Over the past decade, many large prospective studies have investigated the prognostic value of Ki-67 in outcome of cervical carcinoma. Ki-67 has been used in routine cervical pathology to assess cervical intraepithelial neoplasia (CIN) and to differentiate between postmenopausal atrophy and CIN ^4,9. However few studies have investigated the application of Ki-67 in cervical cytology using classic Papanicolaou (Pap) smears ^4,9.

We investigated the possibility of applying Ki-67 and polymerase chain reaction (PCR) to liquid based cytology samples. We aimed to show the relationship between cervical carcinoma, some types of HPV and Ki-67 in prostitute women.

These samples were then stained for Ki-67 and Papanicolaou. All participants were asked to complete a self–administered confidental questionnaire, which included questions about contraception, age at first sexual intercourse, smoking, pregnancies, concurrent genital infections, and number of male partners. Investigations were performed in accordance with the guidelines of the Local Ethics Committee. Although no risks were attributable to our study, informed consent was obtained from all the patients.

Cytology
Cervical cells were collected using the PAPETTE ™ (Thermo Shandon, Pittsburg, USA) cervix brush and placed immediately into the vial of PapSpin Collection Fluid (Thermo Shandon, Pittsburg, USA). Thin layer liquid based cytology (LBC) preparations were made with the Cytospin 4 cytocentrifuge (Thermo Shandon). From a starting volume of 1000 μl for each sample, a 200 μl cell suspension was used to prepare each LBC.

All slides were manually screened by a pathologist after training in the evaluation of thin layer slides. Abnormal or dubious cases were reviewed again by the pathologist for final diagnosis.

The cytological results were classified according to the Bethesda 2001 system ^10, using the following categories: Within normal limits (WNL), atypical squamous cells of undetermined significance (ASC-US), low grade squamous intraepithelial lesions (LSIL), high grade squamous intraepithelial lesions (HSIL), and atypical glandular cells (AGC).

HPV- DNA testing by PCR
As reported previously, from the remaining cell suspension of 800 μl, a 400 μl sample was saved for HPV- DNA detection by PCR. All samples (200 μl) were tested with Qiagen, PureArt DNA Mini Kit (Qiagen GmbH, Hilden/ Germany) for DNA extraction. After that, PCR was performed by using “MY09/11” primers which could amplify approximately 400 base pairs of L1 gene location of the virus. These primers were obtained from LCD-Array HPV 2.5 (Chipron GmbH, Germany) kit and included all types of HPV. Amplification was run as follows: initial denaturation of 96°C for 2 minutes, followed by 45 cycles of 94°C/40 sec, 48°C/40 sec and 72°C/40 sec, respectively. For HPV positive samples, hybridization and staining procedures were performed. Briefly, hybridization was performed to on PCR products reproduced by using type specific probes which were fixed to “LCD-array”. The spots formed after staining procedure that showed hybridization, were evaluated by automatic analyse system “SlideReader software” (LCD-Analysis package, Chipron, Germany) was used for determining HPV types.

Immunocytochemistry
From the remaining 400 μl cell suspension after HPV DNA detection, 50 μl was used to make thin layer preparations with the Cytospin 4 cytocentrifuge (Thermo Shandon) on poly-Llysine coated glass slides. Preparations were fixed in a methanol+acetone mixture at -20°C. Single antigen staining was performed with Ki- 67 protein, and Ultra Vision Polyvalent, Rabbit, HRP-AEC kit (Neomarkers-Biogen, Lab Vision Corp. USA). Cells were considered immunopositive if the nuclei were stained. Evaluation of the immunocytochemistry was done by an Olympus Bx51 light microscope. All cells in one high power field (x40), which was considered to be representative of the whole cell area, were counted, and the fraction of positive cells on the slide was calculated. This fraction was expressed as the number of positive cells per 1000 cells to facilitate comparisons.

The statistical procedures were carried out by the software of Epi INFO version 3.3.2 CDC (Centers for Disease Control and Prevention, USA). p<0.05 was considered statistically significant. For the comparison of the findings t-test and chi-square test were performed.

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Figure 1: The cervical cells with pleomorphic, hyperchromatic nuclei and leucocytosis in a HSIL cytology (Pap x400).

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Figure 2: Ki-67 positive stained cervical cells in a cervical cytology (Ki-67 x100).

The mean Ki-67 values were found as 4.7±5.21 in the study and 2.5±1.31 in the control group, respectively. Leukocytosis was detected as 1.6±826 cells/ml in the study and 1.2±1.206 cells/ml in the control group, respectively (Table 1).

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Table 1: The distribution of Ki-67, age, first sexual experience age and leucocytosis according to groups (Group statistics, T test).

The mean age at the first sexual experience was 16.7±2.31 years in the study and 20.3±2.00 years in the control group, respectively.

There were 2 patients with nuclear enlargement, one patient with LSIL and one patient with HSIL in the study group and also 2 patients in the control group. There was a positive correlation between nuclear changes and HPV positivity (x²=28.8 p<0.001).

Two patients were detected as HPV positive in each group; Type 6 and 32 positivity in the study and Types 6 and 51 positivity in the control group (Figs 3, 4).

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Figure 3: Lane M is molecular size marker 100 bp DNA ladder (Biotools, Ispain).

