HPV-positive Cervical Intraepithelial Neoplasia Research Articles

Abstract 690: Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses

Abstract Many cancer immunotherapies require the strong activation of the T-cell mediated adaptive immune response to elicit their full anti-tumor potential. Antigen processing and presentation is a complex, multistep process that can be significantly enhanced via antigen modification, leading to better antigen-specific immune responses. Using mRNA, we can express exogenous antigens inside the cell and facilitate the subcellular localization of these antigens to enhance antigen processing and presentation. In this work, we introduced endolysosomal trafficking domains from CD1d molecules to drive subcellular localization of antigenic proteins to the late endosomes (LE) to enhance class I and class II antigen loading. CD1d are molecules that naturally traffic between the membrane and the late endosomes, where antigens are processed and loaded to both class II and class I molecules in a TAP-independent manner. We compared the immunogenicity of a novel endolysosomal trafficking domain, human CD1d, coupled to a chimeric HPV16 E6 and E7 (HPV16E6E7-hCD1d) antigen to antigen designs intended to be either a) secreted (Sec-HPV16E6E7), b) retained in the endoplasmic reticulum (ER) by an ER retention signal KDEL (HPV16E6E7-KDEL) or c) unmodified antigen sequence (HPV16E6E7). To ensure intracellular translation and expression, all antigens were encoded and delivered as mRNAs. The immunogenicity of each antigen design was assessed in HPV16 positive cervical intraepithelial neoplasia (CIN) donor derived PBMCs and healthy controls. Using confocal microscopy, we observed that the hCD1d trafficking domain directed the translated E6E7 protein into late endosomes, which was not observed for any of the other antigen designs. Importantly, antigen localization to late endosomes resulted in significantly improved immunogenicity in vitro as well as in vivo. We observed a consistent increase in numbers of activated antigen-specific T cells across multiple CIN donor PBMCs treated with HPV16E6E7-hCD1d compared to the other 3 designs, as measured by Interferon gamma secretion, surface marker expression, and T-cell proliferation. To assess the generalizability of this approach, we also compared immunogenicity in human donor PBMCs using the human cytomegalovirus antigen pp65, and observed similar results. Lastly, the immunogenicity of each antigen design was tested in vivo by intramuscular injection of nanoparticle formulated mRNAs in C56/Bl6 mice, and inclusion of the murine analogue (mCD1d) resulted in significantly increased generation of HPV16-specific T cell responses. Based on these findings, we are developing a novel mRNA-based cancer therapeutic that includes the addition of hCD1d trafficking domain in the HPV16-specific antigen component. Citation Format: Weiqun Liu, Daniel Fernandez, Colin J. McKinlay, Daniel O. Frimannsson, Meredith L. Leong, W. Martin Kast, Samuel Deutsch, Ole Audun W. Haabeth. Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 690.

Vitamin D Receptor Gene Polymorphisms and the Risk of CIN2+ in Shanxi Population.

Cervical cancer is one of the most common malignancies in women with high morbidity and mortality. Human papillomavirus (HPV) infection is the primary cause of cervical cancer, of which HPV 16 is the predominant. Early detection and effective treatment of cervical precancerous lesions are the key to preventing cervical cancer. Vitamin D receptor (VDR) gene polymorphism is considered to be an important cause of cancer development. Here, we studied the association of VDR polymorphisms (FOKI, BsmI, ApaI, and TaqI) in HPV16-positive cervical intraepithelial neoplasia (CIN)2+ patients. HPV16-positive patients who visited the Colposcopy Clinic of Obstetrics and Gynecology, the Second Hospital of Shanxi Medical University for biopsy due to abnormal HPV and/or Thinprep cytologic test (TCT) from September 1, 2020 to October 1, 2021 were grouped by pathological results. The fasting blood samples were collected and VDR polymorphisms were detected using TaqMan fluorescent probes, and the three sites of BsmI-ApaI-TaqI were subjected to haplotype analysis. FOKI ff genotype (OR = 2.01; 95% CI = 1.12 - 3.59; p = 0.019) and f allele (OR = 1.48; 95% CI = 1.10 - 1.98; p = 0.009) were found to be associated with the risk of CIN2+. TaqI Tt genotype (OR = 2.03; 95% CI = 1.20 - 3.43; p = 0.008), tt genotype (OR = 2.09; 95% CI = 1.09 - 4.02; p = 0.028), and t allele (OR = 1.35; 95% CI = 1.01 - 1.80; p = 0.041) were associated with the risk of CIN2+. No haplotype was associated with CIN2+ risk. According to the results, FOKI and TaqI polymorphisms are associated with CIN2+ risk.

