Human Papillomavirus (HPV) And Testing
WHEC Practice Bulletin and Clinical Management Guidelines for healthcare providers. Educational grant provided by Women's Health and Education Center (WHEC).
The early successes in cervical cancer screening were based on the development of morphologic criteria for recognizing cells shed from cervical cancer and its precursors. However, our ability to manage cervical diseases more effectively has been limited by an incomplete understanding of the pathogenesis and biology of cervical neoplasia. Recognition that human papillomavirus (HPV) causes most cervical neoplasia suggests that improved screening and management strategies that reflect the biology and behavior of HPV infections may be possible.
The purpose of this document is to review the structure and biology of HPVs and summarize the clinical applications of HPV testing and areas of ongoing research will be discussed.
Biology of HPV Infection:
Human papillomaviruses are epitheliotropic, circular, double-stranded DNA viruses containing about 8000 base pair. Over 70 HPV types have been characterized, of which more than 20 infect the female genital tract. Genital HPV types are classified as low or high risk based on whether they are commonly associated with invasive cervical carcinoma. Low-risk types, such as HPV-6, 11, 42, 43, 44 and others are strongly associated with condyloma acuminate and are uncommon in low-grade squamous intraepithelial lesions (LSIL). These viruses are found infrequently in high-grade squamous intraepithelial lesions (HSIL) and are almost never found alone in carcinoma. High-risk HPV types, including HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68 and others are found commonly in both LSIL and HSIL and account for the majority of HPV types found in invasive carcinoma. To qualify as a distinctive HPV type, a virus must be cloned and demonstrate less than 90% DNA sequence identity with known HPV types at three specific loci within the HPV genome (1).
Human papillomaviruses, like other viruses, are unable to independently replicate their own DNA. Thus, completion of the HPV life cycle requires the metabolic machinery of infected host cells. HPV infection is initiated in the basal epithelial layer, which contains mitotically active cells that replace mature senescent cells shed from the surface (2). The basal layer is reached, presumably, through microscopic mucosal defects. Infected basal cells contain 20 to 100 copies of episomal HPV DNA. Expression of the late viral genes occurs in mature squamous cells, resulting in the production of capsid (covering) proteins and whole virus particles. At the light microscopic level, this process corresponds to LSIL (CIN I and koilocytotic atypia). In most carcinomas, HPV DNA replication is not entirely episomal. Integration of HPV DNA into human host DNA may represent a crucial step in cervical carcinogenesis, specifically the development of invasiveness.
HPV testing methods have been recently reviewed. The majority of HPV tests detect HPV DNA. Identification of HPV mRNA transcripts is of great research interest, but detection of RNA requires special procedures because RNase is ubiquitous in the environment. HPV DNA testing can be performed on fresh, frozen or fixed cells or tissue. Sensitivity is generally reduced in fixed specimens due to loss of DNA integrity. Cells for HPV testing may be obtained via lavage or by brushing, swabbing, or scrapping the cervix. Each of these strategies has its proponents, but results using these collection methods are similar. A major consideration in testing gynecologic specimens is that over 20 different HPV types infect the cervix. Therefore, HPV tests that employ too few type-specific probes will not detect a significant percentage of infections. Avoidance of specimen-to-specimen contamination in the clinic and the laboratory is essential to avoid false-positive results. This is a critical problem when amplification techniques such as PCR are employed (3).
Southern Hybridization: It is the reference method for HPV typing. When applied to fresh material, this technique is highly sensitive; in fixed material, sensitivity is reduced. In Southern hybridization, DNA extracted from cellular samples is cleaved with restriction enzymes recognizing specific DNA sequences. It is a complex, labor-intensive procedure requiring several days to perform and necessitating the use of radioactive material for optimal sensitivity. Small sample size and fixation may reduce reliability and sensitivity. Since the DNA is extracted from the material, it is unclear which of the cells in the sample the source of the HPV DNA detected is; no tissue localization is possible with this method. Southern hybridization however does provide adequate sensitivity for detection of HPV in SILs and carcinomas and also permits specific HPV typing. The DNA banding patterns resulting from electrophoresis and the signals that are obtained with type-specific probes provide superior typing data compared to that obtained with dot blot hybridization (4).
