Ovarian Cancer Early Detection by Circulating CA125 in the context of Anti-CA125 Autoantibody Levels: Results from the EPIC cohort

Abstract

CA125 is the best ovarian cancer early detection marker to date; however, sensitivity is limited and complementary markers are required to improve discrimination between ovarian cancer cases and non-cases. Anti-CA125 autoantibodies are observed in circulation. Our objective was to evaluate whether these antibodies (1) can serve as early detection markers, providing evidence of an immune response to a developing tumor, and (2) modify the discriminatory capacity of CA125 by either masking CA125 levels (resulting in lower discrimination) or acting synergistically to improve discrimination between cases and non-cases. We investigated these objectives using a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (EPIC) including 250 cases diagnosed within 4 years of blood collection and up to 4 matched controls. Circulating CA125 antigen and antibody levels were quantified using an electrochemiluminescence assay. Adjusted areas under the curve (aAUCs) by 2-year lag-time intervals were calculated using conditional logistic regression calibrated towards the absolute risk estimates from a pre-existing epidemiological risk model as an offset-variable. Anti-CA125 levels alone did not discriminate cases from controls. For cases diagnosed <2 years after blood collection, discrimination by CA125 antigen was suggestively higher with higher anti-CA125 levels (aAUC, highest antibody tertile: 0.84 [0.76–0.92]; lowest tertile: 0.76 [0.67–0.86]; p_het=0.06). We provide the first evidence of potentially synergistic discrimination effects of CA125 and anti-CA125 antibodies in ovarian early detection. If these findings are replicated, evaluating CA125 in the context of its antibody may improve ovarian cancer early detection.

Introduction

CA125 (human mucin 16; MUC16) is produced by normal epithelial tissues, as well as epithelial cancers,¹ and is the most extensively investigated marker for early detection of ovarian cancer. However, in prospective studies, CA125 only discriminates between ovarian cases and controls in blood samples collected maximally 1–2 years prior to diagnosis, as we recently demonstrated in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort,² and as has been observed by others.^{3, 4} Thus, there has been interest in markers that might be complementary to CA125 for ovarian cancer early detection; possible candidates include tumor associated autoantibodies.⁵ These autoantibodies may be formed as a result of conditions including aberrant or over-expression of their corresponding antigen and tumor-related inflammation,⁶ and may reach detectable concentrations prior to the antigen themselves.⁷

Antibodies against CA125 are found in circulation. Autoantibodies to CA125 may serve as early detection markers, or may mask antigen detection by conventional assays, as has been demonstrated for both CA125 and another tumor-associated antigen, CA15.3.^{8, 9} Our aims were to investigate whether circulating anti-CA125 antibodies (1) can serve as early detection markers, providing evidence of an immune response to a developing tumor, and (2) modify the discriminatory capacity of CA125 by either masking CA125 levels (i.e., resulting in lower discrimination) or acting synergistically to improve discrimination between cases and non-cases. We evaluated these aims in an ongoing ovarian cancer nested case-control study within the EPIC cohort.

Methods

EPIC Cohort

The study protocol for EPIC¹⁰ and the full nested case-control study population² used for this study have been previously described. Briefly, the EPIC cohort was established between 1992 and 2000 in 23 centers in 10 European countries: Denmark, France, Germany, Greece, Italy, the Netherlands, Norway, Spain, Sweden, and the United Kingdom. More than 520,000 participants (367,903 women) were recruited at study baseline. Study participants completed questionnaires describing diet, reproductive history, menstrual factors, exogenous hormone use, as well as disease history, smoking, and alcohol use. A total of 226,673 women provided a blood sample at or near baseline. Follow-up for cancer outcomes and death is conducted via linkages with cancer and population registries with the exception of centers in Germany, Greece, and Naples, Italy; these centers utilize a combination of active follow-up, next-of-kin, and population registries. The study was approved by the ethics committee of the International Agency for Research on Cancer (IARC; Project 11-01), the University of Heidelberg (S-542/2012), and the ethical committees at the participating centers.

Case and Control Selection

This study included 250 invasive epithelial ovarian cancer (EOC) cases diagnosed within 4 years of blood collection, plus up to 4 matched controls. Cases eligible for this study were diagnosed with incident invasive epithelial ovarian (International Classification of Disease (ICD) code: C569), fallopian tube (C570) or peritoneal cancer (C480, C481, C482, C488) after study baseline blood collection, and had data on tumor histology available from pathology reports or cancer registries. We restricted the study population for this study to cases diagnosed maximally 4 years after blood collection given the focus on early detection.

Controls were alive and cancer-free (except non-melanoma skin cancer) at the index date of their matched case. Controls were matched to cases on: recruitment center, and, at blood collection: age, time of day, fasting status, menopausal status, and current use of exogenous hormones, as well as menstrual cycle phase for premenopausal women, and were selected using incidence density sampling.

