Best Overall Resident Prize Paper
#32 Gene Expression Profiling of Women with Varying Degrees of Cervical Dysplasia
James E. Kendrick, MD

Best Scientific Paper Award
#47 Autoclave Sterilization of Instruments used on Women with Cervical Neoplasia is an Effective Method of Eradicating Residual HPV DNA: A PCR-Based Evaluation
Jacob M. Estes, MD

The George C. Trombetta, MD Teaching Award
#42 An Interim Analysis of a Prospective Blinded Evaluation of the Reid Colposcopic Index for Grading Cervical Intraepithelial Neoplasia
Kristopher J. Kimball, MD

Thomas V. Sedlacek, MD Prize for Best Clinical Research Paper
#17 p16ink4a is Helpful in Discriminating between CIN1 and Equivocal Lesions of the Cervix
Rachel Redman, MD

The ASCCP/Cytyc Young Investigator's Award
#61 Patient Knowledge about Human Papillomavirus and Abnormal Pap Smears
Briana R. Benning, MD

University of Alabama at Birmingham, Department of Obstetrics and Gynecology, Division of Gynecologic Oncology.

BACKGROUND: Cervical carcinoma is the second most common malignancy in women worldwide and is responsible for over 200,000 deaths each year. In order to further reduce the morbidity and mortality associated with this disease, novel strategies are needed to both identify and effectively treat pre-existing disease. Pre-invasive or cervical intraepithelial neoplasia (CIN) lesions of the cervix have been well-characterized , and it has been previously established that high grade dysplasias have a propensity for progression into invasive disease. Many of these dysplasias may be completely preventable through the use of chemoprevention, appropriate vaccine design and targeted immunotherapy. However, despite the firm establishment of a “pre-invasive to invasive” model, little is known about the genomic and proteomic changes that occur in normal to severely dysplastic tissue. Furthermore, less is known about the genomic differences between dysplastic tissue and invasive carcinoma. Despite similar investigations in other solid tumor types including colon, prostate, and breast cancer, we are not aware of previous reports of other investigators formally evaluating genomic difference between normal tissue and CIN III in the same patient. It is through the identification of these changes that one may be able to identify new targets for future therapeutics in addition to potential novel biomarkers.

OBJECTIVE: To determine genomic microarray differences in patients with normal cervical tissue and CIN III.

STUDY DESIGN: Loop Electrosurgical Excision Procedure (LEEP) specimens from a prospective cohort of women with CIN were analyzed in an attempt to identify candidate genes responsible for aberrant cellular changes.

MATERIALS AND METHODS: Following IRB approval, patients referred to the UAB Colposcopy Clinic for a LEEP were identified. Tissue biopsies of both normal tissue and CIN III from fresh LEEP specimens were obtained and sent to pathology for histologic confirmation. Procurement of these two types of tissue from the same patient controls for different types of HPV infection, smoking and nutritional status, age, immunocompetency, and other microenvironment factors (i.e., the patient serves as their own control). Standard RNA extraction techniques (Qiagen, Valencia, CA, USA) were utilized to prepare the tissue specimens for microarray analysis with the Affymetrix GeneChip&#61666; U133A expression array. Paired samples were eligible for microarray analysis only when both normal tissue and CIN III were confirmed by pathology. The data were then subsequently subjected to a log-like transformation and analyzed with a t-test. For this study, a p<0.001 was determined to be statistically significant.

RESULTS: 21 pairs of both normal and CIN tissue were obtained, and underwent histologic evaluation followed by RNA extraction. Within the “normal“ group, 86% were confirmed as true “normals” and 14% CIN. Within the CIN group, 29% were found to have CIN2, 64% were found to have CIN3, and 7% had no evidence of CIN. Mean RNA content of “normal” samples was 2.0 micrograms, while the mean RNA content of CIN samples was significantly higher at 7.4 micrograms (p=0.006).

Using a cutoff of p<0.001, 24 candidate genes were identified from over 18,000 genes. In the CIN III group, 14 genes were overexpressed and 10 genes were underexpressed. 9 of the 14 overexpressed genes were noted to have identities listed in the National Center for Biotechnology Information (NCBI) public domain. 7 of these 9 genes (78%) were directly related to immunity related pathways. One over-expressed gene was identified as p53.

CONCLUSIONS: The presence of CIN is marked by increased transcriptional activity, evident by an almost 4-fold increase in mean RNA content obtained from our CIN samples versus normal cervical tissue. Furthermore, a number of statistically significant overexpressed genes of interest related to immune function/response and cell cycle control were identified in our pilot microarray study. This data has the ability to direct future research endeavors in the field of cervical neoplasia. Future endeavors include the use of laser capture microdissection to evaluate genomic changes strictly at the epithelial level and as such, exclude stromal response contributions.

