Risk factors for persistent cervical intraepithelial neoplasia grades 1 and 2: managed by watchful waiting.

OBJECTIVE: : This study examines risk factors for persistent cervical intraepithelial neoplasia (CIN) and examines whether human papillomavirus (HPV) testing predicts persistent lesions. MATERIALS AND METHODS: : Women with histologically diagnosed CIN 1 or CIN 2 (n = 206) were followed up every 3 months without treatment. Human papillomavirus genotyping, plasma levels of ascorbic acid, and red blood cell folate levels were obtained. Cervical biopsy at 12 months determined the presence of CIN. Relative risk (RR) was estimated by log-linked binomial regression models. RESULTS: : At 12 months, 70% of CIN 1 versus 54% of CIN 2 lesions spontaneously regressed (p < .001). Levels of folate or ascorbic acid were not associated with persistent CIN at 12 months. Compared with HPV-negative women, those with multiple HPV types (RRs ranged from 1.68 to 2.17 at each follow-up visit) or high-risk types (RRs range = 1.74-2.09) were at increased risk for persistent CIN; women with HPV-16/18 had the highest risk (RRs range = 1.91-2.21). Persistent infection with a high-risk type was also associated with persistent CIN (RRs range = 1.50-2.35). Typing for high-risk HPVs at 6 months only had a sensitivity of 46% in predicting persistence of any lesions at 12 months. CONCLUSIONS: : Spontaneous regression of CIN 1 and 2 occurs frequently within 12 months. Human papillomavirus infection is the major risk factor for persistent CIN. However, HPV testing cannot reliably predict persistence of any lesion.

Human immunodeficiency virus (HIV) antigens and RNA in HIV-seronegative women with cervical intraepithelial neoplasia.

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

Heat shock fusion protein-based immunotherapy for treatment of cervical intraepithelial neoplasia III.

OBJECTIVES: SGN-00101 (HspE7, Nventa, San Diego, CA) is a novel therapeutic vaccine consisting of a fusion protein containing an M. bovis BCG heat shock protein (Hsp65) covalently linked to the entire sequence of HPV 16 E7. This trial was designed to evaluate the efficacy and toxicities of HspE7 in women with CIN III. METHODS: HIV (-) women with biopsy-proven CIN III were eligible. Two cohorts were accrued; one cohort to establish efficacy and a second cohort with a longer follow-up period to improve the precision of the trial to estimate response rates. Each patient underwent 3 monthly subcutaneous vaccinations with 500 microg of HspE7 followed by monthly colposcopic follow-up for 1 month in cohort 1 and an extended observation period (2 months) in cohort 2. All patients then underwent a LEEP or cone biopsy of the cervix. A complete pathologic response (pCR) was defined as no evidence of CIN or CIN I (only HPV changes). A partial response (PR) was defined as colposcopic lesion regression of >50% in size. Cervicovaginal lavage samples were obtained at each visit for HPV typing using MY09/ MY11 HPV PCR. RESULTS: Seventy-two patients were registered and screened, of whom 64 were eligible. Fifty-eight patients completed the trial and were evaluable (31 in cohort 1, 27 in cohort 2). There were no significant epidemiologic or HPV type differences between the 2 cohorts so responses were combined for analysis. Of the 58 evaluable patients, 13 (22.5%) had a pCR; 32 (55%) had a PR and 11 (19%) had stable disease. Two (3.5%) patients in cohort 2 had microinvasive disease and were defined as progressive disease. Thirty-three of 58 (57%) of the patients were infected with HPV 16 prior to vaccination or in subsequent visits. There was no significant difference in regression in women infected with HPV 16 compared to those without HPV 16 infection (88% vs. 70%; p=0.12). Women who had a previous LEEP or ablation for CIN were 2.7 times more likely to have a complete response compared to patients without previous treatment, although the difference was not statistically significant (95% CI for rate ratio: 0.95-6.19, p=0.10). At a cellular level, there was a significant association between local inflammation and response; lower grade of lesional inflammation correlated with a response to HspE7 (p=0.04 using Wilcoxon rank sum test). CONCLUSIONS: HspE7 appeared to demonstrate activity in women with CIN III and met a priori assumptions for efficacy; however, it is unclear whether this response was due to natural regression rather than treatment effect. HspE7, which targets the HPV 16 E7 oncoprotein, had efficacy in patients infected with HPV types other than 16, suggesting cross-reactivity. A larger randomized, controlled trial is needed to better define efficacy and to identify subsets of women most likely to benefit from immunotherapy.

Combined human papillomavirus DNA and human papillomavirus-like particle serologic assay to identify women at risk for high-grade cervical intraepithelial neoplasia.

