**1. Introduction**

It is estimated that approximately every fourth malignancy can be linked to an infectious agent, that is, its contribution to various stages of cancer development (reviewed in [1]). About a third of this contribution is related to the human papillomavirus (HPV) (reviewed in [2]). Today, significant evidence confirms the association of high-risk (HR) HPV as a carcinogen or promoter in developing malignant diseases in different locations: the cervix, vulva, vagina, penis, anus, and certain head and neck regions. In first place are neoplasias of the lower genital tract, such as cervical cancer [3]. According to estimates by the World Health Organization (WHO), that is, by the International Agency for Research on Cancer (IARC), 604,000 new cases and 342,000 deaths were registered around the world in 2020, which makes cervical cancer the fourth most frequently diagnosed cancer in women [4]. In Serbia, organized cervical cancer screening has been conducted since

**Citation:** Nikolic, N.; Basica, B.; Mandic, A.; Surla, N.; Gusman, V.; Medic, D.; Petrovic, T.; Strbac, M.; Petrovic, V. E6/E7 mRNA Expression of the Most Prevalent High-Risk HPV Genotypes in Cervical Samples from Serbian Women. *Diagnostics* **2023**, *13*, 917. https://doi.org/10.3390/ diagnostics13050917

Academic Editors: Fabio Bottari and Anna Daniela Iacobone

Received: 16 December 2022 Revised: 21 February 2023 Accepted: 22 February 2023 Published: 28 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

2012, using the PAP test, based on the cytomorphological examination of cervical samples. Screening is mandatory for women aged 25 to 69. However, despite organized screening, cervical cancer remains one of the most common cancers among women in Serbia [5]. The incidence of cervical cancer in Serbia is still among the highest and is approximately twice the average in Europe (10.7 to 100,000) [6,7]. It is necessary to emphasize that data on the HPV prevalence and genotype distribution among women with normal cervical cytology, precancerous cervical lesions, and cervical cancer are missing in the updated IARC Human Papillomavirus and Related Diseases Report for Serbia [6].

HPV vaccination is a crucial prevention tool against HPV infection and HPV-related precancers and cancers [8]. If vaccination against HPV is carried out before initial sexual activities, it is one of the most effective ways to prevent cervical cancer [9]. Still, in Serbia, vaccination against HPV infection is not part of the mandatory national immunization program, but it is recommended for children aged 9 to 19 years [5].

Strong evidence for HPV as a causative aetiology of cancers of various locations was provided by the IARC, which classified HPV according to its potential to cause malignant cell alteration as follows [10]:

Group 1 (carcinogenic to humans, HR) includes HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59;

Group 2A (probably carcinogenic) includes HPV genotype 68;

Group 2B (potentially carcinogenic) includes HPV genotypes: 26, 53, 66, 67, 70, 73, 82, 30, 34, 69, 85 and 97;

Group 3 (low risk, LR) includes HPV genotypes 6 and 11.

Persistent HPV infection is the most critical risk factor for the development of cervical cancer, which is confirmed by the presence of HR HPV in over 99% of cervical cancer samples. Concerning the oncogenic potential, infection with a particular HR HPV genotype carries a specific risk for cellular transformation and malignancy (reviewed in [3]). Namely, one of the most critical determinants of the degree of pathogenicity of different HPV genotypes is the functional differences between their oncoproteins, E6 and E7 [11].

During viral genome integration into the host cell genome, E1 or E2 are usually disrupted [12]. This gene disruption leads to uncontrolled transcription of E6 and E7 genes as the E2 repression on these oncogenes disappears [13,14]. Their protein products lead to unregulated cell proliferation, differentiation, and loss of the reparative abilities of the host cell, wherefore they are considered the main actors of virus-induced oncogenesis of cervical cancer [15,16]. Thanks to the use of cervical cancer screening tests, this cancer is classified as one of the most preventable malignancies. The most common test for this purpose is the cytological abnormality test, the Papanicolaou (PAP) test. However, considering the etiological role of HR HPV in developing cervical cancer, DNA tests have been incorporated into the primary screening of developed countries. Still, this test is characterized by high sensitivity and low specificity, which indicates the necessity of improving the test's characteristics concerning specificity [17]. In this context, the results of numerous studies state that using the HR HPV mRNA test as a basic or additional test in primary screening would improve these characteristics [18].

Given the above, this research aimed to determine the oncogenic activity of the most commonly diagnosed HR HPVs in cervical smear samples using the mRNA test and compare the results according to the severity of the cervical intraepithelial lesion. Furthermore, it aimed to examine the clinical characteristics and predictive potential in assessing the diagnosis of high-grade cervical intraepithelial lesions of HPV DNA and mRNA tests.
