Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients

Viral and bacterial infections are detected in epithelial ovarian cancer (EOC) tissues. Since the fallopian tubes are often affected by pelvic inflammatory disease (PID) and the majority of serous EOCs appear to originate from dysplastic lesions in the distal tube, it is relevant to consider the potential role that infectious agents may play in ovarian carcinogenesis. We sought to analyze the prevalence of human papillomavirus (HPV) and cytomegalovirus (CMV) in EOC tissue and fallopian tube specimens obtained at tumor resection. Ovarian cancer and fallopian tube tissue samples obtained from patients with EOC were analyzed by both qualitative and quantitative PCR to detect and quantify viral DNA. The presence of CMV and HPV DNA was detected in 70% and 74% cancerous ovarian tissues, respectively, and was significantly higher in EOC than in benign tumor cases (P ≤ 0.01). CMV or HPV infection was observed also in the fallopian tube samples. Infection with HPV16 was determined in 70% of EOC cases. Almost two thirds of EOC patients demonstrated coinfection with CMV and HPV in the pathological samples. The results revealed that the presence of CMV and HPV in EOC samples is common. CMV and HPV infections can be potential risks for EOC development.

Ovarian cancer is the leading cause of cancer-related deaths in women, accounting for almost 300,000 new cancer cases, and 185,000 deaths in 2018 worldwide 1 . It has nonspecific symptoms, causing more than 60% of cases to be diagnosed at the advanced stage 2 . Ovarian cancer is a highly fatal disease, with a global 5-year age-standardized survival rate of 30-40% in women at advanced stages of diagnosis 3,4 . Approximately 90% of all ovarian cancers have epithelial origins and these cancers are more invasive 5 . Ovarian cancer is a heterogeneous disease that can be divided into three most common types: epithelial ovarian cancer (EOC), germ cell tumors, and sex cord-stromal tumors. The most common EOC histologic subtypes are serous, mucinous, endometrioid, clear-cell, or any combination of these subtypes 6 . The various histotypes are associated with distinct molecular alterations with different etiology, epidemiology, treatment, and prognosis. High-grade serous ovarian carcinoma (HGSOC) is the most aggressive and common subtype of EOC, representing approximately 75% of EOC cases 7 . EOCs originate from the ovarian surface epithelium and from serous tubal intraepithelial carcinomas (STICs) from the fallopian tube epithelium [8][9][10][11] . The fimbriae of the fallopian tube, which are in close proximity to the ovarian surface, have been suggested as the primary site of HGSOC origin 12 . The presence of lesions in the fallopian tube that have cytological features similar to those observed in HGSOCs is designated as STIC 13 . STICs harbor clonal mutations in the TP53 gene encoding the tumor suppressor p53, indicating that these molecular alterations are as an early event in the oncogenesis of HGSOCs 14,15 . A large and growing body of literature supports a theory that STIC is a precursor of HGSOC involving uterine adnexa and peritoneum 16 .