The objective of this study was to assess the utility of a second generation human papillomavirus (HPV) virus-like particle (VLP)-based ELISA as an adjunct to HPV DNA testing to identify women at risk for high-grade cervical intraepithelial neoplasia (CIN). Participants provided blood, cervical samples and interviewer-obtained questionnaire information. HPV VLPs for types 16, 18, 33, 45 and 52 were produced using a baculovirus expression system. These highly purified VLPs were used in a polymer-based ELISA test. Cases with biopsy-confirmed CIN (CIN I, n = 237; CIN II, n = 56; CIN III, n = 48) and controls (n = 351) with normal Pap smears were tested for HPV DNA by PCR and serologic response to multiple oncogenic HPV VLPs. 258/341 (76%) of cases and 230/351 (65.5%) of control patients had any type of HPV VLP antibody (OR = 1.63, 95% CI 1.16-2.30). More cases were seropositive than controls for each individual HPV type (p < 0.001 for HPV types 16, 18, 33 and 45; p = 0.06 for HPV 52). Reactivity to an increasing number of different HPV type-specific VLPs are associated with high-grade CIN independent of HPV DNA status. HPV VLP assays may be useful as an adjunct to HPV DNA testing in a subset of patients that needs to be defined by further studies.

Vaccine strategies for human papillomavirus-associated cancers.

PURPOSE OF REVIEW: To review novel immunologic strategies for the prevention and treatment of human papillomavirus infection and associated diseases. Both animal model systems and human protocols are discussed. RECENT FINDINGS: Many vaccine platforms for both prevention of human papillomavirus infection and treatment of associated disease have been developed. Virus-like particle constructs containing human papillomavirus capsid proteins have been shown to protect against human papillomavirus infection. Novel peptide or protein constructs containing modified E6 or E7 proteins, novel adjuvants, fusion proteins such as immunoglobulin-G-heavy chain E7, or heat shock proteins have been made as therapeutic modalities. In addition, many new recombinant vaccine vectors such as vaccinia, Listeria species, adenovirus, Semliki Forest vectors, and others have been developed as carriers of immunotherapeutic agents. Recently, chimeric virus-like particle vaccines have been developed to treat existing human papillomavirus-induced neoplasms. SUMMARY: Immunotherapy protocols using a variety of recombinant vectors and modified human papillomavirus proteins induce in-vitro T cell responses and may lead to tumor regression. Vaccine-induced in-vitro immune reactivity and clinical vaccine effects are often not well associated. Further analysis of ongoing human immunotherapy trials is awaited.

Anogenital neoplasia in AIDS.

PURPOSE OF REVIEW: We review the recent literature on anogenital neoplasms in AIDS, with emphasis on cancers associated with HPV infection. Immune reactivity to HPV as well as novel immunotherapeutic and preventative strategies are discussed. RECENT FINDINGS: Many AIDS-associated neoplasms are associated with HPV infection. Whether cervical cancer is truly an AIDS-associated neoplasm has recently been questioned, while the association of anal cancer with AIDS in both males and females is more convincing. Recent reports cast doubt on the efficacy of HAART therapy for HPV-induced anogenital neoplasms, despite efficacy in improving disease caused by other infectious agents. We include here new data on humoral and cellular immune responses to HPV. VLP serology has been reported to be associated with outcome of cervical cancer. VLP seropositivity has been reported to be a favorable prognostic sign in women with HPV 16 positive cervical carcinoma. Several investigators have questioned the immunogenicity of the oncogenic HPV type 16 compared with other HPV types. It has recently been found that in HIV-infected patients, lymphoproliferative cellular immune responses (CMI) to HPV 16 peptides are not associated with CD4 counts, whereas responses to recall antigens and mitogens are associated with CD4 counts. CD4 + T cells may not be responsible for protective cellular immune responses to HPV. VLP serology and CMI responses may be the future intermediate surrogate biomarkers for HPV-associated anogenital neoplasia trials. Several new therapeutic vaccine strategies for management of HPV-induced neoplasia are reviewed. SUMMARY: Most anogenital neoplasms occurring with increased frequency in patients with HIV/AIDS are associated with HPV infections. Current treatment strategies are not effective in clearing anogenital HPV infection and need improvement. Immunotherapy with novel vaccines will provide both prevention and therapy for these common malignancies.

A phase II trial of interleukin-12 in patients with advanced cervical cancer: clinical and immunologic correlates. Eastern Cooperative Oncology Group study E1E96.

