification of (CA)n repeats in the IGF‑1 P1 region and detection of HPV DNA. The blood serum concentration of IGF was determined by enzyme‑linked immunosorbent assay. The identification of the IGF‑1 protein in the cervical tissues was performed by immunohistochemical analysis. The range of the length of the CA repeats in the study DNA was 11 to 21. However, the most common allele length and genotype in the control and study patients from serum and tissues was 19 CA repeats and a homozygous genotype of CA19/19. Statistically significant differences in the concentration of IGF‑1 in the blood serum were observed between H‑SILs and controls, only (p=0.047). However, the concentration of IGF‑1 in the group of females with CA19/19, CA19<19 and CA19>19 was significantly higher in the group of patients with H‑SIL (P=0.041) and ICC (P=0.048) in comparison with the control group. An association was detected between CA repeat length <19 and/or >19, IGF concentration in blood serum and tissues and the development of cervical cancer.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre-existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4-positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1-derived HPV 16-positive W12 cells via an ELF3-dependent pathway. In organotypic raft cultures, FOXP4-downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte-derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3-dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC.

The present study aims to investigate the expression levels of two critical mammalian target of rapamycin (mTOR) downstream effectors, 4E binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) proteins, in precancerous squamous intraepithelial lesions and cancer of the uterine cervix, and their association with human papilloma virus (HPV) infection status. Uterine cervical biopsies from 73 patients were obtained, including 40 fresh-frozen samples and 42 archival formalin-fixed, paraffin-embedded tissue specimens. Whole protein extracts were analyzed for the expression of 4EBP1 and eIF4E proteins using western blotting. In addition, distribution of 4EBP1 and eIF4E protein expression and 4EBP1 phosphorylation (P-4EBP1) were analyzed by immunohistochemistry in archival tissues and correlated with the degree of dysplasia. The presence of high-risk HPV (HR-HPV) types was assessed by polymerase chain reaction. Using western blot analysis, high expression levels of 4EBP1 and eIF4E were observed in all uterine cervical carcinomas, which significantly correlated with the degree of dysplasia. By immunohistochemistry, overexpression of 4EBP1 and eIF4E was detected in 20 of 21 (95%) and 17 of 21 (81%) samples, respectively, in patients with high-grade dysplasia and carcinomas, compared with 1 of 20 (5%) and 2 of 20 (10%) samples, respectively, in patients with low-grade lesions or normal histology. All 4EBP1-positive cases tested were also positive for P-4EBP1. Furthermore, overexpression of 4EBP1 and eIF4E significantly correlated with the presence of HR-HPV oncogenic types. The present study demonstrated that critical effectors of mTOR signaling, which control protein synthesis initiation, are overexpressed in cervical high-grade dysplasia and cancer, and their levels correlate with oncogenic HPV types. These findings may provide novel targets for investigational therapeutic approaches in patients with cancer of the uterine cervix.

The repressor element 1-silencing transcription factor (REST) is a regulator of gene expression, and the Ras association domain family member 1 A (RASSF1A) is an important tumor suppressor gene involved in cancer development. Although extensive characterization of the roles of REST and RASSF1A in cancer development have been reported in cellular models, the link between them and their possible role in the development of squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) of the cervix have not been explored. The aim of this study was to evaluate the expression of REST and RASSF1A in cervical cytological and histological samples from patients diagnosed with SIL or SCC and in CC-derived cell lines.

