Auro-Human Papilloma Virus (HPV) HC Test

Introduction
Cancer is a growing health problem in developing countries. More than half the cancer deaths in the world in 1997 occurred in developing countries (Van Asten, 1997, Gopalan et al.,1999). Cervical and uterine cancers accounted for more than 50% of all cancers. Infection with oncogenic human papilloma virus (HPV) is the major risk factor in the etiology of cervical cancer. Approximately 30 types cause infection of genital mucosal sites, and these genital types are generally characterized as "high-risk" types (e.g., HPV 16, 18, 31, 33, 35, 39, 45, 51, 52), which are associated with low and high-grade squamous intraepithelial lesions (LSIL and HSIL) and invasive cancer, and "low-risk" types (e.g., HPV 6, 11, 42, 43, 44), which are primarily associated with genital warts, LSIL, and recurrent respiratory papillomatosis (RRP) (Lorincz A,1997). Twenty per cent of all female deaths from cancer in India in 1990 were from cervical cancer, amounting to an estimated 61,000 deaths from this cause. The number of known cervical cancer deaths in women in India is projected to increase to 79,000 by the year 2010 (Murray et al.,1990, Gopalan et al., 1999).

Shortcomings of available diagnostic assays
It is well recognised that effectively identifying and treating precancerous lesions must occur to prevent cervical cancer. Cytological screening by PAP smear allows for the detection of both precancerous lesions and early invasive cancer (Ferenczy A,1989) resulting in the reduction in the incidence of cervical cancer since its introduction in the 1950s.

Despite being hailed as a breakthrough in medical science that has saved countless lives, the Pap Smear has its shortcomings which can lead to both false negative (10-50% of high grade lesions and cancers may go undetected) and false positive (the Pap can falsely suspect disease in 5-10% of cases when there is none) results. Due to the fact that the Pap Smear cannot detect all women with precancerous lesions, and cannot predict which women with low-grade epithelial abnormalities are likely to develop subsequent high-grade epithelial abnormalities, women have had to be rescreened at frequent intervals (Lorincz, 1997).

In an effort to reduce the number of false Pap smear results, much emphasis has been placed on improving cervical screening. So far, focus has been placed on improving the sensitivity of the Pap Smear through computer-assisted screening and rescreening, and/or sampling and preparation of the slide, but the limitations of the Pap Smear still remain. This therefore results in a continued reliance on screening at short intervals at a higher cost to pick up the false negative tests.

HPV DNA Testing by Hybrid Capture II Test
By introducing a standardized, objective HPV detection test, the sensitivity and accuracy of cervical screening is significantly enhanced. HPV Testing has proven itself in many studies as a sensitive and specific test for CIN II/III and cancer.

Infection with oncogenic HPV is the major risk factor in the etiology of cervical cancer.

Since the presence of persistent infection with high risk HPV DNA types is considered to be necessary for cervical cancer to develop, HPV testing is prognostic. The presence of high risk HPV DNA (even in the absence of normal cytology) can identify women both with disease and those who are at particular risk of progressing to disease.

Conversely, the absence of HPV DNA can identify women who are unlikely to develop a cervical lesion, or progress to a high-grade epithelial abnormality from a low-grade epithelial abnormality. As a result, it has been suggested that the screening and follow up interval for women with negative High Risk HPV DNA tests (using Hybrid Capture II) could be increased.

The sensitivity for the detection of high grade cervical lesions using the Digene HPV test is 98%, much higher than conventional cytological screening by PAP test. Among those presenting with equivocal PAP, the negative prediction value of the Digene HPV test for high grade lesion or greater at colposcopy is 99%. Compared with HPV PCR the hybrid capture technology is better because it picks up a long stretch of the entire genome of HPV DNA, therefore ruling out false negatives which may be reported by PCR due to genitic mutations in the virus.

There are four key areas where HPV Testing can be used to enhance cervical screening programs

	Triage of women with Pap smears showing Low Grade Epithelial Abnormalities (LGEA) - i.e. atypical cells, non-specific minor changes, criteria of HPV effect, mild dysplasia and inconclusive reports
	Management of Low Grade Epithelial Abnormalities predicting the progression, persistence or regression of untreated histologically confirmed CIN 1
	Population Screening for cervical neoplasia
	Confirmation of disease and HPV cure

Triage of women with Pap smears showing Low Grade Epithelial Abnormalities - i.e. atypical cells, non-specific minor changes, criteria of HPV effect, mild dysplasia and inconclusive reports (Bethesda Reporting System - ASCUS category)

The US Food and Drug Administration approve the Digene HPV Test for use in this setting. Further, the American Society for Colposcopy and Cervical Pathology endorse HPV testing as being an option entirely within the recent National Cancer Institutes management guidelines for ASCUS (Atypical Squamous Cells of Undetermined Significance) smears.