More than one-fifth of ovarian carcinoma appears to be associated with inherited risk, including mutations, e.g., mutations in the BRCA1/2 and TP53 genes 17,18 . Among other factors, such as hereditary, environment, and lifestyle, chronic inflammation seems to be an important risk factor for EOC development. Epithelial cells are exposed to increased levels of inflammatory mediators including pro-inflammatory cytokines, chemokines and hormones that induce DNA damage via oxidative stress and contribute to increased cell division, genetic and epigenetic changes. The main causes of inflammation in the ovaries and fallopian tubes are ovulation, infection, and endometriosis. The predominant hypothesis on ovarian carcinogenesis suggests that incessant ovulation, ovarian rupture and inflammation cause a pro-oxidative microenvironment and mutagenic DNA damage 19,20 . Pelvic inflammatory disease (PID), an infection of the female reproductive organs, also results in DNA damage and a pro-inflammatory response. The role of environmental factors such as bacterial (e.g., Chlamydia trachomatis, Mycoplasma genitalium) and viral infections is still under investigation [21][22][23][24][25] . Recently, it has been shown that C. trachomatis DNA is highly expressed in tubal serous cancer compared with high-grade serous ovarian cancer 26 . Some reports confirmed the presence of human papilloma virus (HPV) in malignant ovarian cancer 25,[27][28][29][30][31][32][33][34] , while others did not [35][36][37][38] . High-risk HPV (HR-HPV) types 16 and 18 were the predominant genotypes associated with advanced stages 28,30,31,34,39,40 . However, the low-risk HPV (LR-HPV) type 6 was also detected 25 . The HR-HPV E6 and E7 oncoproteins can inactivate the tumor suppressors' p53 and Rb, respectively 41 . Evidence of cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) infection in ovarian cancer tissues was also detected 25,[42][43][44] . These widespread herpesviruses can establish life-long infections and contain oncoproteins promoting malignant transformation and metastasis. CMV can spread to the upper genital tract and persistent infection is usually asymptomatic. The oncomodulatory properties of CMV may play an important role in ovarian carcinogenesis and disease progression.
To elucidate the potential role of viruses in ovarian carcinogenesis, the prevalence of HPV types and herpesviruses in EOC and fallopian tube tissue samples was determined. Variation in the UL55 and the US28 genes was determined in all patients with active CMV infection.    www.nature.com/scientificreports www.nature.com/scientificreports/ Coinfection with both CMV and HPV was observed in samples obtained from seventeen patients with EOC (63.0%), including 16 cases with CMV and HPV16 infection (one case with CMV, HPV6 and HPV16), and one case with CMV, HPV6 and HPV45 infection (Table 2). Moreover, the frequency of CMV and HPV16 coinfection was significantly higher in EOC than in benign tumor cases (P = 0.004; Fisher's exact test). Univariate analysis revealed that the odds of being coinfected were 24 times greater for cases with EOC than those with benign tumor (OR 24.39; 95% CI 1. 28-466.30).

Discussion
In this study, the presence of CMV and/or HPV was detected both in cancer and fallopian tube DNA isolated from patients with EOC. Among the examined patients, especially those with HGSOC, CMV and HPV DNAemia was common and was detected in more than two thirds of patients. Furthermore, more than half of the patients with EOC were coinfected with both CMV and HR-HPV. To our knowledge, this is the first demonstration of HPV/CMV coinfection of the upper genital track and CMV infection of the fallopian tubes in patients with EOC. Low amounts of viral DNA copies indicate that CMV and HPV may be present at low activity in EOC tissues. These results suggest that CMV exists in ovarian and fallopian tube cells in a latent phase and could be reactivated under the influence of the inflammatory tumor microenvironment. Extremely low amounts of viral DNA copies and increased levels of specific IgM antibodies found in the majority of patients confirmed our hypothesis. CMV is suspected to act as an oncomodulator that may infect cancer cells and modulate their malignant properties in a way not involving direct transformation 45,46 . Moreover, CMV infection elicits biological responses that are similar to those in chronic inflammation, leukocyte dysfunction, and angiogenesis. CMV coinfection can promote HPV-induced transformation and cause a more rapid development of cancer. Hence, we propose that coinfection with HR-HPV and CMV may be a risk factor for EOC development.
HPV is a carcinogenic virus and a major cause of cervical, anal, penile, and oropharyngeal cancer. A meta-analysis revealed an association between HPV infection and ovarian cancer, as the virus was detected in 17.5% of cases 47 48 . Although more than 200 different HPV types have been characterized, HPV types 6, 11, 16 and 18 are the most clinically relevant. HPV types 6 and 11 account for approximately 100% of genital wart cases, while 20-50% of lesions also contain coinfections with HR-HPV types 50 . Generally, HPV16, 18, 6, 33, and 45 genotypes were found in ovarian cancer. This study confirmed the common presence of HPV16 in 70% of EOC samples, including ovarian serous carcinomas. HPV16 was the most common genotype followed by HPV18 in other studies, although HPV18 was also identified 25,30,33,40,51,52 . HPV16 and/or HPV18 were detected not only in OC tissue, but also in fallopian tubes 53 .
