The role of human papillomavirus (HPV) testing in the follow-up of patients after treatment for cervical intraepithelial neoplasia (CIN)

Introduction : The aim of this study was to examine the role of human papillomavirus testing in the follow-up after treatment for CIN, as a prognostic sign for residual/recurrent cervical precancerous lesions. Methods : A hospital-based analysis was performed on 460 patients previously treated for CIN with cold knife conization, at the University Clinic for Gynecology and Obstetrics and General Hospital Remedika, in Skopje, Republic of Macedonia, in a period of 3 years. The patients were followed-up with HPV testing in addition to cytology, colposcopy and/or biopsy. The ﬁ rst after treatment HPV testing was performed 8 months after cold knife conization, proceeded by follow-up within 24 months after treatment, at 4 months intervals. Results : Among 460 treated patients, at the ﬁ rst HPV and cytologic testing, 8 months after treat-ment, 69 (15%) were HPV+, and 391 (85%) HPV negative. From the 69 HPV+ patients, 41 (59.4%) were with cytologic abnormalities and 28 (40.6%) without abnormalities. 12 months after treatment, the number of HPV+ patients developing cytologic abnormalities raised to 45/70 (64.29%). Within the 24 months after treatment, the number of patients who had recurrent/ residual CIN from the HPV+ patients reached 50/71 (70.42%); which was 10.87% from all 460 treated patients. is more accurate than cytology at the same follow-up intervals.

The role of human papillomavirus (HPV) testing in the follow-up of patients after treatment for cervical intraepithelial neoplasia (CIN) INTRODUCTION Cervical cancer with an estimated 530 000 new cases in 2008 is the third most common cancer in women, and the seventh overall. More than 85% of these cases occur in developing countries, where it accounts for 13% of all female cancers. Th e highest-risk regions are Eastern and Western Africa [agestandardized rate (ASR) greater than 30 per 100 000], and the lowest are Western Asia, Northern America and Australia/New Zealand, where the rates are less than 6 per 100 000. Above all, in 2008, cervical cancer proved to be the reason for 275.000 deaths with the mortality incidence ratio of 52%, and an estimation of 88% of deaths in developing countries from which 53.000 in Af-rica, 31.700 in Latin America and the Caribbean, and 159.800 in Asia (1,2). Today, it is well-established that Human papillomavirus (HPV) infection is the most important cause of cervical cancer, with a special attention to HPV types 16 and 18, which proved to be the reason in 70% of the world cervical cancer cases. Th e World data show that around 11.4% of women are evaluated to capture cervical HPV infection at a given time. Th e same data presented that the prevalence of HPV 16 and/or 18 ranging from normal cytology is 3.8%; through 24.3% in low-grade cervical lesions; up to 51.1% in high-grade cervical lesion. Th e same types are blamed for about 70.9% of the most invasive cases (3). Th e DNA-HPV detection results of cervical infection are measured in all cervical morphological lesions ranging from normal fi ndings up to invasive cervical cancer, showing that the prevalence of HPV increases with the malice of the lesion. HPV remains the cause of almost 100% of all cases of cervical cancer. Th e vaccine-prventable HPV-16 and -18, are still the reason for more than 70% of all cervical cancer cases in the world, especially in high-grade cervical lesions, 41-67% (4). After HPV-16/18, the six most common HPV types in all world regions, which account for an additional 20% of cervical cancers worldwide are the types: 31, 33, 35, 45, 52 and 58. Th e discovery of Human Papilloma Virus (HPV) infection to be the prime cause for this disease, gives a tremendous chance to prevent and early detect cervical neoplasia (5,6). Recent studies demonstrated that HPV test combined with cytology may improve the early de-tection of both primary cervical neoplasia as well as recurrence of neoplasia after therapy, decreasing the need for more radical treatment (7,8). Cervical conization is defi ned as excision of a coneshaped or cylindrical wedge from the uterine cervix that includes the transformation zone and all or a portion of the endocervical canal. It is used as a de-fi nitive diagnostic and treatment tool for squamous or glandular intraepithelial lesions and for excluding micro-invasive carcinomas. Th ere are several conization techniques: cold-knife (scalpel) conization, laser conization, or electrosurgical loop conization, each with certain benefi ts and disadvantages. Th e cleanest specimen mar-gins for patho-histologic analysis is provided only by cold-knife conization. As an attempt to excise gross cervical tumors per vaginam, in the early 19 th century, similar procedures to conization were used. In the late 20 th century, fi rst conization was used as a diagnostic tool for cervical lesions and later as treatment as well. Today the use of cold-knife conization as a diagnostic tool is reduced since wide spreading the colposcopically directed cervical biopsies combined with endocervical curettage as less invasive procedure with high diagnostic value. However, in selected situations, it is still very important diagnostic tool and accepted modality for management and treatment of CIN (9)(10)(11)(12). Conization site usually heals in 6 weeks. To determine treatment success and avoid possibility of residual or recurrent CIN, Papanicolaou tests should be performed every 4 months during the fi rst and second postoperative years and every 6 months thereafter. A single follow-up Papanicolaou test shows positive results in fewer than 25% of women with residual disease, therefore we designed our study to determine the role of human HPV testing in the follow-up after treatment for CIN, as a valuable prognostic sign for residual/recurrent cervical precancerous lesions.

