Serologic markers of Chlamydia trachomatis and other sexually transmitted infections and subsequent ovarian cancer risk: Results from the EPIC cohort

A substantial proportion of epithelial ovarian cancer (EOC) arises in the fallopian tube and other epithelia of the upper genital tract; these epithelia may incur damage and neoplastic transformation after sexually transmitted infections (STI) and pelvic inflammatory disease. We investigated the hypothesis that past STI infection, particularly Chlamydia trachomatis, is associated with higher EOC risk in a nested case‐control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort including 791 cases and 1669 matched controls. Serum antibodies against C. trachomatis, Mycoplasma genitalium, herpes simplex virus type 2 (HSV‐2) and human papillomavirus (HPV) 16, 18 and 45 were assessed using multiplex fluorescent bead‐based serology. Conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) comparing women with positive vs. negative serology. A total of 40% of the study population was seropositive to at least one STI. Positive serology to C. trachomatis Pgp3 antibodies was not associated with EOC risk overall, but with higher risk of the mucinous histotype (RR = 2.30 [95% CI = 1.22‐4.32]). Positive serology for chlamydia heat shock protein 60 (cHSP60‐1) was associated with higher risk of EOC overall (1.36 [1.13‐1.64]) and with the serous subtype (1.44 [1.12‐1.85]). None of the other evaluated STIs were associated with EOC risk overall; however, HSV‐2 was associated with higher risk of endometrioid EOC (2.35 [1.24‐4.43]). The findings of our study suggest a potential role of C. trachomatis in the carcinogenesis of serous and mucinous EOC, while HSV‐2 might promote the development of endometrioid disease.


| INTRODUCTION
Epithelial ovarian cancer (EOC) is a heterogeneous disease, with distinct histologic subtypes hypothesized to arise via different pathways of carcinogenesis. 1 The low-grade serous, endometrioid and clear cell histotypes have generally accepted origins and/or precursor lesions, 2,3 and are suggested to be more strongly associated with well-known EOC risk factors (eg, oral contraceptive [OC] use, parity, number of life-time ovulations, tubal ligation, hysterectomy and endometriosis), while risk factors for high-grade serous ovarian cancer (HGSC), the most common and lethal subtype, are less well defined and risk factor associations are generally weaker for HGSC than for other subtypes. 4 Evidence is accumulating that the majority of ovarian cancers originate in non-ovarian epithelial tissue, for example, the distal fallopian tubes (HGSC) 1,5

or ectopic endometrial tissue (endometrioid/clear cell).
Given the likely extra-ovarian origin of a proportion of EOCs, exposures associated with tubal pathologies are of increasing interest with respect to EOC risk. Sexually transmitted infections (STIs) are associated with a range of gynecologic sequelae including pelvic inflammatory disease (PID), 6,7 PID has been associated with EOC risk, 8,9 though data to date suggest strongest associations with borderline tumors. 10 Chlamydia trachomatis and Mycoplasma genitalium are two sexually transmitted bacterial causes of PID. C. trachomatis infects the secretory cells of the fallopian tubes in experimental animal models, 11,12 and may promote tumorigenesis by accelerating cell proliferation, inhibiting cell apoptosis (eg, via chlamydial heat shock protein 60 [cHSP60] production), promoting host DNA damage, and inducing chronic inflammation. 13 M. genitalium has been shown to induce chromosomal aberrations and polysomy in benign human prostate cells, and may promote anchorage-independent growth, allowing cells to detach from the surrounding extracellular matrix and metastasize, indicating possible pro-carcinogenic properties. 14 Herpes simplex virus type 2 (HSV-2), a mostly sexually transmitted virus, has been associated with higher risk of cervical cancer. 15 Finally, sexually transmitted human papillomavirus (HPV) has a well-characterized role in carcinogenesis. HPV infection has its greatest impact in the transformation zone of the uterine cervix but is also implicated in the development of anorectal carcinomas originating in, or close to, the anorectal squamocolumnar epithelial junction. 16 Similarly, the distal end of the fallopian tubes harbors the potentially vulnerable junction of the fallopian tubal epithelium and the peritoneal mesothelium. 17 Epidemiologic data on STIs and EOC risk are limited and the results divergent, [18][19][20][21][22][23][24][25] and few studies are prospective. 18,22,25,26 The largest and most recent prospective study (n = 337 cases) observed a twofold higher risk of EOC among women seropositive to chlamydial Pgp3 antibodies, 25 with similar associations between relatively high circulating antibodies in another recent prospective study 18 27 Briefly, information on lifestyle, diet, reproductive and anthropometric factors was collected at baseline. A total of 226 673 women provided a blood sample at recruitment. Participants provided written informed consent at baseline and the Ethical Committee of IARC and the University of Heidelberg approved our study.

