The effects of the LaSota strain of oncolytic Newcastle disease virus vaccine on cervical intraepithelial neoplasia Patients-Clinical cohort study.

BACKGROUND: Cervical cancer is one of the most common malignancies in women, and its treatment has many side effects. Therefore, in this research, the effects of the LaSota strain of oncolytic Newcastle disease virus vaccine on cervical intraepithelial neoplasia (CIN) patients were investigated. METHODS: 15 patients who met the inclusion criteria and diagnosed as CIN II and CIN III were included in the study. The vaccine was injected inside the cervix (neoplasia site) at increasing doses during 21 days, and they were evaluated for adverse events. NDV antibody titer was measured in 90 days and the levels of ki-67 and p16 proteins were studied by immunohistochemistry. Also, the levels of some important inflammatory cytokines in the serum of CIN patients were measured and finally the patients were evaluated according to the final outcomes and the reduction of tumor lesions. RESULTS: Only in the first dose of vaccine some patients showed flu-like symptoms. The accumulation of NDV antibodies started on the 7th day of the study and increased until the 90th day. Administration of LaSota vaccine had no significant effect on the expressions of Ki-67 and p16 proteins. Nevertheless, a decrease in the serum levels of Il-1ß was observed in patients after the administration of the vaccine, but the serum levels of both Il-2 and INF-γ upregulated significantly. Also, vaccine administration had no significant effect in reducing CIN grades and lesions. CONCLUSIONS: In general, we concluded that LaSota strain of NDV vaccine has no therapeutic effectiveness in CIN patients.

Elevated expression of endocan in the development of cervical squamous neoplasia of the uterus.

Accumulated evidence has shown that endocan, which was originally called endothelial cell-specific molecule-1, is an attractive prognostic factor in a variety of cancers. However, the relevance of endocan expression in human malignancies remains to be clarified. In the present study, the expression of endocan in cervical squamous neoplasia of the uterus, including low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively), as well as in invasive squamous cell carcinoma was examined by immunohistochemistry. Endocan was not sufficiently expressed in the normal cervical epithelium. Endocan expression was present in LSIL cases but was limited to basal and parabasal areas of the cells. HSIL cases exhibited strong expression of endocan with widely distributed expression toward the epithelial surface. In contrast, further strong expression of endocan was not observed in patients with invasive carcinoma. This study is the first study showing increased expression of endocan in precancerous dysplastic lesions and malignancy of the cervix. The data suggest that a high expression level of endocan potentially contributes to the development of cervical squamous neoplasia of the uterus.

Increase of the T-reg-recruiting chemokine CCL22 expression in a progressive course of cervical dysplasia.

PURPOSE: An increasing infiltration of FoxP3-positive T-regs is associated with a higher grade of cervical intraepithelial neoplasia. The T-reg-recruiting chemokine CCL22 is expressed in various tumour entities. Aim of our study was to investigate the role of CCL22 in the progression and regression of cervical intraepithelial neoplasias, especially in patients with intermediate cervical intraepithelial neoplasias (CIN II). Furthermore, our aim was to characterize the CCL22-producing cells and explore the role of innate immunity in the process of cells recruitment. METHODS: CCL22 expression was analyzed immunohistochemically in 169 patient samples. The immunoreactive score as well as the median numbers of positive cells were calculated in each slide and correlated with the histological CIN grade and FoxP3 expression. Additionally, CD68/CCL22 as well as CD68/PPARγ and CD68/FoxP3 expression were examined by double immunofluorescence. Statistical analysis was performed by SPSS 26. RESULTS: A significantly higher expression of epithelial CCL22 in CIN II with progression in comparison to CIN II with regression (p = 0.006) could be detected. CCL22 was correlated with FoxP3 (Spearman's Rho: 0.308; p < 0.01). In 88%, CCL22-positive cells were positive for CD68, and 71% of CD68-positive macrophages expressed PPARγ. Colocalization of CD68 and FoxP3 was detected in 12%. CONCLUSION: We could demonstrate that increased expression of CCL22, mainly produced by macrophages, correlates with elevated potential of malignancy. CCL22 expression could act as a predictor for regression and progression in cervical intraepithelial neoplasia, and it may help in the decision process regarding surgical treatment versus watchful waiting strategy in order to prevent conisation-associated risks. Furthermore, our findings support the potential of CCL22-producing cells as a target for immune therapy in cervical cancer patients.