HPV is assembled in the superficial layers and replicates without releasing virions into the bloodstream, and thus there is minimal immune recognition by the host. HPV reaches the ovarian epithelium and can be integrated into the genome of EOC cells 24,48 . This results in increased expression of viral E6 and E7 oncoproteins that inactivate two cellular tumor suppressor proteins, p53 and Rb, and leads to increased cell proliferation and malignant transformation. LR-HPV E6 and E7 proteins have lower binding affinities for tumor suppressor proteins compared with those of HR-HPV types 54,55 . Both oncoproteins target the cell cycle regulators, causing suppression of cell apoptosis that leads to extension of cell life span and increase in HPV replication 56 . Moreover, continuous expression of E6 and E7 in HPV-positive cancer cells is linked to significant alterations in the amounts of intracellular and exosomal miRNAs that are linked to the regulation of cell proliferation, senescence and apoptosis 57 .
The present investigation showed positive amplification in approximately 70% of EOC samples of two CMV genes, UL55 encoding envelope gB and US28 that encodes chemokine receptor US28. Sequence analysis revealed that the CMV gB2 and pUS28 A2 genotype was prevalent in EOC cases, which is in agreement with the genotype distribution in infants and adults from Central Poland 58,59 . Our findings are compatible with the results of Radestad et al., who found the expression of CMV immediate-early (IE) protein in 75% cases, while late CMV tegument protein (pp65) in 67% of ovarian cancer cases in the Gotland (Swedish) population 43 . The results by Shanmughapriya et al. showed the presence of low amounts of CMV DNA in 50% of tumor tissue samples obtained from women with EOC, while low protein expression was found in the majority of tissue sections, including those from patients with serous EOC 25 . We found that active CMV infection may occur both in EOC tissues and fallopian tubes. Earlier studies have shown that CMV infection of the fallopian tubes may be associated with ectopic pregnancy 60 . It should be noted, that CMV infection was low-grade and was only detected by employing sensitive techniques such as nested PCR.
CMV is a widespread opportunistic pathogen that is carried by 40-100% of the world's population, depending on age, socioeconomic status, and geographical location 61 . CMV has developed mechanisms that allow it to survive in latent form in an immunocompetent host, while it reactivates during immunosuppression. CMV can spread to the upper genital tract, and infection is usually persistent, latent and asymptomatic. Several studies have identified high frequency of active CMV infection in tumor tissues, including colorectal cancer 62,63 , malignant glioma 64 , prostatic neoplasia and carcinoma 65 , cervix cancer 66 , and EBV-negative Hodgkin's lymphoma 67 . Recently, CMV DNA and proteins were detected in ovarian cancer, including HGSOC, and in benign cystadenomas 25,43,44 . CMV is not considered to be oncogenic but is rather oncomodulatory in nature, although the mechanisms of its contribution to cancer remain poorly understood [68][69][70] . CMV is regarded as an oncomodulator because it promotes cell proliferation, and cell-cell progression, vascular disease development, and immune evasion that contribute to the development of autoimmune diseases and inhibit apoptosis 69,71 . CMV infection may thus promote malignant transformation by controlling of the cell cycle and dysregulating of physiological processes. The CMV US28 gene was shown to be a key mediator of virus-induced vascular disease and has been implicated in models of CMV-associated glioblastoma 70,72 . The US28 protein activates NF-ĸB, which directly transactivates the CMV major IE promoter 73 and induces caspase-dependent apoptosis in different cell lines 74  www.nature.com/scientificreports www.nature.com/scientificreports/ probably plays a role in CMV-mediated inflammatory diseases and CMV-mediated oncogenesis 69 . CMV encodes four proteins, IE1, IE2, pp71, and pUL97 that can bind or phosphorylate Rb family proteins and inhibit the cell cycle arrest functions of p53. Moreover, CMV induces a mesenchymal-to-epithelial transition (MET) 75 . In contrast, EBV DNA was occasionally detected in EOC tissue (5% of cases) 42,76 , and no association between EOC and EBV antibody levels was found 23 . Taken together, these results indicate that the role of the EBV in ovarian malignancy is probably less important than that of CMV.
