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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2017  |  Volume : 60  |  Issue : 3  |  Page : 328-335
Associations between epidermal growth factor receptor and topoisomerase II-alpha gene copy number variations, human papillomavirus positivity, and cytologic analysis in cervical cell lesions


1 Department of Genetics, Faculty of Medicine, Şifa University, Izmir, Turkey
2 Department of Pathology, Faculty of Medicine, Şifa University, Izmir, Turkey
3 Burç Genetics Diagnostic Center, Istanbul, Turkey
4 Department of Obstetrics and Gynecology, Karşıyaka Government Hospital, Izmir, Turkey

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Date of Web Publication22-Sep-2017
 

   Abstract 

Background: Cervical cancer is the second most common gynecologic cancer worldwide. Human papillomavirus (HPV) infection is a leading etiological factor in cervical carcinoma.The aim of this study was to compare HPV positivity, EGFR and TOP2A gene copy number variations and cervical cytologic findings. Materials and Methods: The study group comprised 100 female volunteers between 21-64 years old. Cytologic analysis was performed using the liquid-based cytology technique. HPV DNA testing was performed in all cases. Copy number variations that belong to EGFR and TOP2A genes evaluated by using FISH analysis. Results: Cytologic analysis of the cervical samples revealed abnormal findings in 13 of the 100 study subjects. ASCUS , LSIL, HSIL were determined in 8, 2 and 3 cases respectively as the result of cytologic analysis on all cases. Forty-one (41%) of the 100 women were HPV-positive. Chi-square analysis confirmed that HPV-positive women showed significantly more abnormal cytology (P = 0.035). EGFR deletion, TOP2A deletion and both EGFR and TOP2A deletion were determined in 1, 8 and 1 cases respectively. We found no statistical difference in abnormal cytologic findings between subjects with these gene deletions and subjects with normal gene copy numbers (P > 0.05 for both). No cases of amplification were determined for either gene. Conclusion: As a result, HPV positivity and the determination of changes that may occur in the gene copy number in patients with abnormal cytology can be an important tool on account of prognosis. Research with more patients may be suggested.

Keywords: Cervical cytology, EGFR gene, FISH, HPV, TOP2A gene

How to cite this article:
Kaya I, Sivrikoz ON, Etlik &, Gök A. Associations between epidermal growth factor receptor and topoisomerase II-alpha gene copy number variations, human papillomavirus positivity, and cytologic analysis in cervical cell lesions. Indian J Pathol Microbiol 2017;60:328-35

How to cite this URL:
Kaya I, Sivrikoz ON, Etlik &, Gök A. Associations between epidermal growth factor receptor and topoisomerase II-alpha gene copy number variations, human papillomavirus positivity, and cytologic analysis in cervical cell lesions. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Dec 7];60:328-35. Available from: http://www.ijpmonline.org/text.asp?2017/60/3/328/215368



   Introduction Top


Cancer continues to be a major health and economic problem throughout the world. Cervical cancer is the second most common gynecologic cancer worldwide.[1],[2],[3] Each year approximately 530,000 new cases are diagnosed, and 275,000 cases are fatal. Widespread cytologic screening using the conventional Papanicolaou (Pap) test has significantly reduced the incidence and mortality of cervical cancer.[4],[5] It has recently been accepted that human papillomavirus (HPV) infection is a leading etiological factor in cervical carcinoma.[2],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17] HPV is associated with a range of clinical conditions ranging from benign lesions to cancer. Genital HPVs are divided into high- and low-risk types. Molecular and epidemiologic studies have demonstrated a strong relationship between high-risk HPV and cervical squamous cell carcinoma.[18],[19] In particular, chronic infection with HPV-16 and HPV-18 has been linked to cervical cancer.[10] Viral DNA replication and activation of the productive phase of the HPV lifecycle are an ongoing process with differentiation of HPV-positive epithelial cells. E6 and E7 genes belong to HPV that play a role in the malignant progression of infected cells.[15] A direct correlation has been observed between the implementation of cervical cytology screening and a reduction in the incidence and mortality of cervical cancer.[7] In conventional cytology, cells collected through smear are quickly spread onto a glass slide.[19] In addition to cell aggregation and overlap, abnormal cells may be obscured by blood, mucous, and other debris, which can potentially lead to false negative or inconclusive results.[20] The liquid-based thin smear Pap test, developed as an alternative to the conventional method, seems more effective in providing adequate sample and detecting a substantial proportion of low- and high-grade intraepithelial lesions. One of the main benefits of liquid-based cytology is that the residual cellular material can be used to detect infectious agents such as HPV through molecular tests.[20],[21],[22],[23]

