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Research Article
 

A Comparison Between Cytological Method and PCR in the Diagnosis of HPV Infection Among Patients with Cervical Cancer



S. Soghra Moosavi, Saeed Soltani and Mojhgan Shaikhpoor
 
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ABSTRACT

Present study is aimed at comparison between cytological method and polymerase chain reaction in the diagnosis of HPV infection among patients with cervical cancer. Cervical cancer is one of the common reproductive system cancers in developing countries that involve a much numbers of women annually. It is believed that human papillomavirus protein products including E6 and E7 cause transformation. Forty five women out of 166 studied ones were infected by HPV (p = 0.1). Among these 45 patients, 24 cases were recognized with HPV 16 (p = 0.2), 14 with HPV 18, 3 with both HPV 16 and HPV 18 and 4 with other types of HPV. PCR indicated 31 samples with HPV infection (24.03%) whereas cytology could only find 4 HPV infected patients (3.11%). The difference was statistically significant (p = 0.003). We conclude that PCR is more sensitive to diagnose HPV infection and also its type than cytology.

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  How to cite this article:

S. Soghra Moosavi, Saeed Soltani and Mojhgan Shaikhpoor, 2008. A Comparison Between Cytological Method and PCR in the Diagnosis of HPV Infection Among Patients with Cervical Cancer. Biotechnology, 7: 798-802.

DOI: 10.3923/biotech.2008.798.802

URL: https://scialert.net/abstract/?doi=biotech.2008.798.802
 

INTRODUCTION

Cervical cancer is the second most common cancer among women next to the breast cancer (Ogunmodede et al., 2007). Cervical infection by human papillomavirus is the most important risk factor developing cervical pre-malignant and malignant lesions (Bozzetti et al., 2000). A number of secondary factors are thought to influence the likelihood that an HPV infection will persist and progress to cervical cancer such as sexual and reproductive factors (Nabaei and Bahiraei, 2001), sexual intercourse at lower age (Dyson et al., 2002), poor socioeconomic conditions, cigarette smoking, long term use of contraceptive pills, nutritional diet and etc (Nabaei and Bahiraei, 2001).

Human papillomaviruses are classified according to their genetic similarities (Swygart, 1997). Up to now, more than 70 types of HPV have been characterized and their DNA sequence were identified (Cubie et al., 2001). They tend to special epithelium. The most common HPV are those tend to the epithelium of genital system. However, these viruses are also able to infect other sites such as upper respiratory tracts, connective tissues, paraunguinal tissues and etc; although, the main source of HPV is male and female lining cutaneous and wet mucosal tissue of genital organ (Anderson et al., 1997). There are 30 types of HPV that may infect the squamous epithelium of the lower tract of male and female reproductive system (anogenital area). The lesions appear in two forms: cauliflower-like and flat warts. Some other types of these viruses lead to asymptomatic or precancerous diseases (Lytwyn et al., 2000). HPV genome is divided into three different regions including early, late and Long Control Region (LCR). Moreover, early region proteins are classified into two groups constitute of E1, E2, E3, E4 proteins and E5, E6, E7 oncoproteins (Dyson et al., 2002). E5 oncoproteins stimulate the growth of epithelial cells and in many cancers, lead to increased cellular mitosis and consequently, cause papilloma lesions (Prayitno, 2006). E6 oncoprotein is the most important HPV proteins, responsible for chromosomal abnormalities and progressing cells to neoplasia (Dyson et al., 2002). E6 oncoprotein destroys P53 (Pei, 1996). In addition, E7 oncoprotein bind to Rb gene product and similar proteins, so inactivate them (Kim et al., 2001). There are several methods to recognize HPV such as cytological evaluation, colposcopy, biopsy, tissue diagnosis using Schiller`s test (Molijn et al., 2005) and molecular methods such as Southern Blot, Dot Blot Hybridization, Polymerase Chain Reaction and so on (Griffin et al., 1997). According to above mentioned introduction, the purpose of present study is to investigate on sensitivity of PCR versus cytological methods in diagnosis of HPV infection in patients with cervical cancer and to evaluation about their application in screening programs.