The Impact of Human Papilloma Viruses, Matrix Metallo-Proteinases and HIV Protease Inhibitors on the Onset and Progression of Uterine Cervix Epithelial Tumors: A Review of Preclinical and Clinical Studies.

Infection of uterine cervix epithelial cells by the Human Papilloma Viruses (HPV) is associated with the development of dysplastic/hyperplastic lesions, termed cervical intraepithelial neoplasia (CIN). CIN lesions may regress, persist or progress to invasive cervical carcinoma (CC), a leading cause of death worldwide. CIN is particularly frequent and aggressive in women infected by both HPV and the Human Immunodeficiency Virus (HIV), as compared to the general female population. In these individuals, however, therapeutic regimens employing HIV protease inhibitors (HIV-PI) have reduced CIN incidence and/or clinical progression, shedding light on the mechanism(s) of its development. This article reviews published work concerning: (i) the role of HPV proteins (including HPV-E5, E6 and E7) and of matrix-metalloproteinases (MMPs) in CIN evolution into invasive CC; and (ii) the effect of HIV-PI on events leading to CIN progression such as basement membrane and extracellular matrix invasion by HPV-positive CIN cells and the formation of new blood vessels. Results from the reviewed literature indicate that CIN clinical progression can be monitored by evaluating the expression of MMPs and HPV proteins and they suggest the use of HIV-PI or their derivatives for the block of CIN evolution into CC in both HIV-infected and uninfected women.

The Transcription Factors TBX2 and TBX3 Interact with Human Papillomavirus 16 (HPV16) L2 and Repress the Long Control Region of HPVs

The minor capsid protein L2 of human papillomaviruses (HPVs) has multiple functions during the viral life cycle. Although L2 is required for effective invasion and morphogenesis, only a few cellular interaction partners are known so far. Using yeast two-hybrid screening, we identified the transcription factor TBX2 as a novel interaction partner of HPV type 16 (HPV16) L2. Coimmunoprecipitations and immunofluorescence analyses confirmed the L2-TBX2 interaction and revealed that L2 also interacts with TBX3, another member of the T-box family. Transcription of the early genes during HPV infection is under the control of an upstream enhancer and early promoter region, the long control region (LCR). In promoter-reporter gene assays, we observed that TBX2 and TBX3 repress transcription from the LCR and that this effect is enhanced by L2. Repression of the HPV LCR by TBX2/3 seems to be a conserved mechanism, as it was also observed with the LCRs of different HPV types. Finally, interaction of TBX2 with the LCR was detected by chromatin immunoprecipitation, and we found a strong colocalization of L2 and TBX2 in HPV16-positive cervical intraepithelial neoplasia (CIN) I-II tissue sections. These results suggest that TBX2/3 might play a role in the regulation of HPV gene expression during the viral life cycle.

