Both patients and healthy subjects were nonpregnant white, African-American, or Hispanic women, aged 18 years and older, and residents of Harris County, Tex., at the time of the study. Other eligibility criteria for both groups included no previous history of cervical neoplasia, of treatment for cervical neoplasia or cancer, or of hysterectomy. Patients with a confirmed histological diagnosis of CIN were identified among women referred to the University of Texas M. D. Anderson Cancer Center Colposcopy Clinic (UTMDACC) between September 1991 and August 1994 for further evaluation of an abnormal Pap smear. Of 640 women, 399 were confirmed with CIN; 325 met the other eligibility requirements and agreed to participate. Healthy women were selected from women attending family planning and screening services at two Harris County Health Department clinics serving large, multiethnic populations. Women were eligible for the healthy group when the cytological smear at the time of recruitment was normal and when they had no history of abnormal Pap smear or cervical biopsy. Of 414 women who met all eligibility criteria, 270 agreed to participate.
Patient data collection.
Patients had a complete physical examination, a repeat Pap smear, a colposcopic examination, colposcopically directed biopsies of abnormal areas, and two cervical samples collected for HPV testing. Cytologically normal women had a complete physical examination, a Pap smear, and two cervical samples collected for HPV testing. Exams and specimen collection for cytologically normal women were performed by nurse practitioners trained at the UTMDACC. The first samples for HPV testing were collected with a cotton swab and preserved in the transport medium provided by the manufacturer (Digene). The samples for PCR analysis were then collected with cervical brushes which were placed in vials and frozen immediately.
Cytological and histological diagnoses.
Cytological and histological specimens for all patients were interpreted at the UTMDACC Department of Pathology. Two independent readers at the UTMDACC reviewed each Pap smear and biopsy. A committee of staff members including the Director of the Colposcopy Clinic (M.F.M.) reviewed discrepant cases monthly and reached a final diagnosis. Cytological specimens for the healthy women were read and interpreted at the San Antonio Chest Hospital, in San Antonio, Tex. A high level of agreement in Pap smear diagnoses between that hospital and the UTMDACC was observed (kappa coefficient, 0.85).
DNA extraction-identification of HPV DNA-positive specimens.
The cytobrush specimens were thawed and vortexed in 1 ml of 0.01 M phosphate-buffered saline–5 mM EDTA, pH 7.4. A contamination control, consisting of 1 ml of water, was inserted after every 10th patient sample and subjected to the entire extraction and DNA detection protocol. Specimens were centrifuged at 1,000 × g for 5 min at room temperature. DNA was isolated from each cell pellet by standard phenol-chloroform extraction. Each supernatant from the DNA extraction was centrifuged in a Centricon 100 microconcentrator (Amicon, Inc., Beverly, Mass.) at 1,000 × g for 30 min. Retentates were collected and diluted to 200 μl with water. Ten microliters of this DNA was used in each fluorogenic PCR.
Each DNA specimen was tested for overall HPV positivity by PCR with L1 consensus primers (13
) followed by electrophoresis in ethidium bromide-containing gels. HPV-positive samples were then tested by the quantitative fluorescent probe assay for HPV types 16, 18, 31, and 45.
Probes and primers.
The fluorogenic probe assay is based on the increase in fluorescent signal which occurs when probes are degraded by the 5′→3′ exonuclease activity of Taq
). After degradation, the reporter dyes, FAM (6-carboxyfluorescein) and HEX (hexachlorofluorescein), present at the 5′ ends of the probes can diffuse away from a quencher dye, TAMRA (6-carboxy-tretramethyl-rhodamine), present on or near the 3′ end of each probe, thereby increasing the fluorescent signal from the reporter dyes. The probe sequences for each of the high-risk HPVs (Table ) were selected and synthesized as described previously (18
). The primer sequences (Table ) were selected by using the Oligo 5.0 primer analysis program (National Biosciences, Inc., Plymouth, Minn.). The primer pairs for each of the HPV types were selected based on having a Tm
of approximately 65°C, predicted lack of cross-hybridization to other common HPV types, no predicted loop formation, and no predicted dimer formation with the other primer.
TABLE 1 Primers and probes used in HPV fluorogenicassay Assay controls.
Control templates for HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56 were prepared by PCR amplification of cloned DNA with L1 type-specific primers (sequences available on request). The DNA concentrations were determined by fluorometry (DyNA Quant 200; Amersham Pharmacia Biotech, Piscataway, N.J.). Assay controls, consisting of a dilution series of the homologous template (1 × 105 to 3 × 101 copies) and a set of heterologous templates (2 × 103 copies each of HPV types 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, and 56 in separate tubes), were included in each run. Each control sample also contained 50 ng of human placental DNA. Significant cross-reactivity was not normally observed with any of the heterologous templates. Data was utilized only from assays in which the controls registered <50 copies of each heterologous template.
The 50-μl PCR mixtures contained 10 mM Tris (pH 8.3), 50 mM KCl, 4.5 mM MgCl2, 200 μM deoxynucleoside triphosphates, 0.3 μM (each) primer, 50 nM (each) fluorogenic probe (FAM-HPV probe and HEX-globin probe), 0.025 U of AmpliTaq Gold DNA polymerase (The Perkin-Elmer Corp., Norwalk, Conn.) per μl, and 10 μl of template DNA. Following Taq polymerase activation and template denaturation for 12 min at 95°C, amplification conditions were as follows: 40 cycles of 30 s at 94°C, 10 s at 60°C, and 2 min at 65°C. Amplification was carried out in a Perkin-Elmer 9600 thermal cycler (Perkin-Elmer), after which the samples were transferred to a MicroFLUOR W, 96-well, white microtiter plate (Dynatech Industries, Inc., McLean, Va.), and the fluorescence was measured in a Perkin-Elmer LS-50B luminescence spectrometer. Data acquisition and analysis were performed with the TaqMan Fluorescence Data Manager (Perkin-Elmer) and Excel 5.0 (Microsoft Corporation, Redmond, Wash.). None of the contamination controls tested positive.
Copy number determination.
The spillover fluorescence from the FAM (HPV) channel into the HEX (globin) channel and vice versa was calculated from two sets of control samples, one containing both probes but only HPV template and the second containing both probes but only globin template. Included with each set of patient specimens were the assay controls and a dilution series of non-HPV-containing human cellular DNA. Plots of the homologous template dilution series fluorescence versus log (template copies) were linear over the range of 50 to 109 copies, thus allowing HPV copy number to be determined from the fluorescence in patient samples. Globin copy numbers in each specimen were determined similarly. All patient samples were assayed at least twice; samples with copy numbers >109 were diluted and retested. Patient copy numbers were the average of at least two determinations.
Deletion of DNA from the HPV genome is known to occur on integration; however, in most cases integration occurs in the E1-E2 region and the L1 open reading frame is retained (17
). In addition, integration occurs only rarely in CIN lesions (6
). Thus, the L1 copy numbers reported here should reflect the complete genome copy numbers.
The positive threshold for each assay was the average signal in all contamination controls plus two standard deviations. The thresholds were different for each assay: HPV16, 37 copies; HPV18, 60 copies; HPV31, 34 copies; and HPV45, 96 copies. The sensitivity of the consensus primer PCR test used to select HPV-positive specimens (see above) was also about 100 copies. Comparison of median copy numbers between types requires that the threshold be set at or above the highest value for any type. The threshold in the present study was set at 100 copies. From the globin and HPV copy numbers, HPV copies per microgram of human cellular DNA were calculated. Determinations of the percentage of infected cells in each sample were not made, and hence copies per infected cell could not be calculated.