High-Risk HPV Testing

Ever since the completion of the landmark ASC-US/Low-grade Triage Study (ALTS) trial in 2001, the role of high-risk HPV (hrHPV) testing has been firmly cemented in the management of women with abnormal Pap smears. The ALTS trial most notably taught us that for women with a diagnosis of atypical squamous cells of undetermined significance (ASC-US), follow-up testing for hrHPV is more sensitive than a repeat Pap test in identifying patients at risk for a high-grade cervical intraepithelial neoplasia (CIN) 3 or worse squamous neoplasia, and is almost as good as colposcopy, the gold standard used in the study.

We also learned that the prevalence of dysplasia (and hrHPV) was high enough in patients with a diagnosis of low-grade squamous intraepithelial lesion (LSIL) and that hrHPV testing was not useful, and these patients needed immediate follow-up colposcopy to detect a possible high-grade CIN (CIN 2/3).

These guidelines were codified by the American Society of Colposcopy and Cervical Pathology in their 2003 "Consensus Guidelines for the Management of Women With Abnormal Cervical Cancer Screening Tests," updated in 2006. They put forth hrHPV testing as an appropriate method for triaging patients with ASC-US on a Pap smear. In 2006, an additional recommendation was added to utilize hrHPV testing as an initial screening test, along with a Pap smear, in women over 30 years of age.

Laboratory Methods

Numerous assays aimed at testing for the presence of hrHPV or for quantitating hrHPV are available to laboratories. The key desirable feature in any hrHPV test is high sensitivity since it is used as a screening test, although clinical validation is essential with any clinically significant test. To detect hrHPV, most tests rely on amplification of the target hrHPV DNA or amplification of signals that are turned on in the presence of the target DNA.

PCR is a classic target-amplification test, which works well for detecting hrHPV. The Roche Amplicor HPV test, widely used in Europe, is a target-amplification PCR-based assay and has been shown to have high sensitivity for the 13 high-risk subtypes it is designed to detect as well as good clinical performance. Two hrHPV tests approved by the FDA for use on ThinPrep Pap specimens are the Qiagen digene High-Risk HPV HC2 DNA Test and the newly approved Hologic Cervista™ HPV HR test.

Digene HC2

The hrHPV test used in the ALTS trial was the digene HC2, noteworthy for its high sensitivity, ease of use and relatively low expense. The HC2 test is an in vitro nucleic acid hybridization assay that uses enhanced chemiluminescence for the qualitative and semiquantitative detection of 13 hrHPV subtypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) in cervical samples. Patient samples are simultaneously tested with positive and negative DNA controls, and results are reported as positive or negative on the basis of a ratio of relative light units to a cutoff value derived from the positive control (RLU/CO).

The HC2 test has proven to be an excellent and reliable test for detecting hrHPV, but the configuration of the test assay has been shown to have two potential flaws. The first regards the subject of cross-reactivity. The HPV-specific RNA molecules used to detect HPV in this test may cross-react with other subtypes of HPV that the assay is not designed to detect.¹A negative impact of the cross reactivity is that low-risk subtypes (e.g., 6 and 11) may be detected as positives if they are present in the sample in high enough copy numbers. This would result in reduced specificity of the test (i.e., false positives).

However, not all of the cross-reactivity is bad; some cross-reactive HPV strains are actually high-risk subtypes, and this cross-reactivity increases sensitivity for hrHPV subtypes. An example is the recently discovered hrHPV subtype 66, detected by HC2.

The HC2 also lacks a loading control to assess specimen adequacy in the assay platform.²The test does not monitor how much genomic DNA is present in the sample and therefore cannot determine if the sample is adequate (e.g., pure water would be found to be negative for HPV, not insufficient).

Despite criticisms of the HC2 test, it has been validated clinically during the ALTS trial, showing a sensitivity of 96.3 percent. Clinical validation is extremely important, as the various hrHPV subtypes present in patient populations as well as other confounding variables unique to the clinical setting can only be assessed by clinical validation. Indeed, many tests have performed well in laboratory and analytical testing environments, only to break down during clinical validation. HC2 has performed well both analytically and clinically.

Cervista

Hologic Cervista™ HPV HR is a newly FDA-approved hrHPV test to detect 14 oncogenic strains of HPV (the same 13 as HC2 plus hrHPV 66) and reduce false-positive results associated with low-risk cross reactivity. In addition, the Cervista test contains an internal control to validate that sufficient DNA is present for testing. The test is marketed to compete with the HC2 test, and FDA approval was achieved in spring 2009 based on extensive clinical validation at multiple centers.

The Cervista test uses patented "Invader" chemistry, a proprietary nucleic acid signal amplification test that includes two stages of signal amplification, resulting in a very sensitive test. The platform may also be used for other nucleic acid tests (e.g., Hepatitis C viral testing), so laboratories that already use the technology may save money by utilizing the same equipment for multiple assays.

Analytic and clinical-based testing of Cervista has been based mostly on comparison with HC2, since HC2 was the only FDA-approved test before Cervista. Analytically, Cervista shows comparable sensitivity and improved specificity to HC2.

Dr. Knoepp is assistant professor, Department of Pathology, University of Michigan, Ann Arbor.

References

1. Johnson LR, Starkey CR, Palmer J, Taylor J, Stout S, et al. A comparison of two methods to determine the presence of high-risk HPV cervical infections. Am J Clin Pathol 2008;130(3):401-8.

2. Ginocchio CC, Barth D, Zhang F. Comparison of the Third Wave Invader human papillomavirus (HPV) assay and the Digene HPV hybrid capture 2 assay for detection of high-risk HPV DNA. J Clin Microbiol 2008 May; 46(5):1641-6. Epub 2008 Mar 26.