The diagnostic value of HPV E6/E7 mRNA test in young women with cervical squamous intraepithelial lesion: A Retrospective Analysis

Background At present, the HPV DNA test is used to triage young female patients with abnormal cytology. Still, it is not suitable to precisely identify the population with persistent HPV infection. The purpose of this study was to evaluate the diagnostic value of HPV E6/E7 mRNA test in young women with abnormal cytology by comparing HPV DNA test. Methods A total of 258 young women aged 20 to 29 years,with squamous cell abnormalities on the cervical cytology, were enrolled in this study between January 2015 and December 2019.All patients were subject to HPV DNA test, HPV E6/E7mRNA test,colposcopy biopsy, and histopathological examination.A comparative analysis of the diagnostic performance of the HPV DNA test and HPV E6/E7mRNA test was conducted according to the histological diagnosis(CIN II and CIN II+were dened as high-grade squamous intraepithelial lesion+(HSIL+)). Results The results showed that HPV E6/E7 mRNA test had a higher specicity of 47.3%(40.0%-55.1%) for HSIL+ compared to HPV DNA test that had specicity of 16.0% (11.0%-22.6%)in young women(P<0.01). The HPV E6/E7 mRNA test presented high rates of specicity,positive predictive value (PPV), and negative predictive value(NPV), which were 92.1%(86.0%-96.0%), 62.1%(42.4%-78.7%), 92.1% (85.9%-95.8%), respectively, compared to that of HPV DNA,which were 15.8%(10.4%-23.2%),14.6% (9.40%-21.9%), and 71.0%(51.8%-85.1%),respectively(P<0.01)in young women with mildly abnormal cytology(ASC-US and LSIL). Yet, with severe abnormal cytology(ASC-H and HSIL), HR-HPV test was

often have no obvious clinical symptoms. Although cytological screening is still the main method for cervical screening in the clinic setting,it has no uniform standard, and it is vulnerable to subjective factors.Therefore,in some cases, its repeatability and accuracy are poor. Due to its low sensitivity, the application of cytological screening alone may miss cervical lesions' diagnosis.
The HPV DNA test has been used as an important auxiliary method for cervical screening and has an important role in clinical application.HPV DNA test has high sensitivity and high NPV, which can make up for the short comings of cytology; however, its speci city and PPV are low [5][6][7][8].Therefore, it is particularly important to explore method with high speci city and good PPV in order to provide accurate and reliable detection for young women with persistent HPV infection.
Current studies have con rmed that the de nite cause of cervical cancer is a persistent infection of HR-HPV. The replication of HR-HPV is directly related to the differentiation of cervical epithelial cells. In the process of HPV infection, the viral genome is integrated into the human genome, in which oncogenes E6 and E7 bind to and regulate cell gene products, alter cell growth cycle and DNA repair, resulting in genomic instability and leading to cell immortalization. The oncogenes E6 and E7 are expressed in cells with later stages of differentiation on the surface and intermediate layers. Therefore, the integration of E6 and E7 genes on the host chromosome caused by persistent infection is necessary for the malignant transformation of certain epicells. The detection of HPV E6/E7 gene can be used to identify the risk of persistent HPV infection and cervical lesions in women [9,10]. HPV E6/E7 mRNA test has been proposed as a biomarker for HPV oncogene expression. Previous studies have shown that the HPV E6/E7 mRNA test has a very good clinical sensitivity and higher speci city compared to the HPV DNA test [11,12].Therefore,HPV E6/E7 gene detection has been researched as a critical clinical auxiliary diagnostic technology by European Genital Infection and Tumor Research Institutions in recent years [13].
A prospective study revealed that E6/E7 mRNA-positive young women(25-34years old) had a high incidence of CIN lesions [14],which suggested that HPV E6/E7 mRNA is valuable for cervical cancer screening in young women.The aim of this study was to examine the application of HPV E6/E7 mRNA test in the diagnosis of cervical lesions in young women under 30 years old with different cytological grades.