MATERIALS AND METHODS

This study was conducted in Cellular and Molecular Biology Laboratory, Khatam University, Tehran, Iran. Samples were prepared from those women who had clinical manifestations or tended to be assured of their health, referred to Shahrara specialized laboratory. In addition, some paraffin embedded tissue blocks were studied and repeated preparations were made.

Cytological method: The swab which had been used to collect endocervical and exocervical sample were placed on a clean glass slide and the specimen was distributed evenly. In order to prevent air drying artifacts, samples were fixed in a fixator. Then the samples were stained with papanicolaou (the slides were placed in ethanol 100°C for 15 min and then washed 0. Moreover they remained in Hematoxylin, ethanol 96°C and EA50 solution for 5, 1-2 and 5 min, respectively. Slides were exposed to ethanol 96°C for 2 or 3 times and then let them dry (Young et al., 1989). Finally, the slides were studied using optical microscope.

PCR method: Sampling swab was put in a microtube containing 1 mL of PBS solution and then rotated. In order to extract DNA, 1 mL of lysing buffer was added [including 10 mM Tris (pH = 8), 1 mM EDTA, SDS 1%, 200 µg mL-1 Proteinkinase K and 1 µL of 2ME (mercaptoethanol)] into microtube and put it in Vortex for 3 min. These tubes were placed for 5 min in heating block with a temperature of about 65°C. Then 130 µL of KCl was added to the contents of tubes and remained in a temperature of about -20°C for 5 min. Samples were centrifuged in 13000 rpm for 10 min, let the proteins deposit. In order to concentrate DNA, 500 µL Isopropanol and 60 µL sodium acetate 3M were added to the samples, then they were left for 10 min in -20°C. Sodium acetate caused DNA to be ionized and decreased its solubility in water (Shibata et al., 1988). Also, adding highly purified ethanol led to decreased water contents of the samples that per se contributed to the appearance of DNA in the form of non-soluble opacities. From samples that were sent to the laboratory in paraffin embedded blocks, we performed sections and omitting paraffin and also the hydration before DNA extraction.

PCR phases: We concerned a 0.5 mL microtube for each of purified DNA samples. Then, 7.2 mL PCR mix (contained PCR buffer including 10 mM Tris HCl in pH = 9, 1 mM MgCl2, 50 mM KCl and 0.2 mM of each dNTP) was added. Additionally, 50 pm of each primer including MY09 (5`GCTCC[C/A]A[G/A][G/A]GGA [T/A]ACTGAT3`) and MY11 (5`GC[C/A]CAGGG[T/A] CATAA[T/C]AATGG3`) was added (Marybeth et al., 1996). At the next step, 2.5 µL purified DNA with respect to the numbering following 0.1 µL (0.5 U) Taq Polymerase enzyme were added. Preventing materials vaporization, 2 drops of paraffin were added finally and caps of microtubes were fastened. The microtubes were placed in Termocycler. First PCR product was used as the target DNA for second PCR. In this phase, each patient two microtubes were concerned (for HPV 16 and HPV 18). All of the mentioned process phases were repeated again, except adding specific primers for HPV 16 and HPV 18 including (Kampion, 1992):

HPV 16
forward primer: 5´-GAACAGCAATACAAVCAAA-3´
HPV 16
reverses primer: 5´-CCATGCATGATTACAGCTGG-3´
HPV 18
forward primer: 5´-TGCCAGAAACCGTTGAATCC-3´
HPV 18
reverses primer: 5´-CAATGTCTTGCAATGTTGCC-3´

Termocycler program constituted of 35 repetitive cycles. First of all, the samples were left in 94°C temperature for 3 min and in 94°C temperature for 30 sec (to separate double strands); then in 50°C temperature for 1 min (to bind the primers to the strands) and in 72°C temperature for 30 sec (to elongate the concerned strand). They were left in 72°C temperature for additional 3 min for assurance. The second PCR phase was similar to the first one. In order to deposit and stain DNA, PCR products that were 11 µL were combined by 2 µL loading buffer and then microfuged (Pernoll and Benson, 1987). Agarose gel was used for electrophoresis of the samples. Samples were studied to observe the specific bands under UV transilluminator.

