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10/23/03
Human Papillomavirus
J. Sanders Sevall, Ph.D. & Stacey B. Weston, Ph.D.
More than 90 different human papillomaviruses (HPVs) have been identified. All are small DNA viruses that can infect epithelial cells and can cause a variety of hyperplastic leisons.^1 Most HPVs are associated with warts (or papillomas), which are benign tumors, either in the skin or in oropharyngeal, esophageal, or genital mucosa. Cutaneous and genital warts can be eradicated by topical therapy, freezing, or surgery if they are unattractive or uncomfortable. If left untreated, warts often regress spontaneously. ^1 Each HPV type has its own tissue specificity, clinical association and risk of malignant transformation. Cutaneous palmar and plantar warts are widespread and contain HPV type 1, 2, 3, 4 or 10 and sometimes type 28 or 29 and type 26, 27 or 28 in immunocompromised patients.^2 The risk for malignant transformation is essentially zero. Epidermodysplasia verruciformis is a rare hereditary skin disorder that develops into skin cancer in sun-exposed areas and is
associated with rare HPV types, particularly types 5 and 8, but also types 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 46, 47 and 50. Laryngeal papillomas are benign epithelial tumors associated with HPV types 6 and 11.
About 20 HPV types are associated with genital warts, or condyloma acuminata, the third most common sexually transmitted disease, and rapidly increasing in incidence. About 90% of exophytic condyloma acuminata are associated with "low risk" HPV-types 6 and 11.^3 Other "low risk" types include 30, 42, 43 and 44.^2,4 Genital malignancies, including moderate to severe dysplasia and invasive carcinoma of the cervix, vulva, anus and penis^5 are associated with the "high risk" HPV types 16, 18, 45 and 56. The "intermediate risk" group includes HPV 31, 33, 35, 39, 51, 58 and 61.^3 HPV DNA is detectable by PCR in 92% of squamous cell carcinomas of the uterine cervix and 75% of adenocarcinomas of the uterine cervix.
HPV type 16 predominates in 50-60% of cervical cancers; HPV type 18 is found in 10-20%. HPV 45 and HPV 31 are the next most common HPV types in invasive cervical carcinoma with frequencies of 5-10% each. Squamous cell carcinoma is associated with HPV type 16 (HPV type 16 [78%] versus HPV type 18 [16%]); whereas, adenocarcinoma is associated with HPV type 18 (HPV type 16 17% versus HPV type 18 [56%]).^6 Phylogenetically, the HPV from the "high" and "intermediate" groups can be divided into three carcinogenic groups: HPV type 16-related (HPV 16, 31, 33, 35, 52 and 58), HPV type 18-related (HPV 18, 39, 45, 59 and 68) and HPV type 51-related (HPV 51 and 56).^7
In situ hybridization (ISH) can be used to detect and type HPV DNA, while at the same time retaining the tissue architecture, thus ensuring that the histopathological lesion is the source of HPV DNA.^8 Partially degenerate non-radioactive biotinylated probes for HPV types 6/11, 16/18 and 31/33/35 show HPV type 6/11 in 100% of condyloma acuminata; flat venereal warts contain both HPV types 6/11 (4%) and 16/18 (62%), and CIN 1-3 contain either HPV types 16/18 or 31/33/35.^9,10 Using ISH probes to HPV types 6, 11, 16, 18, 31, 33 and 51, HPV-DNA is detectable in 18% of CIN I, 30% of CIN II, 28% of CIN III, 84% of flat condyloma, 11% of cervical adenocarcinoma, 21% of squamous cell carcinoma and 13% of normal cervical tissue.^11 The most common consensus primer set for the detection of HPV by polymerase chain reaction (PCR) uses the L1 region of the genome, but its length of 450bp hampers efficient amplification, especially in formalin-fixed tissue, or if L1 is deleted, which
commonly occurs in invasive cancer. The E6 and E7 regions can be used, but type-specific primers are required, which limits the detection range of HPV types.^12Consensus primers to the combined E1/L1 region detect diverse HPV types,^13 and probe hybridization^14,15 or sequencing^16 is used to identify the specific HPV type. PCR is more sensitive than Southern blot, and detects HPV sequences in 90% versus 57% of stage I and II cervical cancer.^17 The discordance is due to the inability of Southern blot to detect low copy number. The likelihood that CIN 2 or worse is present when HPV types 16, 31, or 33 and 18 are detected by PCR is 88%, and 36%, respectively.^18 The presence of HPV 16 correctly predicts CIN 2 or worse in 93% of patients.^19 When dot blot, PCR and cytology are used in combination, the detection rate for any CIN is 91%.^20 HPV is detectable in the urine by PCR in 87% of women with condylomata, CIN 1 or worse.^21
Hybrid capture, where DNA target/RNA probe hybrids are detected by anti-RNA/DNA antibody, distinguishes low-risk HPV types 6, 11, 42, 43, and 44 from intermediate- and high-risk types 16, 18, 31, 33, 35, 45, 51, 52 and 56 has replaced dot blot and Southern blot as the method of choice. It has a sensitivity of detecting high-risk squamous intraepithelial lesions of 74% when used alone, and 91% when combined with cytology of cervical scrapings.^22,23 Hybrid capture correlates with PCR.^24
Of all the molecular methods of detection of HPV DNA, only PCR and hybrid capture have enough sensitivity to be used clinically. The detection limit of PCR is 10 to 100 HPV genomes and for hybrid capture the detection limit is about 5,000 HPV genomes whereas Southern blot has a detection limit of about 100,000 HPV genomes.^4 A series of studies support the use of PCR-based techniques for the diagnosis of high risk HPV infection. The advent of rapid real time PCR in the analysis of high risk HPV allows type 16 to be differentiated from type 18 on the basis of melting point^25,which might prove beneficial in screening programs.
