Distribution of high-risk HPV types in Yugoslav women with cervical neoplasia

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1 Journal of BUON 7: , Zerbinis Medical Publications. Printed in Greece ORIGINAL ARTICLE Distribution of high-risk HPV types in Yugoslav women with cervical neoplasia J. Stojanović 1, Z. Magić 1, M. Milacić 1, D. Nenadić 2, B. Stanimirović 3, D. Vukicević 3 1 Institute of Medical Research; 2 Clinic of Gynaecology, Military Medical Academy; 3 Clinic of Gynaecology and Obstetrics Narodni Front, Beograd, Yugoslavia Summary Purpose: The purpose of this study was to determine the frequency of high-risk types of human papillomavirus (HPV) infection, namely type 16, 18, 31 and 33, among Yugoslav women diagnosed with different grades of squamous intraepithelial lesion (SIL), as well as to investigate the relationship between HPV infection and age, parity, age at first intercourse, number of sexual partners and residence of the patients, all of which are considered risk-factors. Patients and methods: DNA was isolated from cervical swabs of 72 women using phenol/chloroform/isoamylalcohol extraction. Detection of HPV DNA in patients genomic DNA was performed using the polymerase chain reaction (PCR) method with type-specific primer pairs, and amplification products were analyzed using 2% agarose and 10% polyacrylamide gel electrophoresis. Results: Thirty out of 72 (41.7%) patients with cervical intraepithelial neoplasia (CIN) were HPV-positive and 8 of them were double positives. HPV31 was the most frequent high-risk HPV type in this group of patients (13.9%). Eighty percent of the high-grade SIL (HSIL) patients were HPV-positive and 38.8% of the low-grade SIL (LSIL) patients were HPV-positive. Compared to HPV-negative women, the HPV-positive ones were younger, had started sexual activity earlier, and overall had more sexual partners. Conclusion: Our study showed that oncogenic HPV types are responsible for the transition of LSIL to HSIL, and for its further progression to an invasive carcinoma of the cervix. Thus, HPV typing should become a widely used method for identifying women with increased risk for developing HSIL and invasive cervical cancer. We also concluded that sexual behavior is connected with the frequency of HPV infection. Henceforth, introduction of prophylactic measures could reduce the incidence of HPV infected women in our country. Key words: cervical cancer, human papillomavirus, polymerase chain reaction, risk factors, squamous intraepithelial lesion Introduction Received ; Accepted Author and address for correspondence: Jelena Stojanović, MS Institute of Medical Research Military Medical Academy Crnotravska Beograd Yugoslavia Tel/fax: VMAIMI@EUnet.yu Cancer of the uterine cervix is one of the leading causes of cancer death in women worldwide. It is a disease which develops progressively: from low-grade through high-grade CIN to an invasive malignant disease. Thus, one of the ways to prevent cervical carcinoma is early detection and treatment of preinvasive stages. The incidence of cervical neoplasia in Yugoslav women is very high, just behind breast malignancies [1]. Papanicolaou smear screening (Pap test) reduces the incidence and mortality rate of invasive cervical cancer, but it has certain limitations, such as high rates of false-negative and false-positive results, and disconformity of the cytological and histopathological terminology which can lead to confusion. Since fundamental importance of HPV in the etiology of cervical cancer has been clearly demonstrated, it would be necessary to evaluate the HPV typing in cervical cancer screening. There is much interest in the use of HPV testing to improve both the effectiveness and cost of cervical screening. The main role of HPV DNA testing is to provide quality control for cytologic and histopathologic diagnoses, as detection of HPV DNA in the absence of cytological abnormalities can indicate the presence of CIN missed by cytology. Testing for high-risk HPV could also be used to identify