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Figure 4: 1-Schematic diagram of LCD-Array. Arrays were spotted in dublicate. The different regions explored by the probes tiled on the array.

The mean values of Ki-67 were found as 2.7±1.2 and 3.6±4.1 in HPV positive and negative patients respectively. Leucocytosis was calculated as 2.2 ± 0.95 6 cells/ml in HPV positive and 1.3 ± 1.01 6 cells/ml in HPV negative women, respectively.

There was a positive correlation between nuclear changes and HPV positivity (x2=28.8 p<0.001), other results were not statistically meaningful. There was no relationship between HPV, Ki-67 and leukocytosis. Also there was also no relationship between HPV and contraception, smoking, or concurrent genital infections (Table 2).

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Table 2: The distribution of contraception, nuclear changes, smoking and genital infection according to HPV positivity (Chi square test).

Since the introduction of programs for preventing cervical cancer, the incidence of this type of cancer has declined appreciably in developed countries. In contrast, cervical cancer is still a problem in developing countries. Strategies for preventing cervical cancer in these countries should overcome barriers such as inadequate medical infrastructure and poor rates of participations in screening programs ^8,13.

Detection of some specific types of HPV provides strong evidence implicating the role of HPV in cervical carcinogenesis ¹³. Most authors agree with the hypothesis that the integration of the HPV genom occurs very early in the development of cancer ². We also found a positive correlation between nuclear changes and HPV positivity (x²=28.8 p<0.001)

We detected nearly equal HPV-DNA loads either in the control and in the study group which had all risk factors for the development of cervical carcinoma.

The use of Pap test alone is not enough to eliminate cervical carcinoma ¹⁴. Liquid based cytology has some added advantages like immunocytochemistry and PCR besides classical examination and also it has a favourable cost-effectivity ¹⁵. Molecular epidemiologic studies have elucidated the central role that specific HPV types play in the development of cervical neoplasia ¹⁶. The increased sensitivity afforded by liquid based preparations is a peculiar advantage of HPV testing over repeat Pap tests ¹⁷.

We detected HPV 6 and 32 in prostitute women and HPV 6 and 51 in the control group in this study. Different genetic, racial and also environmental factors such as geographical locations may contribute differently to the mechanism of cervical cancer induction by different types of HPV ^8,18. Recent studies have shown that HPV types 16 and 18 were prevalent in Western countries, but HPV 18 has shown profound variations in different geographical egions and HPV 16 is the most prevalent type associated with cervical malignancy worldwide ¹⁸. Other HPV types such as 31, 33, 35, 52b and 58 have been frequently reported specially in Japan ¹⁸.

On the other hand PCR is an extremely sensitive method and it is being applied in diagnosing a wide range of clinical conditions ¹⁹. Therefore PCR technique and laboratory contamination might account for these differences ¹⁸.

Although several studies have been performed to detect HPV in cervical smears in Turkey, this is the first immunocytochemical study conducted in prostitute women in Turkey.

We could not find a statistically significant relationship between contraception, age at the first sexual experience age, smoking, pregnancies and concurrent infections with HPV and Ki- 67 protein expression rates. However this study might have given different results if it were done with a large study population consisting of similar patient groups.

Ki-67 is a nuclear antigen expressed in all phases of the cell cycle excluding the G0 phase ²⁰. The cytological diagnoses of ASCUS or HSIL with a high count of Ki-67 immunopositive cells could be useful for the follow up of these women, whose cell cycles might be disrupted. The detection of deregulation of the cell cycle identifies those infections at risk of progression towards dysplasia and carcinoma⁴.

Ki-67 expression is normally confined to the basal and parabasal layers of the normal cervical squamous epithelium in histologic sections. This expression extends above the basal one third of epithelium and the number of positive cells increases in dysplasia and carcinoma⁴.

The overlap in immunopositive counts in the different groups could be explained by the fact that normal proliferating cells from the basal layer of the epithelium could be assessed as positive cells ⁴. Mean Ki-67 values were detected as 4.7±5.21 and 2.5±1.31 in our study and in the control group respectively. This study reveals that when used alone, Ki-67 is not a satisfactory method for the differential diagnosis of these cases.

Shabeli et al. used Ki-67 marker in liquid based cytology samples and showed strong Ki 67 positive cells in HSIL ve HPV16 positive cases⁴.

We did not detect HPV 16 DNA and there is not a statistically significant relation between Ki-67 and HPV types in our study. This may suggest that Ki-67 immunocytochemistry could be useful as a surrogate marker of HPV-16 infection but not for other types of HPV.

Ki-67 immunocytochemistry is an easy and fast technique which could be an attractive method especially for smaller or less specialized laboratories.

Since the origin of Ki-67 antigen has not been known yet, the false positive results cannot be excluded. At the same time, Ki-67 immunoreactivity is highly dependent on fixation time, specimen storage conditions and antigen retrieval techniques ²⁰.

Ki-67 is a very popular laboratory technique, however, this method has some serious limitations and its cytochemical importance needs to be studied in a prospective situation ²⁰.

A combined approach including routine cytological parameters, immunocytochemistry and other molecular techniques such as PCR may be more useful. However this combined approach should be evaluated in large scale prospective studies.

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