HPV-associated Cervical Intraepithelial Neoplasia Research Articles

Abstract 3297: A cell of origin for cervical cancer

Abstract Background Squamous cell carcinoma of the cervix and its precursor [cervical intraepithelial neoplasia (CIN)] are thought to develop either within, or in close proximity to, the squamo-columnar (SC) junction of the ecto and endocervix. Despite this assumption, one issue unresolved in cervical cancer research is the identity of the normal cervical cell type in the SC junction in which tumorigenesis begins. Methods Using GeneChip Human Exon Arrays (Affymetrix), we compared the gene expression profiles of the ecto and endocervical epithelia with the SC junctional cells. Fetal and adult cervical specimens were studied in situ to identify, characterize and determine the dynamics of junctional cell development. CINs and cancers were stained with junction-specific antibodies to ascertain their relationship to the SC junctional cell population. Cervices in which the transformation zone had been excised were analyzed for evidence of the SC junctional immunophenotype. Results Human adult cervices displayed a unique monolayer of cuboidal cells at the SC junction that displayed a unique 77 gene expression signature. Analysis of fetal and postnatal mouse and human cervices using biomarkers from this unique gene signature revealed broad expression in the lining cells of the lower genital tract that, over time, become concentrated in the region of the SC junction. Junction-specific biomarkers consistently immunostained high-grade CINs and squamous and glandular cancers associated with both carcinogenic human papillomaviruses (HPV) and strong expression of p16ink4. Non-carcinogenic HPV-associated CINs and low-grade CINs predominated in the ectocervical location and were usually SC junctional-marker negative. Cervices previously subjected to surgical excision of the SC junction did not remanufacture junctional cells at the new SC junction. Conclusions This study shows, for the first time, that a discrete, residual embryonic cell population in the cervix is uniquely susceptible to carcinogenic HPV infection and is lost following surgical excision of the SC junction. The negative association between the junctional immunophenotype and noncarcinogenic HPV-related CIN may signify different affinities for the SC junction between carcinogenic and non-carcinogenic HPVs. Because junction-specific biomarkers highlight CINs and all cancers with carcinogenic HPVs, they may facilitate interpretation of cervical cancer precursors as well as propose a novel cell type for studies of experimental papillomaviral carcinogenesis. The possibility that unique populations of junctional cells are also involved in the pathogenesis of other HPV-related cancers (anogenital and oropharyngeal) merits investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3297. doi:1538-7445.AM2012-3297

Intranasal immunization with synthetic peptides corresponding to the E6 and E7 oncoproteins of human papillomavirus type 16 induces systemic and mucosal cellular immune responses and tumor protection

The E6 and E7 oncoproteins of the high-risk HPV type16 represent ideal targets for HPV vaccine development, they being consistently expressed in cervical cancer lesions. Since HPV-16 is primarily transmitted through genital mucosal route, mucosal immune responses constitute an essential feature for vaccination strategies against HPV-associated lesions. We present here evidence showing that mucosal immunization of mice by the intranasal route with a mixture of peptides E7 44–62 and E6 43–57 from the E7 and E6 oncoproteins of HPV-16, respectively, using a mutant cholera toxin adjuvant (CT-2 *), primed strong antigen-specific cellular immune responses in systemic and mucosal tissues. Significant levels of IFN-γ production by both CD4 and CD8 cells were observed along with CTL responses that were effective against both peptide-pulsed targets as well as syngeneic tumor cells (TC-1) expressing the cognate E6 and E7 proteins. Furthermore, mice immunized with the peptide mixture and CT-2 * effectively resisted TC-1 tumor challenge. These results together with our earlier observations that T cell responses to these peptides correlate with recurrence-free survival in women after ablative treatment for HPV-associated cervical intraepithelial neoplasia, support the potential of these E6 and E7 peptides for inclusion in vaccine formulations.

