Uterine cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer death among women in developed countries . Although cervical screening including the human papillomavirus (HPV) test has reduced the incidence and mortality rate of cervical cancer worldwide , there is still little information about the role of less prevalent and rare HPV genotypes, such as HPV68, during cervical carcinogenesis .
Stratified mucin-producing intraepithelial lesion (SMILE) is an uncommon premalignant lesion of the uterine cervix . It is thought to arise from the reserve cells of the transformation zone throughout the full epithelial thickness of a lesion, with some overlap with the architecture of squamous intraepithelial lesion (SIL) or adenocarcinoma in situ (AIS) . SMILE is characterized by several histopathological features, including epithelial stratification, diffuse mucin production throughout the epithelial layers, and an absence of classic gland formation ; nuclear atypia, hyperchromasia, mitosis, and apoptotic bodies are often observed in the lesion, which is similar to other forms of intraepithelial neoplasia including usual-type AIS of the endocervical glandular epithelium. Histochemical staining for mucin [6,7,8] and immunohistochemical detection of Ki-67/Mindbomb E3 ubiquitin protein ligase (MIB)-1 have revealed a high proliferative index . Importantly, diffuse positivity for the cell cycle regulation protein p16INK4a—which is associated with high-risk HPV infection—is also observed ; however, there is limited information available on the involvement of high-risk HPV in the pathogenesis of SMILE [10,11,12].
Studies over the last two decades have shown that persistent HPV infection is the main cause of cervical cancer development. Clinically validated HPV tests are recommended by the U.S. Preventive Services Task Force (USPSTF) and the Japan Society of Obstetrics and Gynecology for cervical pre-cancer screening, triage, and treatment follow-up in clinical practice [13, 14]. About 40 different HPV types can infect the cervix, of which 14 (type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) are classified by the World Health Organization as being associated with a high risk of SIL and cervical cancer development [15,16,17]. Most oncogenic or high-risk HPV types associated with invasive cervical cancer are phylogenetically clustered within the species groups Alphapapillomavirus 9 (Alpha-9: HPV16 along with HPV31, 33, 35, 52, and 58) or Alphapapillomavirus 7 (Alpha-7: HPV18 along with HPV39, 45, 59, and 68) . These two groups account for approximately 75 and 15%, respectively, of all cervical cancer cases worldwide . However, compared with HPV16 and HPV18, the carcinogenicity of other HPV types has not been well investigated, and rare HPV genotypes are poorly understood. It is thought that high-risk HPVs preferentially infect and replicate in the basal layer of the epithelium  with the integration of HPV sequences into the host cell genome leading to SIL progression. On the other hand, it is unclear whether high-risk HPV contributes to the development of SMILE . HPV RNA in situ hybridization (ISH) is an established method for detecting genomically integrated HPV sequences [22, 23]. In the present work, we investigated whether rare, high-risk HPV contributes to the development of SMILE using RNA ISH to assess the integration of viral DNA in cervical cancer lesions.