Patients
A hospital-based analysis was performed on 460 patients previously treated for CIN with cold knife conization, at the University Clinic for Gynecology and Obstetrics and General Hospital Remedika, in Skopje, Republic of Macedonia, in a period of 3 years. Th e patients were followed-up with HPV testing in addition to cytology, colposcopy and/or biopsy. Th e fi rst after treatment HPV testing was performed 8 months after cold knife conization, proceeded by follow-up within 24 months after treatment, at 4 months intervals.

Procedures
During each of these follow-up visits, patients received colposcopy, conventional PAP or liquid based PAP (CYTOFAST by HOSPITEX DIAGNOS-TICS, Sesto Fiorentino, Italy) and HPV test specimens. Biopsy and/or endocervical curretage was performed to prove recurrent/residual lesion only if the previous test suggested low-grade or high-grade cervical lesions. Residual/recurrent dis-ease was defi ned only if the CIN2+ lesion was histologically confi rmed at least 8 months after treatment. After retreatment, women received further follow-up test,s but were dropped out from the study. If women had histologically confi rmed CIN1, follow-up continued without treatment ('wait and see'). Human papillomavirus DNA was detected by Polymerase Chain Reaction (PCR) method in the Laboratory for Molecular Biology, Institute of Biology, Faculty of Natural Sciences and Mathematics, Skopje, Macedonia. Th e material for analysis (exfoliated cells in medium) was analyzed 24-48 hours after sample collection. Th e cervical cells were collected and digested with an appropriate buff er containing Proteinase K and 0,5% SDS. Th e total DNA was isolated with NaCl/chloroform extraction and ethanol precipitation. Th e PCR amplifi cation was performed with 3 pairs of consen-sus primers (MY09/11, GP5+/6+, HPVpU 1M/2R) specifi c for L1 and E6/E7 regions of the HPV genome (thermo-cycler Perkin Elmer Geneamp PCR System 2400). Positive and negative controls were included in each of the tested series. Th e positive primers were genotyped and digested with 7 restrictional endonucleases (AfaI, HaeIII, PstI, AccI, AvaII, BglII, AvaI) specifi c for "low-risk" HPV types (6,11,40,42 . Th e results were analyzed with agarose gel electrophoresis and visualised on UV transluminator. Th e viral genotype was determined through the length of restrictional fragments of the electrophoresis gel (12). Th e results from the conventional PAP or liquidbased smears that were used for cytological analy-sis were interpreted according to the Bethesda classification III System, 2001.

Statistical analysis
All data gathered during the follow-up period, were analyzed using the statistical package SPSS 20. We computed the sensitivity, specifi city, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratio with 95% confi dence intervals (95% CI), for cytology abnormalities development and HPV presence/persistence and the combination of these tests by making use of cross tabs.   (Table 3).

DISCUSSION
It is evidence-based that HPV, especially the high risk types, are the most important cause of cervi-cal cancer (13,14). It is also clear that there is a relation between persistent infection of HPV and CIN2+ lesions.
Th e aim of this study was to examine the role of HPV testing in the follow-up after treatment for CIN, as a prognostic sign for residual/recurrent cervical precancerous lesions. Based on many dif-ferent large studies it was published that at least 95% of patients who have CIN can be cured by diff erent conization techniques. However, cure rates as low as 60% have also been reported. Also, in patients with positive margins for precancerous lesion in the  Our study, in 8, 12 and 24 months after treatment showed similar sensitivity of 89, 92 and 94% respectively; but higher specifi city at the same points of follow-up period (93, 94, and 95% respec-tively) . Specifi city given by other authors of HPV tests for CIN detection are as follows: 88%-Nagai, 86%-Nobbenhuis, and 84%-Paraskevaidis (18,(20)(21)(22). In our study, an increase of specifi city and sensitivity was noticed in the further follow-up period points. Based on these observations, there is a need, the follow-up period to be increased in longer than 12 months. Diagnostic value of HPV testing in the follow-up of patients after treatment for CIN, as a method of detection of recurrent/residual neoplasia, increases with time and seems to be of great significance the hole period of 24 months after treatment. An attention should be drawn to signifi cance of initial viral test (8 months after treatment) for prediction of recurrent/residual CIN, since 59.4% of the patients with CIN recurrence were HPV positive at the fi rst follow-up visit after conization.