Laboratory Assays

CA125 antigen and anti-CA125 antibodies were quantified in serum or plasma in the Brigham and Women’s Hospital Obstetrics and Gynecology Genital Tract Biology Laboratory using electrochemoluminiscence detection (MS600 Reader, Meso Scale Discovery (MSD), Rockville, MD; CA125 antigen (MSD catalog number K151WC) and an anti-CA125 custom assay detecting human IgG1 antibodies to MUC16 purified from a human ovarian cell line (Meridian Life Sciences Inc., Memphis, TN). Anti-CA125 reagents were provided by Fujirebio Diagnostics, Inc. (Malvern, PA). Cases and matched controls were analyzed in the same analytic batch, alongside blinded quality controls; members of the laboratory staff were blinded to the case, control, or quality control status of the samples. The coefficients of variation (CV) for the blinded quality controls with values within the linearity range of each assay were calculated. For CA125, the interplate CV was 19%, the mean intraplate CV was 9%. For anti-CA125 anitbodies, the interplate CV was 35% and the mean intraplate was 14%. To account for variation in anti-CA125 antibody levels by laboratory batch, average batch recalibration was used as proposed by Rosner et al.¹¹

Statistical Analyses

Levels of both CA125 and anti-CA125 antibodies were log transformed to achieve a more normal data distribution, and standardized to mean of 0. Spearman partial correlations, adjusted for matching factors, were used to examine correlations between CA125 and anti-CA125 in cases and controls. Tertiles of the biomarkers were defined in controls. Locally estimated scatterplot smoothing (LOESS) curves were used to describe mean levels of CA125 by anti-CA125 antibody levels in tertiles among cases and controls, by lag-time between blood collection and EOC diagnosis. Adjusted areas under the receiving operator characteristic curve (aAUCs) were calculated using conditional logistic regression models calibrated towards the absolute risk estimates from a pre-existing epidemiological risk model¹² as an offset-variable. Discrimination of antigen was evaluated in tertiles of antibody levels, and vice versa. Heterogeneity in the discrimination of one marker by tertiles of the other marker was evaluated using a likelihood ratio test comparing models with and without an interaction term. Statistical analyses were conducted in SAS v. 9.3 (Cary, NC).

Results

The study population was median age 58 years (range: 30–78) at blood collection, and predominantly postmenopausal (72%). Cases were median 60 years (range: 31–79 years) at EOC diagnosis, and the majority were diagnosed with tumors of serous histology (54%) (Table 1).

Table 1.

Baseline characteristics, n (%) or median (range): EPIC ovarian cancer nested case-control study

	Cases n=250	Controls n=822
Age at blood collection, years*	58 (30–78)	58 (30–77)
Ever full term pregnancy	186 (79%)	686 (88%)
Ever OC use	96 (38%)	386 (47%)
Postmenopausal at blood collection*	181 (72%)	585 (71%)
Ever menopausal hormone therapy use	67 (27%)	232 (28%)
Age at diagnosis, years	60 (31–79)
Time between blood collection and diagnosis, years	1.84 (0–3.99)
Histology
Serous	134 (54%)
Endometrioid	23 (9%)
Mucinous	32 (13%)
Clear Cell	6 (2%)
NOS/Other	55 (22%)
Stage at diagnosis
Localized (Stage I)	39 (17%)
Regional/Metastatic (Stage II or higher)	188 (73%)

^*matching factors

Anti-CA125 antibodies did not discriminate between EOC cases and controls, regardless of time between blood collection and diagnosis (e.g., cases diagnosed <6 months after blood collection, aAUC: 0.62 [95% confidence interval: 0.49–0.74]). Results were similar when we examined anti-CA125 antibodies within tertiles of CA125 (e.g., aAUCs cases diagnosed <2 years after blood collection: lowest tertile of CA125, aAUC: 0.53 [0.27–0.79]; highest tertile of CA125, aAUC: 0.59 [0.51–0.66]; p_het=0.51).

Levels of CA125 and anti-CA125 antibodies were not correlated in controls (Spearman r=−0.02). Correlations in cases differed by time between blood collection and diagnosis. We observed an inverse association between CA125 and anti-CA125 antibodies among cases diagnosed <1 year after blood collection and a weak positive correlation among cases diagnosed 1–4 years after blood collection (Table 2). The inverse association observed among cases with blood collection proximate to diagnosis is visualized on the LOESS plot (Figure 1a) showing that among cases diagnosed 0–1 year following blood collection lowest CA125 levels were observed in women with relatively high antibody levels (tertile 3, red line). CA125 levels among controls were consistent across all anti-CA125 tertiles (data not shown). Among women diagnosed within 2 years of blood collection, CA125 discrimination was strongest among women with relatively high anti-CA125 antibody levels (highest tertile of antibody: aAUC: 0.84 [0.76–0.92]) and lowest among women with low antibody levels (lowest tertile of antibody: aAUC 0.76 [0.67–0.86]; p_het=0.06); Figure 1b). In a sensitivity analysis excluding women diagnosed <6 months prior to blood collection, aAUCs for CA125 were similar among women with high antibody levels (highest tertile: aAUC: 0.83 [0.74–0.92]), but somewhat lower among women with low antibody levels (aAUC: 0.71 [0.60–0.82]; p_het=0.15).