Serologic response to human papillomavirus 16 among Australian women with high-grade cervical intraepithelial neoplasia

This study evaluated the detection of human papillomavirus (HPV) 16 antibody in HPV 16-associated cervical intraepithelial neoplasia (CIN) in Australian women. Seroreactivity to HPV 16 L1 virus-like particles was assessed in patients with CIN 2 (n= 169) and CIN 3 (n= 229) lesions previously tested for the presence of HPV DNA. Seropositivity was significantly commoner in women with HPV 16 DNA-positive lesions (98/184) than in women with no HPV DNA in the lesion (15/47) or with HPV of types other than 16 in the lesion (43/167) (P= 0.0004). In addition, seropositivity was observed in 33% (55/169) of women with CIN 2 and 46% (106/229) of women with CIN 3, in keeping with the lower fraction of CIN 2 (57/169) than CIN 3 (127/229) biopsies positive for HPV 16 DNA. HPV 16 seropositivity is most common in women with HPV 16-associated CIN, but many patients with HPV-associated CIN 3 are seronegative, and HPV 16 seropositivity is common in women with CIN associated with other HPV types. Overall, HPV 16 serology is a poor predictor of presence of HPV 16-associated CIN 3 in patient population studied.

Correlation of human papillomavirus and adeno-associated virus 2 infection in cytology with Korean women

The purpose of our prospective study was to investigate the prevalence of adeno-associated virus (AAV) and human papillomavirus (HPV) 16 and/or HPV 18 infection in Korean women with normal cervical smears and those with HPV-associated cervical intraepithelial neoplasia (CIN) and cancer in cytobrush samples, and to evaluate the correlation between AAV 2 and HPV 16 and/or HPV 18 infection. AAV 2 was detected in CIN I (9.7%), CIN II (20%), CIN III (22.8%), and cancer (10%). HPV 16 was detected in CIN I (42%), CIN II (55%), CIN III (54.3%), and cancer (70%). HPV 18 was detected in CIN I (51.6%), CIN II (50%), CIN III (62.8%), and cancer (43.3%). HPV 16 or HPV 18 was detected in CIN I (18.3%), CIN II (80%), CIN III (71.4%), and cancer (80%). In normal and HPV-infected group, AAV 2 DNA was detected in 16.3% and 4.4% of samples, respectively. HPV 16 was detected in 10.2% of normal patients and in 44.4% of HPV-infected patients, and HPV 18 was detected in 12.2% of normal patients and in 40% of HPV-infected patients. HPV 16 or HPV 18 was detected in 18.3% of normal patients and in 57.7% of HPV infection. The correlation between AAV 2 and HPV 16 was statistically significant in normal and CIN I/II group only, and AAV 2 and HPV 16 and/or HPV 18 showed no correlation. Therefore, the correlation between AAV and HPV were not statistically significant. These data support the previous reports that AAV might not be associated with cervical tumorigenesis.

Serologic response to human papillomavirus 16 among Australian women with high-grade cervical intraepithelial neoplasia

This study evaluated the detection of human papillomavirus (HPV) 16 antibody in HPV 16–associated cervical intraepithelial neoplasia (CIN) in Australian women. Seroreactivity to HPV 16 L1 virus–like particles was assessed in patients with CIN 2 (n = 169) and CIN 3 (n = 229) lesions previously tested for the presence of HPV DNA. Seropositivity was significantly commoner in women with HPV 16 DNA–positive lesions (98/184) than in women with no HPV DNA in the lesion (15/47) or with HPV of types other than 16 in the lesion (43/167) (P = 0.0004). In addition, seropositivity was observed in 33% (55/169) of women with CIN 2 and 46% (106/229) of women with CIN 3, in keeping with the lower fraction of CIN 2 (57/169) than CIN 3 (127/229) biopsies positive for HPV 16 DNA. HPV 16 seropositivity is most common in women with HPV 16–associated CIN, but many patients with HPV-associated CIN 3 are seronegative, and HPV 16 seropositivity is common in women with CIN associated with other HPV types. Overall, HPV 16 serology is a poor predictor of presence of HPV 16–associated CIN 3 in patient population studied.

