SCImago Journal & Country Rank
This journal is a member of, and subscribes to the principles of, the Committee on Publication Ethics (COPE)
2006, Volume 22, Number 3, Page(s) 167-173
[ Abstract ] [ PDF ] [ Similar Articles ] [ E-Mail to Editor ]
HPV-DNA testing and Ki-67 immunocytochemistry in liquid based cervical cytology in prostitute women
Esin ATK1, Bar OTLU2, Meryem ETN3, Devrim TOK4, Rza DURMAZ5
1Department of Pathology, Mustafa Kemal University, Faculty of Medicine, HATAY
2Department of Microbiology, Inonu University, Faculty of Medicine, HATAY
3Department of Microbiology, Mustafa Kemal University, Faculty of Medicine, HATAY
4Department of Obstetric and Gynecology, Mersin University, Faculty of Medicine, HATAY
5Department of Microbiology, Inonu University, Faculty of Medicine, HATAY
Keywords: HPV, Ki-67, thin layer liquid based cytology, immunocytochemistry
Abstract
HPV causes several changes in the function of host genes, and these interactions cause deregulation of the cell cycle manifested by abnormal expression of cell cycle associated proteins, such as Ki-67. The detection of Ki-67 can play a role in screening and diagnosis of HPV infection with risk of progression towards dysplasia and carcinoma. To show this relation in prostitute women, cervical cells were collected in the PapSpin Collection Fluid. A starting volume of 1000 l for each sample, and a 200 l cell suspension were used to prepare each sample for thin layer liquid based cytology and then they were stained by Papanicolaou method. The cytological results were classified according to the Bethesda 2001 system. From the remaining cell suspension of 800 l, a 400 l sample was used for HPV-DNA detection by PCR, a 50 l alliquot was used to make thin layer preparations for immunocytochemistry. Single antigen staining was performed with Ki-67 protein. Cells were considered immunopositive if the nuclei were stained. All cells in one high power field (x400) were counted, and the fraction of immunopositive cells on the slide was calculated. This fraction was expressed as the number of positive cells per 1000 cells to facilitate comparisons of differential cell counts. HPV types 6 and 32 in the study, and HPV types 6 and 51 in the control group were detected. The mean Ki-67 values were 2.71.2 and 3.64.1 in HPV positive and negative cases respectively. There was a positive correlation only with nuclear changes and HPV positivity (x2=28.8, p<0.001). There was not any significant correlation between HPV or Ki-67 and leukocytosis. An association with HPV and contraception, smoking, and concurrent genital infection was not found. The prevalence of HPV types in different geographical locations and races may indicate different etiologies of cervical cancer. Our results suggest that Ki-67 immunocytochemistry is not useful as a surrogate marker for HPV types of 6, 32 and 51.
Introduction
The causal relation between genital human papilloma virus (HPV) infection and cervical dysplasia/carcinoma is well established 1-4. However, even though the prevalence of cervical cells infected with HPV can be as high as 60%, only 10% of infected women will develop cervical dysplasia 4-8. The infection of cervical cells by HPV manifests itself by changes in the function or expression of host genes, and the detection of these alterations can play a role in screening and diagnosis 2,4. These interactions cause deregulation of the cell cycle manifested by abnormal expression of cell cycle associated proteins, such as Ki-67. The detection of abnormal expression can identify clinically important cases of HPV infection with risk of progression towards dysplasia and carcinoma 4.

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.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Methods
    Study population
    There were 20 prostitute women as a study group and 20 ordinary women as a control group sampled from the out- patient clinic of Gynecology Department of Mustafa Kemal University, Medical Faculty with a mean age of 32.65.78 and 32.95.32 respectively. The cervical cytology samples were obtained from all 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 96C for 2 minutes, followed by 45 cycles of 94C/40 sec, 48C/40 sec and 72C/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 -20C. 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.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Results
    Immunoreactive cells showed dark brown homogeneous or punctate nuclear staining by Ki-67 (Figs 1, 2).


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


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

    The mean Ki-67 values were found as 4.75.21 in the study and 2.51.31 in the control group, respectively. Leukocytosis was detected as 1.6826 cells/ml in the study and 1.21.206 cells/ml in the control group, respectively (Table 1).


    Click Here to Zoom
    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 16.72.31 years in the study and 20.32.00 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).


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


    Click Here to Zoom
    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 2.71.2 and 3.64.1 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).


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

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Discussion
    Papanicolaou (Pap) cervical cytology screening has helped to reduce cervical cancer rates dramatically since its implementation in 1950s 11. Pap test reporting classifications have evolved and been refined with the Bethesda system. At the same time there is an increased interest in using the detection of HPV-DNA as an adjunct to classic cytological evaluation 2,11,12.

    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 4.75.21 and 2.51.31 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.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • References

    1) Lin H, Moh JS, Ou YC, Shen SY, Tsai YM, Chien CCC, et al. A simple method for the detection and genotyping of high-risk human papillomavirus using seminested polymerase chain reaction and reverse hybridization. Gynecol Oncol 2005;96:84-91.

