Abstract
Background/Aim: Data on the prevalence of human papilloma virus (HPV) DNA in different subtypes of endometrial carcinomas (EC) are limited. Patients and Methods: We investigated the incidence of HPV16 DNA E6/E7 transcripts in 47 type I (endometrioid-type) tumors and eight type II (non-endometrioid-type) uterine neoplasms applying PCR-based technology. Immunohistochemical staining in HPV16 positive cases was also performed, and seven lymph node metastases were examined for the presence of HPV16 DNA E6/E7. Results: None of the type I ECs was positive for HPV16 E6 gene transcripts; however, four out of 8 (50%) type II ECs (two out of four papillary-serous and two out of four clear-cell carcinomas) were positive for HPV16 E6 transcripts. The difference in HPV16 E6 transcripts between endometrioid and non-endometrioid neoplasms was statistically significant (p=0.0011). Apart from the cancer subtype, none of the EC clinicopathological features were related to HPV16 E6 positivity. None of 55 ECs contained an HPV16 E7 gene transcripts. All slides from gene-positive samples revealed intense immunostaining reactions. Interestingly, the virus was not detected in any of seven lymph node metastases, including four from HPV16-positive primary tumors. Conclusion: HPV16 E6 gene transcripts may be present in ECs, primarily in the non-endometrioid (type II) uterine cancer subtypes. HPV E6/E7 DNA transcripts were not found in lymph node metastases, even when the primary tumors harboured HPV DNA.
Human papilloma virus (HPV) can infect specific genital tract organs in females, including the vulva, vagina, and uterine cervix (1-4). Currently, HPV infection seems to be the most important risk factor for cervical cancer development (2, 4-6). However, data concerning the pooled prevalence and any possible influence of HPV infection on endometrial carcinogenesis in humans are controversial (7-22). An association between HPV DNA, especially HPV16 and HPV18, and endometrial carcinoma (EC) has previously been reported (10, 16, 18). For example, six out of 25 (24%), and five out of 25 (20%) endometrial carcinomas were positive for HPV16 and HPV18, respectively, in a Greek study (18). However, none of the clinicopathological features were related to virus infection (18), similarly to data presented previously by our group (16). However, in another study, HPV DNA was not detected in ECs with or without squamous differentiation, or in normal endometrial samples (21). Furthermore, none of the rare EC subtypes, including primary squamous carcinoma, endometrial mucinous microglandular adenocarcinoma, and endometrial transitional cell carcinoma, showed any signs of HPV DNA (19).
In a systematic review and meta-analysis, Olesen et al. (22) reported that HPV DNA prevalence ranges from 0% up to 61.1%, and it was significantly associated with the molecular technique used for viral detection. Recently, Hareza and co-investigators (3) suggested that HPV infection has only a limited role in the aetiology of EC development despite the close anatomical proximity of the endometrium to the uterine cervix.
In the present study, we aimed to investigate the incidence of HPV16 DNA transcripts E6/E7 in different uterine cancer subtypes (type 1 and type 2). Immunohistochemical (IHC) staining in HPV16 positive cases was also performed, and seven lymph node metastases were examined for the presence of HPV16 DNA transcripts E6/E7.
Patients and Methods
Fifty-five paraffin-embedded slides of anatomopathologically confirmed ECs were collected from the archive files of the Department of Pathology at the Princess Anna Mazowiecka Hospital, Warsaw, Poland. All patients underwent surgery between 2015 and 2020. The mean patient age was 62 years (range 35-86 years), and the clinicopathological data of patients are depicted at Table I. All slides were carefully re-examined by a highly experienced anatomopathologist to confirm the primary diagnosis and to identify the neoplastic material for DNA extraction.
Clinico-pathological features of 55 patients with endometrial cancer.
DNA was extracted from paraffin-embedded slides using the ExCRACTme DNA Tissue kit (BLIRT, Gdańsk, Poland) based on manufacturer’s recommendations. HPV16 E6 and E7 transcripts were amplified using the MyTaqHS Red Mix (BLIRT). The primer sequences and product lengths are shown in Table II. The thermal profiles for both amplification procedures were as follows: initial denaturation at 95°C for 1 min, denaturation at 95°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, and termination extension at 72°C for 4 min. The PCR products were stored at 4°C until analysis. After electrophoresis on 1.5% agarose gel, the PCR products were identified using Midori Green (NIPPON Genetics EUROPE GmbH, Düren, Germany) nuclear staining dyes. For the positive control, DNA from HPV16 positive human cervical cancer was used. DNA from normal human skin was used as a negative control. All amplification procedures were performed in duplicate.
HPV16 E6 and E7 primer sequences and the length of the PCR-products.