Evaluation of variable p16 immunostaining patterns, Ki-67 indices and HPV status in cervical SILs and squamous cell carcinomas: An institutional experience.

Objective: The present study evaluates the immunoexpression of p16 and Ki-67 in cervical squamous intraepithelial lesion (SIL) and carcinomas and correlates their expression with clinicopathological features and HPV-DNA status. Material and Methods: A total 36 included cases of SIL and squamous cell carcinoma (SCC) were subjected to p16 and Ki-67 immunostaining. p16 staining was evaluated depending on grading, distribution, localization pattern, intensity and IHC score. Ki-67 expression was graded based on percentage of positive cells. Results: Incidence of HSIL and SCC cases was found to be significantly increased with parity > 5. p16 grade III diffuse nucleocytoplasmic immunostaining was observed in 62.5% LSIL, 80% HSIL and 87% SCC cases. Significant association of p16 staining intensity, IHC score and Ki-67 indices was noted with increasing grades of SILs and carcinomas. Conclusion: Our experience indicates that a combination of p16 and Ki-67 immunostaining may be useful to determine the severity of dysplastic change.

Quality Control of Biospecimens in a Danish Clinical Cytology Biobank.

Introduction: Based on the experience from a Swedish biobank, we established a clinical cervical cytology biobank and adapted it to a Danish setting. The aim of the present study was to validate the biobank material regarding quality and quantity, to determine the usefulness of the material for future diagnostics and biomarker testing. Methods: Cervical cytology samples collected in ThinPrep were analyzed before and after biobanking using p16/ki-67 dual staining, a human papillomavirus (HPV) DNA test (Cobas), and a test for HPV messenger RNA (mRNA; Aptima). The concordance of the test results before and after biobanking was assessed. We also evaluated the morphology before and after biobanking and did additional tests on the biobanked material to qualify the usefulness of the material (library preparation for next-generation sequencing [NGS], reverse transcription-polymerase chain reaction [RT-PCR], and the Inno-Lipa HPV genotyping test). Results: For the Cobas HPV test, the concordance was 92% (122/133), and for the Inno-Lipa test (30 samples), it was 100%. For the Aptima assay, the concordance was a little lower, 84% (42/50). The morphology of the cell was well preserved, and the concordance of the p16/ki-67 dual staining was 88% (37/42). The functional tests showed that DNA-based NGS libraries (TST15 panel; Illumina) had good quality parameters. However, with the RT-PCR, 12% of the samples showed poor quality and a too low input amount for the analysis. Conclusion: The quality of the biobanked samples is high, and the material is suitable for testing of DNA, RNA, and protein. However, for testing of specific biomarkers, pilot studies are recommended to ensure sufficient input amount and quality of the material, especially for RNA-based studies.

Transcriptomic Network Analysis Using Exfoliative Cervical Cells Could Discriminate a Potential Risk of Progression to Cancer in HPV-related Cervical Lesions: A Pilot Study.