Re-screening of HPV-negative carcinomas from a multinational study show that 99.8% of all cervical squamous cell carcinomas are HPV positive.^26-28 In the multinational study, negative HPV results are explained mainly by a lack of morphological confirmation of cervical cancer in the tissue. Of 930 carcinomas only two did not contain HPV-DNA. This very small percentage renders the question whether a high risk HPV-independent pathway for squamous cell carcinoma of the cervix exists or is clinically irrelevant.^29 Also in cervical adenocarcinomas a prevalence of high risk HPV of more than 90% is found.^30,31
Prevalence of high risk HPV types in women with normal cervical cytology is age dependent, reaching a peak prevalence of about 10% between 20 and 24 years of age and decreasing to about 3% in women over 30 years.^32,33 Moreover, it appears that in a population based cohort (age 34-54 years) in The Netherlands the acquisition of high risk HPV types is about 0.5% per year while the clearance is about 39% per year, which complies with the prevalence of 3%.^34Therefore, high risk HPV testing in population-based screening for women over 30 years of age is advocated.^35-37
Only women with persistent high-risk HPV infections, either in cytological abnormal or normal smears,^ ,34,35,38-40 show progressive CIN. The progression rate of CIN in women with high-risk HPV-positive, cytologically normal or abnormal smears is about 5% per year. Women with a high risk HPV-positive, cytologically normal smear (age over 30 years) have a 116-times higher chance of developing CIN III than women with an HPV-negative, cytologically normal smear.
Although primary screening by cytology with referral to colposcopy has dramatically reduced mortality and morbidity due to cervical cancer, cytology is inherently insensitive. Pap screening for cervical neoplasia has a sensitivity of about 50-60% and a specificity of about 90%. In an attempt to increase the sensitivity of cytology, patients diagnosed with "atypical cells of undetermined significance" (ASCUS, a heterogeneous mixture of cervical pathology from non-specific inflammatory changes, subtle HPV infection to a pre-cancerous lesion) are referred to colposcopy; however, many women with minor smear abnormalities will be negative at colposcopy.^41 The "gold standard" for the final diagnosis is determined by histology at colposcopy, which can be categorized as a high-grade intraepithelial lesion (HSIL), low-grade intraepithelial lesion (LSIL), equivocal (histology negative, but cytology ASCUS or higher) or clearly negative (both histology and cytology). Hybrid capture HPV
DNA preformed on repeat Pap smear cytology is predictive of CIN at colposcopy. HPV DNA-positive cases have a final diagnosis of CIN in 30%, 45% and 80% of cases when the initial repeat Pap smear cytology is negative, ASCUS and SIL, respectively; whereas, HPV DNA-negative cases have a final diagnosis of CIN in 5%, 5% and 25%, respectively, for the same respective initial cytology diagnoses.^42 Of 995 women who had ASCUS and also had HPV testing as part of their routine screening, all the women had had either follow-up colposcopy or repeat Pap tests. Results of the HPV testing and histology were available for 973 of the women and of these, 65 (6.7%) were found to have HSILs or, in the case of one women, cancer.^43 In 89.2% of the women who were later discovered to have HSILs, HPV testing done as part of their first screening had been positive. By comparison, repeat Pap testing of these women had been abnormal in 76.2% of the cases but the difference was not statistically
significant.^43 It was concluded that HPV testing makes it possible to identify most women with ASCUS who are at high risk of a subsequent diagnosis of HSIL on the basis of a single screening procedure.
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