Lack of Significant Effects of Chlamydia trachomatis Infection on Cervical Adenocarcinoma Risk: Nested Case-Control Study

Background A role of Chlamydia trachomatis in HPV-induced cervical carcinogenesis has been reported for cervical cancer but studies on cervical adenocarcinoma are limited. Methods A total of 1,553 cervical smears taken up to 26 years before diagnosis in a large population-based nested case-control study of cervical adenocarcinoma (AC, 132 cases with matched controls), and adenocarcinoma in situ (AIS, 159 cases with matched controls) were tested for C. trachomatis and HPV DNA by a type-specific PCR bead-based multiplex genotyping (TS-MPG) assay. Results Only 1.7% of samples were positive for C. trachomatis, with no significant differences between AC/AIS cases and controls. HPV-positivity was detected in 49.3% of C. trachomatis-negative and 65.4% C. trachomatis-positive samples, respectively. Conclusions A large prospective study did not find any risk for cervical adenocarcinoma and/or AIS conferred by C. trachomatis infection. Impact C. trachomatis appears not to be involved in cervical adenocarcinomas.


Introduction
Persistent infection with human papillomavirus (HPV) is a virtually necessary cause of cervical cancer [1]. One of the most common curable sexually transmitted infections (STI) worldwide Chlamydia trachomatis has been associated with an increased risk of cervical cancer [2][3][4]. While the most of the studies are focused on squamous cervical carcinoma, there was more limited power to study cervical adenocarcinoma, but no association was found [3,5], which is surprising as C. trachomatis primarily infects glandular cells [2]. Some studies relied on seroepidemiology, where it is difficult to rule out residual confounding completely [1]. One study reported no detection of C. trachomatis DNA in 71 archived formalin-fixed paraffin-embedded tissues of cervical adenocarcinoma (5), but cross-sectional studies are less informative than longitudinal studies [1] and C. trachomatis has only been detected in samples taken many years before the cancer, not close to the diagnosis [2].
We previously reported a large prospective study of cervical adenocarcinoma that established a strong association with HPV present many years before cancer diagnosis (6). We now wished to use this large prospective study to investigate possible associations between C. trachomatis and cervical adenocarcinoma.

Results
In total, 1.7% (26/1553) of samples were positive for C. trachomatis ( Table 1). The 26 samples corresponded to only 21 distinct subjects. No major difference in C. trachomatis-positivity was observed between the outcomes and case-control status; the odds-ratios (ORs) for the association between chlamydia infection in the first collected smear and subsequent adenocarcinoma were 1.25 for AIS (95% CI 0.34-4.65) and 3.0 for AC (95%CI 0.31-28.84); ORs associated with chlamydia infection in the last smear before diagnosis were not estimable due to the lack of exposed cases (AIS) and controls (AC), respectively ( Table 2). The full data set is available as Supplementary Information (S1 Table).
HPV-positivity was detected in 49.3% (n = 753) of C. trachomatis-negative and 65.4% (n = 17) C. trachomatis-positive samples. Because of the overall low numbers, a further analysis of possible interaction between C. trachomatis and HPV status was not feasible.

Discussion
In the current largest prospective study to date of C. trachomatis and cervical adenocarcinoma, with a follow-up period of up to 26 years, C. trachomatis was not associated with increased risks of subsequent invasive adenocarcinoma (AC) and its precursor, adenocarcinoma in situ (AIS). The average age of participants in this study was above 30 years, and it is possible that European Commission FP7 Marie Curie Actions -People -Co-funding of regional, national and international programmes (COFUND) (to VS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The other authors have nothing specific to declare.
Competing Interests: The authors have declared that no competing interests exist.
co-infections/interactions between C. trachomatis and HPV, or other STI, may be of more relevance at earlier age and this may require further studying. A higher prevalence of HPV infections have been found in younger Italian women affected with a C. trachomatis chronic infection from a STI centre than in the ones from an assisted reproductive technology clinic [9]. HPV genotype distribution showed that mostly uncommon low risk genotypes were associated with C. trachomatis [10]. However, while Danish women who reported more than one Chlamydia infection had a statistically significantly increased risk of CIN3+, no association was found between C. trachomatis DNA and subsequent risk of CIN3+ among the ones who were HPV-positive or had a persistent HPV infection at baseline [11]. No association between C. trachomatis status, as assessed by DNA or IgG, and risk of cervical premalignancy, after controlling for carcinogenic HPV-positive status was found in a previous study from the United States [12], which suggested that positive associations between C. trachomatis and cervical premalignancy could have been caused, in part, by an increased susceptibility to HPV infection [12]. However, no studies have been done among the women with cervical adenocarcinoma and the present study is the first of this kind. Moreover, the reports of an interaction between C. trachomatis and HPV in squamous cell carcinoma of the cervix are mechanistically unexplained [1] and further studies on the co-factor role of genital microbiota in promoting malignancies, in particular in high-risk and younger populations, may be warranted. a Matched case/control pairs that differ in exposure and contribute to the OR estimation, reported as exposed cases/exposed controls b Could not be calculated due to perfect separation (no exposed cases or controls) Supporting Information S1 Table. The analysis dataset on the detection of Chlamydia trachomatis and human papillomavirus (HPV) infections in women with cervical adenocarcinoma. SubjectID-unique subject identifier, RisksetID-unique risk set identifier, CaseStatus-case-control status (1 = case), AgeAtDiag-age at the time of diagnosis, HPVpos-HPV-positivity (1 = HPV-positive), CTpos-Chlamydia trachomatis-positivity (1 = Chlamydia-positive), AgeAtSmear-age at the time of collecting cervial smear). (ODS)