Instability in the number and structure of chromosomes is characteristic of malignant tumors, especially those originating from epithelial cells. Chromosomal instability develops at early stages of cervical neoplasia and can be detected even in premalignant lesions. Cervical carcinomas include some structural chromosomal changes and numerical chromosomal imbalances.[24],[25] Many biological factors have been suggested as prognostic determinants of cervical cancer.[26] Epidermal growth factor receptor (EGFR) is present in many normal tissues and expressed in a wide variety of solid tumors, including cervical cancer.[27]

EGFR belongs to the erbB family, which includes erbB1 (known as EGFR), erbB2 (human epidermal growth factor receptor 2 [HER2]), erbB3, and erbB4.[28] EGFR is a cell surface receptor tyrosine kinase and is expressed in all epithelial and stromal cells.[29] Overexpression of EGFR has been implicated in the pathogenesis of many human tumors.[28],[29],[30],[31],[32],[33],[34],[35]

DNA topoisomerase II-alpha (TOP2A) is a nuclear enzyme that alters the topology of DNA and is encoded by the TOP2A gene located in the 17q12-21 region. TOP2A amplifications and deletions are indicators of both poor prognosis and greater response to treatment with anthracycline.[36]

The aim of this study was to compare HPV positivity and abnormal cytology findings from routine cervical smear screening with EGFR and TOP2A gene copy numbers to determine whether HPV-positive women with abnormal cytology exhibit significant copy number variations in these genes.


   Material and Methods Top


The study group comprised 100 female volunteers between 21 and 64 years old who underwent routine cervical cytology screening in 2013 and 2014. The study was approved by the Local Ethics Committee. Cervical swab samples were obtained from each of the participants, and slides were prepared for cytologic analysis using the liquid-based cytology technique. An HPV test was performed for all participants, followed by genotyping for positive cases. EGFR and TOP2A gene analyses were also conducted on slides prepared by liquid-based cytology.

Liquid-based cytology

Cervical brush samples were obtained from 100 female volunteers and transferred into gynecologic ThinPrep solution. From each of these solutions, two slides were prepared using the ThinPrep 2000 instrument. One of the slides was stained with Pap stain for microscopic analysis; the other was used for EGFR and TOP2A gene analysis by fluorescence in situ hybridization (FISH). The Pap-stained samples were analyzed twice by pathologists masked to the samples' clinical data. The samples were evaluated according to the Bethesda system.

Human papillomavirus test

DNA from the swab material obtained with the ThinPrep or smear kits was isolated by the salting out procedure. DNA samples were diluted with 50 μl TE buffer. The DNA content of the preparations was assessed by human beta-globin gene amplification. DNA isolation was repeated from any samples not showing a 270 bp PCR product using beta-globin primers GH20 (5'-GAA GAG CCA AGG ACA GGT AC-3') and PC04 (5'-CAA CTT CAT CCA CGT TCA CC-3'). All PCR reactions totaled 50 μl and included 5 μl DNA, 5 μl 10X PCR buffer, 2.0 mmol/l MgCl2, 0.2 mmol/l dNTPs, 0.4 μmol/l primer, 2 U Taq polymerase (MBI Fermentas)(Hanover, MD, USA), and ddH2O. Amplifications were performed in a Mycycler™ (BioRad) thermal cycler for 40 cycles with the following parameters: 94°C for 1 min, annealing temperature for 1 min, and 72°C for 1 min. PCR products (20 μl per sample mixed with 6X loading dye) were separated by electrophoresis on a 2% agarose gel and visualized under ultraviolet (UV) light. A nested PCR protocol was used to detect HPV infection. The nested PCR protocol used in the previous studies has been determined the most sensitive and specific detection method for HPV DNA. The MY09/11 primers specific to the L1 region of the HPV genome were used for the first PCR, the product of which was used in the second reaction. It has been reported in the literature that the MY09/11 primers can also bind to the human genome and yield false positive results. In our laboratory, the MY09/11 PCR product was used in a second PCR using GP5+/6+ primers to prevent false positives and negatives (Primers: My11: 5'-GCMCAGGGWCATAAYAATGG-3', My09: 5'-CGTCCMARRGGAWACTGATC-3', GP5+: 5'-TTT GTT ACT GTG GTA GAT ACT AC-3', and GP6+: 5'-GAA AAA TAA ACT GTA AAT CAT ATT C-3'). The second PCR products were separated by electrophoresis on a 2% agarose gel and visualized under UV light. Samples yielding PCR products of 150 bp in length were considered HPV positive.

