Correlation between abnormal cytological findings and human papillomavirus infection of the uterine cervix in Bulgarian women

ABSTRACT

The aim of this study was to analyse the correlation between cases of human papillomavirus (HPV+/−) infection of the uterine cervix revealed through HPV typing and cytological results from Papanicolaou (Pap)-stained cervical smears. Cervical smears of 421 Bulgarian women attending routine gynaecological examinations during the three-year study period were stained by the Pap technique and classified by the Bethesda system. GenoFlow HPV Array Test Kit was used to analyse the HPV status in the collected cervical samples. The results showed that, of the 421 women, 177 (42%) were HPV(+) and 244 (58%) were HPV(−); 334 (79.3%) Pap smears were with normal morphology and 87 (20.7%) had high/low grade squamous intraepithelial lesion (LSIL/HSIL) changes. Of the 87 women with LSIL/HSIL changes, 54% (47/87) were HPV(−) and 46% (40/87) were HPV(+). There was no statistically significant correlation between the HPV(+) status and the cytological LSIL/HSIL findings (P > 0.05). Koilocytes were found in 30.4% (128/421) of the samples. Of the 128 women with koilocytosis, 59.4% (76/128) were HPV(−) and 40.6% (52/128) were HPV(+). There was no significant correlation (P > 0.05) between the presence of koilocytes in cervical smears and HPV infection. Our results suggest that HPV infection is frequent even in women with negative Pap-smear results, and polymerase chain reaction seems to be the only reliable test to diagnose this infection. However, the results from this study cannot be considered to fully support the replacement of cytology and colposcopy examination in cervical cancer screening with HPV genotyping tests only.

KEYWORDS:

• HPV
• infection
• Pap findings
• HSIL
• LSIL

Introduction

It is very difficult to find evidence for human papillomavirus (HPV) infection based on cervical cytology [1]. The cytological sign that, since the mid-1970s, has been generally considered indicative of an HPV infection is the presence of koilocytes in cervical smears [2,3–5]. However, with the ever-developing advancements in molecular diagnostic tools, it has become evident that cytological and histological features of koilocytosis, or koilocytotic atypia, are not sensitive enough as indicators of the presence of HPV DNA [6,7]. Cytological or histological signs of HPV infection are, in fact, not detected in a majority of women who are positive for HPV DNA [8,9]. Other cytological signs of cervical cancer, such as atypia (high/low grade squamous intraepithelial lesion), have not proved reliable as a sole diagnostic tool for HPV infection either [6,7]. Moreover, as already pointed out [1], the presence of koilocytes in histological sections may be difficult to diagnose, since fixation artefacts or poor dehydration can result in the presence of ‘koilocyte-like’ cells with perinuclear halos.

In our previous study of 421 Bulgarian women [10], we showed that, although a significant dependence was observed between HPV(+) status and development of cervical intraepithelial neoplasia (CIN), the HPV(+) status may not be considered a specific and sensitive predictive factor for the development of CIN. Here, we proceed further with the aim to analyse the correlation between cases with HPV+/− infection of the uterine cervix proved through HPV typing and cytological results from Papanicolaou (Pap)-stained cervical smears in the same cohort of 421 Bulgarian women.

Subjects and methods

Study cohort

The study was retrospective and was conducted using the results database of the Genetic Medico-Diagnostic Laboratory ‘Genica’, Sofia, Bulgaria, and the Clinic of General and Oncogynecology at the Military Medical Academy (MMA), Sofia, Bulgaria. The results from a three-year period (2011–2014) were studied. The study cohort included 421 women aged 18–45 years who voluntarily attended a gynaecological clinic for HPV screening (to determine the presence or absence of infection) or for a routine gynaecological check-up. The patients’ medical history and Pap smear results were collected, and colposcopy and HPV tests were performed in each case. All women gave their informed consent to participate in this study. The study was approved by the MMA Ethics Committee. All data concerning the patients' personal details, age, previous treatment for HPV infection, including surgical or destructive treatment of the cervix, co-morbidities, etc., were complied with the data protection guidelines (Anonymity Clause).

Exclusion criteria were: pregnancy, diagnosis of immune diseases, previous diagnosis of HPV infection, history of previous surgical or destructive treatment of the cervix, precancerous conditions and previous HPV vaccine.