Immunohistochemistry. Immunohistochemistry was performed only in slides that were positive for the presence of HPV16 transcripts, using monoclonal anti-papillomavirus antibody 16, 18 E6 protein, clone C1P5 (catalog number MAB874, EMD Millipore Corporation, Bedford, MA, USA) with a two-step method. Briefly, slides were de-paraffinized and rehydrated using a routine technique. Antigen retrieval by microwave and blockade of endogenous peroxidase activity were conducted before the primary antibody was added. The primary antibody was diluted 1:200, and the slides were incubated overnight at 4°C. The primary antibody was then removed, and the slides were incubated at room temperature for 30 min. with a biotin-free horseradish peroxidase labeled polymer of EnVision® plus detection system (DAKO, Glostrup, Denmark). Following the reaction with DAB (3,3″-diaminobenzidine), the sections were counterstained with hematoxylin, dehydrated, and cover-slipped. Positive controls were performed according to manufacturer’s recommendation, and sections processed without primary antibody were used as negative controls. Immunostained sections were examined under a light microscope at ×20 magnification by a highly experienced anatomopathologist.
The evaluation of the immunohistochemical reaction was carried out based on the immunoreactive score (23). The percentage of cells with a positive reaction (PP: 0 - no cells with a positive reaction; 1 - up to 10% of cells with a positive reaction; 2 - 11-50% of cells with a positive reaction; 3 - 51-80% of cells with a positive reaction; 4 - >80% of cells with a positive reaction), and the intensity of the reaction (SI: 0 - no colour reaction; 1 - weak colour reaction; 2 - medium colour reaction; 3 - intense colour reaction), in at least five fields of view of the light microscope at ×20 magnification were assessed. The product of PP and SI was the final value of the reaction assessment, and the final result was the average values from five fields of view and presented in the form of conventional units. The final values for the assessed parameters ranged from 0 to 12, which encompassed three levels of reactions: 0-2 poor, 3-5 moderate, and 6-12 intense.
Statistical analysis. Statistical analysis was performed applying the Fisher’s exact test (SPSS, version 14.1, SPSS Inc., Chicago, IL, USA), and p<0.05 was considered as indicating a significant difference.
Results
Altogether, 55 primary human ECs with different histological subtypes were investigated, for the presence the HPV16 E6 and E7 transcripts (Table I). There were 47 type I (endometrioid-type) tumors and eight type II (non-endometrioid-type) uterine neoplasms. Of the eight non-endometrioid (type II) neoplasms, four were papillary-serous carcinomas and four were clear-cell carcinomas.
None of the endometrioid-type ECs was positive for HPV16 E6 gene transcripts. However, four out of eight (50%) type II ECs (two out of four papillary-serous and two out of four clear-cell carcinomas) were positive for the HPV16 E6 gene transcript (Figure 1). The difference between endometrioid and non-endometrioid HPV16 E6 gene transcript positivity was statistically significant (p=0.0011). Aside from the histological type, none of other EC clinicopathological features were related to viral positivity (p>0.05; data not shown). HPV16 E7 gene transcript was not found in any of the 55 ECs.
Agarose gel analysis of Type II ECs applying PCR-based technology with HPV16 E6 specific primers. Line M – M500 marker. Line 1 – positive control. Lines 3-6 Type II ECs (lines 4 and 5 displayed viral transcripts).
Finally, immunostaining was performed only in slides that were positive for the HPV16 E6 gene transcripts. All samples showed intense immunostaining for HPV16 E6 (IF scores between 8 and 12) (Figure 2A and B).
Positive immunostaining with the antibody against HPV16/18 E6 protein in A) uterine serous carcinoma (immunoreactive score=11.5) and B) clear-cell carcinoma (immunoreactive score=12) (×20).
Additionally, seven metastatic lymph nodes, three of endometrioid origin, two of papillary-serous origin and two of clear-cell origin, were examined using a PCR-amplification protocol to concomitantly detect the presence of HPV16 E6/E7 gene transcripts. None of the metastatic samples showed HPV16 E6/E7 sequences. Interestingly, four out of seven metastases originated from primary carcinomas harbouring HPV16 E6 transcripts (two from papillary-serous origin and two from clear-cell origin).
Discussion
HPV DNA testing in specific human tissues is used worldwide to investigate the incidence of viral infection and to correlate HPV detection with well-established clinicopathological features (2-3, 7, 24). However, the molecular mechanisms by which oncogenic viruses disrupt the genomic integrity of human host cells are still being investigated. We should also take into account the deficiency of the immune system as a causative mechanism leading to endometrial and cervical carcinogenesis (25).
In the present study, we investigated the incidence of HPV16 E6/E7 in various human EC subtypes. Altogether, we reported that four out of 55 (7%) ECs of different cancer subtypes harboured HPV16 E6 transcripts. Interestingly, when different histological subtypes were separately evaluated, all HPV16-positive cases had non-endometrioid histology. This finding confirms that HPV16 may be tissue specific. None of the ECs was positive for HPV16 E7. Finally, our results also suggest that HPV16 E6/E7 is not involved in tumour spread due to the lack of viral sequences in DNA isolated from EC lymph node metastatic foci.
