Fully sialylated alpha-chain of complement 4-binding protein (A2160): a novel prognostic marker for epithelial ovarian carcinoma

Abstract

Purpose

Fully sialylated alpha-chain of complement 4-binding protein (A2160) is a member of the glycoprotein family and has recently been identified as a diagnostic biomarker for epithelial ovarian cancer. This study examined the utility of A2160 as a prognostic biomarker for this disease.

Methods

This is a retrospective analysis of prospectively collected plasma samples from 93 women with stage I–IV epithelial ovarian cancer who underwent primary cytoreductive surgery between 2009 and 2014. Pretreatment A2160 levels were correlated to clinico-pathological factors and survival outcome.

Results

Women with advanced-stage disease had significantly higher 2160 levels compared to those with early stage disease (stage I–II versus III–IV, median 2.17–2.70 versus 5.31–8.70 U/mL, P < 0.01). Women with high-grade serous ovarian carcinoma had higher A2160 levels compared to other histologies (6.60 versus 3.01 U/mL, P = 0.05). Women who had suboptimal cytoreduction had significantly higher A2160 levels than those who achieved optimal/complete cytoreduction (7.02 versus 2.30–3.17 U/mL, P < 0.01). On univariable analysis, higher A2160 levels were significantly associated with decreased progression-free survival (64–100 versus 1–33%ile, 5-year rates 53.4 versus 78.9%, P = 0.029). After controlling for age, CA-125 level, cytoreductive status, histology, and stage, higher A2160 levels remained an independent prognostic factor for decreased progression-free survival (adjusted-hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.01–6.11, P = 0.049). Similarly, higher A2160 levels were independently associated with decreased cause-specific survival on multivariable analysis (adjusted-HR 3.07, 95% CI 1.19–7.93, P = 0.021).

Conclusion

Our study suggests that A2160 may be a useful prognostic biomarker for epithelial ovarian cancer, and higher pretreatment levels of A2160 predicts poor survival outcome.

Introduction

Epithelial ovarian cancer (EOC) is still the leading cause of death from gynecological cancer in the United States [1]. Accurate diagnosis and early detection of EOC are challenging, so only 15% of EOC patients have early disease and the vast majority present with advanced disease at diagnosis [2]. While the 5-year survival rate of women with early EOC is nearly 90%, it falls to less than 30% for those with advanced disease.

We recently developed a novel biomarker; fully sialylated alpha-chain of complement 4-binding protein (FS-C4BP, namely A2160) through comprehensive analysis of serum glycopeptides by liquid chromatography–mass spectrometry, which is useful for distinguishing early ovarian clear cell carcinoma from endometriomas [3]. C4BP is a member of the complement cascade that suppresses overactivation of the complement system involved in innate immunity [4]. This is of particular interest because it has been speculated that ovarian cancer cells may express A2160 to escape the host innate immune response via inhibition of the membrane attack complex in the complement pathway [3]. A2160 is a molecule with a strong negative charge that facilitates its attachment to the tumor cell membrane [3]. Therefore, we hypothesized that higher circulating levels of A2160 would be associated with worse survival of women with EOC.

Cancer antigen 125 (CA-125) is one of the few prognostic biomarkers for EOC currently available, but it lacks adequate sensitivity and specificity for monitoring the response to treatment and for early detection of recurrence [5, 6]. Imaging modalities such as computed tomography are often used for surveillance, but also lack sufficient sensitivity and are often inconclusive or late in demonstrating tumor progression, so routine systemic imaging is not supported by the current guidelines [7]. Against this background, we evaluated the usefulness of the pretreatment plasma level of A2160 as a prognostic biomarker for EOC.

Materials and methods

Patient samples

This was a retrospective study that employed consecutive tumor and plasma samples prospectively collected in a gynecologic tumor registry at Tokai University Hospital (Isehara, Japan). Eligible women had EOC in stages I–IV and underwent primary cytoreductive surgery followed by adjuvant chemotherapy between June 1, 2009 and August 31, 2014. Women who received neoadjuvant chemotherapy were excluded.

