Racial and Ethnic Disparities in the Time to Ovarian Cancer Surgery in Patients at an Integrated Health Care Delivery System.

BACKGROUND: Disparities in ovarian cancer survival for African American women are multifactorial. We evaluated racial and ethnic differences in time to ovarian cancer surgery in members of an integrated health care system. PATIENTS AND METHODS: In this retrospective cohort study, we identified women diagnosed with invasive epithelial-type ovarian cancer between January 1, 2008, through December 31, 2014, at an integrated health care system in the United States. We extracted data on cancer-related variables and sociodemographic variables from the health care system's cancer registry and electronic health records. We included patients who received ovarian cancer surgery without neoadjuvant chemotherapy. We defined time to surgery as the number of days between diagnostic imaging study and surgery. We used Cox proportional hazards regression to evaluate crude and adjusted association of race and ethnicity with time to surgery. RESULTS: Of 872 patients included, 55.1% were non-Hispanic White (hereafter, White), 24.9% were Hispanic, 14.6% were Asian/Pacific Islander (PI)/Native American, and 5.5% were African American. Median age at diagnosis was 59.0 years. African American patients were diagnosed at an older age and were more likely to come from deprived neighborhoods than other racial and ethnic groups. Median time to surgery was longer for African American patients compared with White, Hispanic, and Asian/PI/Native American patients (median days: 27.5 vs 21.0, 24.5, and 26.0, respectively; P<.0001). In adjusted models, the likelihood of having received surgery at any given time post diagnostic imaging was 31% lower for African American patients compared with White patients (HR, 0.69; 95% CI, 0.51-0.93). This likelihood was also lower for Hispanic and Asian/PI/Native American patients, but not statistically significant. CONCLUSIONS: Our findings showed that patients with ovarian cancer from racial and ethnic minorities had a lower likelihood of having received surgery at any given time post diagnostic imaging compared with White patients, demonstrating that racial and ethnic differences exist in time to ovarian cancer surgery in patients with relatively equal access to care.

Impact of COVID-19 on Cervical Cancer Screening Rates Among Women Aged 21-65 Years in a Large Integrated Health Care System - Southern California, January 1-September 30, 2019, and January 1-September 30, 2020.

On March 19, 2020, the governor of California issued a statewide stay-at-home order to contain the spread of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19).* The order reduced accessibility to and patient attendance at outpatient medical visits,† including preventive services such as cervical cancer screening. In-person clinic visits increased when California reopened essential businesses on June 12, 2020.§ Electronic medical records of approximately 1.5 million women served by Kaiser Permanente Southern California (KPSC), a large integrated health care system, were examined to assess cervical cancer screening rates before, during, and after the stay-at-home order. KPSC policy is to screen women aged 21-29 years every 3 years with cervical cytology alone (Papanicolaou [Pap] test); those aged 30-65 years were screened every 5 years with human papillomavirus (HPV) testing and cytology (cotesting) through July 15, 2020, and after July 15, 2020, with HPV testing alone, consistent with the latest recommendations from U.S. Preventive Services Task Force.¶ Compared with the 2019 baseline, cervical cancer screening rates decreased substantially during the stay-at-home order. Among women aged 21-29 years, cervical cytology screening rates per 100 person-months declined 78%. Among women aged 30-65 years, HPV test screening rates per 100 person-months decreased 82%. After the stay-at-home order was lifted, screening rates returned to near baseline, which might have been aided by aspects of KPSC's integrated, organized screening program (e.g., reminder systems and tracking persons lost to follow-up). As the pandemic continues, groups at higher risk for developing cervical cancers and precancers should be evaluated first. Ensuring that women receive preventive services, including cancer screening and appropriate follow-up in a safe and timely manner, remains important.