Human papillomavirus (HPV) infection is associated with the vast majority of cervical cancer cases as well as with other anogenital cancers. PepCan is an investigational HPV therapeutic vaccine for treating cervical high-grade squamous intraepithelial lesions. The present study was performed to test whether the cervical microbiome influences vaccine responses and to explore host factors as determinants of the cervical microbiome composition in women with biopsy-proven high-grade squamous intraepithelial lesions. In a recently completed Phase I clinical trial of PepCan, histological response rate of 45% (14 of 31 patients), a significant increase in circulating T-helper type 1 cells, and a significant decrease in HPV 16 viral load were reported. DNA, extracted from liquid cytology specimens collected before and after vaccinations, were amplified and then hybridized to a G4 PhyloChip assay to characterize the microbiome. We describe trends that certain bacterial taxa in the cervix may be enriched in non-responders in comparison to responders (Padj = .052 for phylum Caldithrix and Padj = .059 for phylum Nitrospirae). There was no difference in bacterial diversity between the 2 groups. A permutational analysis of variance performed for various demographic and immune parameters showed significant clustering with microbiome beta diversity for race, HPV 16 status, peripheral T-helper type 1 cells, and HLA-B40 (P = .001, .014, .037, and .024, respectively). Further analyses showed significant differences at the empirical Operational Taxonomic Unit level for race and HPV 16 status. As these results are from a small Phase I study, further studies are needed to examine the role of cervical microbiome in response to HPV therapeutic vaccines.

In the course of identifying the molecular mechanism that is related to strong cell-cell adhesion in stratified structures of the squamous epithelium, calmodulin-like protein 5 (CALML5) was identified as a spinous structure-associated protein by producing monoclonal antibodies with the use of the crude intercellular portion of squamous tissue as an immunogen and by subsequent morphologic screening. By electrophoretic mobility shift assay (EMSA) and a series of mutagenesis studies, two transcription factors, ZNF750 and KLF4, by binding in line to the CALML5 gene promoter, were found to play a central role in CALML5 transcription. Knockdown of CALML5 by siRNA in the A431 cell line that expresses high levels of CALML5 resulted in the acceleration of wound confluence in a scratch assay, indicating that CALML5 functions as a tumor-suppressor in uterine cervical cancer. Immunohistochemical evaluation of squamous intraepithelial lesions, carcinoma in situ (CIS) and invasive uterine cancer, revealed a reduction in CALML5 expression during the stages of CIS through various molecular pathways including the blockage of the nuclear translocation of KLF4. Conversely, restoration of the nuclear translocation of KLF4 by inhibiting ERK-signaling reactivated CALML5 expression in ME180 cells expressing low levels of CALML5. Thus, alteration of the p63-ZNF750-KLF4 axis may result in critical functional loss of CALM-related genes during cancer progression. Although the morphological association of CALML5 with the spiny-structure in relation to cell motility is not clear, evaluation of CALML5 expression provides a useful diagnostic indicator of differentiating dysplasia, preinvasive and invasive cervical cancers.

A subgroup of oropharyngeal squamous cell carcinomas (OSCCs) are causally linked to infection with high-risk human papillomaviruses (HR-HPVs). To evaluate the prevalence of simultaneous oral HPV infection in females with cervical high-grade squamous intraepithelial lesions (HSIL), tonsillar- and cervical smears were collected simultaneously from 73 patients and analyzed for HPV using two commercial assays, PapilloCheck (Greiner-Bio-One) and Linear Array (Roche). Only 3/73 (4.1%) tonsillar smears were HPV positive (HPV+), with HPV types 16, 35, and 45, respectively, detected by both assays (100% agreement). Concordant results were also found in 60/66 (91%) evaluable cervical smears. Of specimens, positive by both assays, typing results completely coincide in 71% (all types are identical) and partially coincide in 27% (at least one type is identical). Taken together, results of HPV detection and typing by PapilloCheck and Linear Array are highly congruent and confirm the low prevalence of HR-HPV in tonsillar smears of patients with HSIL of the uterine cervix. Our data indicate low prevalence of oropharyngeal HPV infection in patients with high-grade cervical dysplasia. The low detection rate was confirmed by using two different commercial assays with largely consistent results of HPV detection and typing, but with some variation for particular HPV types. Comparative testing of larger numbers is required to identify the HPV types prone to escape detection with particular assays.

To further characterise the thin variant of high-grade squamous intraepithelial lesions (HSILs) of the cervix defined by the World Health Organization as full-thickness HSILs with nine or fewer cell layers.