Epidermal growth factor receptor and topoisomerase II-alpha gene analysis

Two different probes were used for FISH analysis: EGFR, Her-1 (7p11)/SE 7, and TOP2A (17q21)/SE 17 (Poseidon-Kreatech). EGFR (7p11) specific DNA probe and TOP2A (17q21) are direct labeled with PlatinumBright 550 (red signals). The SE 7 control DNA probe and SE 17 control DNA probe are direct labeled with PlatinumBright495 (green signals).

The preparation was incubated in 2X saline sodium citrate (SSC), 0.5% IGEPAL solution (37°C) for 15 min, dehydrated by sequential 1-min incubations in 70%, 85%, and 100% ethanol, then dried at room temperature.

For probe application, the slide surface was divided into two equal sections with 10 μl EGFR applied to one side and 10 μl TOP2A to the other side. The slide was covered with a 22 mm × 22 mm coverslip and sealed with rubber cement. Denaturation at 75°C for 5 min and hybridization at 37°C overnight were conducted in a ThermoBrite. The rubber cement was removed after hybridization before the washing procedure. The slides were washed in 2X SSC/0.1% IGEPAL (room temperature for 2 min), 2X SSC/0.3% IGEPAL (72°C for 2 min), 2X SSC/0.1% IGEPAL (room temperature for 1 min), then dehydrated with ascending ethanol as described above and dried at room temperature. DAPI/Antifade was applied, and the samples were analyzed using a fluorescence microscope (Olympus BX51). Signals were counted using a DAPI, Texas Red, and FITC filter set to visualize nuclei. Signals from overlapping cells or those with indistinct nuclei were not included in the evaluation. Fifty cells were analyzed for each gene. Signals were evaluated for disomy, amplification, and deletion.

Statistical analysis

SPSS version 15 (SPSS Inc., Chicago, IL, USA) software was used for all statistical analyses. Normality of variable distributions was assessed using visual (histogram) and analytical methods (Kolmogorov–Smirnov/Shapiro–Wilk tests). Chi-square analysis was used to compare groups. Results with P < 0.05 were accepted as statistically significant.


   Results Top


In this study, liquid-based cytology was performed with cervical samples obtained from 100 female patients. Cytologic analysis was done according to the Bethesda system. HPV genotyping was performed in all HPV-positive cases. EGFR and TOP2A gene copy numbers in the cervical samples were evaluated by FISH using the prepared cytologic slides.

The distribution of patients' cytology, HPV testing, and EGFR/TOP2A gene analysis results are presented in [Table 1]. Forty-one (41%) of the 100 women were HPV-positive, with HPV-6 being the most common HPV genotype (22%). Distribution of the patients' HPV positivity and HPV types are shown in [Figure 1].
Table 1: Distribution of study participants based on human papillomavirus positivity, cytologic findings, and epidermal growth factor receptor/topoisomerase II-alpha gene deletions

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Figure 1: Study participants' human papillomavirus positivity and type distribution among human papillomavirus-positive subjects