| Nested case-control study participant selection
Study design and case and control selection of this nested casecontrol study have been described previously. 28 Briefly, cases of epithelial ovarian, fallopian tube and primary peritoneal cancer were identified through linkages with cancer registries, health insurance records, and direct contact with cohort members. Data on histologic subtype and tumor grade were obtained from pathology reports and cancer registries.
Up to four controls per case were randomly selected using incidence density sampling among all women from the cohort having a

What's new?
Sexually transmitted infections (STI) have been linked with pelvic inflammatory disease but their association with ovarian cancer remains unclear. In this large prospective study, serum antibodies against Chlamydia trachomatis were associated with higher epithelial ovarian cancer risk, though some associations were limited to select histotypes. Herpes simplex virus type 2 infection was associated with endometrioid ovarian cancer, a rarer ovarian cancer subtype. These findings underscore that STIs may be important in the etiology of ovarian cancer and may represent a target for primary prevention.
blood sample, with no reported oophorectomy, and alive and free of cancer at the time of diagnosis of the index case. Cases and controls were matched on study recruitment center, age at blood donation, time of the day of blood collection, fasting status, exogenous hormone use at blood donation as well as menstrual cycle phase for premenopausal women. Further details are provided in the Supplemental Methods. A total of 791 cases and 1669 controls were included in the study (85 cases with 1 control; 606 cases with 2 controls, 8 cases with 3 controls and 82 cases with 4 controls).

| Laboratory assays
Prediagnosis levels of antibodies to C. trachomatis, M. genitalium, HSV-2 and HPV were measured using multiplex fluorescent beadbased serology assays and quantified as median fluorescence intensity (MFI) levels. 29 Samples from cases and controls were analyzed within the same analytical batch and laboratory personnel were blinded to case-control status. C. trachomatis infection history was assessed measuring antibodies to Pgp3 18 and cHSP60-1, plus MOMP-D, MOMP-A, MOMP-L2, TARP-F2 and TARP-F1 from serovar D. 30 MOMPs representing all three biovars were included given the high cross-reactivity between MOMP serovars, and the low prevalence of trachoma (serovar A-C) in Western European countries. Antibodies to Pgp3, a chlamydia plasmid-encoded protein, sometimes referred to as the "gold standard" marker of current or previous infection, 18,31,32 as well as antibodies to cHSP60-1, produced with persistent C. trachomatis infection, 33 were the primary C. trachomatis antibodies of interest. The Pgp3 antibody was positive in 79.5% of women reporting C. trachomatis infection in a recent study, 31 and cHSP60-1 antibodies have previously been linked to tubal damage. 33 History of M. genitalium was assessed using MgPa N-Terminus, and rMgPa antibodies. 18 HSV-2 antibodies to 2mgG unique, specific for HSV-2, 34 were assessed. HPV infection was determined using antibodies to types 16, 18 and 45 oncoproteins E6 and E7 and the major capsid protein L1. 29 An STI was defined as seropositive according to cut-off values and rules summarized in Table S1.