BACKGROUND/AIM: Cervical cancer is the fourth most common type of cancer in women worldwide and it is a major cause of cancer-related deaths in developing countries. Despite the marked reduction observed in the rates of the disease as a result of screening programs, it is necessary to develop robust biomarkers that can detect the neoplastic progression early in HPV-related cervical lesions. MATERIALS AND METHODS: We performed comparative mRNA sequencing from exfoliative cervical cytology samples from nine Korean women using the Illumina NovaSeq6000 platform. Each pathological tissue was matched to the corresponding cytological sample. The pathologic diagnosis was scrutinized with ancillary immunohistochemistry and was considered a confirmative (endpoint) diagnosis. The pathological diagnoses consisted of three cases of chronic cervicitis, 2 high-grade squamous intraepithelial lesions (HSILs), 2 squamous cell carcinomas in situ (CIS), and 2 invasive squamous cell carcinomas (SQCCs), respectively. Using bioinformatic analyses, differentially expressed genes (DEGs; fold change ≥1.5; p<0.05) were applied for Gene Ontology (GO), Gene Set Enrichment Analysis (GSEA), and protein-protein interaction (PPI) networks. RESULTS: From a total of 55,882 genes, 438 DEGs were pinpointed; 282 genes were up-regulated and 156 genes down-regulated. These transcriptomic profiles were clearly divided into neoplastic (HSIL, CIS, and SQCC; ≥HSILs) and non-neoplastic lesions. The up-regulated DEGs were HIF-1a, EDN1, PIK3R3, PPP1CA and AKR1C1. GO, GSEA, and PPI network analyses showed marked associations with metabolism, proteolysis, or proteoglycan process pathways in cervical carcinogenesis. CONCLUSION: The transcriptomic analysis using exfoliative cervical cells was more likely representative of its corresponding histopathological diagnosis, thus emphasizing its potential utility in clinical practice. This study provides comprehensive transcriptomic network analyses for robust biomarkers that might present a high potential risk of progression to cancer in the exfoliative cervical cytology; our findings support their clinical utility for improved cervical cancer screening.

GLUT1, LDHA, and MCT4 Expression Is Deregulated in Cervical Cancer and Precursor Lesions.

Metabolic reprogramming is typical in cancerous cells and is required for proliferation and cellular survival. In addition, oncoproteins of high-risk human papillomavirus (HR-HPV) are involved in this process. This study evaluated the relationship between glucose transporter I (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter type 4 (MCT4) expression and cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC) with HR-HPV infection. The protein expression was evaluated in women with CIN I (n=20), CIN II/III (n=16), or ICC (n=24) by immunohistochemistry. The protein expression was analyzed qualitatively by van Zummeren score and quantitatively by Image ProPlus 6 software. LDHA expression increases in HPV-16 infection. In the CIN I group, GLUT1 immunostaining has a 35% protein expression at the membrane level at more than two thirds of the epithelium, which increased by 21.25% more in CIN II/III in more than two thirds of the epithelium. While LDHA and MCT4 in CIN I mostly do not present immunostaining, or this was only limited to the basal stratum, this expression is increased in CIN II/III and ICC cases. The GLUT1, LDHA, and MCT4 expression increased in ICC. The overexpression in high-grade CIN with HR-HPV infection shows a higher risk for cervical carcinoma progression.

Stratified Mucin-producing Intraepithelial Lesions of the Cervix: Clinical Diversity of Cases and Literature Review.

BACKGROUND: This report discusses the current literature on both non-invasive and invasive SMILE ((i)SMILE) lesions of the cervix with focus on the pathology and its related clinical diversity in several cases. Currently, the knowledge on (i)SMILE is limited to single case reports and series. As a consequence, consensus guidelines regarding the management are lacking. Although there is overlap with both high grade squamous intra-epithelial lesion (HSIL) and adenocarcinoma in situ (AIS) on immunohistochemical analyses, it is recommended to treat SMILE like AIS and further excision is needed when surgical margins are positive for SMILE on conization. (i)SMILE, should be considered as a rare subtype of adenocarcinoma of the cervix, and should be treated as such. CASE REPORT: We describe a case with a SMILE lesion undergoing a subsequent robotic hysterectomy after conization and two cases with iSMILE: one case with an early FIGO-stage 1B1 iSMILE tumor, undergoing a robotic radical hysterectomy with sentinel procedure, and one case undergoing a robotic-assisted pelvic/para-aortic lymph node staging dissection, confirming a metastatic FIGO-stage 3C2 (for primary chemo-radiotherapy treatment). CONCLUSION: Here, we report for the first time a few cases of (i)SMILE with different clinical presentations, their management and follow-up. Immunohistochemical characteristics are given for both primary lesions as well as the metastases.

Role of FoxP3-positive regulatory T-cells in regressive and progressive cervical dysplasia.