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Cytologic analysis of the cervical samples revealed abnormal findings in 13 of the 100 study participants [Table 2]. These findings were atypical squamous cells of undetermined significance (ASCUS) in 8 cases, low-grade squamous intraepithelial lesions (LSIL) in 2 cases, and high-grade squamous intraepithelial lesions (HSIL) in 3 cases [Figure 2],[Figure 3],[Figure 4],[Figure 5]. Abnormal cytologic findings were observed in 9 (22%) of the 41 HPV-positive patients versus only 4 (6.8%) of the 59 HPV-negative patients. Of the 9 HPV-positive patients with abnormal cytology, ASCUS was found in 4 patients, LSIL in 2 and HSIL in 3; according to the cervical intraepithelial neoplasia (CIN) classification system, 2 of these patients were determined to be CIN I, 1 was CIN II, and 2 were CIN III. Of the 59 HPV-negative patients, ASCUS was observed in only 4 (6.8%), and LSIL or HSIL were not detected. Chi-square analysis confirmed that HPV-positive women showed significantly more abnormal cytology (P = 0.035).
Table 2: Results of cytologic analysis in human papillomavirus - positive and human papillomavirus - negative patients

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Figure 2: Normal servical smear (PAP, ×200)

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Figure 3: Atypical squamous cells of undetermined significance (red arrow) (PAP, ×200)

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Figure 4: Low-grade squamous intraepithelial lesions (red arrows) (PAP, ×200)

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Figure 5: High-grade squamous intraepithelial lesions (red arrows) (PAP, ×200)

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Gene analysis of the 100 study participants revealed EGFR deletion in 1 case, TOP2A deletion in 8 cases, and both EGFR and TOP2A deletion in 1 case [Table 3]. In EGFR gene analysis, trisomy was observed in a single cell of one patient and monosomy was observed in another patient; in TOP2A analysis, one patient exhibited polysomy in one cell and monosomy in another cell [Figure 6],[Figure 7],[Figure 8]. No cases of amplification were found in either gene.
Table 3: Distribution of study participants based on epidermal growth factor receptor and/or topoisomerase II-alpha gene deletions, human papillomavirus positivity/negativity, cytologic findings

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Figure 6: Disomy in epidermal growth factor receptor gene: epidermal growth factor receptor gene: human epidermal growth factor receptor-1 (7p11) (red signals)/SE 7 (green signals)

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Figure 7: Monosomy in epidermal growth factor receptor gene: epidermal growth factor receptor gene: human epidermal growth factor receptor-1 (7p11) (red signal)/SE 7 (green signal)

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Figure 8: (a) Deletion in topoisomerase II-alpha gene: topoisomerase II-alpha (17q21)/SE 17 (red signal)/SE 17 (green signal). (b) Polysomy in topoisomerase II-alpha gene: topoisomerase II-alpha (17q21)/SE 17 (red signals)/SE 17 (green signals)

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Of the patients with abnormal cytologic findings, one had an EGFR gene deletion, and two had TOP2A deletions. However, no statistical differences in abnormal cytology emerged between patients with an EGFR or TOP2A gene deletion and patients with normal gene copy numbers (P > 0.05 for both genes).

Overall, HPV-6 was the most common genotype, detected in 22 patients (22% of the study group, 53.6% of HPV-positive patients). Of the 13 patients with abnormal cytology, HPV genotyping revealed types 16/18 in 4 patients, type 6 in 4, type 33 in 1, and the remaining 4 patients were HPV-negative.

Abnormal cytologic findings were detected in 4 (44.4%) of the 9 patients with known high-oncogenetic risk HPV types 16 and 18 versus only 9 (9.9%) of the other 91 patients. The rate of abnormal cytology findings was significantly higher in women with HPV types 16 or 18 (Chi-square test, P = 0.015), whereas there was no statistically significant difference in the rate of abnormal cytology findings between women with HPV-6 and those with other HPV types (P = 0.475).

Of the two patients with EGFR deletions, one had HPV-6, and one had HPV-18; of the patients with TOP2A deletions, two had HPV-6, and one had HPV-18. However, no significant differences in deletion rates emerged for either gene based on the presence of HPV type 6 or 18 (P > 0.05). Furthermore, the differences in EGFR and TOP2A gene deletion between HPV-positive and negative patients were also insignificant (P > 0.05 for both).


   Discussion Top


This study analyzed cervical samples obtained from 100 women during routine cervical screening. HPV status, cytology, and EGFR and TOP2A gene copy numbers were evaluated independently for each woman. Genotyping of the 41 HPV-positive patients revealed HPV-6 to be the most common. None of the participants in our study group were diagnosed with cervical cancer.