Estes JM*, Kirby TK*, Huh WK

University of Alabama-Birmingham, Division of Gynecologic Oncology, Birmingham, Alabama

OBJECTIVE: To determine whether autoclave sterilization eradicates HPV DNA on specula and instruments used to treat women with cervical neoplasia.
MATERIALS/METHODS: Specula used in a referral colposcopy clinic were evaluated to determine the PGMY9/11 primer system’s ability to amplify residual HPV DNA. Each speculum and instrument was sampled with a Dacron swab and stored in PreservCyt® solution at 4°C. DNA amplification was performed under standard conditions followed by HPV typing using the reverse line blot test (Roche Molecular Systems, Alameda, CA). Once validated, the same PCR method was utilized on autoclave-sterilized specula and biopsy instruments. Autoclave sterilization involved hand scrubbing with subsequent autoclaving. All results, with appropriate positive and negative controls, were confirmed in triplicate.

RESULTS: A total of 140 instruments (70 used and autoclaved) were sampled for residual HPV DNA. Five samples in the contaminated specula arm were excluded from analysis secondary to insufficient sampling. Of the remaining samples, 52.3% (34/65) of contaminated instruments – both specula and biopsy instruments – had detectable HPV DNA. 55% (11/20) of contaminated biopsy instruments were positive and 51.1% (23/45) of contaminated specula were positive. All 70 autoclaved samples (50 specula and 20 biopsy instruments) were negative for residual HPV DNA or ß –globin.

CONCLUSIONS: The PGMY9/11 primer system is an effective method to detect residual HPV DNA. Autoclave sterilization appears to eradicate HPV DNA to levels undetectable with this sensitive assay. These results suggest that autoclave sterilization is effective when using non-disposable instruments, and should be the method of choice in studies utilizing PCR-based amplification of HPV DNA.

KEY WORDS: HPV, Specula, Autoclave, Cervical neoplasia

Kimball KJ*, Numnum TM*, Estes JM*, Rocconi RP*, Kirby TO*, Straughn JM*, Conner MG*, Waltman EE*, Huh WK

University of Alabama at Birmingham, Birmingham Al, 619 19th St. South 35249-73333

OBJECTIVE: The Reid Colposcopic Index (RCI) is a scoring system commonly utilized to formulate a colposcopic impression of cervical intraepithelial neoplasia (CIN). Although extensively used in clinical trials, there is a paucity of prospective data evaluating the validity of the RCI. The purpose of this study is to prospectively evaluate, in a blinded fashion, the accuracy of the RCI for grading the severity of cervical lesions in women being treated for CIN.

METHODS: This blinded prospective study evaluated women seen at a referral colposcopy clinic. Patients undergoing a loop electrosurgical excisional procedure (LEEP) were eligible for the study. Colposcopists were blinded to patient history including indication for referral, cytology, histopathology, and previous therapy. In order to ensure blinding of the colposcopists, patients referred for routine colposcopy were intermixed with the study patients in a 1:1 ratio. A blinded RCI (range 0-8) was compiled prior to performing the LEEP as indicated by patient history. A single gynecologic pathologist reviewed all LEEP specimens. RCI scores were divided into 3 groups: 0-2 representing HPV changes or CIN 1; 3-5 representing CIN 1 or 2; and finally, 6-8 indicating CIN 2 or 3. Correlations between RCI groups and LEEP pathology were calculated using simple percentages and Kappa statistical analysis.

RESULTS: Fourty-three patients were enrolled in the study and seven were excluded. Four did not meet inclusion criteria, 2 were excluded secondary to failed blinding of the colposcopist, and 1 patient had an inadequate colposcopy. The remaining 36 LEEP patients with adequate colposcopy were evaluated. Final pathology of LEEP specimens revealed 25% CIN 1, 14% CIN 2, and 61% CIN 3. The mean RCI score was 3.11. Approximate histopathology, utilizing categories defined by Reid, was accurately predicted only 38.9% of the time. Only 19.4% of RCI scores exactly correlated with LEEP diagnosis. Furthermore, only 5% of CIN 3 lesions were predicted utilizing the RCI. Cohen’s k statistic for agreement between RCI scores and LEEP diagnosis was 0.17

CONCLUSIONS: Although commonly used to objectively evaluate cervical lesions, the RCI has not been adequately validated. Interim results from this prospective trial reveal a poor correlation between RCI and LEEP pathology. The RCI should be further validated before utilization in future clinical trials.

KEY WORDS: Reid colposcopic index, cervical intraepithelial lesion, colposcopy, accuracy

Rufforny I, Redman R, Wilkinson EJ, Liu C*, and Massoll NA

Department of Pathology, University of Florida, Gainesville, FL

OBJECTIVE: Recent evidence suggests that the protein p16ink4a is over-expressed in cervical lesions associated with high risk human papillomavirus (HRHPV), specifically subtypes 16 and 18, and that those high risk lesions are more likely to progress to cervical carcinomas. It is unclear, however, if p16ink4a expression in equivocal cervical lesions can help distinguish atypical non-HPV changes such as squamous metaplasia from HPV-related changes like low grade cervical intraepithelial neoplasias (CIN 1).