2 252 women with increased risk of developing HSIL or invasive cervical cancer [2], and to improve the therapy of women with low-grade lesions and abnormal Pap smears. HPV testing is also proposed for post-treatment surveillance of preinvasive or early invasive lesions, in order to monitor the complete excision [3]. There are more than 100 types of HPV, and about 40 of them infect the genital tract. HPV types associated with anogenital precancerous and malignant lesions are classified as low-risk or high-risk according to the risk of progression to invasive cancer. HPV6 and HPV11 are low-risk types associated with LSIL (venereal warts or condylomata accuminata), and they rarely progress to cancer, while HPV16 and HPV18 are high-risk HPV types associated with HSIL (CIN II, CIN III) that can progress to cancer. The DNA of high-risk HPV types has been found in over 90% of HSIL and cervical cancers [4]. HPV positivity rate of normal population is variable, ranging from 3 to 20%, or even more [3]. HPV infection is the main risk factor associated with cervical premalignant disorders and is transmitted sexually [5]. Other risk factors are sexual behavior (which disappears as a risk factor if corrections are made for HPV infection), smoking habits, low socioeconomic status [6], oral and barrier contraceptive use, reproductive history, dietary factors, immunosuppression, etc. [7]. Avoiding these risk factors, together with detection and treatment of precancerous lesions, provides the opportunity to fight against cervical malignancies. Consistent implementation of prophylactic measures has resulted in very low incidence of cervical carcinoma in certain countries, but in other countries, including Yugoslavia, the prevalence of this disease is quite high [1]. The purpose of this study was to determine the frequency of infection with various oncogenic types of HPV (HPV16, HPV18, HPV31 and HPV33) in a group of 72 women previously diagnosed with different grades of CIN. We also tried to find the relation between HPV infection and potentional risk factors such as age, number of deliveries, number of abortions, age at first intercourse, number of sexual partners and residence of the patient. We used the PCR method with type-specific primer pairs for the identification of HPV genome in cervical swab cells. This technique was shown to be the most sensitive and efficient compared with hybridization assays [2,8] and PCR assays with general primer pairs [9]. Patients and methods Patient selection The samples of cervical swabs of 72 women with a history of different cervical intraepithelial lesions were obtained from the Clinic of Gynaecology and Obstetrics Narodni Front, Beograd. The women were referred for gynecological examination during the period from October 2000 through May All patients were diagnosed as LSIL or HSIL according to the histopathology of samples taken at colposcopy. Speciment collection Material for HPV testing was obtained from the cervical canal and exocervix using a special plastic spatula, which was subsequently placed in a tube with 0.9% NaCl solution. DNA isolation from the cervical swabs DNA was isolated from the cells of cervical swabs using the phenol-chloroform DNA extraction method [10]. The concentration of the DNA was determined by spectrophotometry. The quality of isolated genomic DNA was checked using 1% agarose gel electrophoresis. The DNA solutions were stored at 4º C. HPV DNA analysis by PCR Integrated viral DNA was detected in genomic DNA of patients using the PCR method. We used typespecific primer pairs for HPV16, HPV18, HPV31 and HPV33, which are specific for the E6, L1, E4 and E1 regions of viral genome, respectively [9]. Each DNA sample (600 ng) was mixed with PCR buffer, 1 unit of thermostable DNA polymerase (Pharmacia, Upsala, Sweden), 10 nmol of dntp mixture (A:G:C:T=1:1:1:1) (Pharmacia Biotech, Sweden) and 20 pmol of sense and antisense primers in a 50 ml of PCR mixture. The same mixture, but without DNA, was used as the PCR control. The mixture was incubated for 10 min at 95º C for DNA denaturation, and then followed by 40 cycles of amplification using Thermal Cycler (Hybaid, United Kingdom). Each cycle included a denaturation step at 95º C for 1 min, an annealing step at 63º C, 61º C, 61º C and 57º C for HPV16, 18, 31 and 33 respectively for 1 min, and an elongation step at 72º C for 2 min. The final elongation step was prolonged for 10 min, to ensure a complete extension. The PCR products were analyzed by 2% agarose gel (containing ethidium bromide) electrophoresis in 0.5 TBE buffer (45 mm Tris-borate, 1 mm EDTA), run on 80 V for 20 min and visualized under ultraviolet light. To confirm these results we used 10% vertical polyacrylamide gels in 0.5n TBE buffer (LKB, Pharmacia, Sweden). The gels were stained with silver nitrate (Serva Heidelberg, Germany). A DNA molecular weight marker (PCR Markers, Promega, Madison, USA) was used to provide a size estimate in base pairs (Figure 1).