The main question is what the role of HPV infection is in endometrial carcinogenesis (18). In a previous study by Fujita et al. (11), a difference was found between HPV16 detection rate in EC samples that originated from Japan (13 out of 47; 28%), and the United States (six out of 38; 16%). Applying PCR-Southern analysis, the researchers suggested that HPV, especially HPV16, may play an etiologic role in a fraction of endometrial adenocarcinomas” (11). An even higher proportion of HPV16 DNA positive EC cases (eight out of 18; 44%) were reported by Lai et al. (10). In contrast, 10% of ECs from Chinese women in Hong Kong were positive for HPV16 (17). In a systematic review of 28 studies published up to 2014 (22), the HPV DNA prevalence differed, and was up to 61% of EC samples worldwide. The pooled HPV incidence differed significantly when various methods of detection were used, including PCR using general primers, type-specific PCR primers and non-PCR based methods. Notably, none of the clinicopathological features was significantly associated with HPV prevalence (22). In addition, it is worth noting that a subset of misclassified endocervical adenocarcinomas may account for some HPV-positive uterine carcinomas reported as primary endometrial neoplasms (20).
Currently, the presence of HPV DNA in the endometrium appears to have a limited or even no role in the etiology of ECs, despite the close anatomical proximity to the cervix (3). The probability of contamination from cervical/endocervical cells may only be considered when the material is collected during surgical intervention. Presently, EC slides were carefully re-evaluated and marked by experienced anatomopathologist before the DNA was extracted. Therefore, the risk of uterine cervix/endocervical cells contamination was low.
In the literature, HPV DNA was not detected in endometrial adenocarcinoma with or without squamous differentiation, or in non-neoplastic endometrium tissues (19). HPV does not appear to play any role in the pathogenesis of EC, since the endometrium may not be a suitable host for HPV replication (19). As reported previously, HPV infection is not linked with rare EC subtypes (21).
It is worth pointing out that in the diagnostic approach, to differentiate primary endocervical adenocarcinoma from endometrial adenocarcinoma, the detection of HPV16 or HPV18, combined with immunohistochemical staining for p16, vimentin, ER, and PR, has important clinical implications (26). One of the main goals of HPV testing is to distinguish between primary adenocarcinomas of endocervical and endometrial origin, particularly in small tissue samples (19, 26-28). This may also be clinically useful for clarifying the possible nature and origin of ECs when morphological features and tumor location are unclear (20). These results suggest that HPV DNA testing could be a useful adjunct in distinguishing between endocervical and endometrial adenocarcinomas in curettage or small biopsy specimens (27).
The literature contains a limited number of studies presenting the existence of HPV DNA in ECs. Interestingly, Greek investigators (29) reported a higher incidence of seven oncogenic HPV subtypes in women with cervical cancer compared to a group including both endometrial and ovarian cancer patients (74.8 and 27.9%, respectively). Surprisingly, the incidence rate of HPV infection in the benign gynaecological diseases was even higher (45.2%) compared with the endometrial/ovarian cancer group altogether (29). Although the endometrium may be not a suitable host for HPV replication and maturation, some evidence suggest that koilocytic-like changes may occur in uterine adenocarcinomas with squamous differentiation, and positivity may be preferentially present at such sites (18). HPV, originating from the lower genital tract, could represent a mere “passenger”, resisting in the columnar epithelium and having no pathogenic role in the development of EC (18). Furthermore, HPV may not be the primary causative factor for the development of primary endometrial squamous cell carcinoma (30).
Finally, it is worth highlighting that none of the seven lymph node metastatic foci were positive for HPV16 E6 DNA, suggesting that HPV may not be involved in tumor spread. It is worth noting that four out of seven metastases originated from primary carcinomas harbouring HPV16 E6 DNA. In the literature, HPV detection and genotyping, performed on pelvic lymph nodes in two patients affected by synchronous gynaecological malignancies, showed homology between primary tumors and metastases (31). Finally, they concluded that “…Human papillomavirus testing on pelvic lymphatic tissue represents a feasible tool to detect the primary site of lymphatic spread in synchronous gynecological malignancies, when uterine cervix is involved…” (31). Moreover, HPV may be applied as a marker of possible cervical cancer micrometastases within the sentinel lymph nodes (32) or may be also used to predict disease recurrence in patients suffered from cervical cancer (33). Appropriate sample collection (primary tumors and corresponding lymph node metastases) and genetic testing are in progress in our laboratory.
In conclusion, HPV16 E6 gene transcripts may be detected in ECs, primarily in the non-endometrioid (type II) uterine cancer subtypes. HPV16 E6/E7 transcripts are not found in lymph node metastases, even when primary tumors harboured HPV DNA.
Acknowledgements
This study was supported by a grant from Lublin Medical University, Lublin, Poland to AS (Dz. St. 326/23).
Footnotes
Authors’ Contributions
WS: Conception, data collection, analysis, interpretation, drafting, editing, revisions; OS: Data collection, analysis, editing, revisions: KC: Interpretation, drafting, editing, revisions; MW: Conception, analysis, drafting, editing, revisions; BG: Analysis, drafting, editing, revisions; TI: Analysis, interpretation, drafting, editing, revisions; WK: Analysis, interpretation, editing; AS: Conception, interpretation, drafting, editing, revisions.
Conflicts of Interest
The Authors declare that they have no conflicts of interest in relation to this study.
- Received July 19, 2023.
- Revision received September 8, 2023.
- Accepted September 11, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