RESULTS AND DISCUSSION

The traditional way of classifying tumors is by histopathology; the staining and analysis of tissue samples. Now, the ability to analyze change in the levels of the transcripts and/or protein products for literally thousands of genes promises interesting possibilities as a research tool-for understanding the underlying molecular mechanisms, but also for automated tissue diagnosis (Drain et al., 2002; Shin et al., 2003). The diagnosis of cancer relies primarily on invasive tissue biopsy, as non invasive diagnostic test are generally insufficient to define a disease process of cancer. Molecular medicine has led to the discovery and application of molecular tumor markers, which make histology more accurate and additionally help to prognosticate cancer. The diagnosis of cancer involves the analysis of tissue and cytology specimens obtained through several procedures, including surgical biopsy, endoscopic biopsy, Polymerase Chain Reaction etc. Target-amplified HPV assays, such as PCR, produce highly concentrated samples of a specific DNA genetic sequence. The DNA samples are then probed to identify which specific HPV genotypes are present. PCR is the most common target amplified technique; its inherent strength lies in its capacity to detect have programmatic implications. While HPV is an objective test with rapid turnaround, the test results are not immediate. In addition, quality control mechanisms for HPV testing need further evaluation. The referring women ranged between 19 and 65 years of age and their mean age was 38.3 years. Forty five women out of 166 studied ones were infected by HPV (p = 0.1). Table 1 shows result of studying HPV Infection by PCR.

Table 1: Result of studying HPV infection by PCR

Among these 45 patients, 24 cases were recognized with HPV 16 (p = 0.2), 14 with HPV 18, 3 with both HPV 16 and HPV 18 and 4 with other types of HPV. Table 2 shows result of studying HPV infection by cytology.

Patients with HPV 16 and HPV 18 might be infected with other types. Frequencies of HPV infection revealed by cytology and PCR are shown by Fig. 1.

HPVs frequently infect humans. They are classified into categories of low-risk types responsible for the most common sexually-transmitted viral infections and high-risk types which are crucial etiological factors for cervical cancer development (McFadden and Schmann, 2001). It is extremely important to detect and genotype HPVs at an early stage of the infections as to direct clinical treatment and reduce the incidence HPV-related carcinomas, especially cervical cancer (Bosch and de Sanjose, 2002). The traditional method for HPV detection, are morphological and immunological methods. Currently, the methods for HPV detection are molecular biological methods, including nucleic acid hybridization-based and PCR-based methods. Since cervical cancer has a long preinvasive period, it is clear that early diagnosis and preventing the onset of invasive form and also its treatment is very important. Previously, cervical cytopathology was used for diagnosis. This method could only confirm HPV infection without the determination of its type. High rate of false negatives is one of the cytology method defects. There are remarkable differences in the results of this test and it has low reproducibility. Its reliability depends on two main factors including expert sampling and interpretation in addition to some problems such as inadequate cell sampling, unsuitable fixation or obscuring blood or inflammation which my lead to cytopathologist misinterpretation (Koss, 1992; Valente et al., 1996).