PURPOSE: The ability to mount lymphoproliferative responses to peptides derived from the human papillomavirus (HPV) E6 and E7 oncoproteins has been associated with regression of dysplastic lesions of the uterine cervix and loss of associated HPV infection. Interleukin-12 (IL-12) is a potent immunopotentiator of T-cell function, and has been shown in phase I clinical trials to be tolerable. EXPERIMENTAL DESIGN: Patients were required to have measurable metastatic, recurrent or inoperable cervical carcinoma. Patients could have had one prior adjuvant regimen and one prior regimen for advanced disease. Treatment consisted of IL-12 administered at 250 ng/kg IV as a rapid push in the outpatient setting daily x 5 every 21 days. Whole blood samples were acquired twice before treatment then approximately every 3 weeks to assess lymphoproliferative response in vitro to HPV type 16 (HPV 16) E4, E6, and E7 peptides. These responses were correlated with demographics and with clinical outcome. RESULTS: Thirty-four patients were enrolled; 29 were evaluable. Over half had received cisplatin-based chemotherapy. The most common serious toxicities were hematologic or hepatic, and all were reversible. There was one partial responder (3%). The median survival was 6.5 months (95% CI: 5.8, 11.5 months). Eighteen of 29 eligible patients had evaluable laboratory data both pre- and post-therapy. There was a statistically significant increase in lymphoproliferative responses for HPV 16 E4, E6, and E7 peptides (P=0.020, 0.020, 0.043). There was a significant association between change in lymphoproliferative response to HPV 16 E6 peptides and number of cycles of treatment administered (P=0.048). There was no correlation between change in lymphoproliferative response to any peptide with age, performance status, race, prior chemotherapy, time from diagnosis to treatment, or with overall survival. CONCLUSIONS: IL-12 treatment was associated with improved lymphoproliferative responses to HPV 16 E4, E6, and E7 peptides. This is the first clinical trial to demonstrate induction of cell-mediated immune (CMI) responses to specific antigens (peptides) following treatment with IL-12 in women with cervical cancer. This improvement in immune response was not associated with enhanced objective response or survival.

Regression of cervical intraepithelial neoplasia and loss of human papillomavirus (HPV) infection is associated with cell-mediated immune responses to an HPV type 16 E7 peptide.

Most human papillomavirus (HPV)-associated cervical intraepithelial neoplasia(CIN) lesions in normal women regress spontaneously, but a small number persist and may progress to invasive cancer. To evaluate the role of immunity to HPV and the outcome of CIN and associated HPV infection, we examined cell-mediated immune (CMI) responses to HPV 16 E6 and E7 peptides. One hundred thirty-six women with biopsy-confirmed CIN I or CIN II were followed for 1 year at 3 month intervals. Study subjects were 58% Hispanic, 36% African American, and 6% of other ethnicity, and were attending a municipal hospital colposcopy clinic. At each visit, cervical cytology and cervicovaginal lavage for HPV detection and typing was done, and blood was obtained for immunological studies. Lymphoproliferative CMI responses to HPV 16 E6 and E7 peptides were tested. An end point biopsy was done after the 1-year follow-up. The association between CMI responses to specific peptides and the outcome of disease was evaluated. CMI responses to E7 peptide (37-54) correlated significantly with regression of disease and with resolution of viral infection within 12 months. The protective effects of CMI to this peptide were not HPV type-specific. CMI responses to several other peptides also showed an association with regression, although not significant at present sample size. E7 peptide 37-54 contains one or more human T-cell epitopes. Identification and mapping of "protective" epitopes in the HPV E6 and E7 proteins could lead to the development of immunological assays to determine the risk of CIN and the development of immunotherapeutic protocols for the management of premalignant and malignant HPV-associated neoplasia and, ultimately, for the prevention of cancer.

Utilization of the human genome sequence localizes human papillomavirus type 16 DNA integrated into the TNFAIP2 gene in a fatal cervical cancer from a 39-year-old woman.

PURPOSE: The purpose of our study was to characterize a human papillomavirus (HPV) 16 DNA integration in the genome of a rapidly progressive, lethal cervical cancer in a 39-year-old woman. EXPERIMENTAL DESIGN: An HPV 16 integration site from cervical cancer tissue was cloned and analyzed using Southern blot hybridization, nucleotide sequencing, fluorescence in situ hybridization analysis for chromosomal localization and comparison with the draft human genome sequence. RESULTS: HPV 16 DNA (3826 bp) was integrated into the genome of the tumor sample and contained an intact upstream regulatory region and E6 and E7 open reading frames. Both 5' and 3' viral-cell junction regions contained direct repeat and palindrome sequences. The chromosomal location of the viral integration and cellular deletion was mapped to chromosome 14q32.3 using both a somatic cell hybrid panel and fluorescence in situ hybridization. Search of the draft human genome sequence confirmed the chromosomal location and revealed a disruption of the TNFAIP2 cytokine/retinoic acid-inducible gene. CONCLUSIONS: On the basis of the lack of sequence homology between the viral and cellular site of integration and the structure of the viral-cell junctions, it seems that HPV 16 DNA integrates into the host genome by a mechanism of nonhomologous recombination. We suggest that, taken together, maintenance of E6 and E7 expression, loss of the E2 gene and disruption of the TNFAIP2 gene through viral integration contributed to the rapid progression of cervical cancer in this patient. Availability of the human genome sequence will facilitate identification of cellular genes involved in cervical cancer by high-throughput analysis of viral integration sites.