PURPOSE: Forkhead Box Protein 3 (FoxP3) is known as a key mediator in the immunosuppressive function of regulatory T-cells (Tregs). The aim of our study was to investigate whether FoxP3-positive Tregs have the potential to act as an independent predictor in progression as well as in regression of cervical intraepithelial neoplasia, especially in patients with intermediate cervical intraepithelial neoplasia (CIN II). METHODS: Nuclear FoxP3 expression was immunohistochemically analysed in 169 patient samples (CIN I, CIN II with regressive course, CIN II with progressive course, CIN III). The median numbers were calculated for each slide and correlated with the histological CIN grade. Statistical analysis was performed by SPSS 26 (Mann-Whitney U test, Spearman's rank correlation). RESULTS: An increased FoxP3 expression in CIN II with progression could be detected in comparison to CIN II with regression (p = 0.003). Total FoxP3 expression (epithelium and dysplasia-connected stroma) was higher in more advanced CIN grades (p < 0.001 for CIN I vs. CIN II; p = 0.227 for CIN II vs. CIN III). A positive correlation could be detected between FoxP3-positive cells in epithelium and total FoxP3 expression (Spearman's Rho: 0,565; p < 0.01). CONCLUSION: Expression of FoxP3 could be a helpful predictive factor to assess the risks of CIN II progression. As a prognosticator for regression and progression in cervical intraepithelial lesions it might thereby help in the decision process regarding surgical treatment vs. watchful waiting strategy to prevent conisation-associated risks for patients in child-bearing age. In addition, the findings support the potential of Tregs as a target for immune therapy in cervical cancer patients.

Maslinic Acid Inhibits Cervical Intraepithelial Neoplasia by Suppressing Interleukin- 6 and Enhancing Apoptosis in a Mouse Model.

BACKGROUND: Cervical Intraepithelial Neoplasia (CIN) directly precedes cervical cancer, and elevated proinflammatory cytokine Interleukins (IL)-6 is implicated in CIN. OBJECTIVE: As maslinic acid exhibits anti-IL-6 property, the present study sought to determine the effect of maslinic acid on CIN in vitro and in vivo using cell cultures and mouse CIN models, respectively. METHODS: The dose-effect of maslinic acid on HeLa cells, a human cervical cancer cell line, was first evaluated, including cytotoxicity, IL-6 secretion, IL-6 receptor (IL-6R) expression, proliferation potential and apoptosis status. A mouse model of CIN was also established, which was then subjected to increasing doses of maslinic acid treatment, followed by assessment of serum IL-6 level, cervical expression of IL-6R, and the proliferation potential and apoptosis of cervical tissues. RESULTS: Maslinic acid dose-dependently inhibited cell growth and proliferation potential, reduced IL-6 secretion, cervical expression of IL-6R and induced apoptosis of HeLa cells in vitro. In the CIN mouse model, serum IL-6 level and cervical expression of IL-6R were elevated, which could be repressed by maslinic acid administration dosedependently. Additionally, maslinic acid treatment in the CIN mice could also restore the otherwise increased proliferation potential and reduced apoptosis in the cervical tissues. CONCLUSION: Maslinic acid exhibits potent anti-IL-6 property in the CIN mouse model, and alleviates the diseaserelated abnormality in proliferation potential and apoptosis state of the cervical tissue cells, demonstrating its usefulness as a promising agent in treating CIN.

p16 is superior to Stathmin-1 and HSP27 in identifying cervical dysplasia.