Study population
This diagnostic accuracy study conforms to the ethical standards and has been approved by the Medical Ethics Association of Quzhou People's Hospital. The study population consisted of women who attended an outpatient gynecological screening at the gynecology department of Quzhou People's Hospital between January 2015 and December 2019. Women meeting these conditions were enrolled in our study: (1)TCT results higher than ASCUS (ASCUS+);(2) 20-29 years old;(3) sexual life history > 3 years; (4)patients agreed to receive the treatment and signed the informed consents. Exclusion criteria: patients with tumors, blood system, immune system, and mental disorders; patients with severe heart, liver, kidney, and other organ dysfunction; those who cannot cooperate with the treatment or follow-up on time.
All women underwent the following procedures: HR-HPV DNA assay, HPV E6/E7 mRNA test, colposcopy biopsy, and histopathological examination.

TCT test
We removed the mucus and secretions from the cervical surface, used a disposable cervical brush to penetrate the cervical canal, rotated 5 to 10 times in the same direction, and put the cervical brush, from which the cells were brushed off into a bottle containing cell preservation solution for rinsing. The The sample collection method was the same as for TCT and HR-HPV DNA detection.We used the QuantiVirus HPV E6/E7 mRNA assay (Kodia, Xinxiang, China) to detect the 14 high-risk types of E6/E7 mRNA, namely,HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,59, 66, and 68,according to the manufacturer's instructions.The assay used a nucleic acid hybridization procedure based on branched-chain DNA hybridization technology.E6/E7 mRNA was quantitatively detected by ve key steps: sample splitting decomposition, making and testing buffer, board distribution, signal ampli cation, and reading board.In each sample, the result of E6/E7 mRNA was marked as a light unit.Special data calculation software can convert the light unit to copy number under the fact of light emission directly related to the amount of HPV mRNA. If the copy number was equal or greater than 1.0, the result of E6/E7 mRNA was positive. If it was less than 1.0, the result was negative [15].

Colposcopy and histological diagnosis
After wiping off cervical secretions, acetic acid white and iodine test were performed, respectively.Three, six, nine, and twelve points were routinely taken from patients with negative acetic acid white and iodine test, while those with abnormal acetic acid white and positive iodine test had a multi-point biopsy in abnormal areas. The tissue specimens, which were xed by formalin and embedded by para n, were stained with H&E for histopathological examination. Histological diagnostic criteria refer to the classi cation system of female genital tumors revised by WHO in 2014 [16].All specimens were independently examined and diagnosed by two senior pathologists. In this study, we set pathological results higher than HSIL((including CIN , CIN +) as pathological positive, and the results lower than LSIL (including CIN , CIN −) were considered negative.
Statistical analysis SPSS 19.0 statistical software was used for statistical analysis. The chi-square test was used to compare the counting data between groups. All reported P-values were two-sided, and P-value of <0.05 was considered statistically signi cant. Screening results of HR-HPVand E6/E7 mRNA in young women First,we analyzed the performances of the HR-HPV and HPV E6/E7 mRNA in all young women(summarized in Tables  , which is of high value for the diagnosis and postoperative follow-up of high-level CIN, and can reduce unnecessary treatment [17][18][19]. HPV E6/E7 expression, which was only present in CIN -diagnosed patients, is a robust indicator of cytological atypia that is better correlated with progressive lesions than HPV DNA assay [20].A prospective study revealed that women positive on the HPV E6/E7 mRNA test have a greater risk of cervical lesions' malignant progression and deserve greater attention and earlier check-ups [21].

