Objective
This study was conducted to determine whether the human papillomavirus (HPV) genotype by the HPV DNA chip test (HDC) is predictive of residual or recurrent high-grade cervical intraepithelial neoplasia (CIN) 2-3 following a loop electrosurgical excision procedure (LEEP).
Study Design
Between January 2001–February 2007, 672 patients with CIN2-3 were treated by a LEEP and followed up with cytology, the hybrid capture II assay, and the HDC.
Results
A total of 37 (5.5%) patients developed a recurrence, and those who developed a recurrence tested positive for the same high-risk (HR) HPV genotype before and after the LEEP. The same HR-HPV genotype by the HDC during the follow-up had a sensitivity and negative predictive value of 100% for detecting residual/recurrent disease. Persistent HPV-16 and HPV-18 were significantly associated with recurrent CIN2-3 ( P < .05).
Conclusion
Persistent infection with the same HR-HPV genotype, especially HPV-16 and HPV-18, should be considered a risk factor for developing residual/recurrent CIN2-3.
High-grade cervical intraepithelial neoplasia (CIN) 2-3 bears a significant risk of developing invasive carcinoma if left untreated. Persistent high-risk (HR) human papillomavirus (HPV) infections are more strongly associated with the development of CIN2-3 and are considered the first step in the progression to cervical carcinoma. Conservative treatment with a loop electrosurgical excision procedure (LEEP) is both a diagnostic and therapeutic procedure that can effectively eradicate CIN2-3. However, residual/recurrent disease after a LEEP varies between 5–30%, requiring follow-up and retreatment once lesions have been identified. The main objective in the follow-up of patients after a LEEP due to CIN2-3 is the early detection of recurrent disease because of the risk of progression to invasive carcinoma if an effective treatment is not rendered. HR-HPV detection techniques have recently been proposed in the follow-up of patients treated for CIN2-3 because of the high sensitivity and negative predictive value for detecting recurrent disease as a good index of disease clearance. Thus, growing evidence indicates that HR-HPV in conjunction with cytology should be useful for monitoring women treated for CIN2-3.
The aim of this study was to determine whether the HPV genotype by the HPV DNA chip test (HDC; MyGene Co, Seoul, South Korea) is predictive of residual or recurrent disease in follow-up of CIN2-3 after a LEEP.
Materials and Methods
We retrospectively reviewed the records of all 681 women with histologically confirmed CIN2-3 who were treated by a LEEP in the Department of Obstetrics and Gynecology of Chonnam National University Hospital between January 2001–February 2007.
In all, 672 women were considered eligible for the study if they fulfilled the following criteria: (1) histologically confirmed CIN2-3 by a LEEP; (2) patients in whom both pre- and post-LEEP HR-HPV test results from the HDC and the hybrid capture II assay (HC2) (Digene Co, Gaithersburg, MD) were available; and (3) patients who were followed up for ≥2 years. We excluded 9 patients who underwent hysterectomy after a LEEP. Epidemiologic data, HR-HPV test data from the HDC and HC2, and pathology data were obtained from medical records.
A LEEP was performed under local anesthesia using wire loop electrodes, with diathermy apparatus set. A section was placed at the 12 o’clock position of the LEEP specimen for orientation, and then specimens were fixed in 10% formalin for pathologic examination.
The patients underwent a postoperative examination at 3, 6, 12, 18, and 24 months during the first 2 years, then yearly thereafter. In every visit after the LEEP, HPV DNA tests (HC2 and HDC) and cytology were performed on all the patients, and colposcopy/biopsy was performed for abnormal cytology or HR-HPV detection by HPV DNA tests. For statistical analysis, the results of cervical biopsies obtained during follow-up were grouped as negative when normal/cervicitis or CIN1 was present and positive when CIN2 or CIN3 was present. Positive histologic results during follow-up were considered as recurrent disease. The study protocol was evaluated and approved by the institutional review board at Chonnam National University Hospital.
HC2
The sample was collected by placing a cytobrush into the exocervix and rotating the brush 3 times; the sample was kept frozen at –20°C in a collection tube (Digene Co) until needed. The denatured single-strand DNA was hybridized with RNA researcher of a mixed HR-HPV group. This reaction mixture was placed in a microtiter well coated with antibodies for the RNA/DNA hybrid. After RNA/DNA hybrid-antibody bonding, the mixture was reacted with alkaline phosphatase-conjugated antibodies, washed, and lumi-Phospho 530 (Lumigen, Southfield, MI) was added to react with the dioxetane-based chemiluminescent substrate. Alkaline phosphatase was added to obtain luminescent light, which was measured with a luminometer and expressed in a relative light unit (RLU). The solution containing 1 pg/mL of HPV-16 DNA was used as the positive control group for the HR-HPV group. The RLU for all the samples was set to the degree of relative brightness compared with the positive control group. This ratio was considered positive when it was ≥1.0 and negative when it was ≤1.0. The samples were analyzed for the presence of the 13 types of HR-HPV group (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68).