Gemogenovatucel-T (Vigil) immunotherapy as maintenance in frontline stage III/IV ovarian cancer (VITAL): a randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Gemogenovatucel-T is an autologous tumour cell vaccine manufactured from harvested tumour tissue, which specifically reduces expression of furin and downstream TGF-ß1 and TGF-ß2. The aim of this study was to determine the safety and efficacy of gemogenovatucel-T in front-line ovarian cancer maintenance. METHODS: This randomised, double-blind, placebo-controlled, phase 2b trial involved 25 hospitals in the USA. Women aged 18 years and older with stage III/IV high-grade serous, endometrioid, or clear cell ovarian cancer in clinical complete response after a combination of surgery and five to eight cycles of chemotherapy involving carboplatin and paclitaxel, and an Eastern Cooperative Oncology Group status of 0 or 1 were eligible for inclusion in the study. Patients were randomly assigned (1:1) to gemogenovatucel-T or placebo by an independent third party interactive response system after successful screening using randomly permuted block sizes of two and four and stratified by extent of surgical cytoreduction and neoadjuvant versus adjuvant chemotherapy. Gemogenovatucel-T (1 × 107 cells per injection) or placebo was administered intradermally (one per month) for a minimum of four and up to 12 doses. Patients, investigators, and clinical staff were masked to patient allocation until after statistical analysis. The primary endpoint was recurrence-free survival, analysed in the per-protocol population. All patients who received at least one dose of gemogenovatucel-T were included in the safety analysis. The study is registered with ClinicalTrials.gov, NCT02346747. FINDINGS: Between Feb 11, 2015, and March 2, 2017, 310 patients consented to the study at 22 sites. 217 were excluded. 91 patients received gemogenovatucel-T (n=47) or placebo (n=44) and were analysed for safety and efficacy. The median follow-up from first dose of gemogenovatucel-T was 40·0 months (IQR 35·0-44·8) and from first dose of placebo was 39·8 months (35·5-44·6). Recurrence-free survival was 11·5 months (95% CI 7·5-not reached) for patients assigned to gemogenovatucel-T versus 8·4 months (7·9-15·5) for patients assigned to placebo (HR 0·69, 90% CI 0·44-1·07; one-sided p=0·078). Gemogenovatucel-T resulted in no grade 3 or 4 toxic effects. Two patients in the placebo group had five grade 3 toxic events, including arthralgia, bone pain, generalised muscle weakness, syncope, and dyspnea. Seven patients (four in the placebo group and three in the gemogenovatucel-T group) had 11 serious adverse events. No treatment-related deaths were reported in either of the groups. INTERPRETATION: Front-line use of gemogenovatucel-T immunotherapy as maintenance was well tolerated but the primary endpoint was not met. Further investigation of gemogenovatucel-T in patients stratified by BRCA mutation status is warranted. FUNDING: Gradalis.

Primary Human Papillomavirus Screening: Women's Perceptions of New Cervical Cancer Screening Recommendations.

Background: Current clinical guidelines recommended primary human papillomavirus (HPV) screening for cervical cancer testing. Previous studies reported patient-level barriers (e.g., limited knowledge and attachment to Pap test) that may hinder wide adoption of primary HPV screening. We assessed these women-level factors following the implementation of primary HPV screening (July 2020) at Kaiser Permanente Southern California (KPSC). Methods: We administered a patient survey (mail and on-line) to female KPSC members aged 30-65 years who received primary HPV screening between October and December 2020. Those who preferred English vs. Spanish language were sampled separately. The survey included domains on knowledge about HPV and HPV screening, awareness of screening guidelines, and attitudes about HPV testing. Demographic data were collected using electronic health records. We used weighted multivariable logistic and modified Poisson regressions for associations between language preference and survey responses. Results: In total, 3,009 surveys were returned (38.0% response rate). Few women (7.0%) found HPV testing as an acceptable screening method. The majority of women (92.2%) remained unaware that HPV testing can replace Pap test for screening. The Pap test was the most preferred screening approach for 33.2% Spanish-speaking women vs. 19.9% English-speaking women. Only 20.6% knew that women aged 30-65 years can be screened every 5 years with cotest or primary HPV screening. Most women (96.4%) did not perceive stigma about taking the HPV test. Conclusion: Proactive patient education will help improve women's knowledge about primary HPV screening, which may facilitate its implementation in additional health care settings.

Cervical Cancer Screening: Patient Perspectives on Transitioning to Primary High-Risk Human Papillomavirus Testing Alone.