BACKGROUND: The aim of this study was to determine how Stathmin-1 and Heat Shock Protein 27 (HSP27) can be used as adjunctive biomarkers to differentiate high-grade dysplasia from benign/reactive lesions in cervical tissues. In addition, we aimed to see if any of these markers can differentiate endometrial from endocervical adenocarcinomas. METHODS: Fifty cases including benign cervical tissue, low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), adenocarcinoma in situ of the endocervix, invasive endocervical adenocarcinoma, and endometrial adenocarcinoma were selected. Stathmin-1 and HSP27 immunohistochemistry (IHC) were performed for each case and the results were compared to the previously available p16 IHC stains. RESULTS: p16 stained positively in 100% of HSIL, endocervical adenocarcinoma in situ, and invasive endocervical cases. Stathmin-1 stained positively in 43% of HSIL and 90% of endocervical adenocarcinoma in situ and all invasive endocervical cases. Stathmin-1 and p16 were negative in all benign cervical samples. Stathmin-1, HSP27, and p16 stained 100% of LSIL cases. HSP27 stained indiscriminately, including 100% of benign cervical tissue. 87% of the endometrial adenocarcinomas stained positively for p16, Stathmin-1, and HSP27. CONCLUSION: p16 remains superior to both Stathmin-1 and HSP27 in differentiating dysplasia from benign, reactive changes of the cervix.

Stratified Mucin-Producing Intraepithelial Lesion (SMILE) of the Uterine Cervix: High-Risk HPV Genotype Predominance and p40 Immunophenotype.

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.

Wnt3a/ß-Catenin/CBP Activation in the Progression of Cervical Intraepithelial Neoplasia.

Piwil2 reprograms HPV-infected reserve cells in the cervix into tumor-initiated cells (TICs) and upregulates Wnt3a expression sequentially, which leads to cervical intraepithelial neoplasia (CIN) and ultimately squamous cell carcinoma (SCC). However, little is known regarding Wnt signaling in the maintenance of TIC stemness during the progression of cervical lesions. We herein investigated the expression of canonical Wnt3a signaling and related genes by microarray data set analysis and immunohistochemical (IHC) staining of samples obtained by biopsy of normal cervix, low- and high-grade CIN, and invasive SCC tissue. Array data analyzed by GEO2R showed higher expression levels of Wnt signaling and their target genes, significant upregulation of stemness-associated markers, and notably downregulated cell differentiation markers in CIN and SCC tissues compared with those in the normal cervix tissue. Further, Gene Set Enrichment Analysis (GSEA) revealed that Wnt pathway-related genes significantly enriched in SCC. IHC staining showed gradually increased immunoreactivity score of Wnt3a and CBP and notable translocation of ß-catenin from the membrane to the cytoplasm and nucleus during the lesion progression. The intensity and proportion of P16, Ki67 and CK17 staining also increased with the progression of cervical lesions, whereas minimal to negative Involucrin expression was observed in CIN2/3 and SCC. Therefore, canonical Wnt signaling may contribute to the progression of CIN to SCC and may be a potential therapeutic target.

WAPL induces cervical intraepithelial neoplasia modulated with estrogen signaling without HPV E6/E7.

Since cervical cancer still afflicts women around the world, it is necessary to understand the underlying mechanism of cervical cancer development. Infection with HPV is essential for the development of cervical intraepithelial neoplasia (CIN). In addition, estrogen receptor signaling is implicated in the development of cervical cancer. Previously, we have isolated human wings apart-like (WAPL), which is expected to cause chromosomal instability in the process of HPV-infected precancerous lesions to cervical cancer. However, the role of WAPL in the development of CIN is still unknown. In this study, in order to elucidate the role of WAPL in the early lesion, we established WAPL overexpressing mice (WAPL Tg mice) and HPV E6/E7 knock-in (KI) mice. WAPL Tg mice developed CIN lesion without HPV E6/E7. Interestingly, in WAPL Tg mice estrogen receptor 1 (ESR1) showed reduction as compared with the wild type, but cell growth factors MYC and Cyclin D1 controlled by ESR1 expressed at high levels. These results suggested that WAPL facilitates sensitivity of ESR1 mediated by some kind of molecule, and as a result, affects the expression of MYC and Cyclin D1 in cervical cancer cells. To detect such molecules, we performed microarray analysis of the uterine cervix in WAPL Tg mice, and focused MACROD1, a co-activator of ESR1. MACROD1 expression was increased in WAPL Tg mice compared with the wild type. In addition, knockdown of WAPL induced the downregulation of MACROD1, MYC, and Cyclin D1 but not ESR1 expression. Furthermore, ESR1 sensitivity assay showed lower activity in WAPL or MACROD1 downregulated cells than control cells. These data suggested that WAPL increases ESR1 sensitivity by activating MACROD1, and induces the expression of MYC and Cyclin D1. Therefore, we concluded that WAPL not only induces chromosomal instability in cervical cancer tumorigenesis, but also plays a key role in activating estrogen receptor signaling in early tumorigenesis.