Treatment of Cervical Intraepithelial Neoplasia With Topical Imiquimod

Cervical intraepithelial neoplasia (CIN) is a common disease among women of reproductive age. High-grade CIN lesions (CIN 2–3) are precancerous and can progress to cervical cancer. The most important risk factor for the development of CIN and progression to cervical cancer is persistent high-risk human papillomavirus (HPV) infection. The preferred treatment in most countries is surgical excision using conization. There is high need for a medical treatment alternative to surgical therapy for CIN 2–3 largely because of the risk of preterm birth and other long-term sequelae of cervical conization in future pregnancies. No medical therapy has been established in clinical practice for patients with CIN 2–3. In 2008, a study reported on the efficacy of imiquimod, a topical immune response modulator, against HPV-related vulvar intraepithelial neoplasia. Earlier this year, a randomized controlled trial found that combined use of imiquimod and conization did not improve CIN 2–3 recurrence rates compared with use of conization alone. However, the study had a number of design flaws. A major problem was the application of imiquimod only 5 times before surgery, an uncommon and short treatment regimen. The aim of the present randomized, double-blind, placebo-controlled phase 2 trial was to estimate the therapeutic efficacy of vaginal, self-applied imiquimod in women with high-risk HPV-positive CIN 2–3. Study subjects were 59 patients with untreated, histologically proven, high-risk, HPV-positive, and newly diagnosed CIN 2–3. Eligible women were randomized to receive either treatment with self-applied vaginal suppositories containing imiquimod or placebo for 16 weeks. The primary study outcome was efficacy, defined as histologic regression to CIN 1 or less following treatment. Secondary outcomes included complete histologic remission, HPV clearance, and treatment tolerability. A sample size of 24 patients per group was calculated to detect a 35% absolute increase in CIN 2–3 regression (imiquimod vs. placebo), assuming a 2-sided 5% significance level and a power of 80%. All patients tested positive for high-risk HPV at baseline. After 16 weeks of treatment, histologic regression to CIN 1 or less was achieved in 73% in the imiquimod group compared with 39% in the placebo group (P = 0.009). There was a higher complete histologic remission in the imiquimod group (47%) than in the placebo group (14%) (P = 0.008). Human papillomavirus clearance rates after treatment were 60% in the imiquimod group and 14% in the placebo group (P < 0.001). Complete remission rates among patients with HPV-16 infection were 47% in the imiquimod group and 0% in the placebo group (P = 0.003). Three of the 59 patients (5%), all within the placebo group, were found to have microinvasive cervical cancer after treatment. Topical imiquimod had no high-grade adverse effects; treatment was well tolerated. These findings suggest that vaginal imiquimod therapy is an efficacious, feasible, and well-tolerated medical treatment for patients with CIN 2–3.

Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial

Human papillomavirus (HPV) testing is more sensitive for the detection of high-grade cervical lesions than is cytology, but detection of HPV by DNA screening in two screening rounds 5 years apart has not been assessed. The aim of this study was to assess whether HPV DNA testing in the first screen decreases detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse, CIN grade 2 or worse, and cervical cancer in the second screening. In this randomised trial, women aged 29-56 years participating in the cervical screening programme in the Netherlands were randomly assigned to receive HPV DNA (GP5+/6+-PCR method) and cytology co-testing or cytology testing alone, from January, 1999, to September, 2002. Randomisation (in a 1:1 ratio) was done with computer-generated random numbers after the cervical specimen had been taken. At the second screening 5 years later, HPV DNA and cytology co-testing was done in both groups; researchers were masked to the patient's assignment. The primary endpoint was the number of CIN grade 3 or worse detected. Analysis was done by intention to screen. The trial is now finished and is registered, number ISRCTN20781131. 22,420 women were randomly assigned to the intervention group and 22 518 to the control group; 19 999 in the intervention group and 20,106 in the control group were eligible for analysis at the first screen. At the second screen, 19 579 women in the intervention group and 19,731 in the control group were eligible, of whom 16,750 and 16,743, respectively, attended the second screen. In the second round, CIN grade 3 or worse was less common in the intervention group than in the control group (88 of 19 579 in the intervention group vs 122 of 19,731 in the control group; relative risk 0·73, 95% CI 0·55-0·96; p=0·023). Cervical cancer was also less common in the intervention group than in the control group (four of 19 579 in the intervention group vs 14 of 19,731; 0·29, 0·10-0·87; p=0·031). In the baseline round, detection of CIN grade 3 or worse did not differ significantly between groups (171 of 19 999 vs 150 of 20,106; 1·15, 0·92-1·43; p=0·239) but was significantly more common in women with normal cytology (34 of 19,286 vs 12 of 19,373; 2·85, 1·47-5·49; p=0·001). Furthermore, significantly more cases of CIN grade 2 or worse were detected in the intervention group than in the control group (267 of 19 999 vs 215 of 20,106; 1·25, 1·05-1·50; p=0·015). In the second screen, fewer HPV16-positive CIN grade 3 or worse were detected in the intervention group than in the control group (17 of 9481 vs 35 of 9354; 0·48, 0·27-0·85; p=0·012); detection of non-HPV16-positive CIN grade 3 or worse did not differ between groups (25 of 9481 vs 25 of 9354; 0·99, 0·57-1·72; p=1·00). The cumulative detection of CIN grade 3 or worse and CIN grade 2 or worse did not differ significantly between study arms, neither for the whole study group (CIN grade 3 or worse: 259 of 19 999 vs 272 of 20,106; 0·96, 0·81-1·14, p=0·631; CIN grade 2 or worse: 427 of 19 999 vs 399 of 20,106; 1·08, 0·94-1·24; p=0·292), nor for subgroups of women invited for the first time (CIN grade 3 or worse in women aged 29-33 years: 102 of 3139 vs 105 of 3128; 0·97, 0·74-1·27; CIN grade 2 or worse in women aged 29-33 years: 153 of 3139 vs 151 of 3128; 1·01, 0·81-1·26; CIN grade 3 or worse in women aged 34-56 years: 157 of 16,860 vs 167 of 16 978; 0·95, 0·76-1·18; CIN grade 2 or worse in women aged 34-56 years: 274 of 16,860 vs 248 of 16 978; 1·11, 0·94-1·32). Implementation of HPV DNA testing in cervical screening leads to earlier detection of clinically relevant CIN grade 2 or worse, which when adequately treated, improves protection against CIN grade 3 or worse and cervical cancer. Early detection of high-grade cervical legions caused by HPV16 was a major component of this benefit. Our results lend support to the use of HPV DNA testing for all women aged 29 years and older. Zorg Onderzoek Nederland (Netherlands Organisation for Health Research and Development).

E6 and E7 from Human Papillomavirus Type 16 Cooperate To Target the PDZ Protein Na/H Exchange Regulatory Factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.

Co-Expression of HSV2 and Chlamydia trachomatis in HPV-Positive Cervical Cancer and Cervical Intraepithelial Neoplasia Lesions Is Associated with Aberrations in Key Intracellular Pathways

Objective: Oncogenic human papillomaviruses (HPVs) are the etiological agents of cervical cancer. Different cofactors might be needed for malignant transformation, but they still remain elusive. Methods: To delineate the role of Chlamydia trachomatis (CT) and herpes simplex virus type 2 (HSV2) in HPV-positive cervical intraepithelial neoplasia (CIN) lesions and cervical carcinoma a series of 149 cervical cancer and CIN biopsies were analyzed for CT and HSV2 DNA by PCR, and HPV genotyped by InnoLipa. Monitoring of aberrations in key intracellular pathways due to CT/HSV2 and HPV co-expression were analyzed with 13 biomarkers. Results: Of the 149 samples tested, 136 were HPV DNA positive; 32/136 contained also CT DNA and 29 HSV2 DNA. Detection of CT was significantly (p = 0.0001) related to multiple-type HPV infections, while HSV2 was of borderline significance (p = 0.053). Of the 13 biomarkers tested, cytoplasmic and nuclear NF-ĸB and VEGF-C were significantly increased in CT+/HPV+ lesions; p = 0.023, p = 0.045, and p = 0.020 as well as survivin, p = 0.026. Survivin was the only marker that was overexpressed also in HSV2+/HPV+ lesions, p = 0.027. Conclusions: CT infection favors the entry and persistence of multiple HR-HPV types, which leads to viral integration, inhibition of apoptosis, overexpression of E6/E7 oncogenes and cell transformation.