Dot Blot Hybridization: It is well suited to analyzing large numbers of fresh specimens. Spots of denatured DNA that is loaded onto filters or members are tested with HPV type-specific DNA or RNA probes. The material is not digested with restriction endonucleases or electrophoresed. Information obtained from banding patterns is unavailable, and high background related to non-specific adherence of probes to the filter may limit interpretation. Radiolabeling or enzymatic detection methods can be used. The simplicity of the procedure facilitates testing many samples at once and yields results within two days (5).
In Situ Hybridization: It combines detection of HPV nucleic acids with morphology, thus permitting the localization of HPV infection to specific cells or tissues. Reliable in situ hybridization kits are commercially available for clinical use. Signal detection is achieved using either (a) an avidin-biotin system similar to that employed in immunoperoxidase procedures or (b) radiolabled probes. Use of biotin probes provides results in one day instead of four, allows the storage of probes for up to one year, and avoids logistical problems related to the handling of radioactive material (6). The sensitivity of in situ hybridization is less than that of blotting techniques, with detection in the range of 1-20 HPV copies per cell. This level of sensitivity is useful in studies of SIL, but some carcinomas may contain too few viral copies for detection, and the majority of HPV infections in cytologically normal subjects cannot be identified. In addition, multiple slides are required to test with multiple probes and controls.
Hybrid Capture: It is a newly devised liquid-based HPV test that permits rapid detection and quantitation of HPV DNA in large number of samples. In cytology, this technique has been used primarily to test exfoliated cells suspended in ViraPap buffer (Digene Diagnostics) and other liquid buffers. The sample of DNA is denatured and then mixed with cocktails of 14 type-specific HPV RNA probes in a microtiter tray. The wells in which the reaction occurs are coated with an antibody that binds DNA-RNA hybrids. The recent introduction of a genomic probe to measure the total cellular DNA in the specimen may permit the calculation of the ratio of the amount of type-specific HPV DNA to total cellular DNA in the sample, thus providing a crude measure of viral load. Results if Hybrid Capture testing show good inter-and intra-laboratory reproducibility and correlate will with conventional HPV tests such as Southern blot. The test is rapid, and probes recognize all the major pathogenic HPV types that infect the female genital tract. The test is somewhat less sensitive than PCR and is prone to rare sporadic false-positive results (7). FDA-approved test for risk HPV is Hybrid Capture II test.
Polymerase Chain Reaction (PCR): It is not an HPV DNA detection technique, but rather a method for specifically amplifying DNA, which can then be tested for the presence of HPV DNA using Southern blot, dot blot, or another technique. As a result of DNA amplification, PCR may detect as few as one HPV copy per million cells. In PCR, a DNA primer set is used to initiate multiple rounds of sequence-specific DNA replication. PCR-based HPV tests are especially important in epidemiologic studies and clinical studies of older women in which detection of low-level infection in normal subjects is important. The added sensitivity of PCR-based methods is less advantageous in studies of prevalent cervical neoplasia because most HPV-related lesions contain enough viral copies to be detected without amplification (8).
HPV Testing Strategies To Improve Pathologic Diagnosis and Clinical Management:
In 1996 the National Cancer Institute began a prospective randomized trial comparing three management strategies for women with cervical cytology reports showing atypical squamous cells of undetermined significance (ASCUS) and LSIL (9). The patients were randomized into a conservative (repeat cytology every 6 months only) group, a colposcopy (immediate colposcopy of all patients) group, and a secondary triage (HPV testing for types of HPV known to place a woman at high risk for cancer) group. The LSIL arm was closed early when it was determined that greater than 80% of the women with LSIL results were HVP positive. The recommendation of the ASCUS/LSIL Triage Study (ALTAS) trial is that HPV testing is not useful for women with LSIL test results. The ASCUS arm of the ALTS trial has been completed, and the results were included in the report of the American Society of Colposcopy and Cervical Pathology (ASCCP) Consensus Conference (10). The recommendation of the consensus conference was that any of the three management strategies (a) repeat cytology [repeated at 4-6 month intervals until two consecutive "negative for intraepithelial lesion or malignancy" results are obtained], (b) immediate colposcopy, or (c) HPV triage is an acceptable approach for the management of patients with ASCUS cytology.