Comparison of Cervical Levels of Interleukins-6 and -8 in Patients with and without Cervical Intraepithelial Neoplasia.

INTRODUCTION: Interleukins-6 and -8 are two pro-inflammatory cytokines increasing in serum and local levels under malignant conditions. There are limited evidences on the association between cervical level of these two factors and cervical intraepithelial neoplasia (CIN). So, this study aimed to explore the association between cervical levels of IL-6 and IL-8 with cervical premalignant lesions. METHODS: The present case-control study was conducted on married women undergone Pap smear for routine screening in two groups as the group with CIN (n=100) and the healthy control group (n=100). Cervical secretions were collected using sterile swab and the levels of IL-8 and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). The obtained data were analyzed by SPSS software. RESULTS: The mean cervical IL-6 level was 568.66±594.62 pg/ml in the patients with CIN and 212.7±213.9 pg/ml in the controls (P <0.001). The cervical IL-8 levels in the case and control groups were measured to be 1320.43±876.5 pg/ml and 1053.59±747.64 pg/ml, respectively (p=0.02). By modifying the confounding size effect of the age and marital duration, it was determined that cervical levels of IL-6 and IL-8 were both associated with CIN. CONCLUSION: Our results showed that the cervical levels of IL-6 and IL-8 are associated with CIN independent of age and marital dura.

Classification of high-grade cervical intraepithelial neoplasia by p16ink4a , Ki-67, HPV E4 and FAM19A4/miR124-2 methylation status demonstrates considerable heterogeneity with potential consequences for management.

High-grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a , Ki-67 and host-cell DNA methylation) could provide guidance for clinical management in women with high-grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0-3) and Ki-67 (Scores 0-3), referred to as the "immunoscore" (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124-2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0-2 (6.0%), 151 lesions within IS group 3-4 (30.4%) and 316 lesions within IS group 5-6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). Methylation positivity increased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5-6 (38/316). Notably, in a minority (43/497, 8.7%) of high-grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high-grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a "wait and see" policy to reduce overtreatment of high-grade CIN lesions.

TOP2A/MCM2, p16INK4a, and cyclin E1 expression in liquid-based cytology: a biomarkers panel for progression risk of cervical premalignant lesions.

BACKGROUND: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. METHODS: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. RESULTS: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). CONCLUSIONS: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.

Expression profiles of metallothionein-I/II and megalin/LRP-2 in uterine cervical squamous lesions.

Metallothioneins (MTs) are phylogenetically old cysteine-rich proteins, which are implicated in a variety of physiological and pathological processes. Their growth-regulating, anti-apoptotic and anti-inflammatory functions have been attributed not only to intracellular free radical scavenging and to zinc and copper regulation but also to the ability of secreted MT to bind on surface lipoprotein receptor-megalin/LRP2, which enables the endocytosis of MT-I/II and a wide range of other functionally distinct ligands. In the present study, we analysed the expression pattern of both proteins in 55 cases of premalignant transformation of cervical squamous cells, i.e. in low- and high-grade squamous intraepithelial lesion (LSIL and HSIL). The data showed that in LSIL (cervical intraepithelial neoplasia CIN1; N = 25) MTs were present only in basal and parabasal cells and that megalin was only weakly expressed. In HSIL (CIN2; N = 15 and CIN 3/carcinoma in situ; N = 15), however, overexpression and co-localization of MT with megalin were found in the entire hyperplastic epithelium. Moreover, megalin immunoreactivity appeared on the glandular epithelium and vascular endothelium, as well as on lymphatic cells in stroma. Besides, multiple megalin-positive cells expressed phosphorylated Akt1, implying that MT- and/or megalin-dependent prosurvival signal transduction pathways might contribute to the development of severe cervical dysplasia. The data emphasize the diagnostic power of combined MT/megalin analysis in pre-cancer screening.

