Role of human papillomavirus (HPV) testing in the follow-up of patients after treatment for cervical precancerous lesions

European Journal of Obstetrics & Gynecology and Reproductive Biology 118 (2005) 229–234 www.elsevier.com/locate/ejogrb

Role of human papillomavirus (HPV) testing in the follow-up of patients after treatment for cervical precancerous lesions Zolta´n Herna´dia,*, Krisztina Szo˝keb, Tama´s Sa´pya, Zoa´rd T. Krasznaia, Gyo¨rgyike Soo´sc, Gyo¨rgy Veressb, Lajos Gergelyb,d, Jo´zsef Ko´nyab a

Department of Gynecologic Oncology, Medical Health Science Center, University of Debrecen, Debrecen, Hungary Department of Medical Microbiology, Medical Health Science Center, University of Debrecen, Debrecen, Hungary c Department of Pathology, Medical Health Science Center, University of Debrecen, Debrecen, Hungary d Tumor Virus Research Group, Hungarian Academy of Sciences and University of Debrecen, Debrecen, Hungary

b

Received 28 April 2004; received in revised form 22 June 2004; accepted 30 June 2004

Abstract Objective: To evaluate the role of human papillomavirus (HPV) testing in post-treatment follow-up of patients after therapeutic excision of the cervix due to positive screening tests. Study design: A hospital-based retrospective analysis was performed with prospective collection of patient data of women screened for cervical cancer at a Gynecologic Outpatient Clinic. Patients after therapeutic excision due to positive screening results were identified and followed up with HPV testing and serial cytology. Results: After 61 treatment for cervicalis intraepithelialis neoplasia (CIN), high-risk HPV infection was detected during the post-treatment follow-up at 18 cases (29.5%), 10 of them had persisting cytological atypia (positive predictive value (PPV): 56%), 5 developed CIN (PPV: 28%). When the HPV test was negative (43 patients) in the post-treatment period, neither CIN nor persisting cytological atypia developed (negative predictive value (NPV): 100%) during 1201 patient months (median 26 months). Conclusions: A negative HPV test eliminates the risk of recurrent disease after treatment for CIN. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Recurrence of CIN, Post-treatment HPV infection; HPV screening

1. Introduction Persistent infection of the female genitalia by oncogenic human papillomavirus (HPV) types is the major cause of the precancerous, squamous intraepithelial lesions (SIL) and the invasive cancer of cervix uteri [1–3]. However, the nature of HPV infection limits the diagnostic use of HPV testing, since transient infection is common among young adults [4], while above the age of 30, the infections by high-risk HPV types tend to persist longer [5]. Though the single point detection of HPV infection itself does not distinguish the frequently occurring transient infections from the less frequent persistent infections, the addition of HPV testing to the cytology-based cervical screening improves the screening efficiency. For patients with * Corresponding author. Tel.: +36 52 417171; fax: +36 52 417171. E-mail address: [email protected] (Z. Herna´di).

equivocal cytologic results, HPV testing as a secondary screening assay has a good sensitivity and a high negative predictive value for underlying high-grade cervicalis intraepithelialis neoplasia (CIN) [6–8]. Due to the limited sensitivity of cytological screening, HPV testing was recommended as a part of the primary screening for cervical cancer [9]. Primary screening protocols using both cytology and HPV testing identify the cases at risk with high sensitivity [10], while the duration of the protection – if both tests are negative – is under current investigations [11]. After therapeutic excision of the CIN lesion, the patient is to be followed thoroughly for recurrent disease. Margins free of neoplastic cells at the histologic examination of the removed lesions predict a good prognosis [12,13], while incomplete excision exposes the patient to a markedly increased risk of recurring disease. Nevertheless, only a fraction of patients with marginal involvement will have

