High-risk HPV testing in women with borderline and mild dyskaryosis: long-term follow-up data and clinical relevance.

In The Netherlands and most other European countries, women with two serial cervical smears with borderline or mild dyskaryosis (BMD) within 6 months are referred for colposcopy-directed biopsies. Only about 10% of these women have high-grade cervical intraepithelial neoplasia (CIN). This study therefore investigated whether human papillomavirus (HPV) testing could identify which women with smears read as BMD are most likely to have high-grade CIN, either at referral or during follow-up and the relationship was determined between clearance of high-risk HPV and regression of abnormal cytology. Women with smears read as BMD (n=278) were referred to the gynaecologist for colposcopy. They were subdivided into two groups; group A comprised women with a single smear (n=172) and group B women with two sequential smears (n=106) read as BMD before referral. High-risk HPV detection with Hybrid Capture II (HC II) was performed on a cervical scrape taken at the first visit before colposcopy (i.e. baseline smear) and during follow-up. Biopsies were taken when lesions suspected for CIN were seen at colposcopy. High-risk HPV DNA was present in the baseline smears of 126 (45.0%) women; 26 (20.6%) of them had histologically confirmed CIN 2/3 at the first visit and another 14 (11.1%) during follow-up. Only one of the 152 women (0.7%) with a negative high-risk HPV test had a CIN 2 lesion at the first visit and no CIN lesions were detected during follow-up of these women. After exclusion of women who were treated for prevalent high-grade CIN, the median follow-up times were 1.3 years (range 0.0-4.3 years) and 1.6 years (range 0.0-4.5 years) for women with HPV-negative and HPV-positive baseline smears, respectively. The sensitivity of a positive high-risk HPV test for CIN 2/3 at the first visit was 96.3%, the specificity 60.2%, the positive predictive value 20.6%, and the negative predictive value 99.3%. These values did not change markedly when stratified for group A or group B. Thus, a high-risk HPV positive test was strongly associated with the presence at the first visit and the development of CIN 2/3 lesions during follow-up. Moreover, regression of abnormal cytology in women with a positive high-risk HPV test at baseline was strongly associated with viral clearance and occurred 0.3 years (range -1.2 to 1.7 years) later than HPV clearance. This study establishes the value of a high-risk HPV positive test for women at risk of high-grade CIN, with virtually no risk for missing CIN 2/3. Addition of a test on high-risk HPV in women with BMD could prevent 55% of the referrals and/or repeat smears.

HPV presence precedes abnormal cytology in women developing cervical cancer and signals false negative smears.

In a retrospective case-control study, we investigated high-risk HPV DNA presence by general primer GP5+/6+ PCR in the last normal cervical smear in the patient archives (i.e. baseline smear) of 57 women who later developed cervical cancer. Also, normal cervical smears of 114 age-matched control women were analysed. High-risk HPV DNA was detected in 37 of the 57 (65%) baseline smears of the case women, and 7 (6%) of 114 smears of the control women (OR 28, 95% Cl 11-72). The HPV positive subsequent smears and cervical cancer biopsies of the case women contained the same HPV type as was detected in the baseline smear. After cytological revision, the baseline smears of 48 case women (84%) were reclassified as abnormal, 33 (69%) of which scored high-risk HPV DNA positive. Ultimately, an undisputable normal baseline smear was found in only 10 case women. In 7 (70%) of them this smear was HPV positive, whereas only 7 (7%) of 104 revised, undisputable normal smears of control women were high-risk HPV positive (OR 32, 95% Cl 6.8-153). The results showed that (1) high-risk HPV presence precedes abnormal cytology in women who develop cervical cancer, and (2) high-risk HPV testing signals false-negative smears of women at risk of cervical cancer.

A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears.

The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the beta-globin gene appeared to be superior for the GTC-based assays. Using competitive beta-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 x 10(5) to 3 x 10(5) genome equivalents in smears containing 5 x 10(5) to 20 x 10(5) nucleated cells, indicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3-6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 +/- 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears.

Correlation between serum alkaline phosphatase and localization of alkaline phosphatase in the liver.

1. A good correlation exists between histochemically judged and biochemically determined activity of alkaline phosphatase in the liver. 2. Normal localization of alkaline phosphatase in the human differs from that in the rat. 3. In Wistar rats normal localization is: low or no activity in bile canaliculi preferently in the peripheral part of the lobule. 4. Normal localization in the human is: moderate to strong activity in the sinusoidal wall, in the central and peripheral part of the lobule. 5. A relation exists between histochemical localization and serum value of alkaline phosphatase in rat and in human. 6. Raised activities at localizations where "normally" no activity is present, e. g. bile canaliculi in human, sinusoidal wall in rat, do correlate the best with raised serum activities.