Dual staining for p16/Ki-67 to detect high-grade cervical lesions: Results from the Screening Triage Ascertaining Intraepithelial Neoplasia by Immunostain Testing study.

We compared clinical performance of p16/Ki-67 dual-stained cytology and human papillomavirus (HPV) genotyping, via different algorithms-alone, or in combination with cytology-to identify cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) in women referred to as colposcopy. We included 492 cervical specimens (134 normal, 130 CIN1, 99 CIN2, 121 CIN3, 8 cancers) randomly selected from 1158 specimens with valid conventional cytology, HPV (cobas 4800 HPV test) and biopsy results. Dual-stained cytology was retrospectively performed (CINtec PLUS assay) on PreservCyt material; slides were read by a cytologist and confirmed by two pathologists, blinded to cytology, biopsy and genotyping results. Sensitivity and specificity (95% confidence intervals in parentheses) of dual-stained cytology to detect CIN2+ and CIN3+ were compared to other screening tests available for the same women. Positivity rate for dual-stained cytology increased with histological severity: 30.6% in normal, 41.5% in CIN1, 72.7% in CIN2, 86.8% in CIN3 and 87.5% in cancer. Dual-stained cytology alone had lower sensitivity than HPV testing for CIN2+ [80.7% (75.0-85.6) vs 89.9% (85.3-93.5)] and CIN3+ [86.8% (79.7-92.1) vs 92.3% (86.2-96.2)]. However, corresponding specificity values were higher [64.0% (57.9-69.8) vs 56.1% (49.8-62.1) for CIN2+; 54.0% (48.7-59.2) vs 44.4% (39.2-49.6) for CIN3+]. Combining dual-stained cytology with an ASC-US abnormality threshold decreased specificity to 31.4% (25.9-37.4) for CIN2+ and 24.2% (19.9-29.0) for CIN3+. The corresponding values considering low squamous intraepithelial lesion threshold values were 42.8% (36.8-49.0) and 35.0% (30.1-40.1). Dual-stained cytology and HPV testing exhibited similar performance, although the former improved the specificity by 7.9% and 9.6% for CIN2+ and CIN3+, respectively.

Determinants of p16/Ki-67 adequacy and positivity in HPV-positive women from a screening population.

BACKGROUND: The objective of this study was to describe the determinants of adequacy and positivity of the p16/Ki-67 assay in a human papillomavirus (HPV)-positive screening population enrolled within the New Technologies for Cervical Cancer 2 (NTCC2) study. METHODS: ThinPrep slides were immunostained for p16/Ki-67; each slide had 3 reports from different laboratories. The authors included population-related, sampling-related/staining-related, and interpretation-related variables in the analyses. Adequacy and positivity proportions were stratified by variables of interest. Univariate and multivariate logistic models were used to identify determinants of adequacy and positivity. RESULTS: In total, 3100 consecutive HPV-positive cases were analyzed. Because every slide was interpreted by 3 centers, 9300 reports were obtained, including 905 (9.7%) that were inadequate and 2632 (28.3%) that were positive. The percentage of cases in which all 3 reports were inadequate increased with increasing age of the women and with inadequate cytology. The highest percentage of adequacy in all 3 reports and of cases with all 3 reports positive was observed in specimens from women who had grade ≥2 cervical intraepithelial neoplasia (CIN2+), atypical squamous cells of undetermined significance or more severe (ASC-US+) cytology, or mRNA positivity. The number of inadequate reports was significantly associated with increasing age, inadequate cytology, mRNA negativity, and scant cellularity. A positive p16/Ki-67 report was associated with an ASC-US+ result and with a positive mRNA result in cases both with and without CIN2+ but was associated with an HPV type 16 and/or 18 infection only in CIN2+ cases. The presence of CIN2+ was strongly associated with dual staining positivity. CONCLUSIONS: The interpretation of p16/Ki-67 results may be influenced by several different variables, all of which are part of the steps in the procedure, and by the characteristics of the screened population.