The main limitation of this study was the small sample size and the limited availability of some clinical materials; therefore, HPV and CMV positivity might not reflect a true statistical distribution. Further studies using larger patient groups are needed to confirm our findings. Despite a limited number of specimens in this study, our findings may indicate an important role for HPV, CMV and coinfection with both in the development of ovarian cancer.
In conclusion, HPV is probably responsible for some cases of ovarian cancer, while CMV may act as oncomodulatory virus and may promote disease progression. A detected coinfection of CMV and HPV in ovarian cancer and/or fallopian tube indicates a potential oncogenic interplay between the two viruses. A randomized control trial is needed to clarify whether anti-viral therapy is beneficial to EOC patients with CMV and/or HPV detected in the tumor. The results may provide new insight into the pathogenesis of ovarian cancer and new strategies for the use of antiviral therapy in oncology patients.

Methods
Study design and participants. Between November 2017 and June 2019, 27 women aged 34-85 years with presumed epithelial ovarian cancer were enrolled in the study. The inclusion criterion was referral for surgery to a specialized center on suspicion of EOC. Women were considered ineligible to participate if they met any of the following criteria: synchronous cancer other than EOC and ovarian cancer of nonepithelial origin. Patients underwent cytoreductive surgery at the Department of Surgical and Oncological Gynecology, Medical University of Lodz, or at the Tomaszow Health Center, Poland performed by the same experienced oncological gynecologist. After a midline longitudinal incision of the abdominal wall, meticulous inspection of the peritoneal cavity was performed, and the tumor was excised for intraoperative pathological examination. Samples of both the tumor tissue and distal part of the fallopian tube (including tubal fimbriae) unilateral to the tumor site were obtained and immediately secured for laboratory tests after confirmation of EOC by a pathologist. Then, cytoreductive surgery was performed with the intention to obtain a complete cytoreduction protocol. The control group consisted of 8 patients aged 37-88 years who underwent surgery for uterine fibroids or benign ovarian tumors. Finally, the tissue material consisted of 20 HGSOCs, 7 ovarian cancers of other histologic types, and 4 metastatic ovarian cancers. Because metastatic ovarian cancer is histologically different from primary ovarian cancer, the 4 patients with metastatic tumors were excluded from the study group and incorporated into a distinct group for comparison. The basic tumor characteristics are presented in Table 1. Tumor and blood samples from patients with benign ovarian cystadenoma were included as a control group. All diagnoses were confirmed by gynecological pathologist at the Department of Pathology, either of the Medical University or the Polish Mother's Health Center Research Institute, Lodz, Poland. All subjects were of European descent, and there were no ethnic differences between the EOC cases and the control group.
The study protocols were approved by the Bioethics Committee of the Medical University of Lodz (RNN/346/17/KE from 21 November 2017). All procedures performed in the studies were in accordance with the Helsinki declaration. All patients provided written informed consent for participation in this study. tissue collection and cultures of primary ovarian cancer cells. Tumor and blood samples were obtained from 27 patients with EOC, 4 patients with metastatic cancer, and 8 patients with benign tumors. In addition, fallopian tube tissue samples were obtained from 10 females diagnosed with EOC and 3 women with metastatic cancers. Tumor and fallopian tube tissue samples were collected at the time of surgery in ice-cold Dulbecco's Phosphate Buffered Saline (DPBS; Sigma-Aldrich Co. Ltd., Ayrshire, UK) and processed within 1 hour. The solid ovarian cancer specimens were washed with ice-cold DPBS to remove contaminating blood and then minced into small pieces (approximately 1-2 mm 3 ). Primary ovarian cancer cells were isolated from solid specimens using the modified method described by Pribyl et al. 77  www.nature.com/scientificreports www.nature.com/scientificreports/ manufacturer's instructions. The concentration and purity of DNA were assessed using a NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA). DNA was extracted from 200 µl of blood, 5 × 10 6 cancer cells, and 25 mg tissue, eluted in 100 µl of elution buffer, and then stored at −20 °C.