METHODS: One hundred and ninety-one cervical lesions, (81 CIN 1, 52 squamous metaplasia, 33 cellular features suggestive of HPV-related change, 9 reserve cell hyperplasia, 4 microglandular hyperplasia, and 12 inflammatory cervicitis) were randomly selected from archival cervical biopsy specimens. All 191 samples were studied with p16ink4a (JC8 monoclonal antibody, DAKO Cytomation) to determine if p16ink4a reactivity was related to the presence of HPV only. P16ink4a reactivity was scored on a three-tier system as follows: negative, 0-5% cells reactive; focal/scattered positive, </= 80% cells reactive; diffuse positive, >80% cells reactive. P16ink4a reactivity was based on normal/reactive cervical specimens where anti-p16 antibody was negative or weakly reacted with a subset of non-cervical epithelial cells. All CIN 1 lesions not reactive for p16ink4a were investigated for the presence of HRHPV subtypes 16 and 18 by real-time polymerase chain reaction (RT-PCR) technique.

RESULTS: Expression of p16ink4a was not detected in normal squamous epithelium, equivocal cervical lesions with non-specific atypical changes including immature squamous metaplasia, reserve cell hyperplasia, microglandular hyperplasia, reactive changes associated with inflammation or lesions with cellular features suggestive of HPV-related change. Thirty of 81 CIN 1 lesions expressed p16ink4a (11 had focal/scattered reactivity and 19 had diffuse reactivity). The 51 CIN1 lesions negative for p16ink4a were also RT-PCR negative for the presence of HPV subtypes 16 and 18.

CONCLUSIONS: These data support the use of p16ink4a immunohistochemical evaluation of cervical biopsies for better discrimination of non-HPV associated lesions from HPV-related lesions. Better discrimination will help to reduce false-negative interpretations and improve cervical pre-cancer diagnosis as well as reduce false-positive interpretations, thus optimizing patient management and care.

KEY WORDS: p16ink4a, CIN 1, HPV; Equivocal cervical lesions

Benning BR*, Lund MR

The Medical College of Wisconsin, St Joseph Regional Medical Center, Columbia St Mary’s Hospital, Milwaukee, WI

OBJECTIVE: The objective of the study is to assess patient knowledge about human papillomavirus (HPV) and abnormal Pap smears among women attending obstetrics and gynecology clinics in an urban area, and to determine if self-report of a prior abnormal Pap smear improves knowledge.

BACKGROUND: HPV is the most prevalent sexually transmitted infection in the United States and other areas of the world. Infection with high-risk HPV types is strongly associated with cervical dysplasia and cancer. Previous studies in the United States have found that women’s awareness of HPV is low, both inside and outside of the healthcare setting.

METHODS: An IRB approved survey was designed and offered to all patients age 18 and over presenting to the obstetrics and gynecology clinics at three locations in an urban area. The surveys were confidential and anonymous, and assessed demographic factors, sexual behaviors, and history of sexually transmitted infections or abnormal Pap smears. Each patient was also given a 13-question knowledge assessment test about HPV and cervical cancer. Surveys were made available in English and Spanish. Comparisons of categorical data were made with a Chi-square test, or with the Mantel-Haenszel Chi-square trend test when the response was an ordered category. Exact methods for test statistics were used wherever small expected cell counts were present. In some cases the exact p-values were determined through Monte Carlo simulation using 15000 trials. Variables such as age and number of years which had reasonably continuous responses were compared with t-tests, Wilcoxon tests, or the Kruskal-Wallis test.

RESULTS: 363 surveys were collected. The mean patient age was 29 years, with a wide range of socioeconomic backgrounds and sexual behaviors. Of women surveyed, 53% reported a history of sexually transmitted or vaginal infection, and 38% reported a lifetime history of an abnormal Pap smear. 44% of patients reported that they had heard of HPV. Women who reported a history of sexually transmitted or vaginal infection were significantly more likely to have had an abnormal Pap smear (RR 3.5, p<0.01), but not any more likely to report that their health care provider had talked to them about HPV (p=0.08). Women who reported a history of abnormal Pap smear were no more likely to have heard of HPV (p=0.52), or to know that it is the main cause of cervical cancer (p=0.46), than those who did not report a history of an abnormal Pap smear. Factors associated with having heard of HPV include Caucasian or Asian race (p<0.01), commercial insurance (p<0.01), seeing a faculty physician (p<0.01), or knowing someone who has had an abnormal Pap smear (p<0.01). There was no significant difference in scores on a 13-item knowledge assessment test about HPV and cervical cancer between women who reported a history of abnormal Pap smear and women who did not report a history of abnormal Pap smear.

CONCLUSIONS: There is no significant difference in knowledge about HPV between patients who report a history of abnormal Pap smear and patients who do not report a history of abnormal Pap smear.

KEY WORDS: Papillomavirus, human; Papanicolaou smear; uterine cervical neoplasms; cervical intraepithelial neoplasm