Monoclonal Expansion with Integration of High-Risk Type Human Papillomaviruses Is an Initial Step for Cervical Carcinogenesis: Association of Clonal Status and Human Papillomavirus Infection with Clinical Outcome in Cervical Intraepithelial Neoplasia

To define the natural history of cervical intraepithelial neoplasia (CIN) as related to clonal status, we evaluated 20 cases of CIN1 and 18 cases of CIN2 that had been clinically followed for 7 to 48 months at Osaka University Hospital. These included 10 cases that progressed, 15 cases that persisted, and 13 cases that regressed. We analyzed the clonal status of each case by analysis of the pattern of X-chromosomal inactivation. Human papillomavirus (HPV) infection was detected by PCR-RFLP analysis. CINs that are monoclonal or infected by high-risk HPVs are more likely to progress or persist than cases that are polyclonal or negative for high-risk HPVs (p = 0.009 for monoclonal vs polyclonal, p = 0.024 for high-risk HPV positive vs negative p = 0.024). Eighteen (90%) of 20 monoclonal, high-risk HPV-associated CINs progressed or persisted, whereas 9 (60%) of 15 polyclonal or high-risk HPV-negative CINs regressed. Therefore, the combination of clonality status and high-risk type HPV infection was significantly correlated with clinical outcome (p = 0.003). The physical status of the HPV genome was evaluated in 17 cases of HPV-16 positive CINs by real-time PCR. Of those, the HPV viral genome was present in both episomal and integrated forms in 14 CINs (84%), and 12 of these cases (86%) were monoclonal in composition. By contrast, all three CINs in which the HPV genome was present in episomal form were polyclonal. In one CIN1 that was polyclonal, HPV-16 was originally present in episomal form but after 24 months, the patient developed a monoclonal CIN3 in which the HPV-16 genome was present in mixed form. These results may imply that HPV viral integration into the host genomic DNA is associated with progression from polyclonal to monoclonal status in CIN. These events may play a fundamental role in the progression from low-grade to higher grade dysplasia of the cervical mucosa.

Evaluation of the Possible Protective Role of Adeno-Associated Virus Type 2 Infection in HPV-Associated Premalignant Disease of the Cervix

Objective. Our objective was to examine the prevalence of adeno-associated virus (AAV) infection in women with normal cervical smears and those with HPV-associated cervical intraepithelial neoplasia (CIN).Methods. HPV typing was performed on DNA from cervical smears of 211 women with CIN (CIN 1 = 83, CIN 3 = 128) and 433 healthy women who had a normal cervical smear. HPV typing was performed on all cases and controls using type-specific oligonucleotide primers (HPV 16, 18, 31, 33). AAV DNA was amplified by nested PCR from the same samples. The amplified DNA were separated on 2% agarose gels, blotted, and hybridized to AAV-2 DNA labeled by random priming with [α-32P]dCTP to confirm specificity of amplification.Results. A total of 131 cases of CIN were positive for one of the HPV types either alone or in combination. HPV 16 was present in 120 (57%) cases, HPV 18 in 15 (7%), HPV 31 in 27 (13%), and HPV 33 in 15 (7%) and there were multiple HPV types detected in 34 (16%) cases. All of the controls were selected to be negative for HPV. A total of 6/433 (1.4%) control cervical smears and 4/211 (1.9%) of CIN (CIN1 = 2; CIN3 = 2) contained AAV DNA. No correlation between AAV and any clinical feature was observed.Conclusions. These results are different from some that have been previously published and suggest that AAV DNA is not frequently present in either normal control cervical samples or cervical intraepithelial neoplasia. This does not support the hypothesis that AAV may be protective against cervical cancer. Further research is necessary to understand the natural history of AAV infection and its role in human disease.