0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.06.029

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recurrent disease, which can be controlled by cytological and colposcopic follow-up [14,13]. This follow-up is usually longitudinal with multiple visits, while detecting the same type of human papillomavirus after the surgical wound has healed is a feasible, single point assessment of the residual disease [15]. The expression of papillomaviral oncogenes is essential for most, if not all, premalignant lesions of squamous cell origin with a potential to progress into invasive cancer, but HPVs are not found in the surrounding healthy epithelium [16]. However, after eradication of the CIN lesion, HPV DNA persists in a relevant proportion of cases with a tendency to disappear during a prolonged follow-up [17]. The levels of the anti-HPV antibodies decline, as well [18,19]. Persistence of the same HPV type in cytological smears taken after the excisive treatment should be considered as a strong predictor of the residual or recurrent disease [20]. Negative HPV tests within 2 years after treatment for high-grade CIN excludes the residual or recurrent lesion with high probability, which can help to establish the guidelines when to return the patient back to the population-based screening programs [21]. On a hospital-based patient population, we conducted an analysis of the diagnostic value of secondary HPV testing. The predictive force of the HPV testing for residual or recurrent disease was assessed in patients after excisive treatment of precancerous lesions of the epithelium of the uterine cervix. The recurrence of CIN was analyzed in women who had HPV test after treatment.

of the cervical epithelium that is the cytology proved to be pseudo-positive. Cytological results in this period were still given by the Papanicolaou classification. The grade P3 (mild to moderate dyskariosis) indicated equivocal cytologic abnormalities corresponding to ASCUS and LGSIL while the P4 grade was equivalent to HGSIL by the Bethesda classification system [23]. The distribution according to the pre-excision cytology at patients on the post-treatment follow-up was as follows: 2 patients with ASCUS, 7 patients with LGSIL and 52 patients with HGSIL (Table 1). The starting point of the follow-up period relevant for the study was the surgical removal of the cervical lesion. The type of the surgical excision was LEEP at 13 patients and cold knife cone biopsy at 48 patients. Both in case of the cone excision and in case of the LEEP the lesion seen at colposcopy was excised totally with a safety zone. The distribution of patients according to the grade of CIN was as follows: CIN1: 12, CIN2: 18, CIN3: 31 patients, respectively (Table 1). As the main focus in this study was on the recurrence of CIN after surgical excision of the lesion, those patients with CIN histology (61 patients.) were followed-up both with HPV testing and serial cytology (median interval: 5 months, range: 1.5–12 months). The endpoint of the follow-up was either the last registered visit or the surgical removal of the recurrent cervical lesion. The outcome of the post-treatment follow-up was assessed by the recurrence of persistent cytological atypia and CIN. 2.2. HPV detection and typing

2. Material and methods 2.1. Study design A retrospective study with prospective collection of data was performed on patient data of women screened at the Gynecologic Outpatient Clinic at the Medical Center of the University of Debrecen, which is a regional center for Debrecen town and Hajdu´ -Bihar county in Hungary. The routine diagnostic HPV tests and further HPV typing were done at the Department of Medical Microbiology while the histopathological evaluation of tissue specimens removed at excision from the cervix were performed at the Department of Pathology. Based on the archived data of screening, we identified patients who had excisional biopsy between May 1997 and December 1999. The electronic patient registry of the Medical Center was used to collect anamnestic data and to register prospective data from visits after treatment. Colposcopic examinations were performed using OPTON colposcope (Karl Zeiss, Germany). The examinations were done using acetic acid application, and the terminology used is equivalent to the 1990 Colposcopy Nomenclature of the IFCPC [22]. Out of the 76 patients treated by surgical excision due to positive cytology, 61 patients had a CIN histology while at 15 patients the histology excluded the precancerous lesions

HPV DNA was detected with the Digene Hybrid Capture Tube test (Digene Diagnostics, Inc. Beltsville, MD USA) according to the manufacturer’s instructions. A high-risk HPV-positive result by the Hybrid Capture Tube test is interpreted as an infection by one or more of the following HPV types: 16, 18, 31, 33, 35, 51, 52, 56, while the low-risk Table 1 Characteristics of patients followed-up after treatment for CIN Characteristics