Data collection

Information on the age at surgery, the International Federation of Gynecology and Obstetrics (FIGO) stage, tumor histology, and the effectiveness of the cytoreductive procedure (residual tumor volume) was collected from the medical records. A gynecologic pathologist (H.K.), who was blinded to the prognosis and clinical information, reviewed the archived pathology slides to assess tumor histology and tumor grade [8]. Survival data were updated in December 2016. Disease progression after initial therapy was evaluated according to the guidelines of the Gynecology Cancer Intergroup (GCIG) [9], and progression was documented on the basis of either objective RECIST criteria or the CA-125 criteria [10]. When disease progression was documented by both methods, the date of progression was defined as the earlier of the two dates. The date and cause of death were also obtained from the medical records.

Preoperative measurement of A2160 and CA-125

In all subjects, a blood sample was collected in a tube containing EDTA on the day of primary cytoreductive surgery prior to the operation. After processing, plasma samples were stored frozen at − 80 °C. Plasma samples were not thawed before being used for measurement in this study. The plasma level of A2160 was measured by the method described previously [3]. Using the same sample, the plasma CA-125 level was measured with a commercial microparticle enzyme immunoassay employing a monoclonal antibody for CA-125 (Abbott Laboratory, Chicago, IL, USA). The laboratory technician who performed measurement of A2160 and CA-125 was blinded to the prognosis of the patients and other clinical information.

Definitions

Optimal cytoreduction was defined as a maximum residual tumor diameter no larger than 1 cm [11]. Because there was no validated cut-off value for A2160 as a prognostic indicator for EOC, we grouped plasma A2160 levels in a trichotomized fashion: 1–33, 34–66, and 67–100%ile. Age (< 60 versus ≥ 60 years) and CA-125 levels value (< 35 versus ≥ 35 IU/L) were grouped based on historical cutoff. High-grade serous ovarian carcinoma was defined as moderately and poorly differentiated serous ovarian carcinomas, whereas low-grade serous cancer was defined as well-differentiated serous ovarian carcinoma. Progression-free survival (PFS) was defined as the time interval between the date of cytoreductive surgery and the date of the initial disease progression/recurrence, or the most recent follow-up visit if alive. Cause-specific survival (CSS) was defined as the time interval between the date of primary cytoreductive surgery and the date of death from ovarian cancer or to the last follow-up visit if the patient remained alive.

Statistical analysis

The primary objective of this study was to examine correlations between the pretreatment plasma level of A2160 and the characteristics of EOC. The secondary objective was to evaluate the prognostic significance of the pretreatment A2160 level in women with EOC. The Mann–Whitney U test or Kruskal–Wallis H test was used to compare A2160 levels among clinico-pathological factors as appropriate. Spearman’s correlation coefficients were used to assess continuous variable (age, CA-125 and A2160). The Kaplan–Meier method was used to estimate the survival curves of groups with different A2160 levels, and the statistical significance of differences between the curves was assessed by the log-rank test for univariable analysis.

In the association model, Cox proportional hazards models were employed to assess the association between the plasma A2160 levels and survival outcomes on multivariable analysis, adjusted for a priori survival factors (age, CA-125 level, cytoreduction, histology, and stage). Magnitude of statistical significance was expressed with adjusted-hazard ratio (HR) and 95% confidence interval (CI). All statistical analyses were two-tailed, and a P value of less than 0.05 was considered to indicate statistical significance. The Statistical Package for the Social Sciences (IBM SPSS, version 24.0, Armonk, NY, USA) and our original statistical software were used for these analyses.

Ethical approval

The study protocol was approved by the Research Ethics Committee of Tokai University hospital (IRB registration number: 09R-082), as was the use of clinical information and plasma samples. Written informed consent was obtained from all participants in the study.

Results

Patient demographics are shown in Table 1. The mean age of the EOC patients was 57.1 years. All patients were Asian. The most common histologic subtype was clear cell carcinoma (n = 43, 46.2%), followed by endometrioid (n = 19, 20.4%) and high-grade serous carcinomas (n = 15, 16.1%). The majority of our study population had early disease (stage I–II in 57.0%), and 75.3% underwent complete/optimal cytoreductive surgery. In the entire cohort of 93 women, the 5-year PFS rate was 61.4% and the 5-year CSS rate was 68.8%. Median follow-up time of women without any survival event was 53.4 months. In this cohort, there were 35 women (37.6%) who had disease progression/recurrence, and there were 26 (28.0%) women who died of ovarian cancer.

Table 1.