Table 2: Result of studying HPV infection by cytology

Fig. 1: Frequencies of HPV infection revealed by cytology and PCR

In this study, PCR indicated 31 samples with HPV infection (24.03%) whereas cytology could only find 4 HPV infected patients (3.11%). The difference was statistically significant (p = 0.003). This result is in accordance with previous research. For example, Naucler et al. (2004) using PCR method found that HPV-16 and-18 are the most frequent HPV infections associated with cervical cancer in Mozambique and PCR is more sensitive than cytological method in diagnosis of HPV infections. Guerrero et al. (1992) by comparison of Viral Pap, Southern Hybridization and PCR for HPV identification, suggest that PCR-based HPV identification is the method of choice for future epidemiological investigation. We believe that low quality Pap smear especially with inadequate cell sample is etiologic. On the other hand, cytology disability for diagnosis of unclear HPV infection may result in false negative results. Unlike to the cytology, PCR is able to recognize symptomatic and asymptomatic HPV infection and also its type. There are two major restrictions that may obstruct the use of PCR in cervical cancer screening programs: (1) the methods and instrumentation required to process cervical specimens and (2) the technical equipment requirements for interpreting test results. Regarding the first restriction, it is possible that instrumentation and processing of samples may be simplified by developments in isothermal amplification of the target HPV DNA. As implied by its name, isothermal amplification does not require the constant change in temperature generally needed to separate, hybridize and amplify target DNA. Instead, enzymes catalyze the formation of daughter strands identical to the targeted section of DNA. These enzymes are effective in all three phases of amplification, which can proceed at room temperature. This technology is still in development. The second restriction ultimately may be addressed through adaptations of current approvals and/or development of simple, rapid, endpoint read-out systems using a lateral flow (immunochromatographic) technology.

In conclusion, PCR sensitivity for diagnosis of HPV infection is 4 fold higher than cytology, immunohistochemistry, histopathology and colposcopy. Among different molecular methods, PCR is the most sensitive method diagnosing HPV infection. It is recommended to apply polymerase chain reaction with specific pair primers for detection of HPV infection and related types of it, rather than conventional methods. However there is some limitation such as need to expensive laboratory equipments and reagents in polymerase chain reaction to detection of HPV in cervical cancer. Use of other complementary method of nucleic acid amplification such as isothermal amplification may be result in more convince in cervical cancer screening programs.

REFERENCES
1:  Anderson, M., J. Handley, L.H. Wood, S. Murant, M. Stower and N.J. Maitland, 1997. Analysis of prostate tissue DNA for the presence of human papillomavirus by polymerase chain reaction, cloning and automated sequencing. J. Med. Virol., 52: 8-13.
PubMed  |  

2:  Bosch, F.X. and S. de Sanjose, 2002. Human papillomavirus in cervical cancer. Curr. Oncol. Rep., 4: 175-183.
PubMed  |  

3:  Bozzetti, M., B. Nonnenmacher, I. Mielzinska, L. Villa and A. Lorincz et al., 2000. Comparison between Hybrid Capture II and polymerase chain reaction results among women at low-risk for cervical cancer. Ann. Epidemiol., 10: 466-466.
CrossRef  |  PubMed  |  

4:  Cubie H.A., A.L. Seagar, E. McGoogan, J. Whitehead, A. Brass, M.J. Arends and M.W. Whitley, 2001. Rapid real-time PCR to distinguish between human papillomavirus types 16 and 18. J. Clin. Pathol. Mol. Pathol., 54: 24-29.
PubMed  |  

5:  Drain P.K., K.K. Holmes, J.P. Hughes and L.A. Koutsky, 2002. Determinants of cervical cancer rates in developing countries. Int. J. Cancer, 100: 199-205.
PubMed  |  

6:  Dyson N., M.P. Howley, K. Munger and E.D. Harlow, 2002. The humanpapilloma virus 16 oncoprotein is able to bind to the retinoblastoma gene products. Science, 243: 934-936.
CrossRef  |  PubMed  |  

7:  Griffin, N.R, D. Dockey and F.A. Lewis, 1997. Demonstration of low frequency of human papillomavirus DNA in cervical adenocarcinoma and adenocarcinoma in situ by the polymerase chain reaction and in situ hybridization Int. J. Gynecol. Pathol., 10: 100-106.
PubMed  |  

8:  Guerrero, E., R.W. Daniel, F.X. Bosh, X. Castellsague, M. Nubia, M. Gilli and P. Viladuli, 1992. Comparison of viral Pap, southern hybridization and polymerase chain reaction for Human papillomavirus identification in an epidemiological investigation of cervical cancer. J. Clin. Microl., 30: 2951-2959.