Detection and assessment of cMV DnA. The CMV DNA was quantified using a qRT-PCR method with the genesig Standard Kit (Primerdesign Ltd, York House, UK) according to the manufacturer's instructions. The DNA corresponding to the CMV UL55 gene was quantified using the 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). To validate any positive findings, a negative control without template DNA or RNA was included in each run.
The hypervariable fragments of the CMV genes, UL55 and US28, were amplified with DreamTaq DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA, USA) using a nested and heminested PCR method, as described previously 58 . DNA isolates from MRC-5 cells infected with CMV laboratory strain AD-169 was used as a positive control, and nuclease-free water was used as a negative control. Positive and negative controls were included with each run. All amplifications were carried out in a Veriti 96-Well Thermal Cycler (Applied Biosystems). The amplicons were separated and analyzed using the QIAxcel system (Qiagen). The retention time of the PCR fragments relative to the QX Alignment Marker 15 bp/1 kb fragments was calculated using the BioCalculator software (Qiagen). The PCR product sizes were determined by comparing the retention time with the QX DNA Size Marker 50-800 bp. The PCR products were sequenced using the MiSeq system (Illumina, San Diego, CA, USA). The obtained sequences were analyzed and verified with the Chromas-Win95/98/NT/2000/XP and Basic Local Alignment Search Tool (BLAST). The results were compared with reference sequences in the GenBank database.
Detection of other herpesviruses. The human DNA herpesviruses, including Epstein-Barr virus (EBV), and herpes simplex type 1 (HSV-1) were quantified using a qRT-PCR method with the genesig Standard Kits (Primerdesign Ltd.) as described above. The PCR test has been used for detection of EBV DNA in specimens as described previously 78 . Detection and assessment of HpV DnA. The HPV16 and HPV18 copy numbers were quantified using a qRT-PCR method with the genesig Standard Kits (Primerdesign Ltd.) as described above. The test amplifies the region that codes HR-HPV E6 oncoprotein. High risk Human Papillomavirus Multiplex screening genesig kit (Primerdesign Ltd.) was used to simultaneously detection of DNA of 14 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The DNA was quantified using the LightCycler 96 System (Roche Diagnostics GmbH, Mannheim, Germany). AmpliSens HPV16/18-FRT PCR kit was used for qualitative and quantitative detection and differentiation of HPV types 16 and 18 DNA using real-time hybridization-fluorescence detection of amplified products (InterLabService Ltd., Moscow, Russian Federation). The HPV DNA was quantified using the 7900HT Fast Real-Time PCR system (Applied Biosystems).
DNA isolates from Ca Ski and HeLa cells infected with HPV16 and HPV18, respectively, were used as a positive control, and nuclease-free water was used as a negative control. Positive and negative controls were included with each run. The amplicons were separated using the QIAxcel DNA Screening Kit (Qiagen). The results were validated by direct sequencing of selected PCR products using the 96-capillary 3730xl DNA Analyzer (Applied Biosystems) to confirm the detected genotypes.
Statistical analysis. GraphPad Prism 5.00 software (GraphPad Software, San Diego, CA, USA) was used for all analyses. Differences between groups were examined using the Fisher's exact probability test according to the characteristics of data distribution. Logistic regression was used to calculate the ORs and their corresponding 95% CIs by comparing the case group to the control group. A P-value less than 0.05 was considered statistically significant. The number of samples (n) and the P-values of significant differences are given in tables.