Results
There are many studies on the performance of HPV E6/E7 mRNA test in diagnosing cervical diseases. Some reports have suggested that HPV E6 / E7 mRNA test is more sensitive and speci c than the HPV NDA test [22][23][24]. It has also been suggested that the HPV E6/E7 mRNA test is only more speci c than the HPV DNA test,having no difference in sensitivity [25]. Nonetheless, some studies suggest that HPV E6 / E7 mRNA test is more sensitive for the diagnosis of high-level CIN [26].The research results of each scholar are different due to the different research backgrounds, sample, and research methods.
In this study, the positive rate of HPV DNA assay was 85.3%, which was much higher than that of the HPV E6/E7 mRNA test (61.6%).This relatively high percentage of HPV DNA infection in cases with positive cervical cytology is consistent with some previously published studies [22,23,27].There was an association between E6/E7 mRNA expression and histology (OR = 106.12) [28]. Data analysis from our study showed an association between the results of HPV E6/E7 mRNA, histological diagnosis, and the TCT grade.The positive rate of HPV E6/E7 mRNA and histological diagnosis increased with the upgrade of TCT. However, there was no signi cant correlation between HPV DNA positive rate and TCT grade.
The performances of the HPV DNA assay and HPV E6/E7 mRNA test were analyzed in young women.HPV DNA was similar to HPV E6/E7 mRNA in sensitivity,PPV, and NPV,while there was no signi cant difference between the two methods.Still, the speci city of HPV E6/E7 mRNA(47.3%)was signi cantly higher than that of HPV DNA(16.0%) (P<0.01).In the study of Munkhdelgeret al that included 188 women with the squamous intraepithelial cervical lesion, HPV E6/E7mRNA testing was more speci c than HPV DNA assay (85.00% versus 40.83%) [29].In their study,DrageDabeski et al [27]screened a group of 128 sexually active women aged 20 to 59 years with squamous cell abnormalities on the cervical cytology, reporting a greater speci city (88.89%)and greater positive predictive value (93.59%)of HPV E6/E7 mRNA test.The speci city of the HPV E6/E7 mRNA test in our study was signi cantly lower than in the above studies, probably because of the differences in age and number of cases.
Benevoloet al suggest that HPV E6/E7 mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and LSIL because of being more speci c than HPV DNA assay (82% in ASC-US, 76% in LSIL,versus 29% in ASC-US,13% in LSIL) [30].Zhuetal pointed out that HPV E6/E7 mRNA testing could be used as a valuable test in such a setting with higher speci city for the detection of highgrade cervical neoplasia compared toHPV DNA detection without losing sensitivity in women with ASCUS [25].Based on the above research, we further analyzed the performances of the HPV DNA assay and HPV E6/E7 mRNA test in young women with mildly abnormal cytology(ASU-US and LSIL). The HPV E6/E7 mRNA test presented high rates of speci city, PPV, and NPV, which were 92.1%, 62.1%, 92.1%, respectively, compared to that of HPV DNA,which were 15.8%, 14.6%,71.0%,respectively(P<0.01).Yet, with severe abnormal cytology(ASC-H and HSIL), HR-HPV DNA assay is similar to HPV E6/E7 mRNA testing in sensitivity(χ 2 =0.98,P=0.322),speci city(χ 2 =0.938,P=0.333),PPV(χ 2 =0.074,P=0.786) and NPV(χ 2 =0.00,P=1.000);and there was no signi cant difference between the two methods.Our results are similar to the above results. Still, in addition to speci city, the PPV and NPV were higher than that of HPV DNA, which further highlighted the advantage of HPV E6/E7 mRNA in young women with mildly abnormal cytology.
The present study has a few limitations.None of the women in this study had a diagnosis of SCC by TCT, and there were a limited number of women with severe abnormal cytology as well as HPV DNA positive. Therefore, future studies with a larger sample size are warranted.

Conclusions
The results of our study suggested that the HPV E6/E7 mRNA test was more speci c than HPV DNA assay in young women and that the HPV E6/E7 mRNA test presented high rates of speci city, PPV, and NPV compared to HPV DNA assay,especially in those with mild abnormal cytology.Based on the abovereported data, we believe that HPV E6 /E7mRNA testing has signi cant clinical value in screening cervical cancer and predicting the risk of HSIL+in young women, especially those with mild abnormal cytology.

Declarations
Ethics approval and consent to participate: This study conforms to the ethical standards and has been approved by the Medical Ethics Association of Quzhou People's Hospital and written informed consent was obtained prior to the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent for publication: Not applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.