9:  Kampion, M.J., 1992. The adequate cervical smear: A modern dilemma. J. Fam. Prac., 34: 271-292.
Direct Link  |  

10:  Kim, Y., S.Y. Hur, T.E. Kim, J. Lee, E.S. Namkoong, I.K. Kim and J.W. Kim, 2001. Protein kinas C modulates telomerase activity in human cervical cancer cell. Exp. Mol. Med., 33: 156-163.
PubMed  |  

11:  Koss, L.G., 1992. Diagnostic Cytology and its Histopathologic Bases. 4th Edn. Lippincott, Philadelphia.

12:  Lytwyn, A., J.W. Sellors and J.B. Mahony, 2000. Comparison of human papillomavirus DNA testing and repeat Papanicolaou test in women with low-grade cervical abnormalities: A randomized trial. Can. Med. Assoc. J., 163: 701-707.
CrossRef  |  

13:  Marybeth, W., M.J. Carmody, H. Tarraza and P.H. Calvin, 1996. Use of the polymerase chain reaction to specifically amplify integrated HPV 16 DNA virtue of its linkage to interspersed repetitive DNA. Mol. Cel. Probes, 10: 107-116.
CrossRef  |  

14:  McFadden, S.E. and L. Schumann, 2001. The role of human papillomavirus in screening for cervical cancer. J. Am. Acad. Nurse Pract., 13: 116-125.
PubMed  |  

15:  Molijn, A., B. Kleter, W. Quint and J.L. van Doorn, 2005. Molecular diagnosis of human papillomavirus (HPV) infections. J. Clin. Virol., 32S: S43-S51.
CrossRef  |  PubMed  |  

16:  Naucler, P., F.M. Costa, O. Ljungberg, A. Bugalho and J. Dillner, 2004. Human papillomavirus genotypes in cervical cancers in Mozambique. J. General Virol., 85: 2189-2190.
CrossRef  |  

17:  Ogunmodede, F., H.S. Yale, B. Krawisz, G.C. Tyler and A.C. Evans, 2007. Human papillomavirus infections in primary care. Clin. Med. Res., 5: 210-217.
CrossRef  |  PubMed  |  

18:  Pei, X.F., 1996. The human papillomavirus E6|E7 genes induce discordant changes in the expression of cell growth regulatory proteins. Carcinogrnesis, 17: 1395-1401.
PubMed  |  

19:  Pernoll, M.L. and R. Benson, 1987. Current Obstetrics and Gynecology Diagnosis Treatment. 10th Edn. Appleton and Lange, California, ISBN 0071439005 / 9780071439008.

20:  Poljak, M., I.J. Marin, K. Seme and A. Vince, 2002. Hybrid capture II HPV test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail. J. Clin. Virol., 25: 89-97.
PubMed  |  

21:  Prayitno, A., 2006. Cervical cancer with human papillomavirus and epstein barr virus positive. J. Carcinog, 5: 13-13.
CrossRef  |  

22:  Shibata, D.K., N. Arnheim and W.S. Martin, 1988. Detection of human papillomavirus ion paraffin embedded tissue using the polymerase chain reaction. J. Exp. Med., 167: 2000-2008.

23:  Shin, H.R., D.H. Lee, R. Herrero, J.S. Smith, S. Vaccarella and S.H. Hong, 2003. Prevalence of human papillomavirus infection in women in Busan, South Korea. Int. J. Cancer, 103: 413-421.
PubMed  |  

24:  Swygart, C., 1997. Human papilloma virus: Disease and laboratory diagnosis. Br. J. BioMed. Sci., 54: 229-303.
PubMed  |  

25:  Valente, P., D. Schantz and J.F. Trabal, 1996. The determination of papanicolaou smear adequacy using a semiquantitative method to evaluate cellularity. Diagnostic Cytopathol., 7: 576-586.
PubMed  |  

26:  Young, L.S., I.S. Bevan, M.A. Johnson, P.I. Blomfield, T. Bromidge, N.J. Maitland and C.B.J. Woodman, 1989. The polymerase chain reaction: A new epidemiological tool for investigating cervical human papillomavirus infection. BMJ., 298: 14-18.
PubMed  |  


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