Pap test

The Pap test was performed at the Department of Pathology at MMA, Sofia. The Pap test was taken from the exocervix and the cervical canal. From the samples, cytological smears were prepared by fixation on a microscope slide and staining by the method of Pap [11]. After staining, microscopic analysis (Optika B-382-ALC, Optika SRL, Ponteranica, Italy) was performed, in which signs of benign and malignant cervical cells and other pathological changes were detected based on the morphological characteristics of the cells. The results were reported according to the 2001 Bethesda System terminology: squamous-cell abnormalities were classified as atypical (i.e. atypical squamous cells of undetermined significance (ASCUS) or atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) or carcinoma. Glandular-cell abnormalities are classified as atypical glandular cells, adenocarcinoma in situ or adenocarcinoma [12]. In the Bethesda System, koilocytes (koilocytosis) and cervical intraepithelial neoplasia (CIN 1) are considered to be LSILs. HSILs include CIN 2 and CIN 3 [13]. Smears with ASCUS, LSIL and HSIL were subjected to further analysis.

HPV typing

HPV typing was performed at ‘Genica’ LTD, Sofia, Bulgaria, to detect HPV infection. Cervical swabs were taken and then HPV DNA polymerase chain reaction (PCR) test and Flow-through™ hybridization were performed as a highly reliable, highly specific and sensitive test for high-risk HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68; moderate-risk genotypes: 53, 73, 81 and 82; and low-risk genotypes: 6, 11, 42, 43, 44, 40, 61, 54, 55, 70, 57, 71, 72, 84 and 26.

Sample collection and storage

Cervical cell samples were stored in liquid transport medium, PreservCyt solution (Hologic Inc., Marborough, MA, USA). Specimens were stored at 5–30 °C during transportation and were kept at room temperature up to two weeks.

DNA preparation

Approximately 1–2 mL of specimen was used for each test. The samples were centrifuged at 1200 r/min (Sorvall Legend XT Centrifuge, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min and the pellets were re-suspended with 200 µL phosphate buffered saline buffer. For DNA preparation, the QIAapm Blood Mini kit (QIAGEN, Valencia, CA, USA) was used. Eluted DNA in QIAGEN Buffer AE (50–200 µL) was stored at −15 to −20 °C until further analysis.

Amplification of target DNA

GenoFlow HPV Array Test Kit (FT-PRO, GF assay; Diagcor Bioscience Inc., Hong Kong) was used for the performance of this step. The PCR amplification was performed in a final volume of 25 µL and the thermal profile suitable for GeneAmp PCR System 9700 (Applied Biosystems, Foster city, CA, USA) was as follows: 95 °C for 9 min; 43 cycles at 95 °C for 20 s, 55 °C for 30 s, 72 °C for 30 s and finally, a 72 °C step for 5 min. To monitor the test performance, a negative control (DNase-free water), amplification control and positive control (non-infectious DNA containing an HPV sequence) procedures had been made.

‘Flow-through’ hybridization

GenoFlow HPV Array Test Kit (FT-PRO) was used for the performance of this step using an FT-PRO Flow-through System Set-up (Diagcor Bioscience Inc.). After denaturation of the PCR products at 95 °C for 5 min, they were immediately placed in an ice bath for at least 2 min. Hybridization and colorimetric detection of the samples were according to the manufacturer's protocol (GenoFlow HPV Array Test Kit, FT-PRO).

Data interpretation

Interpretation of the results was performed by using data analysis strip and Capture Image Capture System for scanning, analysis and storage of the membrane images.

Data analysis

All data were statistically analysed using IBM SPSS statistics. Contingency tables were used for the main variables, e.g. HPV+/− infection, age, Pap test results and LSIL/HSIL for each patient, with the values considered separately and in pairs. The correlation between the variables was evaluated by Pearson's chi-square test.

Results and discussion

Many authors state the connection of cervical Pap koilocytosis with HPV(+) infection [2,4]. The results obtained in our study (Table 1) showed that, one or more HPV genotypes were identified in 177 (42%) of the 421 women included in this study, and 244 women (58%) were HPV(−). Based on the results from the cytological analysis, koilocytes were identified in 52 women (12.3%) in the HPV(+) group and in 76 women (18.1%) in the HPV(−) group. In our study, there was no statistically significant linear correlation between the HPV(+) status detected by specific PCR amplification of HPV DNA and the presence of Pap-detected koilocytes (P > 0.05). These results are in accordance with a growing body of evidence that in many cases there is no full correspondence between cytological and DNA-based test results [6,7]. For example, Richardson et al. [14] recently reported a reduction in the diagnostic accuracy of Pap cytology when evaluating patients' cervical HPV status, possibly due to a heightened awareness of potential abnormalities, which led to more false-positive results. The authors compared original cytotechnicians' Pap assessments in which the HPV status was concealed versus Pap assessments in which the HPV status was revealed in three screening populations [14].

Table 1. Correlation between HPV+/− genotyping and Pap (koilocytes+/−) patients.