INTRODUCTION: In 2018, the US Preventive Services Task Force updated cervical cancer screening recommendations to allow for screening every 5 years with primary human papillomavirus (HPV) testing in combination with cytology (cotesting) or every 5 years with primary HPV screening alone. Despite these changes, the uptake of primary HPV screening has been lower than expected. The purpose of this study was to evaluate the patient perspective of an integrated health system transition from cotesting to primary HPV testing among a 30- to 65-year-old cohort. METHODS: Semistructured phone interviews were conducted from July to December 2023 at Kaiser Permanente Colorado with 16 members aged 30-65 years. Interviews asked about reactions to the forthcoming change in cervical cancer screening, personal concern about cervical cancer risk, feedback on patient-facing education materials, and preference on communication timing and modality. RESULTS: Participants reported concerns about cervical cancer screening intervals, primarily the reduction in frequency leading to underdiagnosis of sexually transmitted infections (STIs). Participants recommended defining the rationale for the change to primary HPV testing in the patient education materials. Participants preferred communication about the change in-clinic between practitioner and patient. DISCUSSION: The interviews identified key themes, including the differentiation between cervical cancer and STI screening methodologies, potential underdiagnosis of STI and cervical cancer, and the rationale supporting primary HPV testing and associated screening intervals. CONCLUSION: These qualitative findings can inform health systems of potential patient concerns to address when considering the transition from cotesting every 3 years to primary HPV testing every 5 years for cervical cancer screening.

A study protocol for a cluster randomized pragmatic trial for comparing strategies for implementing primary HPV testing for routine cervical cancer screening in a large health care system.

BACKGROUND: Limited guidance exists regarding implementation strategies that best facilitate cancer screening practice substitution and achieve optimal stakeholder-centered outcomes. Here we describe the protocol for a randomized pragmatic trial comparing two implementation strategies to facilitate substitution of primary HPV screening for Pap and HPV co-testing to perform routine cervical cancer screening of women aged 30-65 years at Kaiser Permanente Southern California (KPSC). METHODS: Twelve service areas within KPSC will be randomized to a "centrally-administered system-wide implementation + local-tailored implementation" strategy or a "centrally-administered system-wide implementation only" strategy. The centrally-administered strategy comprises clinician and staff educational activities. Sites in the local-tailored arm will then conduct a structured local needs assessment followed by site-specific selection and deployment of implementation interventions. Surveys and interviews will be conducted among women and providers from the primary care and ob/gyn departments prior to the system-wide transition, shortly after the transition, and after the completion of local-tailored interventions. A stakeholder advisory committee will assist with study design, defining stakeholder-centered outcomes, and developing data collection tools. RESULTS: The primary outcome of interest is uptake of primary HPV screening. Secondary provider-centered outcomes include provider knowledge, delivery of patient education, satisfaction with the practice substitution process, and resistance to primary HPV screening. Secondary patient-centered outcomes include patient knowledge, stigma, and satisfaction with the screening process. Intervention fidelity will also be measured via surveys. CONCLUSIONS: Findings from this study will help inform future use of a local-tailored implementation strategy for adopting primary HPV screening at large health care systems. Findings may also be applicable to other types of practice substitution.

De-implementation and substitution of clinical care processes: stakeholder perspectives on the transition to primary human papillomavirus (HPV) testing for cervical cancer screening.

BACKGROUND: New cervical cancer screening guidelines recommend primary human papillomavirus (HPV) testing for women age 30-65 years. Healthcare organizations are preparing to de-implement the previous recommended strategies of Pap testing or co-testing (Pap plus HPV test) and substitute primary HPV testing. However, there may be significant challenges to the replacement of this entrenched clinical practice, even with an evidence-based substitution. We sought to identify stakeholder-perceived barriers and facilitators to this substitution within a large healthcare system, Kaiser Permanente Southern California. METHODS: We conducted semi-structured qualitative interviews with clinician, administrative, and patient stakeholders regarding (a) acceptability and feasibility of the planned substitution; (b) perceptions of barriers and facilitators, with an emphasis on those related to the de-implementation/implementation cycle of substitution; and (c) perceived readiness to change. Our interview guide was informed by the Consolidated Framework for Implementation Research (CFIR). Using a team coding approach, we developed an initial coding structure refined during iterative analysis; the data were subsequently organized thematically into domains, key themes, and sub-themes using thematic analysis, followed by framework analysis informed by CFIR. RESULTS: We conducted 23 interviews: 5 patient and 18 clinical/administrative. Clinicians perceived that patients feel more tests equals better care, and clinicians and patients expressed fear of missed cancers ("…it'll be more challenging convincing the patient that only one test is…good enough to detect cancer."). Patients perceived practice changes resulting in "less care" are driven by the desire to cut costs. In contrast, clinicians/administrators viewed changing from two tests to one as acceptable and a workflow efficiency ("…It's very easy and half the work."). Stakeholder-recommended strategies included focusing on the increased efficacy of primary HPV testing and developing clinician talking points incorporating national guidelines to assuage "cost-cutting" fears. CONCLUSIONS: Substitution to replace an entrenched clinical practice is complex. Leveraging available facilitators is key to ease the process for clinical and administrative stakeholders-e.g., emphasizing the efficiency of going from two tests to one. Identifying and addressing clinician and patient fears regarding cost-cutting and perceived poorer quality of care is critical for substitution. Multicomponent and multilevel strategies for engagement and education will be required. TRIAL REGISTRATION: ClinicalTrials.gov, # NCT04371887.