Expansion of the CD8 + CD45RA + CD27 - CD28 - T-Cell Subset in Patients with HPV-Associated Cervical Cancer and its Precursors - Anstieg der CD8 + CD45RA + CD27 - CD 28 - T-Zell-Subpopulation bei Patientinnen mit HPV-assoziiertem Zervixkarzinom und seinen Vorstufen -

Objective: More than 90% of cervical cancers and precursors are caused by infection with high-risk human papillomavirus (HPV). Cellular immune responses associated with HPV may be detectable in peripheral blood. The purpose of the present study was to assess the immune status in patients with HPV-infection and to identify T cell subsets which may serve as a surrogate marker for immune activation induced bei HPV. Methods: Subsets of peripheral blood lymphocytes in women (n = 30) without signs of HPV-infection were compared with those of patients with histologically verified HPV-associated cervical intraepithelial neoplasia (CIN) (n = 22) or invasive cervical carcinomas (n = 16) using four-color flow cytometric analysis. Results: One of the major changes in the T cell repertoire was a significant expansion of T cells characterized by the CD8+ CD45RA+ CD27- CD28- immune phenotype in patients with HPV associated CIN and cancer compared with the control group (p < 0.01 and 0.0001, respectively). This immune phenotype also differed significantly (p < 0.04) between patients with CIN and CC. Conclusion: The significant T cell response in patients with HPV associated CIN and cancer suggests a proliferative expansion of HPV-antigen-experienced cytotoxic T cells. Further studies are necessary to determine the viral specificity and the validity of lymphocyte subset analysis for monitoring immune function in patients with HPV-associated neoplasia.

Cytokine production patterns in cervical intraepithelial neoplasia: association with human papillomavirus infection.

Genital infection with certain strains of human papillomavirus (HPV) is associated with a high risk of malignant transformation, and HPV-associated cervical intraepithelial neoplasia (CIN) can become invasive cancer. Host factors are critical in regulating tumor growth, and cytokines that modulate immunologic control may be of particular importance. The type 1 cytokines interleukin 2 (IL-2) and interferon gamma (IFN gamma) are immunostimulatory and are thus capable of limiting tumor growth. The type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10) are immunoinhibitory and are thus capable of stimulating tumor growth. We analyzed the production of cytokines by peripheral blood mononuclear cells (PBMCs) in women with CIN associated with localized or extensively spread HPV infection. Thirty women diagnosed with CIN and 10 age- and sex-matched healthy control subjects were enrolled in the study conducted at Istituto Nazionale Tumori, Milan, Italy. The following parameters were analyzed: 1) HPV infection of the cervix and other sites of the lower genital tract by colposcopic, cytologic, and histologic examinations; 2) HPV typing; 3) in vitro production of IL-2 by PBMCs in response to stimulation with soluble antigen (influenza [FLU] antigen) or to cell-associated human leukocyte antigen (HLA) alloantigen; and 4) in vitro production of the type 1 cytokines IL-2 and IFN gamma and of the type 2 cytokines IL-4 and IL-10 by PBMCs in response to mitogen stimulation. Statistical significance was determined by nonparametric tests (two-sided). High-grade CIN associated with HPV infection was detected in all case patients, and HPV type 16 or 18 infection was detected in cervical tissue of 21 (70%) of 30 case patients. HPV infection that had spread to other sites of the lower genital tract, thus resulting in more extensive disease, was detected in 16 (53%) of the 30 individuals with CIN, whereas HPV infection was limited to the portio in 14 (47%). IL-2 production by PBMCs in response to stimulation with soluble antigen or HLA alloantigen was reduced in the group with extensive disease compared with that in the group with localized disease or with that in healthy control subjects. In contrast, IL-4 and IL-10 production in response to mitogen stimulation was elevated in the group with extensive disease compared with that in the group with localized disease or with that in healthy control subjects. The highest production of IL-4 and IL-10 was detected in patients with HPV infection that had extended beyond the genital tract. CIN is characterized by different immunologic profiles, in which HPV infection is or is not confined to the portio. Production of cytokines that mainly enhance potentially protective cell-mediated immunity is defective in the women in whom extended HPV infection was observed. A pronounced shift from type 1 to type 2 cytokine production is associated with more extensive HPV infection. These data reinforce the need for detailed analyses of immune dysregulation in CIN patients. They also suggest the potential usefulness of the cytokine assays for determining prognosis or deciding whether cytokine-based therapy is indicated.