Patient demographics

Characteristic	N = 93
Age (years)	57.1 (SD ± 12.1)
< 50	23 (24.7%)
50–59	29 (31.2%)
60–69	27 (29.0%)
≥ 70	14 (15.1%)
CA-125 (IU/L)	192 (IQR 1047)
< 35	19 (20.4%)
≥ 35	74 (79.6%)
Cytoreduction
Suboptimal	23 (24.7%)
Optimal	27 (29.0%)
Complete	43 (46.2%)
Histology
Clear cell	43 (46.2%)
Endometrioid	19 (20.4%)
HGSOC	15 (16.1%)
Mucinous	8 (8.6%)
LGSOC	4 (4.3%)
Unclassified	4 (4.3%)
Grade
1	28 (30.1%)
2	38 (40.9%)
3	27 (29.0%)
Stage
I	45 (48.4%)
II	8 (8.6%)
III	32 (34.4%)
IV	8 (8.6%)

Mean (± standard deviation) or median (interquartile range) is shown CA-125 cancer antigen 125, HGSOC high-grade serous ovarian carcinoma, LGSOC low-grade serous ovarian carcinoma

Correlations between the plasma A2160 levels and patient demographics or tumor characteristics are shown in Table 2. The median pretreatment A2160 level was 3.09 U/mL. Plasma A2160 level was associated with cancer stage, and its plasma level was significantly higher in advanced-stage disease compared with early stage disease (stage I–II versus III–IV, median 2.17–2.70 versus 5.31–8.70 U/mL, P < 0.01). The plasma A2160 level was also higher in patients with high-grade serous carcinoma compared to other histology types although it did not reach statistical significance (6.60 versus 3.01 U/mL, P = 0.05). Moreover, patients with suboptimal cytoreductive surgery had significantly higher A2160 levels compared to those with complete/optimal surgery (7.02 versus 2.30–3.17 U/mL, P = 0.01). The plasma A2160 level was significantly associated CA-125 level (r = 0.37, P < 0.01).

Table 2.

Correlation of FS-C4BP (A2160) and clinico-pathological factors

Characteristic	Median (U/mL)	1–33%ile	34–66%ile	67–100%ile	P value
FS-C4BP (A2160)	3.09	0.44–2.29	2.30–4.89	5.68–44.0
Age (years)					0.16*
< 50	3.08	10 (43.5%)	6 (26.1%)	7 (30.4%)
50–59	2.74	9 (31.0%)	10 (34.5%)	10 (34.5%)
60–69	3.19	9 (33.3%)	8 (29.6%)	10 (37.0%)
≥ 70	9.09	3 (21.4%)	7 (50.0%)	4 (28.6%)
CA-125 (IU/L)					< 0.01*
< 35	2.53	8 (42.1%)	9 (47.4%)	2 (10.5%)
≥ 35	3.36	23 (31.1%)	22 (29.7%)	29 (39.2%)
Cytoreduction					< 0.01
Suboptimal	7.02	4 (17.4%)	6 (26.1%)	13 (56.5%)
Optimal	3.17	5 (18.5%)	12 (44.4%)	10 (37.0%)
Complete	2.30	22 (51.2%)	13 (30.2%)	8 (18.6%)
Histology					0.17†
Clear cell	2.77	14 (32.6%)	18 (41.9%)	11 (25.6%)
Endometrioid	2.27	10 (52.6%)	3 (15.8%)	6 (31.6%)
HGSOC	6.60	2 (13.3%)	5 (33.3%)	8 (53.3%)
Others	4.20	5 (31.3%)	5 (31.3%)	6 (37.5%)
Grade					0.10
1	2.56	13 (46.4%)	5 (17.9%)	10 (35.7%)
2	3.05	13 (34.2%)	16 (42.1%)	9 (23.7%)
3	4.59	5 (18.5%)	10 (37.0%)	12 (44.4%)
Stage					< 0.01
I	2.70	19 (42.2%)	19 (42.2%)	7 (15.6%)
II	2.17	4 (50.0%)	1 (12.5%)	3 (37.5%)
III	5.31	7 (21.9%)	9 (28.1%)	16 (50.0%)
IV	8.70	1 (12.5%)	2 (25.0%)	5 (62.5%)

Univariable analysis with Kruskal–Wallis H test or Spearman’s correlation coefficient (*) for P values. Significant P values were emboldened.