Performance of the cobas HPV Test for the Triage of Atypical Squamous Cells of Undetermined Significance Cytology in Cervical Specimens Collected in SurePath.

OBJECTIVES: Determine performance of the cobas human papillomavirus (HPV) test for triage of atypical squamous cells of undetermined significance (ASC-US) in SurePath. METHODS: Women presenting for routine screening had cervical specimens collected in SurePath and specimen transport medium (STM); those with ASC-US cytology underwent colposcopy. Performance of cobas HPV in SurePath specimens that had undergone a preanalytic procedure to reverse possible cross-linking of HPV DNA was compared with Hybrid Capture 2 (hc2) specimens in STM. RESULTS: Among 856 women, HPV prevalence was 45.8%; HPV 16 and HPV 18 prevalences were lower than expected in the 21- to 29-year-old group in this highly vaccinated population. cobas HPV performance in SurePath was comparable to hc2 in STM. Sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 3 or worse were 87.5% (95% confidence interval [CI], 71.9%-95.2%) and 55.5% (95% CI, 52.1%-58.9%) for cobas and 85.3% (95% CI, 69.9%-93.6%) and 54.7% (95% CI, 51.4%-57.9%) for hc2. Sensitivity was negatively affected by random biopsies performed at colposcopy; comparable sensitivities were achieved in the nonvaccinated and vaccinated populations with disease determined by directed biopsy only. CONCLUSIONS: Performance of cobas HPV for ASC-US triage in pretreated SurePath specimens meets criteria for validation. Preliminary data indicate reliable performance of HPV testing in a highly vaccinated population.

Targeted Treatment of a Rare Vaginal Sarcoma With an Anaplastic Lymphoma Kinase Inhibitor.

BACKGROUND: Anaplastic lymphoma kinase is a tyrosine kinase receptor that can become oncogenic. Crizotinib is a tyrosine kinase inhibitor that shows activity in patients with anaplastic lymphoma kinase rearrangements that have failed conventional therapies. CASE: A 34-year-old woman presented with a painful 3-cm left vulvar-vaginal mass, which was excised and determined to be a sarcoma with positive surgical margins. Fluorescence in situ hybridization testing of her tumor was conducted and demonstrated anaplastic lymphoma kinase gene rearrangements. A 3-cm mass recurred 1 month later. Treatment with 250 mg crizotinib orally twice daily resulted in complete regression of all visible or palpable tumor within 3 weeks. CONCLUSION: Molecular evaluation techniques can be used to direct targeted therapy for select malignancies. Future technologic advances will expand the number of malignancies for which these treatment approaches can be used.

Long-term survival advantage and prognostic factors associated with intraperitoneal chemotherapy treatment in advanced ovarian cancer: a gynecologic oncology group study.

PURPOSE: To determine long-term survival and associated prognostic factors after intraperitoneal (IP) chemotherapy in patients with advanced ovarian cancer. PATIENTS AND METHODS: Data from Gynecologic Oncology Group protocols 114 and 172 were retrospectively analyzed. Cox proportional hazards regression models were used for statistical analyses. RESULTS: In 876 patients, median follow-up was 10.7 years. Median survival with IP therapy was 61.8 months (95% CI, 55.5 to 69.5), compared with 51.4 months (95% CI, 46.0 to 58.2) for intravenous therapy. IP therapy was associated with a 23% decreased risk of death (adjusted hazard ratio [AHR], 0.77; 95% CI, 0.65 to 0.90; P = .002). IP therapy improved survival of those with gross residual (≤ 1 cm) disease (AHR, 0.75; 95% CI, 0.62 to 0.92; P = .006). Risk of death decreased by 12% for each cycle of IP chemotherapy completed (AHR, 0.88; 95% CI, 0.83 to 0.94; P < .001). Factors associated with poorer survival included: clear/mucinous versus serous histology (AHR, 2.79; 95% CI, 1.83 to 4.24; P < .001), gross residual versus no visible disease (AHR, 1.89; 95% CI, 1.48 to 2.43; P < .001), and fewer versus more cycles of IP chemotherapy (AHR, 0.88; 95% CI, 0.83 to 0.94; P < .001). Younger patients were more likely to complete the IP regimen, with a 5% decrease in probability of completion with each year of age (odds ratio, 0.95; 95% CI, 0.93 to 0.96; P < .001). CONCLUSION: The advantage of IP over intravenous chemotherapy extends beyond 10 years. IP therapy enhanced survival of those with gross residual disease. Survival improved with increasing number of IP cycles.

