ervicovaginal screening for squamous-cell carcinoma (SCC) has been one of the success stories in pathology. The morbidity and mortality of a deadly and disfiguring disease among women in the United States has become rare. Countries that do not have screening cytology programs still manifest the devastating effects of cervicovaginal SCC. Human papillomavirus (HPV) infection is the proven oncogenic agent for SCC1 and is an example of a cancer caused by an infectious agent. Screening cytology carries a high specificity for detecting high-grade and low-grade squamous intraepithelial neoplasia (HSIL and LSIL), which leads to proper treatment.
Because cervical SCC is aggressive and metastasizes frequently, high screening-test sensitivity is important because early treatment reduces the morbidity of disease. Measures to increase the Pap screening-test sensitivity have addressed the working conditions and preparedness of cytotechnologists (CTs) who perform the screening, the preparation method, and ancillary testing, including molecular testing. Government intervention, specifically through the advent of Clinical Laboratory Improvement Amendments in 1988 (CLIA '88) has improved the safety and working conditions of CTs. Limitation of the number of slides reviewed (100 slides per eight-hour shift, maximum 200 slides in 24 hours), and the implementation of proficiency testing and encouragement of laboratory education programs have increased the accuracy of the Pap test.
Transitioning from smear preparations to monolayer preps (e.g., Thinprep and Surepath) make highly cellular, bloody, or inflammatory specimens easier to screen and lead to increased detection of single HSIL. Automated screening instruments have improved the sensitivity of cytologic screening. Computer-assisted automated imaging screens for dark, hyperchromatic cells which are reviewed by CTs ensure the worrisome areas on each slide are examined so further diagnostic information can be provided. Cases can then be signed out as negative or sent to a cytopathologist for confirmation of cellular findings.
Cytologic cases with relatively clear-cut morphology that correlates with the follow-up biopsy findings have defined management guidelines as outlined by the American Society of Colposcopy and Cervical Pathology, or ASCCP. The equivocal cytologic cases, however, are more difficult to handle clinically. Initially, the Bethesda diagnostic category of atypical squamous cells of undetermined significance (ASC-US) was redefined in 2001 to allow the cytologist to flag a case as atypical without committing to a diagnosis of SIL. Squamous-cell nuclei no more than 2.5 times of an intermediate cell nucleus or squamous cells with an inconspicuous perinuclear halo fall into the diagnostic category of ASC-US. These parameters, however, are poorly reproducible.2 However ASC-US cases require follow-up because they carry a high risk of developing to a high grade squamous intraepithelial lesion or HSIL (see Figure 1).
While cytology has proven its worth as a screening test, ancillary testing using molecular methods to detect high-risk HPV (HRHPV) has become an important triage tool in equivocal cases. ASC-US patients with HRHPV infection are more likely to show dysplasia on biopsy. In addition, HPV testing can be performed on the same vial used for that Pap test, which saves the patient another visit to the clinician.
Table 1. Test accuracy for detecting CIN2 or higher in patients with ASC-US.
There are many options for HRHPV testing, and there are even more on the horizon with the development of more cutting-edge molecular diagnostic testing. Ideally, new testing methods would have high sensitivity and negative predictive value (NPV) to complement the high specificity and positive predictive value (PPV) of cytology. In this article, we will discuss some of the more commonly used methods, as well as some newer techniques in development.
Digene Hybrid Capture 2
The Digene Hybrid Capture 2 is a nucleic-acid hybridization assay probe set that detects the DNA of 13 of HRHPV types, including HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). The target HRHPV DNA hybridizes to a specific RNA probe which reacts with an antibody conjugated to alkaline phosphatase. A chemoluminescent substrate is cleaved by the complex and measured by a luminometer, which confirms the presence or absence of HRHPV DNA. This is widely available to both large reference labs, academic centers, and many hospital labs. For detecting moderate dysplasia or higher, the sensitivity is 85% to 90%, and the specificity is 70% to 75%. While this test provides information that one of the HRHPV types is present, it does not specify which types, specifically HPV16 and 18. Ahead, we will discuss two methods that provide this information.
Polymerase chain reaction- (PCR-) based testing
The Cobas 4800 PCR-based HPV DNA-detection system (Roche Molecular Systems, Pleasanton, CA) has recently been validated by Stoler, et al. This test uses PCR to detect the pool of 14 HRHPV DNA serotypes but has specific probes for detecting HPV 16 and 18 at the time of the test. Cervista testing (Hologic, Bedford, ME) also is PCR based; however, HPV16 and 18 must be offered separately. The Cobas tests everything at once rather than offering a separate test for HPV 16 and 18. These genotypes have been proven to carry the highest risk for developing high-grade squamous dysplasia, and Gardasil is specifically tailored to vaccinate against these serotypes.