^†P = 0.05 between HGSOC versus non-HGSOC

FS-C4BP fully sialylated alpha-chain of complement 4-binding protein, CA-125 cancer antigen 125, HGSOC high-grade serous ovarian carcinoma

P values of less than 0.05 were indicated bold

The results of survival analysis are shown in Table 3. According to univariable analysis, patients with a high A2160 level had significantly decreased PFS compared to those with a low level (64–100 versus 1–33%ile, 5-year rates 53.4 versus 78.9%, P = 0.029; Fig. 1a). The tumor stage (I–II versus III–IV), tumor histology (high-grade serous versus others), cytoreductive status (suboptimal versus complete/optimal), and CA-125 level (< 35 versus ≥ 35 IU/L) were also significantly associated with PFS (all P < 0.05). Similarly, a high level of A2160 was significantly associated with decreased CSS (64–100 versus 1–33%ile, 5-year rates 57.8 versus 100%, P < 0.01; Fig. 1b). For CSS, stage, histology, cytoreduction, and CA-125 level were significant prognostic factors on univariable analysis (all, P < 0.05).

Table 3.

Multivariable analysis for progression-free analysis

	Number	5-year (%)	Adjusted-HR (95% CI)	P value
A2160 level (%ile)
1–33	31	78.9	1
34–100	62	53.4	2.48 (1.01–6.11)	0.049
Age (years)
< 60	52	60.8	1
≥ 60	41	62.2	0.58 (0.29–1.17)	0.13
Histology
Non-HGSOC	78	69.0	1
HGSOC	15	25.9	1.09 (0.49–2.50)	0.83
Stage
I	45	86.5	1
II-IV	48	39.8	3.94 (1.37–11.3)	0.011
Cytoreduction
Suboptimal	23	20.2	1
Optimal	70	69.0	0.28 (0.12–0.66)	< 0.01
CA-125 level (IU/L)
< 35	19	73.7	1
≥ 35	74	59.0	0.54 (0.19–1.57)	0.26

Association of A2160 and progression-free survival was adjusted with age, histology, stage, cytoreductive status, and CA-125 level on a Cox proportional hazard regression model. Grade was not entered as integrated to histology. Significant P values were emboldened

HR hazard ratio, CI confidence interval, HGSOC high-grade serous ovarian carcinoma, CA-125 cancer antigen 125

P values of less than 0.05 were indicated bold

Fig. 1.

A2160 level and survival of women with epithelial ovarian cancer. Log-rank test for P values. a Progression-free survival and b cause-specific survival stratified by the preoperative plasma level of FS-C4BP (A2160) are shown

When multivariable analysis was performed adjusting for age, CA-125 level, cytoreductive status, histology, and stage (Table 3), a high plasma level of A2160 still showed a significant association with decreased PFSS compared to a low A2160 level (adjusted-HR 2.48, 95% CI 1.01–6.11, P = 0.049). Advanced-stage was also independently associated with a decreased CSS (adjusted-HR 3.94, 95% CI 1.37–11.3, P = 0.011). Complete/optimal cytoreduction was associated with improved CSS (adjusted-HR 0.28, 95% CI 0.12–0.66, P < 0.01.

Because there was no death from ovarian cancer in the lowest one-third level of A2160, multivariable analysis was performed by dichotomizing A2160 levels to lower-half versus higher-half based on the median to examine the independent association of A2160 level and CSS (supplemental Table S1). After controlling for age, CA-125 levels, cytoreductive status, histology, and stage, higher A2160 levels remained an independent prognostic factor for decreased CSS (adjusted-HR 3.07, 95% CI 1.19–7.93, P = 0.021).

Discussion

This study demonstrated that the preoperative plasma level of A2160 was significantly associated with the prognosis of EOC, and a high plasma level of A2160 was an independent prognostic indicator for ovarian cancer mortality (primary endpoint). In addition, the A2160 level was well correlated with known prognostic factors such as the tumor stage, grade, and histology, indicating that A2160 is an indicator of the aggressiveness of EOC. We also observed that CA-125 was not an independent prognostic factor for cause-specific survival after adjustment for the plasma level of A2160, suggesting that A2160 may possibly be a more promising prognostic biomarker for EOC than CA-125.