Surgical cytoreduction and hormone therapy of an advanced endometrial stromal sarcoma of the ovary.

BACKGROUND: Primary endometrial stromal sarcomas of the ovary are rare gynecologic malignancies. We report a disseminated case of this tumor arising from ovarian endometriosis. CASE: A 45-year-old woman presented with an abdominal pelvic mass and an elevated CA 125. Exploration showed extensive tumor spread from the ovaries to the upper abdomen. Surgery included a total hysterectomy, bilateral salpingo-oophorectomy, splenectomy, partial gastrectomy, partial pancreatectomy, transverse colectomy, appendectomy, and omentectomy. Final pathology showed a low-grade endometrial stromal sarcoma of the ovary arising from foci of endometriosis. Megestrol acetate was initiated, and she is currently without evidence of disease. CONCLUSION: This is an advanced case of a primary low-grade endometrial stromal sarcoma of the ovary arising from endometriosis managed by total resection and progestational therapy.

Activity of trabectedin (ET-743, Yondelis) in metastatic uterine leiomyosarcoma.

BACKGROUND: The ongoing search for more effective agents in the treatment of uterine leiomyosarcomas is warranted because of the poor prognosis related to these tumors. CASE: A case of advanced, recurrent and refractory uterine leiomyosarcoma is presented that responded to trabectedin (ET-743) 1.2 mg/m2 intravenously over 24 h every 3 weeks after failing four prior regimens. A durable objective response lasting at least 8 months was documented. CONCLUSION: Trabectedin (ET-743) has activity in uterine leiomyosarcoma and warrants further investigation.

Gene expression profiling of in vitro radiation resistance in cervical carcinoma: a feasibility study.

OBJECTIVE(S): To determine the feasibility of integrating an in vitro chemo-radiation response assay (IVRRA) with a gene microarray system to investigate the molecular patterns of expression that contribute to radiation resistance in cervical cancer. METHODS: Viable primary untreated cervical cancer specimens were obtained and exposed to gamma irradiation at a dose of 3 Gy in the IVRRA to determine in vitro radiation sensitivity. RNA was purified for microarray analysis with the Affymetrix Human Genome U95A Array carrying more than 12,000 gene probes. Gene expression analysis was performed, and specimen transcript patterns were correlated with radiation response using an iteration analysis model and Pearson's correlation coefficient. RESULTS: A feasibility set of eight tumor specimens was studied. Tumors were classified into 4 extreme (ERR), 2 intermediate (IRR) and 2 low radiation resistance (LRR) categories. An intrinsic radiation response gene set of 54 genes transcripts with 100% accuracy for the classification of each tumor's radiation response category was identified. CONCLUSION(S): Gene sets associated with in vitro radiation response profiles in cervical cancer can be generated using the IVRRA and microarray technology. This has direct applications to the study of the biological pathways contributing to radiation resistance and may lead to the development of alternative treatment modalities. The potential of these technologies for cancers in which radiotherapy is employed warrants further investigation.

Gemcitabine and cisplatin chemotherapy is an active combination in the treatment of platinum-resistant ovarian and peritoneal carcinoma.

The treatment of recurrent ovarian cancer with the combination of gemcitabine and cisplatin chemotherapy has recently been shown to be an active regimen. But the majority of positive responses have been observed in patients considered either platinum-sensitive or who have had extended platinum-free intervals. The purpose of our study was to review our experience with this regimen in women with platinum-resistant ovarian and peritoneal carcinoma with more recent exposure to platinum. We studied twenty-two patients who had relapsed within six months of their most recent platinum-based regimen and were treated with gemcitabine (450-600 mg/m(2)) and cisplatin (30 mg/m(2)) on days 1 and 8 of a 21-day cycle. The overall response rate was 64% (95% C.I. 42-85%) with seven (32%) complete and seven (32%) partial responses. The median progression-free interval was 6.7 months for responding patients and 3.9 months for the entire study group. Median survival for responders was 15.8 months compared to 8.8 months for non-responders. Overall survival was 11.4 months. Grade 3 or 4 toxicity was encountered in 59% of treatments. We conclude from this limited review that the combination of gemcitabine and cisplatin chemotherapy is an active regimen in platinum-resistant ovarian and peritoneal carcinoma and warrants consideration in the management of patients with recurrent disease.