The sensitivity and specificity of the Cobas detection system is comparable to the Digene Hybrid Capture 2 (see Table 1). Recently, in the ATHENA HPV study (Addressing the Need for Advanced HPV Diagnostics) validation data, women harboring HPV16/18 had a relative risk (RR) ratio of 32.5 for moderate cervical intraepithelial neoplasia, or CIN2, or higher versus HPV-negative patients, and an RR of 3.7 versus patients harboring the other HPV types.
The Cobas 4800 molecular system is currently for investigational use only and is not for sale in the United States. Identification of women infected with HPV16/18 may warrant a different treatment strategy in the future. The value of the Cobas 4800 may lie in future studies that assess the efficacy of Gardasil in eliminating HPV 16 and 18 as a cause of squamous dysplasia. The analytic sensitivity of PCR-based HPV testing is very high and can test for a variety of HPV types; however, the presence of HPV alone does not confirm disease. As with all PCR testing, care to avoid contamination is necessary. These methods perform as well as Hybrid Capture assay, however, with the added benefit of being able to discriminate which HPV subtype is present.
Quantification of intracellular E6/E7 mRNA expression
Intracellular MicroRNA detection of the E6/E7 domain of the HPV genome is yet another novel development in HPV testing. Integration of these oncogenes into the host DNA is a significant molecular event preceding the development of squamous dysplasia.3 In contrast to the aforementioned methods that carry very high NPV, this test is exciting because of the higher specificity and PPV for detecting dysplasia in a normal patient population compared to other HPV tests.
In situ hybridization for single colored probes that are specific for HPV E6 and E7 mRNA are quantified using flow cytometry. Micro RNA detection of E6/E7 is still under development but could potentially become a game changer for HPV screening and triage strategy. The benefit of Thinprep Pap screening is the high specificity, and PPV and HPV testing that performs in a similar manner, without losing specificity, may function as a useful adjunct to screening cytology.
Immunocytochemistry for p16/Ki67
Immunocytochemistry for a combined panel P16 (CINtec MTM Laboratories, Heidelberg, Germany) serves as a surrogate biomarker for HPV infectivity in cervical neoplasia.4 P16 protein plays a role in the mechanism of regulation of the cell cycle. It is associated with the retinoblastoma protein mediated control of the cell cycle and is upregulated in cells infected by HRHPV. The immunostain can also come as a dual stain with Ki 67, a proliferation marker. P16 staining in the cytoplasm as well as Ki67 staining in the nucleus is highly sensitive for detecting dysplasia. The strength of using this is that the cytologist can visualize the specific cells that are staining.
The sensitivity for the dual stain is 92%, and the specificity is 80%. Many cytology labs are not validated to perform immunocytochemistry, so this test may require referral to a larger laboratory. Cutoffs for how many cells are positive and how intense the staining presents may require training of those who interpret the test. While the test gives information on the presence or absence of a dysplastic cell, it does not confirm the presence of HRHPV DNA.
Since HPV testing offers increased sensitivity and NPV, it has helped to stratify women carrying an equivocal diagnosis of ASC-US into low-risk and high-risk groups, Until HPV testing provides a similar high PPV and specificity. A possible new model of cervical screening in the future may see molecular HPV testing as the screening test, with cytology performed on HPV positive patients.
With the widespread implementation of Gardasil, dysplasia caused by HPV 16 and 18, may be significantly reduced. At this point, length of time the vaccine remains effective is uncertain. In addition, other HPV DNA types cause dysplasia, and the biologic behavior of each subtype is also unknown. Some new methods that test for specific HPV types will be instrumental in helping gynecologists, cytopathologists, and infectious-disease scientists answer these questions.
Figure 1. Pap stain 400x. The image on the right shows an example of ASC-US. The highlighted cell shows a squamous cell with an ill-defined nuclear halo and an increased nuclear size. This patient was shown to be positive for high-risk HPV DNA and, subsequently, progressed to high-grade squamous intraepithelial lesion on follow-up cytology. The cells show large, hyperchromatic nuclei with nuclear membrane irregularities and an increased nuclear to cytoplasmic ratio.
Jordan P. Reynolds, MD
, formerly a cytology fellow at the Mayo Clinic in Rochester, MN, is currently on staff at the Cleveland Clinic in Cleveland, OH.
- Walboomers JMM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;89:12-19.
- Schmidt JL, Henriksen JC, McKeon DM, et al. Visual Estimates of Nucleus-to-Nucleus Ratios Can We Trust Our Eyes to Use the Bethesda ASCUS and LSIL Size Criteria? Cancer Cytopathol. 2008;114:287-293.
- Duensing S, Lee LY, Duensing A, et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proceedings of the National Academy of Sciences of the United States of America. 2000;97:10002-10007.
- Keating JT, Cviko A, Riethdorf S, et al. Ki-67, cyclin E, and p16(INK4) are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia. Am J Surg Pathol. 2001;25:884-891.