3 253 Figure 1. Polyacrylamide gel electrophoresis of the HPV16, HPV18, HPV31 and HPV 33 after PCR amplification. Statistical analysis The statistical significance of the association between HPV infection and the patients age, and the age at first sexual intercourse was determined by the Student s t-test, while the association between HPV infection and the number of deliveries, number of abortions and number of sexual partners of the patients was determined by the x 2 test. Results Demographic and clinical characteristics of 72 analyzed patients are presented in Table 1. The patients age ranged from 19 to 56 years (median 31 years). The age group of years was the largest. About half of the patients had children; the majority of them (93%) had one or two children. The number of women who had had and who had not had abortions was similar (35:37). All patients were sexually active, with a median age of 19 years at first intercourse (range years). Most of the analyzed women had had less than 5 sexual partners (83.3%), with a median of 2. The histopathological diagnosis was LSIL in 67 patients (93%) and HSIL in the remaining 5 (6.9%) patients. Thirty of 72 patients were HPV-positive (41.7%), and 8 of them were double positives. Among various types of HPV, infection with HPV31 was the most frequent (13.9%). Four out of 5 patients diagnosed with HSIL were HPV-positive (80%), and 26 out of 67 LSIL patients were HPV-positive (38.8%) (Table 2, Figure 2). By comparing certain data on HPV-positive and HPV-negative patients, we may come to conclusions connected with sexual behavior as a risk factor for HPV infection, and by that for CIN and cervical cancer (Tables 3 and 4). Discussion Results of previous investigations indicate that PCR is the most sensitive method in the detection of high-risk Table 1. Demographic and clinical characteristics of SIL patients (n= 72) Characteristic n (%) Age (years) 19 1 (1.4) (41.7) (26.4) (22.2) 50 6 (8.3) Deliveries (no) 0 32 (44.4) 1 16 (22.2) 2 21 (29.2) 3 3 (4.2) Abortions (no) 0 35 (48.6) 1 17 (23.6) 2 6 (8.3) 3 8 (11.1) 4 6 (8.3) Partners (no) 1 25 (34.7) 2 13 (18.1) 3 14 (19.4) 4 8 (11.1) 5 7 (9.7) 6 5 (6.9) Age at first intercourse (years) 16 6 (8.3) (34.7) (36.1) (16.7) 23 3 (4.2) LSIL Con.pl. 67 (93.1) 13 (18.1) HP diagnosis CIN I 54 (75) HSIL 5 (6.9) HP: histopathological; LSIL: low-grade squamous intraepithelial lesion; HSIL: high grade squamous intraepithelial lesion; CIN: cervical intraepithelial neoplasia, Con. pl: condyloma plana HPV in cervical swabs when comparing with other techniques, such as the hybrid capture assay [8]. Absolute specificity is achieved by using type-specific primer pairs. Conversely, general primer pairs are not specific for different HPV types such as GP5/6, CPI/IIG, MY09/11;

4 254 Table 2. Results of HPV detection and typing HPV typing LSIL+HSIL LSIL HSIL n % n % n % Negative HPV HPV HPV HPV HPV HPV HPV Total Table 3. Mean values of age and age at first intercourse of HPVpositive and HPV-negative SIL patients HPV-positive Figure 2. Results of HPV detection and typing. HPV-negative Age (years) 32.8± ±10.7 Age at first intercourse (years) 18.6± ±2.2 Table 4. Contribution of potentional risk factors (no. of deliveries, abortions and lifetime sexual partners) in HPV-positive and HPVnegative CIL patients HPV-positive HPV-negative Potential risk factor n % n % Deliveries (no) Abortions (no) Sexual partners (no) they amplify conserved regions of HPV genome, which do not differ between HPV types. Conventional typespecific primer pairs amplify regions of 200 and more base pairs [9]. As sensitivity of PCR amplification increases with the decrease of the amplifier length, new type-specific primer pairs for HPV16, HPV18, HPV31 and HPV33, which amplify only 96, 115, 100 and 114 base pairs respectively, as those we used in the present study for HPV DNA detection, contribute to a higher sensitivity of the method. HPV detection by PCR has not been conducted on the population of Yugoslav women until now. Our investigation showed that HPV31 is the most frequent oncogenic type of HPV in our population, in contrast to literature data showing that HPV16 represents the most common high-risk HPV type. It might be that women from our country are genetically more susceptible to HPV31 than to other high-risk HPV types. It is also possible that the group of women we analyzed was not large enough to elicit general conclusions. Previous investigations [3] showed that over 95% of cervical cancers, 75-95% of high-grade CIN lesions, and 25-40% of low-grade CIN lesions are associated with a positive HPV test on exfoliated cervical cells. That coincides with our finding that 80% of HSIL patients and 38.8% of LSIL patients are positive for high-risk HPV. Since oncogenic HPV types have been significantly more often detected in HSIL than in LSIL, we may consider them responsible for the onset of high-grade lesions and their further progression to invasive cervical carcinoma. Numerous epidemiological studies have linked cervical cancer to sexual behavior, suggesting a venereal

