Introduction: Endocrine treatment combined with CDK4/6 inhibitors is the preferred treatment strategy in patients presenting with ER-positive/HER2-negative breast cancer, but the clinical course remains highly variable among individual patients. There is an unmet need for prognostic or predictive biomarkers in this important group of patients. Recently, we have identified circulating glypican-4 (GPC4) as a new biomarker of inferior outcomes in patients with metastatic colorectal cancer. The impact of plasma GPC4 levels on the survival of breast cancer patients is unknown and has been addressed in the present study. Methods: Our study included 47 patients with ER-positive/HER2-negative metastatic breast cancer prior to treatment with CDK4/6 inhibitors combined with endocrine therapy. The endpoint was overall survival (OS) at 24 months. GPC4 levels were measured in plasma using an enzyme-linked immunosorbent assay. Results: Increased circulating GPC4 levels were significantly linked to advanced age, postmenopausal state, visceral metastases, and invasive lobular carcinoma. During the 2-year observational follow-up period, 25.5% of patients died. The area under the receiver operating characteristic curve (ROC-AUC) analysis revealed an AUC of 0.713 [0.555–0.871]; p = 0.029 for OS; and an optimal cutoff value of GPC4 for predicting OS of 4.77 ng/mL. No patient showing GPC4 values below this cutoff died during the observational period. Cox regression analysis showed a hazard ratio of 2.14 [95% confidence interval: 1.24–3.67]; p = 0.006 for one standard deviation change of plasma GPC4. Conclusions: Our study suggests circulating GPC4 as a significant predictor of poor survival in metastatic breast cancer patients.
Breast cancer is the most common type of cancer diagnosed in women globally . With approximately 70% of diagnoses, ER-positive/HER2-negative (ER+/HER2−) tumors represent the most common subset of breast cancer and are responsible for most breast cancer-related deaths . Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors in combination with endocrine therapy have significantly improved the management of advanced ER+/HER2− breast cancer and have become standard of care [3, 4]. Despite the substantial progress in breast cancer therapy, several patients do not respond to this treatment, and metastatic breast cancer (MBC) still remains virtually incurable. Survival prognosis is of particular interest to many cancer patients and critical for clinical decision-making. Unfortunately, most biomarkers tested were unsuccessful despite considerable efforts and a strong scientific rationale [5, 6].
Glypicans, together with syndecans, belong to the family of cell-surface heparan sulfate proteoglycans, members of which have been recently identified as critical drivers for malignant transformation, cancer cell growth, and progression [7, 8]. The glypican subfamily contains six members, namely, glypican-1–glypican-6 (GPC1–GPC6). They exhibit differential expression in several cancers, acting as both tumor promoters and inhibitors in a cancer-type-specific manner . Glypicans are attached to the cell membrane via a glycosylphosphatidylinositol anchor but can be released from the cell surface into the bloodstream by proteolytic shedding or glycosylphosphatidylinositol-anchor cleavage mechanisms [9, 10]. Activated shedding of membrane glypicans and other proteoglycans is observed under various acute and chronic clinical conditions [11‒13]. Consequently, circulating proteoglycans have been proposed as valuable diagnostic and prognostic biomarkers in many diseases, including cancer . Recently, we have identified circulating GPC4 as a biomarker of poor survival in patients with metastatic colorectal cancer . In addition, we showed significant associations between shed GPC4 levels and reduced mortality in coronary angiography patients  as well as patients with peripheral artery disease . However, studies on GPC4 in breast cancer are limited [17, 18], and the association between circulating GPC4 and the prognosis of patients with MBC is unknown so far. In the current study, we, therefore, aimed to investigate the association between circulating GPC4 and overall survival (OS) in a consecutively recruited population of ER+/HER2− MBC patients.
The present analysis included 47 patients with ER+/HER2− MBC who started first-line or second-line treatment with CDK4/6 inhibitors combined with endocrine therapy. Patients were consecutively enrolled at the “Oncology Study Center Ravensburg” (Ravensburg, Germany) from October 2017 through November 2019. Only patients with available clinical and laboratory data and complete follow-up were included in the present study. Baseline clinicopathological parameters including age, menopausal state, tumor histopathology (invasive carcinoma of no special type [NST] versus invasive lobular carcinoma [ILC]), metastatic status, and scheduled medical treatments were obtained from medical records. Patients were followed up until death or the end of the observational period at the end of September 2022. The primary endpoint was OS at 24 months. Secondary endpoints were baseline clinicopathological characteristics. The study protocol was approved by the Ethics Commission of the State Chamber of Medicine of Baden-Württemberg (Germany) and is in accordance with the 1964 Helsinki declaration and its later amendments or equivalent ethical standards. Written informed consent was obtained from all patients for participation in this study.