	HPV infection
		Negative		Positive		Total
		n	%	n	%	n	%
Pap-koilocytes	No	168	39.9	125	29.7	293	69.6
	Yes	76	18.1	52	12.3	128	30.4
Total		244	58	177	42	421	100

Note: n, number of patients. P = 0.69. This result is not significant at P ≤ 0.05.

It has been suggested that a positive high-risk HPV DNA test is not consistently associated with a precancerous or cancerous Pap cytology [15,16]. In the study of Lee et al. [16], a negative Pap cytology was found in nearly 40% of HPV 31-positive samples to 71% of HPV 45-postitive ones and only about 20% of HPV 16-positive samples detected in a single test at the time of entrance were found to have HSIL cytology, which is considered an indication for immediate colposcopic biopsy to rule out cancer or an advanced precancerous CIN 3 lesion. Based on these findings, Lee et al. [16] speculate whether a reliable negative HPV test could eliminate the need for a Pap cytology test. They also note that a single positive HPV test alone is not a good indication for colposcopic biopsy, since HPV 16-positive and HPV 18-positive samples can more often than not be associated with a negative Pap cytology or a largely reversible to normal Pap cytology in the category of ASCUS or LSIL [16]. Others have also highlighted that more than 95% of referrals to colposcopic biopsies for diagnostic workup based on a single positive high-risk HPV DNA test result have been found to be unnecessary and potentially excessive [17]. Bruni et al. [18] performed a meta-analysis of 194 studies published between 1995 and 2009 that used PCR or Hybrid Capture 2 for HPV detection in a total of 1,016,719 women with normal cytological findings. They obtained an estimated global HPV prevalence of 11.7% (95% confidence interval, 11.6%–11.7%) [18]. Another study that aimed to estimate the impact on follow-up of HPV testing after the first-time ASCUS diagnosis in 287 cases demonstrates that HPV infections are common in adolescents and suggests that a positive HPV test cannot predict which women will develop carcinoma [19]. That is why it has been recommended to adhere to current guidelines in this population [19]. The review study of Arbyn et al. [20] compared the accuracy of HPV testing against that of repeat cytology for detection of underlying CIN of grade 2 or worse (CIN 2+) or grade 3 or worse (CIN 3+) in women with ASCUS or LSIL. The authors suggest that HPV testing can be recommended to triage women with ASCUS because it has higher accuracy (significantly higher sensitivity and similar specificity) than repeat cytology. They also point out that, in triaging women with LSIL, an HPV test yields a significantly higher sensitivity, but a significantly lower specificity, compared to a repeat cytology [20]. In line with these observations, in our study, we did not find significant correlation (P < 0.05) between HPV(+) status detected by PCR amplification of HPV DNA and cytological LSIL/HSIL changes (Table 2). This observation is in accordance with the results from our previous study on the same cohort of women, which suggested that there is not always a clear-cut causative relation between HPV infection and CIN [10]. We speculate that, although HPV persistence is commonly recognized to play an essential role in the progression of pre-neoplastic lesions and the development of cervical cancer, HPV is probably not the sole reason behind abnormal cytological findings and development of CIN.

Table 2. Correlation between HPV+/− genotyping and Pap (LSIL/HSIL+/−) patients.

	HPV infection
		Negative		Positive		Total
		n	%	n	%	n	%
Pap-LSIL/HSIL	No	197	48.6	137	32.5	334	79.3
	Yes	4	11.2	40	9.5	87	20.7
Total		244	58	177	42	421	100

Note: n, number of patients. P = 0.40. This result is not significant at P ≤ 0.05.

In April 2014, the United States Food and Drug Administration approved an HPV DNA test as a primary screening method in women aged 25 and older [21]. Identification of HPV infection in asymptomatic women may allow the implementation of appropriate prophylactic measures which may have a direct impact on the natural history of the disease and the subsequent development of cervical malignancy [22]. However, it has also been argued that it is not clear whether the HPV DNA test reduces the incidence of cervical cancer compared to cervical cytology and whether the harms from false-positivity increase [23]. This calls for careful in-depth analysis of the benefits and any potential harms from diagnosis based solely on HPV DNA screening.

Conclusions

The results from this study showed that HPV infection is very frequent even in women with negative Pap smear results and that HPV PCR seems to be the only reliable test to detect this infection in samples. However, the obtained results still do not support a replacement of cytological tests and cervical colposcopy in the screening for cervical cancer with HPV-genotyping tests only. Further research is needed to determine whether the benefits truly outweigh the potential harms from false positivity in HPV DNA testing.

Disclosure statement

No potential conflict of interest was reported by the authors.