Our previous study showed that A2160 was more useful for diagnosing ovarian cancer than partially sialylated C4BP and that A2160 is probably produced by ovarian cancer cells [3]. It is speculated that expression of C4BP by ovarian cancer cells assists with evasion of host innate immune response via inhibition of the membrane attack complex in the complement pathway by C4BP [4]. Because sialic acid is a negatively charged molecule, it is reasonable for the fully sialylated form of the alpha-chain of C4BP to show stronger affinity for ovarian cancer cells compared to the partially sialylated form. As a consequence, ovarian cancer cells expressing A2160 may be more effectively protected from the complement-related antitumor immune response of the host. This concept may be supported by our current findings that A2160 was an independent prognostic factor for EOC and that high expression of A2160 was associated with worse survival of women with EOC. Further investigation of the functions of A2160 in relation to post-translational glycosylation may provide useful information for new molecular-targeting therapy.

A diagram of the proposed interaction between A2160 and anticancer immunity is shown in Fig. 2 based on the current and previous studies [3]. The complement system has long been considered to have a role in immune surveillance against cancer because complement is activated on the surface of tumor cells [4]. However, tumor cells can develop mechanisms to inhibit the complement cascade, thus avoiding complement-mediated cytotoxicity. Prolonged activation of the complement system promotes chronic inflammation and an immunosuppressive microenvironment, as well as inducing angiogenesis and activating cancer-related signaling pathways [4]. Deregulation of the complement system is also currently thought to have a role in the process of carcinogenesis [12, 13]. It has been suggested that C4BP is an activating ligand for CD40 that establishes an interface between complement and B-cell activation, through which it may be involved in the pathogenesis of ovarian cancer [14]. From the physiological perspective, the most obvious implication of C4BP binding to the cell membrane is increased complement-regulatory activity on the cell surface. C4BP is a regulator of the C3 convertase, which acts early in the classical pathway, so it can effectively control most complement activation events. It has been reported that binding of C4BP to ovarian adenocarcinoma cell lines was stronger than binding to other tumor cell lines such as teratocarcinoma [15]. Thus, ovarian cancer may use this protein to augment resistance to the humoral and phagocytic immune responses of the host. This speculation might help to explain our finding that the plasma level of A2160 was the best clinical prognostic indicator for EOC.

Fig. 2.

Proposed schema of host anti-immunity via FS-C4BP (A2160) mechanism. Sialic acid is negatively charged, and thus, the fully sialylated form of the alpha-chain of C4BP is expected to hold stronger affinity to ovarian cancer cells compared to the partially sialylated form. As a consequence, ovarian cancer cells covered with FS-C4BP (A2160) on the cell surface membrane is protected from the host immune system via complement pathway (inhibition of the membrane attack complex in the complement pathway by C4BP)

Human epididymis 4 (HE-4) is another biomarker that has been extensively assessed for early detection of EOC and also for its prognostic value, with previous studies suggesting that the preoperative serum level of HE-4 may have prognostic significance for patients with EOC [16, 17]. In the present study, we did not obtain data on HE-4, suggesting that further investigation is warranted to examine the diagnostic utility and prognostic significance of three markers: A2160, CA-125, and HE4.

A limitation of this study is that all of the patients were Asian, and thus the generalizability of our results to other patient populations is unclear. Also, the majority of tumors had clear cell histology, probably due to the ethnic characteristics of Asian patients [18, 19]. Therefore, validation of our findings is required to determine whether the independent association between A2160 and survival of patients with EOC also exists for other histologic subtypes. Lastly, our study probably lacked the power to detect statistical differences of PFS due to the small sample size (type II error). We found that the association of A2160 with PFS was significant in univariate analysis, but not multivariate analysis. Because there were only 33 disease progression events, there may have been over-adjustment in the multivariate model.

A2160 was originally identified by comprehensive glycopeptide analysis based on liquid chromatography mass spectrometry. A novel mass spectrum method is currently under development to improve the quality of A2160 measurement and simplify the process. If A2160 is confirmed to interact with the host immune system, it could be a potential therapeutic target in patients with recurrent EOC. For example, women whose tumors show high expression of A2160 could be treated with a monoclonal antibody targeting this protein or the host immune system could be enhanced by immune checkpoint inhibitor therapy.

In summary, the fully sialylated alpha-chain of C4-binding protein, A2160, is a new biomarker that could be valuable for early monitoring of ovarian cancer. The potential role of A2160 in antitumor immunity could also have an impact on the management of this cancer, with better understanding of the functions of A2160 having therapeutic implications in terms of overcoming tumor resistance to host immunity.