• Age;
  • CIN;
  • conisation;
  • human papillomavirus;
  • recurrence


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Objectives  The aim of this study was to examine the accuracy of the presence of high-risk human papillomavirus (HR-HPV) DNA (HR-HPV DNA test) postconisation as prediction of recurrent or residual cervical intraepithelial neoplasia (CIN) after treatment of high-grade cervical intraepithelial lesions (CIN2+) in a prospective study and to compare this with follow-up cytology and the marginal status of the excised tissue.

Design  Prospective follow-up study.

Setting  Unselected women presenting at colposcopy clinic of University Hospital Gasthuisberg, Leuven.

Population  Seventy-two women treated with conisation for CIN2 or CIN3.

Methods  Women were followed by HR-HPV DNA test (Hybrid Capture II test of Digene®) every 3 to 6 months. The same vial was used for cytology and the HR-HPV DNA test (SurePath™). All women were further followed by colposcopy and cytology for 24 months at 6-month intervals. The outcome of the study was presence of >CIN2, proven with colposcopy-directed biopsy occurring within 24 months after treatment. HR-HPV status was correlated with recurrent or residual CIN2+.

Main outcome measures  Sensitivity, specificity, predictive values and diagnostic odds ratios to predict treatment failure or cure were computed for HR-HPV testing, marginal status and follow-up cytology. HR-HPV status was also correlated with section margins postconisation and with the first cervical smear.

Results  In 6 of the 72 treated women (8%), residual or recurrent CIN occurred. Women with recurrence were significantly older than women without a recurrence (51.5 ± 9.6 versus 39.8 ± 12.2 years, P= 0.007). All six women with recurrence were HR-HPV positive, four had a positive follow-up smear (≥atypical squamous cells of uncertain significance = ASCUS+) and only two had involved section margins. Among the 66 cured women, 15 were HR-HPV positive, 6 had an abnormal smear and 12 had positive section margins. Sensitivity of cytology, positive section margins and HR-HPV DNA positivity was 66.7, 33.3 and 100% to predict treatment failure. Specificity of the three tests was, respectively, 90.9, 81.8 and 77.3%. Women with HR-HPV DNA at 3 to 6 months showed recurrent or residual CIN in 15% (2/13) if they had normal follow-up Pap smears and in 50% (4/8) if they had abnormal Pap smears. Margin status was not statistically significantly associated with human papillomavirus status.

Conclusion  Persistence or clearance of HR-HPV DNA is an early valid prognostic marker of failure or cure after treatment for CIN2+ and is more accurate than cytology or section margin status at the time of conisation. The absence of HR-HPV DNA has a 100% negative predictive value. Higher age at conisation may be a previously unrecognised risk factor for recurrence.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Cervical cancer is preventable by early detection and treatment of cervical intraepithelial neoplasia (CIN) 2 and CIN3 (high-grade CIN). Conservative treatment with large loop excision of the transformation zone (LLETZ) or conisation is the standard procedure for treatment of high-grade CIN.1,2

The risk of recurrence is increased in case of positive section margins, but the accuracy of positive section margins is far from perfect.3–5 Therefore, the optimal management in case of positive section margins remains controversial.

Frequent follow up with cytology and colposcopic evaluation of the cervix is the preferred strategy at present, on the other hand, if a cone biopsy has CIN-free section margins, the risk on recurrences is still in the range of 5–35% of women.6–8

The role of high-risk human papillomavirus (HR-HPV) infections is well established in the pathogenesis of cervical dysplasia and cervical carcinoma. Recent prospective and retrospective studies confirm the importance of HR-HPV status after conisation treatment for CIN2+.9

We introduced human papillomavirus (HPV) DNA testing during follow up after treatment for high-grade CIN in our department. In this prospective cohort study, we aimed to examine the role of HPV DNA testing to detect residual/recurrent disease in the follow up of high-grade CIN after conisation.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Between February 2000 and February 2003, 72 women were treated for high-grade CIN with LLETZ at the Department of Gynaecology of the University Hospital Gasthuisberg, Leuven, and had follow up including HPV DNA testing.

Cervical cellular material was collected with a Cervex-brush™ (Rovers Medical Devices B.V., Oss, the Netherlands) 3 to 6 months after the procedure and placed in a vial with preservation fluid for preparation of a liquid-based smear using the SurePath™ technology (TriPath Imaging Inc., Burlington, NC, USA). The liquid was split into two samples for cytological evaluation and HR-HPV testing using the B-probe of the Hybrid Capture II (HC2) assay (Digene® Corporation, Gaithersburg, MD). Procedures for collecting and transporting specimens for HPV DNA testing were processed according to the manufacturer’s instructions. The HC2 detects 13 different carcinogenic HPV types.