Although most LGEA's spontaneously regress, there is an increasing pressure to refer these for further investigations because 7-20% of these patients will really have a CIN II/III (HGEA)[8].

A positive High Risk HPV Test, when combined with a pap report of this nature is predictive of two things:

	CIN II/III or cancer which was not detected on the Pap smear (7-20%)
	The risk for subsequent high-grade epithelial abnormalities.

Women with a Pap Smear showing atypical cells, non-specific minor changes, HPV effect and inconclusive (ASCUS) together with a positive test for High Risk HPV DNA should proceed directly to colposcopy.

US FDA approved Digene HPV test for use for women with PAP smear showing low grade epithelial abnormalities.

Conversely, a negative High Risk HPV Test means these women are unlikely to have a high-grade epithelial abnormality that was missed on the Pap Smear (Negative Predictive Value = 99%), or are at low risk for subsequent high-grade epithelial abnormalities.

Michele Manos from the Kaiser Foundation Research Institute, Oakland, California demonstrated that by using HPV as a secondary test in addition to the initial Pap smear in cervical cancer screening, there was an increased sensitivity for detecting High Grade Squamous Intra-epithelial Lesions (HSIL) while reducing the number of colposcopies women would have undertaken to get the same result.

Cervical Specimens from 46,000 women presenting themselves for routine Pap smears were collected for conventional cytology, liquid based cytology and High Risk HPV DNA detection by Digene's Hybrid Capture II System. Both ASCUS (Atypical Squamous Cells of Undetermined Significance) and AGUS (Atypical Glandular cells of Undetermined Significance) smears were investigated. The initial results indicated that Undetermined Significance) smears were investigated. The initial results indicated that on colposcopic investigation, 81% of the equivocal Pap smears were classified as normal. The study showed that by using the Hybrid Capture HPV DNA test to manage the equivocal smears, 88% of women with HSIL (High Grade Squamous Intra-epithelial Lesion) were identified. In comparison a repeat Pap Smear identified only 75% of HSIL women. The data was even more compelling in the group of women under 30, where HPV was detected in 100% of those women who really had HSIL. The repeat Pap was abnormal in 64% of the cases with HSIL (Van Asten, 1997)

Dr Walter Kinney, of the Division of Gynaecological Oncology at the Permanente Medical Group Inc, California discussed his experience in a study of the same group of women, using the Hybrid Capture II Test alongside the Pap smear, at the European HPV Clinical Summit Meeting in Vienna, January 1998. In the "critical borderline" group the HPV Test was found to be positive in 39%. The negative predictive value was 99% for histologically defined CIN II/III at colposcopy. Dr Kinney believes that up to 50% of women with mild cellular changes viewed on the Pap Smear could avoid colposcopy altogether.

	Management of Low Grade Epithelial Abnormalities predicting the progression, persistence or regression of untreated histologically confirmed CIN I. Up to 60% of CIN I lesions will spontaneously regress, and up to 25% of CIN I lesions will progress over a two year period if left untreated (NHMRC,1995) High Risk HPV DNA allows for predicting the likelihood of a woman progressing to high-grade epithelial abnormalities. Treatment of CIN I is indicated when a positive High Risk HPV Test is obtained. Conversely a negative result for High Risk HPV means that the women is unlikely to develop high-grade epithelial abnormalities. These women could be followed conservatively by repeat Pap Smears. Once again it has been suggested that the interval of the repeat Pap Smears could be increased from 6 months, to 12 months interval. Several studies have now demonstrated that essentially all low grade CIN that is progressive to high grade CIN is positive for high risk HPV types (Campion et al., 1986 and Greenber et al., 1997) Cox has cited a study by Remick et al., which has shown that all women presenting with abnormal cervical cytology who were persistently high risk HPV positive for up to 36 months of follow up progressed to CIN III. On the other hand, none of the women who were high-risk HPV negative progressed. Prospective studies by Koutsky et al., 1992, have also shown that despite normal cytology 15-28% of HPV DNA positive women developed SIL within 2 years, compared to only 1-3% of HPV DNA negative women. The risk of progression for high-risk HPV types was greater (40%) than for other HPV subtypes (Koutsky et al.,1992). The Portland Prospective Study [4] set out to determine the risk of new SIL following a single positive test for HPV DNA and to clarify the natural history of HPV infection in cytologically normal women. They identified that up to about 65% of HPV positive women (normal cytology) had developed SIL within 5 years.
	Population Screening for cervical neoplasia Modern HPV Tests are sensitive for CIN II/III and cancer. Professor Cuzick of the Imperial Cancer Research Fund in London, has reported that in a study of 3000 women aged >35 years, the Digene HPV Test was better than the Pap Smear, correctly identifying 90% of women with CIN II or higher compared with 75% of women correctly identified using the Pap Smear alone (Cruzick et al., 1998). This work is now being expanded to investigate the screening utility of the Digene HPV Test in a greater sample of 10 000 women. Professor Cuzick estimates that in the UK, £30 million could be saved by reducing the number of colposcopies and extending the screening interval where appropriate. Data from a screening study conducted in Costa Rica, was presented by Lorincz at a recent conference in Montreal. The Hybrid Capture II HPV Test detected 100% of the cervical cancers and 90% of the CIN II/III cases in a single visit compared with detection of 90% of cervical cancers and 78% of CIN II/III using the conventional Pap Smear.
	Confirmation of disease and HPV cure It is commonly assumed that once a woman is infected with HPV it stays for life. It has been determined that although it is possible that the virus does not entirely disappear, that HPV becomes undetectable by the most sensitive tests in about 50% of women within a year of initial detection. The immune system suppresses or eliminates most HPV, and persistent infection with HPV is the risk factor for malignant progression (Ho GYF et al, 1995). Thus, testing women for HPV after treatment could be a way of determining cure and could replace the multiple repeat Pap smears and colposcopies now required to ensure the patients safety. Elfgren et al., have shown that HPV DNA disappeared in 14 of 18 HPV positive women who underwent conization for high grade CIN or suspected microinvasion. The 4 HPV positive women post treatment were shown to have CIN on follow up examinations.