Secondary triage using HPV testing is recommended as the preferred strategy when liquid-based preparations were used for cervical cytology or when co-collection for HPV DNA testing was done. If the HPV test result is negative. It is not necessary to repeat cytology for 12 months. If it is positive, the patient should have colposcopy. If the original cytology report was ASC-H (atypical squamous cells, cannot rule out HSIL), the patient should undergo colposcopy without HPV testing. Algorithms describing these ASCCP-recommended management strategies are available (11).
Pregnant women with ASCUS or ASC-H cytology results do not require different management. Postmenopausal women with ASUS can have repeat cytologic evaluation after treatment with a topical estrogen if there is no contraindication. If the result is again ASCUS, colposcopy is suggested. Immunocompromised women with any cytology abnormality should undergo colposcopy (12).
Recognition of the central role of HPV in cervical neoplasia may have immediate implications for the diagnosis and treatment of cervical disease. If HPV testing proves to be clinically useful, the traditional Pap smear may be enhanced by a new approach that would combine cytologic screening with ancillary testing of samples. It is likely that methods of cervical cytology collection and interpretation will continue to change in the future. It will be necessary for the clinicians to remain informed of new technologies and to access the appropriateness of their use in their individual practices. Currently, HPV DNA testing is being examined as a screening tool for the detection of women with CIN in developing countries where facilities able to interpret cervical cytology are lacking. All women with high-risk positive results would be sent for colposcopy. This approach is unlikely ever to be useful in countries where cervical cytology screening is already well established because of the cost of a single HPV test and the high prevalence of HPV, especially in women younger than 30 years.
- Cervical cytology screening. ACOG Technology Assessment in Obstetrics and Gynecology No. 2. American College of Obstetricians and Gynecologists. Obstet Gynecol 2002;100:1423-7
- Dunne EF, Unger ER, Sternberg M et al. Prevalence of HPV infection among females in the United States. JAMA 2007;297:813-819
- Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial. The Atypical Squamous Cells of Undermined Significance/ Low-grade Intraepithelial Lesions Triage Study (ALTS) Group. J Natl Cancer Inst 2000;92:397-402
- Kuhn L, Denny L, et al. Human papillomavirus DNA testing for cervical cancer screening in low-resource settings. J Natl Cancer Inst 2000;92:818-25
- Wright TC Jr, Cox JT et al. Consensus Guidelines for the management of women with cervical cytological abnormalities. JAMA 2002;287:2120-9
- Gravitt PE, Peyton CL, Alessi TQ et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000;38:357-361
- Centers for Disease Control and Prevention. Genital HPV Infection Fact Sheet. Rockville MD: CDC National Prevention Information Network; 2004
- World Health Organization (WHO) Initiative for Vaccine Research. Human Papilloma Infection and Cervical Cancer. Geneva, Switzerland: WHO; 2008. http://www.who.int/vaccine_research/diseases/hpv/en/ Accesses May 10, 2008
- Bonnez W. Papillomavirus, In: Richman DD, Whitley RJ, Hayden FJ, eds. Clinical Virology. 2nd ed. Washington, DC: American Society for Microbiology Press; 2002:557-596
- Stoler MH, Sciffman M. Interobserver reproducibility of cervical cytologic and Histologic interpretations: realistic estimates from the ASCUS-LSIL Triage Study. JAMA 2001;285:1500-5
- Braaten KP, Laufer MR. Human papillomavirus (HPV), HPV-related Disease, and the HPV vaccine. Rev Obstet Gynecol 2008;1(1):2-10
- Kahn JA, Rosenthal SL, Jin Y et al. Rates of human papillomavirus vaccination, attitudes about vaccination, and human papillomavirus prevalence in young women. Obstet Gynecol 2008;111:1103-1110
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