Mean woman age was 40 years (22–78 years), with a median of 37 years. High-grade CIN was confirmed with histology, 12 women had CIN2 and 60 had CIN3. Two women with CIN3 also had a glandular lesion (one with glandular intraepithelial neoplasia (GIN1) and another with GIN3), but these women did not show recurrence after treatment.

LLETZ procedures were carried out after application of 3% acetic acid and Lugol’s iodine solution under local anaesthesia with Lignospan® (Septodont, Saint-Maur-des-Fossés Cedex, France) as described by Prendiville et al.1 Special attention was given to the presence of CIN at the level of the section margins.

All 72 women had a first control after 3 to 6 months, consisting of colposcopy, HC2 testing and a Pap smear. Women were further followed at 6-monthly visits for up to 24 months after the LLETZ procedure. Pap smears were taken at each visit. Colposcopy was carried out at each visit on all 72 women and once more if cytology results were abnormal. Abnormal Pap smears were defined as equal to or more severe than ≥atypical squamous cells of undetermined significance according to the Bethesda guidelines.10 Colposcopic directed punch biopsies of the cervix were taken if any suspected area was seen after application of 3% acetic acid. Histologically confirmed recurrent or residual CIN2+ was considered as treatment failure. The accuracy of the prediction of treatment failure by the presence of CIN in the section margins, the HC2 assay at 3–6 months and the cytological status was verified by computing the sensitivity, specificity, predictive values and diagnostic odds ratios (DOR). The Fisher’s exact test was used to test for differences in proportions.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Women with recurrence or residual disease did not differ from cured women for menopausal state, use of tobacco, method of contraception, history of previous cytological abnormalities, sexually transmitted disease (STD), HIV status and CIN grade at conisation (Table 1). However, the mean age was significantly higher in women with treatment failure (P= 0.007). Menopausal state was not significantly associated with recurrence, with mean age at recurrence of 51.5 years.

Table 1.  Women characteristics
 Recurrence (n= 6)No recurrence (n= 66)P value
  1. ASCUS, atypical squamous cells of undetermined significance; n.s., not significant; p/day, package per day; HSIL, high-grade squamous intraepithelial neoplasia; LSIL, low-grade squamous intraepithelial neoplasia.

Mean age (SD)51.5 (9.6)39.8 (12.2)0.007
Menopause, n (%)3/6 (50)12/66 (18)n.s.
Current tobacco use
Nihil, n (%)3/6 (50)49/66 (74)n.s.
≤1 p/day, n (%)3/6 (50)16/66 (24)n.s.
>1 p/day, n (%)01/66 (2)n.s.
Current hormonal use, n (%)2/6 (33)43/66 (65)n.s.
Previous abnormal Pap smear
ASCUS or LSIL, n (%)2/6 (33)20/66 (30)n.s.
HSIL, n (%)2/6 (33)4/66 (6)0.075
STD (ever)00n.s.
Immunosuppression01 (Crohn)n.s.
CIN at conisation
CIN2, n (%)1 (17)11 (17)n.s.
CIN3, n (%)5 (83)55 (83)n.s.

Of the 72 women in follow up, 6 developed recurrence (8%).

One in five women had a positive section margin at the moment of LLETZ procedure (14/72, 19.4%), but only two of them had a recurrence (14.2%) versus 4 of the 58 margin-free women (6.9%), DOR = 2.25 (95% CI: 0.18–17.7) (Table 2).

Table 2.  Sensitivity, specificity, predictive values and DOR of the different tests to predict residual or recurrent CIN2+ (95% CI)
  1. NPV, negative predictive value; PPV, positive predictive value.

Cytology66.7% (22.2–95.7%)90.9% (81.3–97.0%)40.0% (12.2–73.8%)96.8% (88.8–99.6%)20.0 (2.2–243.3)
Section margin33.3% (4.3–77.7%)81.8% (70.4–90.2%)14.3% (1.8–42.8%)93.1% (83.3–98.1%)2.3 (0.18–17.7)
HR-HPV DNA100.0% (54.1–100%)77.3% (65.3–86.7%)28.6% (11.3–52.2%)100.0% (93.0–100%)43.2 (2.3–810.4)

A better prediction could be made by follow-up cytology: 4/10 (40%) women with an abnormal Pap smear out of any of the 6-monthly intervals had a recurrence versus 2/62 (3.2%) women with normal Pap smears (DOR: 20.0 [2.2–243.3]).