HDC
We used the HDC, a polymerase chain reaction (PCR)-based DNA microarray system, as an HPV genotyping method for HPV typing. The HDC contains 24 type-specific probes; 15 probes are HR types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) and 9 probes are low-risk types (6, 11, 34, 40, 42, 43, 44, 54, and 70). Briefly, DNA was isolated from a swab sample using a DNA isolation kit (MyGene Co), and target L1 regions of HPV DNA were amplified and labeled by a single dye (indocarbocyanine-dUTP; NEN Life Science Products Inc, Boston, MA). The PCR products of all samples were detected by electrophoresis with a 2.5% agarose gel. The samples were mixed with a hybridization solution (MyGene Co). Hybridization was performed at 43°C for 90 minutes. The hybridized HPV DNA was visualized using a DNA chip scanner (Scanarray Lite; GSI Lumonics, Ottawa, Ontario, Canada). Fifteen types of HR-HPV positivity were used to assess HDC performance.
Statistical analysis
The correlation between recurrence with post-LEEP HPV status and other clinical factors was determined by a Student t test or Fisher’s exact test. All P values reported are 2-sided, and P values < .05 were considered statistically significant. Agreement between tests was assessed by Cohen kappa statistic, and the P value was calculated using McNemar test, with values between .00–.20 indicating poor agreement, .21–.40 fair agreement, .41–.60 moderate agreement, .61–.80 substantial agreement, and .81–1.00 near-perfect agreement. A receiver operating characteristic curve was used to determine the clinically most useful cutoff value of HR-HPV viral load to predict residual/recurrent disease. The data were analyzed using software (SPSS for Windows, Version 17.0; SPSS, Inc, Chicago, IL). The 95% confidence intervals were calculated.
Results
The mean age of the patients was 39.7 years (range, 21–62 years). High-grade CIN was confirmed by histology; 120 patients had CIN2 and 552 patients had CIN3. Of the 672 patients, 37 (5.5%) developed recurrences during the follow-up; histology revealed 9 cases of CIN2 (24.3%) and 28 cases of CIN3 (75.7%). The mean lag time between the LEEP and the diagnosis of recurrent disease was 16.8 months (range, 3–58 months). The HPV detection rates of the HDC were nearly comparable to HC2. Of the 672 patients, the HC2 was positive in 652 patients (97.0%) and the HDC was positive in 654 patients (97.3%). Patients with recurrent disease did not differ from the cured patients with respect to age, previous cytologic abnormalities, CIN grade, and HR-HPV status at the time of the LEEP. Cone margin involvement, positive endocervical cytology, follow-up cytology, and HR-HPV detection by the HDC and HC2 after the LEEP were associated with a significantly higher risk of residual/recurrent disease. Genotyping of 1 HC2-negative/HDC-positive patient with recurrent disease revealed HPV-53 ( Table 1 ).
| Characteristic | No recurrence, n = 635 | Recurrence, n = 37 | P |
|---|---|---|---|
| Age, y | .36 | ||
| Mean ± SD | 39.5 ± 8.3 | 40.8 ± 7.0 | |
| Range | 21–62 | 27–57 | |
| Initial cytology | .19 | ||
| ASCUS | 100 | 2 | |
| LSIL | 64 | 3 | |
| HSIL | 471 | 32 | |
| CIN at LEEP | .83 | ||
| CIN2 | 113 | 7 | |
| CIN3 | 552 | 30 | |
| Pre-LEEP HC2 | > .99 | ||
| Negative | 19 | 1 a | |
| Positive | 616 | 36 | |
| Pre-LEEP HDC | .62 | ||
| Negative | 18 | 0 | |
| Positive | 617 | 37 | |
| Cone margin | < .01 | ||
| Negative | 543 | 18 | |
| Positive | 92 | 19 | |
| Endocervical cytology | < .01 | ||
| Negative | 614 | 28 | |
| Positive | 21 | 9 | |
| Follow-up cytology | < .01 | ||
| Negative | 576 | 11 | |
| ≥ASC | 59 | 26 | |
| Post-LEEP HC2 | < .01 | ||
| Negative | 591 | 1 | |
| Positive | 44 | 36 | |
| Post-LEEP HDC | < .01 | ||
| Negative | 589 | 0 | |
| Positive | 46 | 37 |
The concordance and discordant results between the 2 HPV tests are summarized in Table 2 . The overall agreement between the 2 tests was 97.3%, with a kappa value of 0.815. Of 652 HR-HPV-positive specimens by the HC2, the HDC was positive in 644 specimens (98.8%). Among 20 specimens that were negative by the HC2, the HDC was negative in 10 specimens (50.0%). When the HDC was compared with the HC2, discordant results were observed among 18 (2.7%) specimens, comprising 8 HC2-positive/HDC-negative and 10 HC2-negative/HDC-positive results. Genotyping of 10 HC2-negative/HDC-positive cases revealed 4 HPV-53, 3 HPV-16, 2 HPV-66, and 1 HPV-18.