| Statistical analyses
Conditional logistic regression was used to calculate odds ratios, as estimates of relative risks (RRs), and 95% confidence intervals (95% CIs) comparing participants seropositive to those seronegative for the individual infections using the laboratory cut-off levels. Further, a recent study observed significant associations between C. trachomatis and EOC in one population using the laboratory cut-off, but in another population only after applying a higher cut-off level to define C. trachomatis seropositivity using the Pgp3 antibody. 18 Therefore, we also evaluated associations between the individual C. trachomatis antibodies and EOC risk comparing "low positive" (laboratory cut-off < MFI individual < median in positive women) and "high positive" (MFI individual ≥ median in positive women), to seronegative. History of infection with different STIs could potentially lead to worse tissue damage, and a tendency toward higher risk of EOC with antibodies to C. trachomatis plus a second infection was found in one study. 25 Thus, infection with C. trachomatis plus any other infection (M. genitalium, HSV-2, HPV), relative to women negative to all infections, was investigated. The following were evaluated as potential confounders/covariates: ever menopausal hormone therapy (MHT) use (never, ever), number of full-term pregnancies (FTP; continuous), duration of OC use (continuous) and smoking status (never, former, current; further evaluated as never, former quit 20+ years, former quit 11-20 years, former quit ≤10 years, current occasional, current 1-15 cigarettes/day, current 16-25 cigarettes/day, current 26 cigarettes/day). Final multivariable models include duration of OC use and number of FTPs. Missing values (OC duration, 3%; number FTPs, 10.6%) were imputed to the most frequently observed value for that variable (OC use = never, FTP = 2); results were unchanged when restricted to women with data on OC use and parity. RRs changed <10% after including the remaining variables.
We investigated STIs and EOC overall, by tumor histology (serous, mucinous, endometrioid, clear cell and NOS), and for HGSC disease (tumor grades 2 or 3). Heterogeneity in the associations between the STIs and EOC risk by disease subtype was assessed comparing models assuming the same association across subtypes to a model assuming different associations across subtypes using the likelihood ratio test. 35 We conducted a sensitivity analysis restricted to parous women. Associations between STIs and EOC risk were evaluated by age at blood donation (<60, ≥60 years), lag time between blood donation and diagnosis (<5, ≥5 years), OC use (ever, never), menopausal status at blood collection (premenopausal, perimenopausal and postmenopausal); the Wald test was used to assess heterogeneity in associations.
Statistical analyses were conducted using SAS software, version 9.3 (SAS Institute, Cary, NC). P values are two-sided and P < .05 was considered statistically significant.

| RESULTS
Median age at blood collection was 56.5 years (range = 29.9-80.7) and the majority of cases and controls were postmenopausal at blood collection (cases, 69.1%; controls, 69.6%) and reported at least one full-term pregnancy (cases, 83.1%; controls, 88.8%; Table 1). Cases were diagnosed at median age 62.9 years (range = 30.6-86.5), a median of 6.3 years (range = 0.04-16.0) after blood collection ( Table 2). The majority of cases were of serous histology (54.7%) followed by "not otherwise specified" (16.7%), endometrioid (11.8%), mucinous (7.3%) and clear cell (   RRs estimated from ORs from a conditional logistic regression model; Cases and controls were matched on study recruitment center, age at blood donation (±6 months), time of the day of blood collection (±1 hour), fasting status (<3, 3-6, >6 hours); exogenous hormone use at blood donation (no/yes) as well as menstrual cycle phase for premenopausal women ("early follicular" (days 0-7 of the cycle), "late follicular" (days 8-11), "periovulatory" (days 12-16), "midluteal" (days 20-24), "other luteal" (days 17-19 or days 25-40), or missing). Adjusted for number of full term pregnancies and duration of use of oral contraceptives (years); results for mucinous disease robust to additional adjustment for smoking. b +, MFI < median indicates positive serology using lab cut-off, and MFI value below median among women seropositive according to lab cut-off ("low positive"); +, MFI ≥ median indicates positive serology using lab cutoff, and MFI value above median among women seropositive according to lab cut-off ("high positive"). .08 .05 a RRs estimated from ORs from a conditional logistic regression model; Cases and controls were matched on study recruitment center, age at blood donation (±6 months), time of the day of blood collection (±1 hour), fasting status (<3, 3-6, >6 hours); exogenous hormone use at blood donation (no/yes) as well as menstrual cycle phase for premenopausal women ("early follicular" (days 0-7 of the cycle), "late follicular" (days 8-11),  P het by histotype = 0.08; Table 4). No associations were observed for the HPV-related markers stated above; results were similar when the L1 antibodies were evaluated (Table 4, footnote). Seropositivity to C. trachomatis plus a second STI was not associated with EOC risk in any subgroup (data not shown).