Cytokine modulation of immune defenses in cervical cancer.

Human papilloma viruses (HPV) are major factors in the etiology of cervical cancer. Natural killer (NK) lymphocytes, an important defense against viral diseases, are present in most HPV-associated lesions and cervical intraepithelial neoplasia. HPV positive cervical cancer cells and HPV-immortalized human cervical epithelial cells which possess properties similar to cervical dysplasia, however, are resistant to NK but are sensitive to lymphokine-activated killer (LAK) lymphocyte lysis. Sensitivity can be enhanced by treatment of cervical cells with leukoregulin, a cytokine secreted by lymphocytes. Combination treatment with leukoregulin and a chemotherapeutic drug, e.g. cisplatin, further enhances sensitivity of HPV-infected cells to LAK lymphocyte lysis. In contrast, gamma-interferon treatment of cervical cells can result in decreased sensitivity to LAK lysis illustrating the potential balance cytokines can exert in the immunologic control of cervical cancer.

Intralesional Recombinant Alpha 2B Interferon in the Treatment of Human Papillomavirus-associated Cervical Intraepithelial Neoplasia

Twenty-four patients with human papillomavirus (HPV)-associated cervical intraepithelial neoplasia (CIN); of whom 13 had CIN 1, 8 had CIN 2, and 3 had CIN 3; were treated with recombinant alpha 2b interferon (IFN) by intraperilesional injections. The dosage given was 3 x 10(6) international units per day, 3 days per week for 3 weeks. In situ hybridization was carried out for HPV types 6/11 and 16/18. One year after treatment, complete response was observed in 8 (33%) cases, partial regression in 14 (58%) cases, persistence in 2 (8%) cases (in which case traditional surgical therapy was performed), and progression in none of the cases. Adverse effects (asthenia, fever, chills, and headache) were observed in 20 (83%) cases. None of the patients had myelodepression. Intraperilesional treatment of HPV-associated CIN with recombinant alpha 2B IFN does not appear to be a valid substitute for traditional treatment, but it could be considered in certain cases when surgery is not advised.

Genital Human Papilloma Virus Infection in Males. A Clinico-Pathologic Study

From March 1987 to December 1988, 402 male sexual partners of 317 women with human papilloma virus (HPV) infection of the lower genital tract and 85 with HPV-associated cervical and/or vulvar intraepithelial neoplasia (CIN and/or VIN) were submitted to clinical examination and peniscopy. The latter was performed at a 6-15 X magnification after a 3 min exposure to 5% acetic acid solution. Visible lesions were biopsied. Thirty-one patients had clinical evidence of HPV infection in the glans, penile shaft or urethra, and 222 had peniscopic evidence of subclinical aceto-white lesions. Of 31 patients with clinical lesions, 11 showed also aceto-white subclinical lesions. Of 253 peniscopically positive males, 237 were biopsied and 191 of these were histologically ascertained. Three patients had penile intraepithelial neoplasia, one with clinical appearance of a Buschke-Löwenstein tumor. The incidence of HPV infection in male sexual partners of women affected by HPV infection of the lower genital tract associated or not with intraepithelial neoplasia is lower than expected. However, clinically negative males should not be considered disease free; in fact, 12 patients, negative at the first examination, showed histological evidence of HPV infection at subsequent controls. Therefore, follow-up of at least 6 months should be allowed to identify HPV bearing males. The reported low frequency of HPV infection may be due to the fact that the males may harbour the virus in the urethra, prostate or seminal vesicles or penis without any clinical evidence of disease. Although research for HPV-DNA in intraurethral and penile scraping material might be useful for diagnosis, peniscopy with a 5% acetic acid application remains the clinical test for evaluating HPV infection in males. The importance of peniscopy should be viewed with respect to the prevention of infection or reinfection of the female sexual partners, in addition to the specific diagnostic purpose in male patients.