P16INK4A Immunoexpression and HPV In Situ Hybridization Signal Patterns

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16 immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16 immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P<0.001). p16 immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P<0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16 staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16 staining showed no evidence of viral integration. Both increased p16 immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16 immunostaining in CIN 1 appears independent of HPV punctate signal type.

Human papillomavirus genotyping and p16INK4a expression in cervical intraepithelial neoplasia of adolescents

Adolescents have high rates of human papillomavirus (HPV) infection, and persistent high-risk HPV infection can lead to the development of cervical cancer. The cyclin-dependent kinase inhibitor, p16INK4a is overexpressed in cervical intraepithelial neoplasia (CIN), probably due to a persistent and integrated HPV infection. This study investigated p16INK4a expression, grades of CIN, and high-risk HPV infection in adolescent cervical biopsies. Biopsies were immunohistochemically stained for p16INK4a. The presence of wide-spectrum, low-risk, or high-risk HPV was determined by amplifying DNA extracted from the cervical biopsies. Biopsies were classified as cervicitis, 15 cases; CIN 1, 48 cases; CIN 2, 46 cases, and CIN 3, 52 cases. The distribution of p16INK4a staining was graded as patchy, diffuse basal, and diffuse full thickness. Pearson's χ2 tests analyzed the relationships between p16INK4a staining, HPV infection, and CIN. Biopsies of cervicitis were negative for HPV and for p16INK4a expression. High-risk HPV 16, 18, and 31 increased from 18% in CIN 1 to 66% in CIN 2/3 (P<0.001). In CIN 1, p16INK4a was positive in 44% of biopsies with 35% showing patchy, 7% diffuse basal, and one case (2%) showing diffuse full thickness staining. In CIN 2/3, p16INK4a was positive in 97% of biopsies with 23% showing patchy, 21% diffuse basal, and 53% diffuse full thickness staining. The difference in the proportions of biopsies showing patchy p16INK4a staining in CIN 1 and diffuse full thickness staining in CIN 2/3 was significant (P<0.001). In CIN 1, 61% of high-risk HPV-positive biopsies were p16INK4a negative, while all high-risk HPV-positive CIN 2/3 biopsies were p16INK4a positive. Diffuse, full thickness p16INK4a expression discriminated low-grade from high-grade CIN and appears to be a marker of persistent high-risk HPV infection.

Relationship between the presence of oncogenic HPV DNA assessed by polymerase chain reaction and Ki-67 immunoquantitative features in cervical intraepithelial neoplasia.