Table 2.

Spearman partial^* correlations between CA125 and anti-CA125 antibody levels in cases, by lag-time, and controls: EPIC ovarian cancer nested case-control study

	Time between blood collection and diagnosis	Spearman r
Cases	<0.5 year	−0.35
	<1 year	−0.22
	1–2 years	0.24
	2–4 years	0.12
Controls	n/a	−0.02

^*Adjusted for matching factors

Figure 1.

(a) LOESS plot showing distribution of log-transformed CA125 among controls [blue] and, among cases, with cases classified in tertiles of anti-CA125 antibody levels. Tertiles of antibody level are represented by green (tertile 1; lowest), pink (tertile 2), and red (tertile 3; highest); (b) aAUC for CA125 by antibody tertiles for cases diagnosed within 2 years of blood collection (c) and between 2 and 4 years of blood collection. P_het represents the heterogeneity between aAUCs in the first and third tertiles.

We observed suggestive differences in discrimination with longer time between blood collection and diagnosis (e.g., 2 to 4 years between blood collection and diagnosis, lowest tertile of antibody, aAUC: 0.57 [0.44–0.70]; highest tertile of antibody, aAUC: 0.69 [0.58–0.81]; p_het=0.10; Figure 1c). Results were similar among cases with regional or metastatic disease (Stage II or higher) at diagnosis (data not shown); case numbers precluded an investigation among cases with localized disease (Stage I) at diagnosis (n=39).

Discussion

Tumor associated autoantibodies may be produced as an anti-tumor immune response to tumor-associated antigen (over)expression, mutations, and/or the products of post-translational processing, as well as the well-described inflammatory milieu of the tumor microenvironment.⁶ These antibodies are attractive as diagnostic biomarkers as generally they circulate at higher levels than their corresponding antigen, demonstrate higher stability over time,^{13, 14} and may be detectable at an earlier disease stage.⁷

In this exploratory study, we provide the first evidence that when circulating CA125 levels are examined in the context of anti-CA125 antibodies, their discriminatory ability for the early detection of EOC may be improved, with higher aAUCs for CA125 observed among women with higher anti-CA125 antibody levels. Among cases with short lag-time between blood collection and diagnosis, we observed lower circulating CA125 levels among women with higher anti-CA125 antibody levels, consistent with the hypothesis that higher antibody levels may mask detection of circulating antigen. Circulating immune complexes (CIC) to CA125 have been identified, and potential interference with conventional assays for CA125 demonstrated previously.⁸ This has also been observed for another mucin, MUC1 (CA15.3), with lower levels of circulating CA15.3 observed among ovarian cancer cases with the highest levels of MUC1 circulating immune complexes.⁹ In the current study, we evaluated IgG1 antibodies to CA125. Future studies should consider CA125/anti-CA125 CICs, and other immunoglobin isotypes such as IgM.

By themselves, anti-CA125 antibodies provided no discrimination in early detection models. This is consistent with one prior investigation in which CA125-IgM levels did not discriminate between prevalent cases and cancer-free controls.¹⁵ In the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) study, antibodies against epitopes of another mucin MUC1, were evaluated with respect to OC early detection in samples taken on average 1 year before diagnosis.¹⁶ As independent markers, none of the anti-MUC1 antibodies predicted OC in the UKCTOCS; antibodies were not examined in the context of antigen levels.

We demonstrate that for CA125, the combination of antigen and antibody may offer better discrimination for OC early detection than CA125 alone. While novel, this investigation was limited by relatively small case numbers in each tertile of antibody level (e.g., <2 years between blood collection and diagnosis, n=41–50 cases per tertile). Additional investigations in prospective settings are required to confirm these findings.

Novelty and Impact.

In this prospective investigation using pre-diagnosis blood samples, anti-CA125 levels did not discriminate between ovarian cases and controls. However, discrimination of CA125 differed by levels of its antibody, with the highest discrimination among women with the highest antibody levels. CA125 and anti-CA125 may act synergistically for ovarian cancer early detection. If these findings are replicated, evaluating CA125 in the context of its antibody may improve ovarian cancer early detection.

Abbreviations

aAUC

adjusted areas under the receiver operating characteristic curve

CA125

cancer antigen 125

CA15.3

cancer antigen 15.3

CI

confidence interval

CV

coefficient of variation

EOC

epithelial ovarian cancer

EPIC

European Prospective Investigation into Cancer and Nutrition cohort

IARC

International Agency for Research on Cancer

MSD

Meso Scale Discovery

MUC1

human mucin 1

MUC16

human mucin 16

UKCTOCS

United Kingdom Collaborative Trial of Ovarian Cancer Screening