Lokale Immunreaktionen bei CIN und Zervixkarzinom - Eine histologische und immunhistochemische Studie*

50 conisation or hysterectomy specimens with different degrees of cervical intraepithelial neoplasia (CIN), carcinoma in situ (CiS) or invasive carcinoma, as well as controls, were studied by conventional histological methods as well as by immunohistochemistry. In CIN I and II there is a significant reduction of T lymphocytes, macrophages and T accessory cells, whereby CIN III, CiS and invasive carcinoma show a noticeable increase in the number of immunocompetent cells. The depletion of immunocompetent cells in CIN probably represent a local immunodeficiency state which permits progression of HPV-associated CIN, whilst the marked increase of accessory and effector cells in the stroma of high grade CIN and invasive carcinoma does not appear to exert control over the neoplastic cells, due to an ineffective tumour-specific activation.

Natural history of cervical human papillomavirus (HPV) infections based on prospective follow‐up

To assess the natural history of human papillomavirus (HPV) lesions in the uterine cervix, a prospective follow-up of untreated lesions has been conducted since late 1981. The present report summarizes the data on 343 women with cervical HPV lesions currently followed-up for a mean of 18.7 (SD 15.2) months by colposcopy and PAP smears (group B) or by additional punch biopsy (group A). Initially these two groups were classified on the first PAP smears, presenting with HPV-induced cytopathic changes, and either with (group A) or without (group B) concomitant changes suggestive of cervical intraepithelial neoplasia (CIN). The clinical course of the HPV lesions could not be predicted adequately from the findings of the first PAP smears, as evidenced by the higher progression rate (15.4%) in the 214 women initially classified in group B, compared with 11.6% in the 129 women classified in group A. Furthermore, the number progressing to carcinoma in situ requiring conization was equal (seven patients) in the two groups. This necessitated a more flexible approach to follow-up, permitting transfer of patients between groups, which resulted in a final allocation of 261 women to group A, and 82 to group B. To date, 25% of the total of 343 HPV lesions have regressed, 61% have persisted, and 14% have progressed. Of the latter, a total of 14 (4.1%) have been coned due to progression to carcinoma in situ. The rate of regression seems to be inversely related, and progression directly related, to the degree of HPV-associated CIN.(ABSTRACT TRUNCATED AT 250 WORDS)

Human papillomavirus (HPV) type as an important determinant of the natural history of HPV infections in uterine cervix.

The present report summarizes our current observations on the natural history of cervical HPV (Human papillomavirus) infections, based on data from 418 women prospectively followed-up in our clinic for a mean of 20 +/- 15 (M +/- SD) months. On each attendance at the clinic (at 6-month intervals), the patients are subjected to colposcopy accompanied by PAP smears and/or punch biopsy, both being analysed for the cytopathic changes of HPV, and for concomitant CIN (cervical intraepithelial neoplasia). In the biopsies, the expression of HPV structural proteins was assessed using an indirect immunoperoxidase (IP-PAP) technique. HPV typing was accomplished by spot hybridization with the DNA probes for HPV 6, 11, 16 and 18. During the follow-up, 24% of the HPV lesions regressed, 55% remained persistent, and 21% progressed, 10.6% having been coned due to progression into CIS. The clinical progression was significantly associated with the grade of HPV-associated CIN. On DNA hybridization, HPV 6 was found in 8%, HPV 11 in 36%, HPV 16 in 11% and HPV 18 in 8% of the 103 lesions typed for HPV DNA so far. HPV-CIN lesions were more frequently than HPV-NCIN associated with HPV 16 and HPV 18, as was the expression of HPV structural proteins. The progression rate was highest (45.5%) in HPV 16 lesions, followed by that (27.3%) in HPV 18 lesions, as contrasted to 0% and 13.3% for HPV 6 and 11, respectively. The natural history of cervical HPV lesions seems to be identical with that of classical CIN lesions.(ABSTRACT TRUNCATED AT 250 WORDS)