CIN cases

Number Median age (range)

61 33 (20–53)

Follow-up time (patient months) Cumulative Median (range)

1626 25 (3–62)

Pap smear before excision ASCUS (%) LSIL (%) HSIL (%)

2 (3.3) 7 (11.5) 52 (85.2)

Excision technique LEEP (%) Cold knife conisation (%)

13 (21.3) 48 (78.7)

Histology CIN1 (%) CIN2 (%) CIN3 (%)

12 (19.6) 18 (29.5) 31 (50.8)

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probe cocktail can detect HPV types 6, 11, 42, 43, 44. The Hybrid Capture results were confirmed by PCR amplification of the consensus MY region [24]. DNA was extracted from the specimens processed previously for the routine Hybrid Capture test and was subjected to the MY09–MY11 PCR amplification and typing as described previously [25]. 2.3. Histology Serial section analysis of the tissue specimen excised was performed according to the standard protocol. 2.4. Statistics Exact confidence intervals (95%) of the specificity and the negative and positive predictive values were calculated with Clopper–Pearson method using Quantitative Parasitology statistical software package at http://bio.univet.hu/QP/ QP.htm [26].

3. Results Of the 72 surgical biopsies of patients with positive cytology, 61 proved to contain CIN lesions. Post-treatment cytology and HPV test was done in all of the 61 CIN cases. After CIN treatment, 18 of 61 patients (29.5%) were HPVpositive (Table 1). The patients were classified according to the Hybrid Capture HPV test results, since this method was used in the clinical routine. All HPV-positive Hybrid Capture specimens and 32 HPV-negative Hybrid Capture specimens were available for PCR amplification of the MY region. The PCR results agreed with the Hybrid Capture results in all but one specimen (98%): one cervical sample tested negative with Hybrid Capture HPV test but tested positive with PCR. At four patients out of those, HPVpositive after primary treatment a repeated cone biopsy was performed and the HPV persisted even after this secondary excision. The clinical outcome of the post-treatment follow-up was evaluated by development of persisting cytological atypia

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and in case of reconization, the histology was also considered. It was found that a negative HPV test in the post-treatment period excluded not only the recurring CIN but also the development of persisting cytological atypia (negative predictive value (NPV) = 100%) (Table 2). Negative HPV results were detected at median of 6 month (range: 1–24) after therapeutic excision. The median followup time for negative cytology was 4 months (range: 2–12). It is of note that 10 (23%) of the 43 patients in the HPVnegative follow-up group had equivocal cytology (P3) at single visits during a cumulative 1201 patient months follow-up. However, none of these changes persisted until the subsequent visits. In the background of the transient cytological findings, inflammation was found in six patients (three of them had Candida vaginitis), one patient developed transient cytological atypia in the first trimester of pregnancy. For three patients, no underlying disease could be retrieved from the patient’s files. Recurrent disease was detected among the patients with positive HPV test during the post-treatment follow-up (Table 3). Four of the primary cervical biopsies, two cone biopsies with CIN3, one cone biopsy with CIN2 and one LEEP biopsy with CIN2 had marginal involvement. One of the patients with incompletely removed CIN3 had four negative HPV tests and remained free of recurrent disease during the 29 months of the follow-up. The other three patients with histologic evidence of incompletely excised cervical lesion had high-risk positive HPV test (type16) and cytological atypia developed during the post-treatment follow-up. Two of these patients underwent further operations within 1 year due to CIN2 and CIN3. In both cases, reconization revealed a higher grade diseases than in the previous biopsies. Recurrent disease developed in two more patients, whose primary lesions had been removed completely by the histology. Both of them had high-risk HPV infection after the surgical removal. Both recurring lesions were of less advanced grade (CIN1, CIN2) than the primary lesions and were diagnosed 21 and 32 months after the primary disease, respectively. Altogether 5 of the 61 post-treatment periods ended with histology proven recurrence of CIN, which were preceded