Most sensitive was a positive HR-HPV DNA test at one single instance at 3 to 6 months after conisation. All six women who developed a recurrence tested positive for HR-HPV, while none of the 51 HR-HPV-negative women developed a recurrence (OR: 43.2 (2.3–810.4) P= 0.0003). Hence the negative predictive value, the absence of HR-HPV DNA, was 100% in this series.

No correlation could be made when comparing section margins and HPV status (confidence interval around DOR includes unity) (Table 3).

Table 3.  Correlation of recurrence and section margin, cytology and high-risk HPV status
 HPV positive (n= 21)HPV negative (n= 51)
Recurrence (n= 6)No recurrence (n= 15)Recurrence (n= 0)No recurrence (n= 51)
Section margins
Involved (n= 14)20012
Not involved (n= 58)415039
Odds ratio17 (0.7–428), P= 0.07 
Follow-up cytology
Abnormal (n= 10)4402
Normal (n= 62)211049
Odds ratio5.5 (0.7–42), P= 0.15 

Women who had a normal follow-up smear but were positive for HR-HPV DNA at 3 to 6 months showed recurrent or residual CIN in 15% (2/13) and in 50% (4/8) if they had abnormal Pap smears at follow up.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

The relationship between a persistent HR-HPV infection and CIN2+ is well established.11–13

We studied the value of the presence of HR-HPV DNA on cytological material 3 to 6 months after conisation in predicting recurrences.

Women with HR-HPV DNA after treatment had a 29% risk of recurrence, whereas none of the women with negative HR-HPV DNA suffered from recurrence. Therefore, sensitivity and negative predictive value of the HR-HPV test are 100%. Lin et al.14 also described an excellent sensitivity and negative predictive value of HPV DNA testing after conisation for predicting recurrences. Similar to our data, specificity and positive predictive value were less optimal.

As this is not a screening setting, but follow-up management of a potentially lethal disease, sensitivity and negative predictive value are valued higher than specificity and false-positive tests because the main goal is not to miss active disease.

Several publications describe the high false-negative rate of Pap smears during follow up after treatment for high-grade CIN.15,16 We had recurrence rates of high-grade CIN of 2.3% in women with repeatedly normal Pap smears. On the contrary, if HR-HPV DNA was positive, there was a high chance of disease recurrence, whatever the cytology findings are, and when HR-HPV DNA was negative, the chance of recurrence was negligible, even if the cytology after treatment had been abnormal.

The accuracy in predicting recurrence in cases of abnormal cytology is higher if HR-HPV DNA is present (50 versus 0%). Bar-Am et al.17 demonstrates that adding HPV testing to cytology serves as an extra safety measure in predicting recurrences and helps to select women for colposcopy. Paraskevaidis et al.16 demonstrated that HPV DNA testing predicts treatment failure more accurately than either the first post-treatment Pap smear or positive cone margins. As demonstrated in our study, a positive cytology result can increase the specificity of a positive HPV DNA test. These results are in accordance with a recent review by Arbyn et al.18

Women with recurrences had a higher mean age at conisation than women without recurrences. Although only borderline significant, these women also seemed to have a small increased risk of having higher grade CIN at conisation. We were not able to explain why older women may have more aggressive disease, but altered immunity or positive selection over time towards viruses with a higher oncogenic risk may be possible.

The mean age of the six women with recurrent disease is 51.5 years. The small number may explain why we did not find an association between residual/recurrent disease and menopausal state although higher age is correlated with recurrence.

We conclude that women with positive HR-HPV DNA after conisation need intense follow up since they are at increased risk for recurrent or residual/recurrent CIN. This may be even more important for older women. If HR-HPV DNA is absent, then risk of recurrence is low; in our series, the rate of recurrence was 0%. Nevertheless, in other studies, some rare recurrent cases that were HPV negative could be detected by cytology only (Zielinski et al.).19