Primary invasive vaginal cancer in the setting of the Mayer-Rokitansky-Kuster-Hauser syndrome.

BACKGROUND: The Mayer-Rokitansky-Kuster-Hauser syndrome occurs in 1 in 4000 to 5000 female births. Primary vaginal cancer constitutes less than 2% of all malignancies of the female genital tract. A report of the first case of the unlikely occurrence of both of these developments in the same patient is presented. CASE: A 34-year-old nulligravid Philippine woman with a history of Mayer-Rokitansky-Kuster-Hauser syndrome presented with a 5-month history of bleeding from a blind vaginal pouch. Vaginal biopsy identified a moderately differentiated endometrioid adenocarcinoma. Exploratory laparotomy, bilateral salpingo-oophorectomy, pelvic and iliac lymph node samplings, and excision of a mullerian remnant were performed with no evidence of disease. A FIGO Stage I vaginal cancer was assigned and pelvic irradiation was given. Disease recurred 4 months later and the patient underwent total pelvic exenteration. More than 1 year since the exenteration procedure, she is without evidence of disease. CONCLUSION: This is the first reported case of a primary vaginal cancer in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome. It is a reminder that routine gynecologic examinations are still warranted as these patients are at risk for malignant changes in residual mullerian tissues.

Transepithelial sampling of the uterine ectocervix with a stiff-bristled, spiral-shaped brush.

OBJECTIVE: To measure the depth of sampling of the uterine ectocervix with the use of a stiff-bristled, spiral-shaped brush (SpiraBrush Cx; Trylon Corporation, Torrance, CA). MATERIALS AND METHODS: Eligible hysterectomy specimens were identified. The ectocervix was brushed with an inked SpiraBrush Cx in four quadrants with either light (gentle) or heavy (forceful) pressure. The depth of sampling was determined histologically. RESULTS: Four normal uteri were sampled a total of 15 times. The entire epithelium was removed to the epithelial-stromal junction in three specimens and in one sample only a few basal cells remained with light pressure. The depth of sampling ranged from 0.2 mm to 0.4 mm. Heavy pressure resulted in the entire removal of epithelium in six samples, with the depth of sampling ranging from 0.2 mm to 0.7 mm. The stroma was disrupted in one sample. Results were uninformative in five samples. CONCLUSIONS: Sampling of the uterine ectocervix with the SpiraBrush Cx seems to be transepithelial in most cases.

Conserved methylation patterns of human papillomavirus type 16 DNA in asymptomatic infection and cervical neoplasia.

DNA methylation contributes to the chromatin conformation that represses transcription of human papillomavirus type16 (HPV-16), which is prevalent in the etiology of cervical carcinoma. In an effort to clarify the role of this phenomenon in the regulation and carcinogenicity of HPV-16, 115 clinical samples were studied to establish the methylation patterns of the 19 CpG dinucleotides within the long control region and part of the L1 gene by bisulfite modification, PCR amplification, DNA cloning, and sequencing. We observed major heterogeneities between clones from different samples as well as between clones from individual samples. The methylation frequency of CpGs was measured at 14.5%. In addition, 0.21 and 0.23%, respectively, of the CpA and CpT sites, indicators of de novo methylation, were methylated. Methylation frequencies exceeded 30% in the CpGs overlapping with the L1 gene and were about 10% for most other positions. A CpG site located in the linker between two nucleosomes positioned over the enhancer and promoter of HPV-16 had minimal methylation. This region forms part of the HPV replication origin and is close to binding sites of master-regulators of transcription during epithelial differentiation. Methylation of most sites was highest in carcinomas, possibly due to tandem repetition and chromosomal integration of HPV-16 DNA. Methylation was lowest in dysplasia, likely reflecting the transcriptional activity in these infections. Our data document the efficient targeting of HPV genomes by the epithelial methylation machinery, possibly as a cellular defense mechanism, and suggest involvement of methylation in HPV oncogene expression and the early-late switch.