Research Article: HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR Assay for Detecting High-Grade Cervical Lesion with Microscope Slides

Date Published: January 14, 2019

Publisher: Hindawi

Author(s): Geehyuk Kim, Jemberu Taye, Kwangmin Yu, Sunyoung Park, Jungho Kim, Sunghyun Kim, Dongsup Lee, Hye-young Wang, Kwang Hwa Park, Hyeyoung Lee.

http://doi.org/10.1155/2019/9365654

Abstract

After breast and colon cancer, cervical cancer is the third most common cancer of women worldwide. Since human papillomavirus (HPV) infection is known to be the predominant cause of cervical cancer, molecular HPV screening is currently used along with cytological and histological examination methods for precancer diagnosis. Nevertheless, the sensitivity of the current HPV test is less than 80%; thus, many cervical cancer cases are not able to be diagnosed by HPV screening alone, and likewise, patients with cervical cancer are often determined to be HPV-negative by the current screening methods. Therefore, human telomerase reverse transcriptase (hTERT) and Ki67 previously identified as cancer markers were attempted. And cervical exfoliated cells of high-grade squamous intraepithelial lesion (HSIL), the most severe precancerous lesion of cancer, were used in the study. However, it takes a long time to collect enough specimens to conduct statistical analysis. Therefore, in the present study, microscope slides, cervical exfoliated cells on glass slides, were attempted. The results of the analysis demonstrated that hTERT and Ki67 expression levels were useful in distinguishing between cancerous and normal specimens, exhibiting a higher sensitivity and specificity than conventional HPV E6/E7 testing. And the study suggests clinical slide cell samples could be effectively used in the context of retrospective studies to identify novel biomarkers.

Partial Text

Cervical cancer is the third most common cancer of women worldwide, after breast and colon cancer [1]. The World Health Organization (WHO) estimates that approximately 528,000 women are diagnosed with cervical cancer each year and that the disease results in approximately 266,000 deaths annually.

The previous study was conducted with FFPE clinical tissue samples [16]. It was appropriate to determine the availability of the biomarker by tissue samples because tissue samples collected precisely only cancerous region through microscopy and IHC test. We also confirmed that hTERT and Ki67 mRNA expression could be complementary biomarkers in diagnosing cervical lesions with histological samples. In the present study, microscope slides with exfoliated cervical cell samples were collected from subjects diagnosed as either healthy or with HSIL and screened for HPV genotypes, assessing the usefulness of hTERT and Ki67 expression as diagnostic markers of cervical cancer. Exfoliated cervical cells before being placed onto a slide are currently used for screening test specimen because they accompanied with less invasive and less labor-intensive procedure than other tests [17, 18]. They are exposed to the air and need refrigeration condition, so their storage period is limited. Above all, precancerous lesion samples are relatively infrequent compared to normal samples, and there is no idea how much time needed to collect enough samples for statistical analysis. In fact, HSIL samples, which are the most severe precancerous lesion, were collected over three years in Korea and China, but there were about 50 samples [16, 19]. In contrast, microscope slides are sealed with Canada balsam in a vacuum state, so their storage period is longer relatively. In fact, there was no difference in the housekeeping gene expression between samples by 5 years. And the slides were always prepared for cytology test as routine screening test. These factors enable to collect large number of HSIL samples over 100 easily and immediately. Thus, the slides are adequate specimen for retrospective cohort study. There were several retrospective studies with the slides; however, they are primarily limited to inspect staining of appearance so far [20–23]. For the first time, molecular tests were performed with microscope slides in this study. To verify nucleic acid degradation and variation, the assays were performed with endogenous control genes during the experiment.

 

Source:

http://doi.org/10.1155/2019/9365654

 

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