    2) Gallo G, Bibbo M, Bagella L, Zamparelli A, Sanseverino F, Giovagnoli MR, et al. Study of viral integration of HPV-16 in young patients with LSIL. J Clin Pathol 2003;56:532-536.

    3) Kulasingam SL, Hughes JP, Kiviat NB, Mao C, Weiss NS, Kuypers JM, et al. Evaluation of human papillomavirus testing in primary screening for cervical abnormalities comparison of sensitivity, specificity and frequency of refferal. JAMA 2002;288:1749-1757.

    4) Sahebali S, Depuydt CE, Segers K, Vereecken AJ, Van Marck E, Bogers JJ. Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool? J Clin Pathol 2003;56:681-686.

    5) Lee NW, Kim D, Park JT, Kim A. Is the human papilomavirus test in combination with the Papanicolaou test useful for management of patients with diagnoses of atypical squamous cells of undetermined significance/ low-grade squamous intraepithelial lesions. Arch Pathol Lab Med 2001;125:1453-1457.

    6) Mikaelsdottir EK, Benediktsdottir KR, Olafsdottir K, Arnadottir T, Ragnarson GB, Olafsson K, et al. HPV subtypes and immunological parameters of cervical cancer Iceland during two time periods, 1958-1960 and 1995-1996. Gynecol Oncol 2003;89:22-30.

    7) Infantolino C, Fabris P, Infantolino D, Biasin MR, Venza E, Tositti G, Minucci D. Usefulness of human papilloma virus testing in the screening of cervical cancer precursor lesions: a retrospective study in 314 cases. Eur J Gynecol Reprod Biol 2000;93:71-75.

    8) Riethmuller D, Gay C, Bertrand X, Bettinger D, Chaal JP, Carbillet J. et al. Genital human papillomavirusinfection among women recruited for routine cervical cancer screening or for colposcopy determined by hibrid capture II and polymerase chain reaction. Diagn Mol Pathol 1999;8:157-170.

    9) Koyamatsu Y, Yokoyama M, Nakao Y, Fukuda K, Saito T, Matsukuma K, et al. A comparative analysis of human papillomavirus types 16 and 18 and expression of P53 gene and Ki-67 in cervical, vaginal and vulvar carcinomas. Gynecol Oncol 2003;90:547-551.

    10) Solomon D, Davey D, Kurman R, Moriarty A, O\'Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287:2114-2119.

    11) Manos MM, Kinney WK, Hurley LB, Sherman ME, Ngai JS, Kurman RJ, et al. Identifying women with cervical neoplasia using human papillomavirus DNA testing for equivocal papanicolaou results. JAMA 1999;281:1605-1610.

    12) Krger-Kuer S, Van Den Brule AJC, Svare EI, Engholm G, Sherman ME, Poll PA, et al. Different risk factor patterns for high-grade and low-grade intraepithelial lesions on the cervix among HPV-positive and HPV-negative young women. Int J Cancer 1998;76:613-619.

    13) Sellors JW, Lorincz AT, Mahony JB, Mielzynska I, Lytwyn A, Roth P, et al. Comparison of self collected vaginal, vulvar and urine samples with physician-collcted cervical samples for human papillomavirus testing to detect high grade squamous intraepithelial lesions. Can Med Assoc J 2000;163:513-518.

    14) Finan RR, Irani-Hakime N, Tamim H, Sharide HE, Daccache JL, Almawi WY. Detection of human papillomavirus (HPV) genotypes in cervico-vaginal scrapes of women with normal and abnormal cytology. Clin Microbiol Infect 2001;7:688-692.

    15) Herrington CS. Does HPV testing have a role in primary cervical screening? Cytopathol 2001;12:71-74.

    16) Venturoli S, Cricca M, Bonvicini F, Giosa F, Pulvirenti FR, Gali C. Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results. J Clin Virol 2002;25:177-85.

    17) Hughes SA, Sun D, Gibson C, Bellerose B, Rushing L, Chen H, Harlow BL, et al. Managing atypical squamous cells of undetermined significance (ASCUS): Human papillomavirus testing, ASCUS subtyping, or follow- up cytology. Am J Obstet Gynecol 2002;186:396- 403.

    18) Hwang T. Detection and typing of human papillomavirus DNA by PCR using consensus primers in various cervical lesions of Korean women. J Korean Med Sci 1999;14:593-599.

    19) Manavi M, Bauer M, Pischinger K, Czerwenka K. Detction of human papillomavirus on papanicolaou stained cervical smears using indirect in situ polymerase chain reaction hybridization. Arch Pathol Lab Med 2001;125:353-357.

    20) Atik E, Serarslan Y, Bakaris S, Savas N. The importance of proliferative activity and angiogenesis in meningioma and glioma. Turkish J Neurosurg 2005;15:176- 181.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • [ Top ] [ Abstract ] [ PDF ] [ Similar Articles ] [ E-Mail to Editor ]