5 255 cause. It has been also shown that HPV is the strongest risk determinant of the entire range of cervical intraepithelial lesions. The finding that either female promiscuity, having a promiscuous sexual partner, or early age at onset of sexual activity confers increased risk for cervical cancer supports the concept of a sexually transmitted agent. The peak incidence of HPV, as a sexually transmitted disease, is in the age group of years, which gradually declines up to about years. There is an inverse relationship of HPV infection with the age of CIN patients. Shlay et al. [11] found age-related differences in the HPV testing, with greater sensitivity in younger women. It was suggested that HPV lesions of more recent onset exfoliate more virus, whereas lesions associated with persistent infection and immune response might be more localized and have lower and less easily detectable mucosal viral load. Among women with CIN, a higher prevalence of HPV DNA was reported in women under 35 years of age than in older women. It also might be that these age differences in HPV testing are consequences of sexual behavior: younger women are more sexually active and have more sexual partners (who are also more promiscuous), and the risk of infection is increased. Young age of the first male partner also acts as a measure of the likelihood of early HPV infection in the women and thus is a possible risk factor for cervical neoplasia. Among the studied patients, the age group of years was the largest, which illustrates the fact that the cervical SIL is rising in younger women. The average age was lower in the group of women found to be HPV-positive, although the difference in age between HPV-positive and HPV-negative women was not statistically significant. A significant risk factor for high-grade and lowgrade cervical lesions is the lifetime number of sexual partners. This can be explained by a greater probability of the contact with a person who carries the virus. In our group of CIN patients 26.7% of HPV- positive women, but only 9.5% of HPV-negative women had 5 sexual partners. In the group of HPV-negative patients, the maximum number of lifetime sexual partners was 5, and in the HPV-positive group it was 10. Young age at first intercourse and early age of parity are also markers of promiscuity and represent risk factors. The cervix is particularly susceptible to a specific agent such as HPV, and the association with the woman s age at first intercourse depends on the first partner(s) being high-risk males (i. e. HPV-infected) or low-risk males (i. e. non-infected) [5]. Beginning sexual activity during adolescence may also be a risk factor on the basis of an altered hormonal environment [12]. In our study, the average age at first intercourse in HPVpositive women was lower than in HPV-negative women, although this difference was not statistically significant. It is also interesting to note that age at first intercourse ranged from years in HPV-positive, and from years in HPV-negative women. Some early studies disregarded reproductive factors as risk determinants, as these factors were considered only as indicators of sexual behavior. In contrast, later studies have shown an independent association between risk of cervical lesions and increasing parity. Kjær et al. [13] reported that parity is related to the risk of SIL (LSIL+HSIL) in both HPV-negative and HPVpositive women. There is no certain biological explanation for this relation, but it may be that chronic irritative processes and lacerations in the cervix that have developed during delivery contribute to the onset of neoplasia. The same authors found that differences concerning parity between HPV-negative and HPV-positive women were not statistically significant [13]. The residence of patients could reflect socioeconomic status, educational level and sexual hygiene of patients, and these might be some of the strongest risk factors. Urban/rural residence ratios in the HPV-positive and HPV-negative group of our patients were similar. These findings are not presented and their interpretation is limited by the small number of the analyzed cases. HPV infection is sufficient by itself to cause small dysplastic lesions, but in the absence of some unknown factors these lesions would disappear after a short period of time. In the case of persistent HPV infection, with an altered immune response or repeated HPV infections (due to many partners and no contraception), the lesion would tend to persist and progress to higher-graded lesion. Early age at first infection and other co-factors of HPV infection determine whether a HPV-caused lesion would regress, persist or progress to higher-grade lesion and cervical cancer. There is a clinical dilemma regarding how to treat women with abnormal Pap smears, and 3 options have been proposed: immediate colposcopy, accelerated repeat Pap testing and testing for HPV. Testing for HPV can clarify the nature of an equivocal Pap result: women with a test positive for HPV are at risk, and the anxiety and cost of colposcopic referral is justified, while those with a negative test should be followed routinely. Despite the close association between high-risk HPV and the development of cervical disease, testing for these viruses has not been incorporated into clinical practice [2]. HPV typing, in addition to colposcopy, cytodiagnostics and histopathology, should become a routine method for the detection and treatment of SIL. The use of HPV testing must be accompanied by complete understanding of the, in most cases, benign nature of HPV infection, and the diagnosis of LSIL made on this basis is not of excessive concern [14].

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