ProEx C Immunocytochemistry and High-Risk Human Papillomavirus DNA Testing in Papanicolaou Tests With Atypical Squamous Cell (ASC-US) Cytology: Correlation Study With Histologic Biopsy

Archives of Pathology & Laboratory Medicine, Oct 2008 by Siddiqui, Momin T, Hornaman, Kelly, Cohen, Cynthia, Nassar, Aziza

Context.-Papanicolaou tests with atypical squamous cells of undetermined significance (ASC-US) cytology and adjunct testing for high-risk human papillomaviruses (hr-HPV) are helpful in detecting high-grade disease. Detection of disease may be further improved with molecular markers known to be overexpressed in cervical carcinoma. ProEx C detects 2 such molecular markers, minichromosome maintenance protein 2 and topoisomerase II, which are associated with abnormal cell cycle regulation.

Objective.-To determine the utility of ProEx C as a marker for high-grade cervical intraepithelial neoplasia 2α disease when compared with hr-HPV status in Papanicolaou tests with ASC-US cytology.

Design.-A SurePath slide was prepared on all ASC-US cases from the residual SurePath vial pellet and stained using the ProEx C reagent prediluted with water-bath antigen retrieval, using a Dako autostainer. Nuclear staining of cytologically atypical squamous cells was considered a positive result. Adjunct testing for hr-HPV used Digene Hybrid Capture 2. Follow-up biopsy results were available for review following the Papanicolaou test.

Results.-Two hundred patients with ASC-US diagnoses were part of this study. The sensitivities of ProEx C and hr-HPV testing in detecting high-grade cervical intraepithelial neoplasia 2 disease were 98.04% and 82.35%, respectively, whereas the specificity for detecting high-grade disease was 74.50% and 73.15%, respectively.

Conclusions.-ProEx C staining is a more sensitive and specific biomarker for detecting cervical disease than adjunct testing for hr-HPV status in Papanicolaou tests with ASC-US.

(Arch Pathol Lab Med. 2008;132:1648-1652)

The advent of the Papanicolaou (Pap) test has resulted in the increased detection of cervical carcinoma and has resulted in decreased morbidity and mortality from these malignancies in female patients.1 However, cervical carcinoma is still the second most common malignancy in women worldwide and is the most common female malignancy in developing countries.2 In the year 2000, approximately 450 000 women worldwide developed cervical carcinoma, and more than 200 000 died from the disease.2 More than 50% of these cervical carcinoma patients were never screened, and of these unscreened patients, 10% to 20% were not screened in the previous 5 years.3-5 Approximately 30% of cancer patients had at least 1 false-negative Pap test because of errors in either sampling or cytologic interpretation before the development of invasive cervical carcinoma.6 Thus, any single Pap test event is only 50% sensitive for detecting high-grade squamous intraepithelial lesion (HSIL) or invasive carcinoma.7 Although independent, recurring Pap tests contribute to a successful screening program, nevertheless, even with perfect compliance, the system has its limitations because of less-thanideal sensitivity, interpretive variance, and morphologic subjectivity.

The Atypical Squamous Cells of Undetermined Significance (ASC-US)-Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS) found that 12% of women with positive ASC-US cytologic results had an underlying high-grade squamous intraepithelial lesion (HSIL).8,9 This finding would result in an estimated 3 million American women receiving abnormal Pap test results that would require colposcopy to exclude HSIL.10

The limitations encountered with the Pap test are now beginning to be addressed by ancillary molecular testing of cervical/vaginal samples. High-risk human papillomavirus (hr-HPV) DNA testing can separate women with an ASC-US diagnosis into 2 groups, with each group having a different risk for HSIL. One group has negative results for hr-HPV DNA and carries a very low risk of HSIL, similar to the risk for a woman with negative results for intraepithelial lesion.11 Hence, a woman with a negative finding for intraepithelial lesion on the Pap test and a negative result from an HPV DNA test is at very low risk of HSIL and can have her screening interval increased.12-14 Women in the other group, with positive results from an HPV DNA test, however, have an increased likelihood of HSIL.15

The most helpful molecular marker for HSIL should have the negative predictive value of a negative HPV DNA test and a higher positive predictive value. One such biomarker being studied is ProEx C (Tripath Imaging, Burlington, NC), which is an immunocytochemical assay that targets the expression of topoisomerase II-α (TOP2A) and minichromosome maintenance protein 2 (MCM2).16,17 Both TOP2A and MCM2 have been identified by transcriptional profiling as genes that are overexpressed in cervical carcinoma. 18-20 Overexpression of TOP2A and MCM2 is a result of aberrant S-phase induction. The HPV oncoproteins are directly involved in the development of cervical dysplasia and carcinoma, E6 through its interaction with tumor suppressor protein p53, and E7 through interaction with hypophosphorylated retinoblastoma proteins pRb, p107, and p130.21 This induces the aberrant transcription of S-phase proteins, such as TOP2A and MCM2.21 In DNA synthesis and proliferation, TOP2A causes enzymatic unlinking of DNA strands during replication, and MCM2 functions during DNA replication by loading the prereplication complex into DNA and unwinding DNA through helicase activity to permit DNA synthesis.22