Modern HPV tests are sensitive for CIN II/III & Cancer
A study by Cox et al., cited in Lorincz (Lorincz A, 1997) reported 20 of 24 women with HPV positive CIN who were treated by cryotherapy became HPV negative at the 4-6 month follow up colposcopy, and that only one of these HPV negative women had Carcinoma In Situ I (CIN I). In contrast all four women who remained HPV positive for high risk HPV types had colposcopy or biopsy proven CIN.

Sampling & Collection and Transportation
Specimens for HPV testing may be obtained using the Digene Cervical Sampler. This comprises a cervical brush and a tube containing specimen transport medium into which the brush is inserted after specimen collection.

Specimens taken with other sampling devices or transported in other transport media have not been qualified for use with this assay. Cervical specimens must be collected prior to the application of acetic acid or iodine if colposcopic examination is being performed.

Cervical Brushes*
The HPV Test is designed for use with specimens collected and transported using the Digene Cervical Sampler (Digene Cervical Brush and Specimen Transport Medium). Specimens may be held for up to two weeks at room temperature, after which specimens can be stored an additional week at 2-8°C. A preservative has been added to the Specimen Transport Medium to retard bacterial growth and to retain the integrity of DNA.

Cervical Biopsies*
Freshly collected cervical biopsies up to 5 mm in cross-section may also be analyzed with the Digene HPV Test. The biopsy specimen must be placed immediately into 1.0 ml of Specimen Transport Medium and stored frozenat -20°C. Biopsy specimens may be shipped at 2-30°C for overnight delivery to the testing laboratory and stored at -20°C until processed. Biopsies less than 2 mm in diameter should not be used.

Specimens in Cytyc PreservCyt Solution
Specimens collected using a broom-type collection device and placed in Cytyc PreservCyt Solution for use in making ThinPrep Pap Test slides can be used in the Digene HPV Test. Specimens should be collected in the routine manner, and the ThinPrep Test slides should be prepared according to Cytyc instructions.

There must be at least 4 ml of PreservCyt Solution remaining for the Digene HPV Test. Samples with less than 4 ml after the Pap Test has been prepared may contain insufficient material and could be falsely negative in the Digene HPV Test.
PreservCyt Solution specimens may be held for up to three weeks at temperatures between 0°C and 3°C.

*NOTE: To prevent caps from popping off specimens that are shipped or stored frozen (for STM specimens or converted PreservCyt specimens):
1. Cover caps with Parafilm prior to shipping specimens previously frozen. Specimens may be shipped frozen or ambient.

References
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	Cuzick J, Beverley E, Ho L, Terry G, Soutter P, Chan W-K, Sapper H. 1998. HPV Testing in Primary Screening of Older Women Presented at European HPV Clinical Summit Meeting.
	Koutsky LA, Holmes KK, Critchlow CW, Stevens CE, Paavonen J, Beckmann AM, et al. 1992. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. N Engl J Med;327:1272-1278.
	Manos MM, et al. 15th International Papillomavirus Workshop, November, 1996 at Gold Coast (Abstract)
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	Elfgren K, Bistoletti P, Dillner L, Wallboomers JMM, Meijer CJLM, Dillner J. 1996. Conization for cervical Intra-epithelial neoplasia is followed by disappearance of human papillomavirus deoxyribonucleic acid and a decline in serum and cervical mucus antibodies against human papillomavirus antigens. Am J Obstet Gynecol;174:937-942.
	Murray CJL, Lopez AD.1996. Global burden of disease : a comprehensive assessment of mortality and disability from diseases, injuries and risk factors in 1990 and projected to 2020. Global burden of disease and injuries series, vol 1. Boston: Harvard school of public health;.
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