In conclusion, when post-treatment HR-HPV DNA is absent at 3 to 6 months after conisation, follow up can be relaxed and a normal screening programme could be considered when a follow-up smear at 12 months is cytologically normal as well. We need more studies that follow women for more than 2 years after treatment of high-grade CIN and investigate the role of HPV testing, age or other risk factors to predict relapse of disease before the optimal long-term follow-up strategy can be established.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References
  • 1
    Prendiville W, Cullimore J, Norman S. Large loop excision of the transformation zone (LLETZ): a new method of management for women with cervical intraepithelial neoplasia. Br J Obstet Gynaecol 1989;96:105460.
  • 2
    Paraskevaidis E, Koliopoulos G, Malamou-Mitsi V, Zikopoulos K, Paschopaulos M, Pappa L, et al. Large loop excision of the transformation zone for treating cervical intraepithelial cervical neoplasia: a 12-year experience. Anticancer Res 2001;21:30979.
  • 3
    Orbo A, Arnesen T, Arnes M, Straume B. Resection margins in conization as prognostic marker for relapse in high-grade dysplasia of the uterine cervix in northern Norway: a retrospective long-term follow-up material. Gynecol Oncol 2004;93:47983.
  • 4
    Paterson-Brown S, Chappatte OA, Clark SK, Wright A, Maxwell P, Taub NA, et al. The significance of cone biopsy resection margins. Gynecol Oncol 1992;46:1855.
  • 5
    Felix JC, Muderspach LI, Duggan BD, Roman LD. The significance of positive margins in loop electrosurgical cone biopsies. Obstet Gynecol 1994;84:9961000.
  • 6
    Gardeil MD, Barry-Walsh C, Prendiville W, Clinch J, Turner MJ. Persistent intraepithelial neoplasia after excision for cervical intraepithelial neoplasia grade III. Obstet Gynecol 1997;89:41922.
  • 7
    Narducci F, Occelli B, Boman F, Vinatier D, Leroy JL. Positive margins after conization and risk of persistent lesion. Gynecol Oncol 2000;76:31114.
  • 8
    Nagai Y, Maehama T, Asato T, Kanazawa K. Persistence of human papillomavirus infection after therapeutic conization for CIN 3: is it an alarm for disease recurrence? Gynecol Oncol 2000;79:2949.
  • 9
    Paraskevaidis E, Arbyn M, Sotiriadis A, Diakomanolis E, Martin-Hirsch P, Koliopoulos G, et al. The role of HPV DNA testing in the follow-up period after treatment for CIN: a systematic review of the literature. Cancer Treat Rev 2004;30:20511.
  • 10
    Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287:211419.
  • 11
    Ter Harmsel BT, Smedts F, Kuijpers J, Van Muyden R, Oosterhuis W, Quint W. Relationship between human papillomavirus type 16 in the cervix and intraepithelial neoplasia. Obstet Gynecol 1999;93:4650.
  • 12
    Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, Rozendaal L, Remmink AJ, Risse EK, et al. Relation of human papillomavirus status to cervical lesions and consequences for cervical cancer screening: a prospective study. Lancet 1999;354:205.
  • 13
    Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J Natl Cancer Inst 1995;87:796802.
  • 14
    Lin CT, Tseng CJ, Lai CH, Hsueh S, Huang KG, Huang HJ, et al. Value of human papillomavirus deoxyribonucleic acid testing after conization in the prediction of residual disease in the subsequent hysterectomy specimen. Am J Obstet Gynecol 2001;184:9405.
  • 15
    Jain S, Tseng CJ, Horng SG, Soong YK, Pao CC. Negative predictive value of human papillomavirus test following conization of the cervix uteri. Gynecol Oncol 2001;82:17780.
  • 16
    Paraskevaidis E, Koliopoulos G, Alamanos Y, Malamou-Mitsi V, Lolis ED, Kitchener HC. Human papillomavirus testing and the outcome of treatment for cervical intraepithelial neoplasia. Obstet Gynecol 2001;98:8336.
  • 17
    Bar-Am A, Gamzu R, Levin I, Fainaru O, Niv J, Almog B. Follow up by combined cytology and human papillomavirus testing for patients post-cone biopsy: results of a long-term follow up. Gynecol Oncol 2003;91:14953.
  • 18
    Arbyn M, Paraskevaidis E, Martin-Hirsch P, Prendiville W, Dillner J. Clinical utility of HPV DNA detection: triage of minor cervical lesions, follow-up of women treated for high-grade CIN. An update of pooled evidence. Gynecol Oncol 2005;99:S711.
  • 19
    Zielinski GD, Bais AG, Helmerhorst TJ, Verheijen RH, De Schipper FA, Snijders PJ, et al. HPV testing and monitoring of women after treatment of CIN 3: review of the literature and meta-analysis. Obstet Gynecol Surv 2004;59:54353.