Abnormal cytologic findings are observed more frequently in the cervical smear samples of HPV-positive patients.[1],[37],[38] In our study group, HPV-positive women showed significantly more abnormal cytology compared to HPV-negative women (P = 0.035).

In the literature, HPV-16 and HPV-18 are most commonly associated with cervical cancer.[11],[39] Clifford et al. reported that HPV-16 was the most common genotype in cervical lesions in all over the world.[37] We observed a significantly higher rate of abnormal cytology findings in women with HPV types 16 or 18 (P = 0.015).

In the current study, we found no statistical difference in abnormal cytologic findings between patients with EGFR and TOP2A gene deletions and patients with normal gene copy numbers (P > 0.05 for both).

EGFR was the first tyrosine kinase transmembrane receptor to be directly associated with cancer.[40] Various studies have demonstrated EGFR overexpression in cervical cancer.[17],[41],[42],[43] EGFR/centromere enumeration probe (CEP) 7 ratio >2 in FISH analysis is considered gene amplification. Some studies have provided evidence that EGFR gene amplification is correlated with the severity of cytologic findings in cervical cancer.[44],[45] Furthermore, it has been proposed that detection of abnormal EGFR activation in early cervical cancer may serve as a biological marker in cervical cancer screening, enabling early diagnosis, and prevention of disease progression.[45] However, Scambia et al. found no correlation between EGFR expression levels and cervical cancer prognosis, and Marzano et al. did not detect EGFR expression in patients who developed cervical cancer.[46],[47] Ngan et al. and Gaffney et al. found increased EGFR expression in cervical cancers but reported that the finding had no prognostic significance.[48],[49] In the current study, none of the participants exhibited EGFR amplification.

Trisomy was detected in one of our study participants. Mian et al. observed trisomy 7 in a large proportion of HSIL patients and claimed that trisomy 7 may be an early finding in cervical carcinogenesis.[50] In contrast, we detected trisomy 7 in just one of the analyzed cells of a single patient.

Many studies have reported TOP2A amplification in breast cancer in particular.[51],[52],[53],[54],[55] HER2-positive patients with TOP2A gene amplification are more responsive to anthracycline treatment than HER2-positive patients without TOP2A amplification, while individuals with normal TOP2A gene copy numbers or deletions show resistance to anthracycline.[51] In a study of early stage breast cancer, Zaczek et al. defined TOP2A amplification as a TOP2A/CEP17 ratio >1.25 or observation of large gene copy clusters in at least 30% of analyzed cells. Their criterion for polysomy 17 was at least three copies of CEN17 in over 30% of evaluated cells.[52] Using these criteria, none of the participants in our study exhibited TOP2A amplification, and polysomy 17 was detected in a single cell of one patient.

Olsen et al. defined TOP2A gene deletion as a TOP2A/CEP17 ratio <0.8, while Järvinen et al. used a ratio of 0.7 or less.[53],[56] We evaluated gene deletions in accordance with the literature. Overall, TOP2A deletions were detected in 8 of our study participants, 2 of whom exhibited both TOP2A gene deletion and abnormal cytologic findings. There was no significant association between TOP2A deletions and abnormal cytologic findings (P > 0.05).


   Conclusion Top


We observed that abnormal cytologic findings were more frequently in the cervical smear samples of HPV-positive patients. We did not determine amplifications of EGFR and TOP2A genes. We did not find statistical difference in abnormal cytologic findings between cases with these gene deletions and cases with normal gene copy numbers. In addition, there was not significant association between gene deletions and HPV positivity. However, we think that identifying variations in gene copy numbers that can appear in HPV-positive individuals with abnormal cytology may be an important tool in terms of prognosis of cervical cell alterations. Therefore, we recommend that screening of larger patient populations and investigation of EGFR and TOP2A gene copy number variations in larger numbers of patients showing abnormal cytologic findings.

Acknowledgments

This investigation was supported by the Scientific Research Unit, Şifa University.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Correspondence Address:
Işın Kaya
Department of Genetics, Faculty of Medicine, Şifa University, Kazım Dirik M. Sanayi Cd. No 735100, Izmir
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_275_16

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