Table 2 Diagnostic value of human papillomavirus testing for recurrent disease Post-treatment HCT result

No. of patients

Outcome

Diagnostic performance [CI95%]

Persistent cytologic atypia HPV-positive HPV-negative

18 43

10 0

PPV: 56% (30.8–78.5) NPV: 100% (91.8–100)

SP: 84% (71.4–93) SE: 100%

CIN1, CIN2, CIN3 HPV-positive HPV-negative

18 43

5 0

PPV: 28% (9.7–53.5) NPV: 100% (91.8–100)

SP: 90% (77.3–96.5) SE: 100%

CIN2, CIN3 HPV-positive HPV-negative

18 43

4 0

PPV: 22% (6.4–47.6) NPV: 100% (91.8–100)

SP: 91% (79.6–97.6) SE: 100%

HCT: Hybrid Capture Tube HPV test, CIN: cervicalis intraepithelialis neoplasia, CIS: carcinoma in situ, PPV: positive predictive value, NPV: negative predictive value, SP: specificity, SE: confidence intervals were not calculated for sensitivity due to low number of patients with recurrent disease.

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Table 3 Patients with positive post-treatment HPV test Patient age at entry

HPV test before excision

Histology

HPV test after excision

Cervical atypia after excision (months (mo))

End of post-treatment follow-up At month

Outcome

41 42 49 26 32 23 22 23 34 27 50 31 42 32 37 29 29 22

nt HPV16 nt nt nt nt nt nt HPV31 HPV16 HPV16 HPV16 nt HPV16 HPV16, 52 HPV16 HPV16 HPV16

CIN3 CIN2 CIN1 CIN2 CIN2 CIN2 CIN2 CIN1 CIN1 CIN3 CIN3 CIN3 CIN3 CIN3 CIN2 CIN3 CIN3 CIN2

HPV16 HPV16 HPV16 HPV16 HPV31 HPV16 HPV16 HPV31 LR-HPVa HPV16 LR-HPVa HPV56 CP8304 HPV66 HPV33 MM4 HPV16, 58 HPV33

P3b from mo 12 mosaic at mo 31 P3b from mo 5 P3b from mo 11 P3a at mo 10 P3a from mo 7 leuokoplakia from mo 12 P3a from mo 7 mosaic from mo 5 P3a from mo 11 P3b at mo 1, 13 mosaic from mo 11 P3a from mo 14 P3b leuokoplakia at mo 5 – – – – – – – P3 at mo 3

32 10 14 11 21 17 31 17 21 12 30 48 46 19 25 29 27 15

CIN2b CIN2, koilocytosisb CIN3b CIN3b CIN1b Koilocytosis, condylomab Koilocytosisb Parakeratosis, cervicitisb Persistent cytological atypia Negative histologyb Disease-free Disease-free Disease-free HPV-negative by mo 18 Disease-free HPV-negative by mo 7 Disease-free HPV-negative by mo 7 Disease-free Disease-free Disease-free

a b

LR-HPV: low-risk HPV infection. Histologic result.

by high-risk HPV infection and persistent cytological atypia in all cases. Five more patients with positive HPV test had equivocal cytology at consecutive visits, four of them had reconization, but the histology revealed condyloma, koilocytosis, no disease and cervicitis, respectively. Another patient with positive HPV test in the post-treatment period had transient cytological atypia, which was associated with HPV infection of other type than in the primary lesion. In conclusion, a positive HPV test indicated a significant risk for the recurrence of persistent cytological atypia and CIN with high sensitivity. However, as consequence of cases mentioned above the positive predictive values are relatively low, 56% (10/18) for persisting cytological atypia and 28% (5/18) for CIN (Table 2). Of the 72 patients with cytological atypia at screening, 15 women were identified, who underwent surgical excision, but no CIN lesions were found in the biopsy specimens. In the post-treatment period, only two of them had positive HPV results, one infection by low-risk HPV was associated with koilocytosis, while the other infection by HPV16 did not cause cytological changes. Only one of the 15 ‘‘no CIN’’ patients had equivocal cytological result at two consecutive visits 22 and 26 months after the surgical excision (15 and 19 months after the negative HPV test). However, the cytological alterations regressed for the rest of the 34month follow-up.