The aims of this study were firstly to determine which Ki-67 immunoquantitative parameters correlate with the presence of oncogenic human papillomavirus (HPV) in cervical intraepithelial neoplasia (CIN) lesions; and secondly to compare prospectively the routinely assessed CIN grades with the Ki-67 quantitative pathological CIN grade, the expert revised grade, and the presence of oncogenic HPV DNA. HPV polymerase chain reaction (PCR) and Ki-67 immunoquantitation were performed on 90 consecutive biopsies (16 CIN 1, 35 CIN 2, and 39 CIN 3). CIN grade was assessed routinely by six different pathologists. The presence of the lesion was confirmed in a histological section following the material used for PCR and Ki-67 analysis. In a second prospective routine test set analysis, 66 more CIN lesions (14 CIN 1, 15 CIN 2, and 37 CIN 3) were routinely graded (also by six different pathologists, routine CIN grade=CIN(ROUT)), studied for oncogenic HPV DNA, and graded by quantitative Ki-67 features (quantitative pathological CIN grade=CIN(QP)). These latter cases were blindly revised by one of the authors (reference CIN grade=CIN(REF)). Eight of the nine Ki-67 immunoquantitative features showed a significant difference between the oncogenic HPV-positive and -negative cases. The best single discriminator was the 90th percentile of the stratification index (SI90). All 61 cases with Ki-67 SI90>0.60 were HPV-positive (68% of the total group studied). Of the 29 cases with SI90< or =0.60, 16 were negative and 13 positive for oncogenic HPV and none of the Ki-67 features (either single or combined) could distinguish them. Using stepwise multivariate analysis, the best discriminating combination of features was SI90 and the percentage of Ki-67-positive nuclei in the deep third layer of the epithelium (PERC DL). The combination of SI90 and the percentage of Ki-67-positive nuclei per 100 microm basal membrane was nearly as strong as that of SI90 and PERC DL. With these two features, 86% of the cases were correctly classified. The subjective estimate of SI90 (>0.60 or < or =0.60) by two independent observers was not accurate and not reproducible. In the prospective routine test set analysis of 66 cases, the 37 CIN(ROUT)=3 all had CIN(QP) and CIN(REF)=3 and all these cases were oncogenic HPV-positive. Eight of the 14 original CIN(ROUT)=1 grades were oncogenic HPV (=HPV)-positive and five of these eight were upgraded by CIN(QP) to CIN 2 and CIN 3. These upgrades were in agreement with the blind reference revisions. The six HPV-negative CIN(ROUT)=1 cases were CIN 1 both by CIN(QP) and by CIN(REF). Thirteen of the 15 original CIN(ROUT)=2 grades were HPV-positive and seven of these were CIN(QP)=3. All six HPV-positive CIN(ROUT)=2 cases that were CIN(QP)=2 were also CIN(REF)=2 at blind revision. In conclusion, this study has shown firstly, that in CIN lesions, Ki-67 immunoquantitative features and the presence of oncogenic HPV are highly correlated, and also within one subjective CIN grade; secondly, that subjective impressions of SI90 are not as accurate or reproducible as quantitative image analysis results; and thirdly, that the routine application of QP CIN-grading gives results that are in very good agreement with CIN grades assessed by an expert. Thus, routine QP-grading may be used to correct the subjective grade assessed by non-expert pathologists.

Decreased synthesis and expression of TGF-beta1, beta2, and beta3 in epithelium of HPV 16-positive cervical precancer: a study by microdissection, quantitative RT-PCR, and immunocytochemistry.

Cervical carcinogenesis is a multistep process initiated by 'high-risk' human papillomaviruses (HR-HPVs), most commonly HPV 16. Transforming growth factor-beta (TGF-beta) inhibits epithelial proliferation and down-regulates transcription of E6/E7 genes of HPV. Altered TGF-beta expression may be important in carcinogenesis. Quantitative RT-PCR was used to investigate TGF-beta1, beta2, and beta3 mRNA levels in nine specimens of normal cervix and 15 cervical precancers (eight HPV-positive, including five HPV 16-positive). Immunocytochemical expression of TGF-beta1, beta2, and beta3 was examined in cervical intraepithelial neoplasia (CIN) positive for HPV 16 (26), and in HPV-negative, normal ectocervical epithelium (9); reserve cell hyperplasia (12); and immature (7) and mature (15) squamous metaplasia. The intensity of staining for TGF-beta1 was measured using grey-scale image analysis. Microdissection was used to investigate epithelial and stromal (excluding crypts) levels of TGF-beta1 mRNA in HPV 16-positive cervical precancer. Normal cervix, including reserve cells and immature and mature metaplasia, showed strong immunocytochemical expression of all TGF-beta isoforms. Expression was decreased in the basal third of the epithelium in CIN 1, in the basal and middle thirds in CIN 2, and in all layers in CIN 3. Quantitative analysis of TGF-beta1 expression showed that the changes in CIN compared with normal ectocervix and mature metaplasia were statistically highly significant (p<0.001, ANOVA). TGF-beta1, beta2, and beta3 mRNA levels showed a significant decrease only in the five HPV 16-positive CIN samples when compared with normal (p=0. 0034, 0.0033, and 0.029, respectively). TGF-beta mRNA levels in HPV 16-positive epithelium also decreased from normal through low-grade to high-grade precancer. Stromal TGF-beta1 was absent or very low compared with epithelial production and was not altered in HPV 16 precancer. Progressive loss of epithelial TGF-beta expression and synthesis may be important in HPV 16-associated human cervical carcinogenesis.