| Sensitivity and subgroup analyses
Results were not materially different in sensitivity analyses restricted to parous women (data not shown). We observed limited heterogeneity in associations by age at blood collection (<60 vs ≥60 years; Table S5). In the analysis considering "high" and "low" positive antibody levels to C. trachomatis cHSP60-1 antibodies and EOC risk, women with high positive antibody levels had higher risk of EOC only among women <60 at blood collection (P het = 0.04; <60 years,  [18][19][20]22,24,25 In the current study, we provide a detailed investigation by histotype, observing suggestive heterogeneity by tumor histology. Pgp3 serology indicating current or past infection with C. trachomatis was not associated with EOC risk overall in contrast to two recent studies, 18,25 but was associated with significantly higher risk of mucinous EOC; this has not previously been described. This result was robust to statistical adjustment for smoking, in addition to OC use and parity; however, the number of mucinous ovarian cancer cases was limited (n = 58) and this result should be interpreted with caution.
Positive serology for cHSP60-1 antibodies was associated with higher risk of EOC overall, and serous disease, as were relatively high levels of the MOMP-A, MOMP-D and MOMP-L2 antibodies.
cHSP60-1 IgG antibodies were associated with Type II EOC in a subgroup analysis in a previous study, 22 and cHSP60-1 antibodies were associated with EOC using higher cut-off levels in a retrospective casecontrol, but not the prospective component of the study by Trabert et al. 18 Chlamydial HSP60 is produced by the chlamydia bacteria to induce a persistent state of infection in the host cell, thereby escaping immune defense. 36 This leads to inhibited cell apoptosis increasing the risk for a DNA-damaged cell to survive, and being further exposed to an inflammatory environment with cytotoxic substances. This state of persistent chlamydia infection with increased cHSP60 production is one pathogenic mechanism by which chlamydia could cause ovarian cancer. 37 Given that cHSP60 is associated with persistent chlamydia infection, cHSP60-1 antibodies may be a marker of persistent M. genitalium is a small intracellular bacterium known to cause PID. Serum antibodies to M. genitalium were not associated with EOC overall or any histotypes in our study, in line with the findings of prospective studies, 18,22 although an association was reported in a retrospective case-control study population 18 and parous women in another study. 25 Positive HSV-2 serostatus was associated with a higher risk of endometrioid EOC. A higher, but not statistically significant, risk of EOC overall was also found in the recent study by Trabert et al. 18 Women with positive serology for HSV-2 had higher risk of endometrial (uterine) cancer in the NHANES (National Health and Nutrition Examination Survey). 15 Ovarian cancer of the endometrioid subtype shares several characteristics with endometrial cancer and is synchronous with endometrioid cancer of the uterus in 15%-20% of cases. 39 Similar to previous studies that found no association of HPV serostatus with ovarian cancer, 18,25,26,38 no associations were observed in our study.
The hypothesis that HGSC would be the ovarian cancer subgroup most likely associated with STI serum markers was not supported by the results of our study, though these analyses were limited by the availability of data on grade (available for 59% of all cases, 66% of serous cases) and, thus, a limited number of HGSC cases (n = 268).
One explanation for the lack of association observed may have been due to limited power to detect an association in this subgroup. A possible biologic interpretation is that the secretory cells of the distal fallopian tube might not be vulnerable to neoplastic transformation caused by infectious agents, even though one recent study suggests that C. trachomatis has long-term impact on the tubal epithelium by altering the phenotype and inducing heritable changes in the epigenome. 40 Only one prospective seroepidemiological study has analyzed the association of STI antibodies and Type II EOC finding a significant association of C. trachomatis antibodies with Type II. 22 Another explanation could be that analyses of serum antibodies do not reflect the previous infections that are crucial in malignant transformation of the secretory cells.
Our study has several strengths. First, the present study included the largest number of cases among seroepidemiological studies focusing on STI antibodies and risk of EOC published to date, allowing a more detailed evaluation by histological subtypes and grade than previous studies. Second, the prospective design excluded disease-related effects on serologic antibody levels; however, no data are available on the timing of infection prior to the blood collection, nor infections in the interval between blood collection and diagnosis/selection as a control. Third, a validated multiplex Luminex assay allows us to evaluate antibodies to several candidate STIs in the study. Seroconversion appears to occur in relatively close temporal proximity to diagnosis, with a total of 92% of women with a positive Pgp3 serology at chlamydia diagnosis and ≥1 day to 6 months after diagnosis, and positive serology was observed in 72% of individuals >4 years after diagnosis in one study. 41  Experimental studies delineating the mechanisms linking STIs to EOC, and the primary prevention potential of STI prevention, are tasks for future experimental, translational and epidemiological research to resolve.