4. Discussion The introduction of HPV testing into the post-treatment follow-up of CIN can help to establish the guidelines when to return the patient back to the population-based screening

programs [9,21]. Cytological abnormalities are not rare after treatment for CIN and although the cytological abnormalities and the positive HPV tests overlap greatly, the sensitivity of cytological screening is lower than that of the high-risk HPV testing [21,27]. In this study, the occurrence of cytological atypia after the excisive therapy was associated with positive post-treatment HPV results. The HPV-associated atypia tended to be persistent, while all cytological abnormalities that developed in the absence of HPV infection were transient. The newly acquired HPV infections as determined by altered HPV type tended to be transient, too and were weakly or not associated with cytological atypia and recurrent disease. It is known from the study of Costa et al. [17] that the incidence of the positive HPV tests decreases in function of the time relapsed after excisional therapy. They demonstrated that factors such as age, lesion grade, volume of the cone and involvement of endocervical margin may have a significant influence on the rate HPV clearance. On the other hand, newly acquired infections increase the actual incidence of the positive HPV tests during the posttreatment follow-up. The findings by us are in good correlation with all these observations. Though a positive HPV test is a sensitive marker its predictive value for persisting cytological atypia and CIN is relatively low. The negative post-treatment HPV test seemed to have a higher predictive value providing a clear-cut distinction of the patients with no excess risk for recurrent CIN. This interpretation of a negative posttreatment HPV test is in agreement with the studies of Bollen et al. [27] and Nagai et al. [28]. HPV testing may miss neoplasias that are HPV-negative or contain HPV types not detected in the routinely used tests.

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There are chronic virus carriers in whom the molecular test results are the only evidence of HPV persistence but who do not have the morphological evidence of CIN [17]. Therefore, patients to be returned to the population-based screening program must have both negative HPV test and normal smear [17,21]. This implies that the post-treatment followup of patients should include both cytology and HPV detection. With regard to the timing of the post-treatment survey, the patients in the study by Costa et al. [17] attended the clinic at 6-month intervals with a maximum follow-up of 39.2 months. Nobbenhuis et al. [21] showed that cytological examinations together with high-risk HPV testing at 6 and 24 months after the treatment are sufficient for the detection of recurrent dysplasia, while patients free of dysplasia during the 2-year post-treatment period are to return to the general screening program. Our results showed that regular cytologic control (6-month interval) with one HPV test preferably between 6 and 12 months after the treatment were efficient, too. Patients with negative posttreatment HPV test did not develop CIN for a median followup of 26 months. On the basis of our findings in this study, which are in good consonance with the data of the literature, we conclude that the inclusion of the HPV testing in the post-treatment follow-up protocol of CIN patients can improve the diagnostic accuracy. The positive HPV test as consequence of its high sensitivity, assured the early detection of patients at increased risk for disease recurrence and progression. However, the positive predictive values were relatively low for persisting cytologic atypia and for CIN/carcinoma in situ (CIS), 56% (10/18) and 28% (5/18), respectively. The negative HPV test in the post-treatment period excluded the persisting cytologic atypia and the recurring CIN (NPV = 100%) and allowed the patient to return back to the population-based screening programs.

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Acknowledgment The technical assistance of Ms. Sa´ ndorne´ Czegle´ di at the cytological registry is gratefully appreciated. This study was supported by research Grant OTKA T038416 and ETT 748/ KO/203.

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