Balance of IgG Subclasses toward Human Papillomavirus Type 16 (HPV16) L1-Capsids Is a Possible Predictor for the Regression of HPV16-Positive Cervical Intraepithelial Neoplasia

Human papillomavirus type 16 (HPV16) is known to be a major causative agent of cervical cancer. To test the hypothesis that an enhanced Th1 response favors the natural course of cervical intraepithelial neoplasia (CIN), we measured IgG subclasses toward HPV16 L1-capsids because IgG1/IgG2 balance reflects Th2 and Th1 responses, respectively. We examined IgG2/IgG1 ratios in sera from 67 anti-HPV16 L1-positive women; 18 were cytologically normal women, 29 were CIN patients, and 20 were cervical cancer patients. The IgG2 dominance (IgG2/IgG1 ratio >1) was observed in 94, 48, and 5%, respectively (p< 0.001). The regression rate of CIN lesions was significantly different between patients with and without IgG2 dominance: 83.3% (5/6) versus 16.7% (1/6), respectively (p< 0.05). These findings raise the possibility that IgG2 dominance toward HPV16 L1-capsids, i.e., Th1 dominance, may be a useful marker to predict viral clearance or the regression of HPV16-positive CIN.

Combination therapy with podophyllin and vidarabine for human papillomavirus positive cervical intraepithelial neoplasia.

Our aim was to establish an effective non-surgical treatment for cervical intraepithelial neoplasia (CIN) through inactivation of human papillomavirus (HPV), the major etiological agent for this disease. We show that vidarabine, a DNA polymerase inhibitor, suppressed growth and HPV gene expression in human cervical keratinocytes immortalized by HPV or in cervical cancer cell lines. Expression of HPV-16 E6 and E7 proteins in normal cervical keratinocytes sensitized cells to apoptosis in the presence of podophyllin or vidarabine. We applied vidarabine ointment and/or podophyllin to cervical epithelium in 28 cases of CIN I-II to evaluate the therapeutic effectiveness of these agents. Co-application of vidarabine and podophyllin in six treatments caused regression of lesions cytologically and histologically, and disappearance of HPV-16 or -18 DNA in 17 of 21 (81%) women. Our results suggest that the combination of vidarabine and podophyllin therapy is an effective non-surgical treatment for HPV-positive CIN.

Susceptibility to human papillomavirus-associated cervical intra-epithelial neoplasia is determined by specific HLA DR-DQ alleles.

Human papillomaviruses (HPV) play a causative role in the aetiology of cervical intra-epithelial neoplasia (CIN) and cervical cancer. Human leukocyte antigens (HLA) are important in the regulation of immune response to foreign antigens. The role of genetic variation at the HLA class II loci (DR and DQ) in CIN (HPV 16, 18, 31 and 33 typed) was investigated by PCR DNA amplification and oligonucleotide probe typing of cervical smears from British Caucasian patients (n = 176) and controls (n = 416). The alleles of the DQB1*03, DRB1*04 and DRB1*11 loci were strongly associated with susceptibility to CIN. Specifically, the haplotypes DRB1*0401-DQB1*0301 and DRB1*1101-DQB1*0301 were significant and indicated susceptibility. The DQB1*03 locus was more contributory to this association than the DRB1 loci. A weak protective effect was shown for the haplotype DRB1*0101-DQB1*0501. Positive correlation was also observed for HPV-positive CIN, suggesting that specific HLA class II alleles may be important in determining the immune response to HPV antigens and the risk for CIN after HPV infection. Our results should help in the rational design of vaccines against HPV.