At baseline, blood samples were collected, and plasma samples were stored at −80°C until used for GPC4 analysis. Plasma GPC4 levels were determined via a commercially available enzyme-linked immunosorbent assay (ELISA; Cloude-Clone; Houston, TX; product number: SEA998Hu) following the manufacturer’s manual. The kit had a detection range of 0.031–2.000 ng/mL and showed a minimum detectable dose of 0.013 ng/mL; the intra-assay coefficient of variation was below 10% and the inter-assay coefficient of variation was below 12%.
Normal distribution was assessed using the Kolmogorov-Smirnov test and the Shapiro-Wilk test indicating that GPC4 values were normally distributed (p = 0.200 and p = 0.081, respectively). The relationship between continuous variables was measured by calculating Pearson’s correlation coefficient (r). Statistically significant differences between continuous GPC4 levels and categorical variables were determined by the Student’s t test or analysis of variance (ANOVA). The distribution of GPC4 values between groups was illustrated as box plot diagrams using the Tukey method for plotting the whiskers and outliers. Survival curves were generated using the Kaplan-Meier method and compared using log-rank Mantel-Cox tests. Hazard ratios (HRs) were derived from univariable and multivariable Cox proportional hazards models. HRs were given together with the 95% confidence intervals in square brackets and the respective p value. Continuous variables were z-transformed for Cox regression analyses. In addition, area under the receiver operating characteristic curve (ROC-AUC) analyses were performed. The optimal cutoff value of GPC4 for predicting OS was assessed using Youden’s index method . Statistical significance was defined as a p value <0.05. Statistical analyses were performed with SPSS 28.0.0 (IBM, Armonk, NY, USA), R statistical software v. 3.2.3 (http://www.r-project.org), and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Furthermore, a post hoc power calculation was performed using G*Power 3 software version 184.108.40.206 .
The clinicopathological characteristics of the study cohort are shown in Table 1. Plasma GPC4 values were significantly associated with age, the menopausal state, the histological type of breast cancer (NST vs. ILC), and the metastatic site (visceral vs. bone-only). Corresponding box plots are displayed in Fig. 1a–d. Notably, plasma GPC4 values were highly significantly increased in ILC compared with NST (8.12 ± 2.18 ng/mL vs. 5.69 ± 2.07 ng/mL, p < 0.001). No significant associations were found between GPC4 levels and the number of metastatic sites or scheduled lines of therapy (p = 0.272 and p = 0.919, respectively).
During the 2-year observational follow-up period, 12 (25.5%) patients died. Baseline GPC4 plasma levels were significantly increased in patients who died during the follow-up period compared to subjects who survived the follow-up period (p = 0.005). Respective mean GPC4 values were 7.83 ± 2.66 ng/mL and 5.85 ± 2.03 ng/mL, resulting in an effect size of 0.84. A corresponding box plot is shown in Figure 1e. A post hoc power calculation demonstrated that our study comprising 35 patients who survived the follow-up period and 12 patients who died during the follow-up period achieved a power of 0.8 given an alpha error probability value of 0.05 and an effect size of 0.84 and, therefore, was sufficiently powered to show significant differences in GPC4 values between survivors and deceased patients.
ROC analysis revealed an AUC of 0.713 [0.555–0.871], p = 0.029, a Harrell’s C of 0.703, and a Somers’ D of 0.405 for GPC4 and overall mortality. Time-dependent ROC curves for GPC4 and age are displayed in online supplementary Figure 1 (for all online suppl. material, see www.karger.com/doi/10.1159/000529547), indicating a higher prognostic value of GPC4 compared to age. The optimal cutoff value of GPC4 for predicting OS was 4.77 ng/mL (Youden’s index = 0.400). Kaplan-Meier survival curves for OS according to the optimal cutoff value of GPC4 are shown in Figure 2a. No patient showing GPC4 values below the optimal cutoff (29.8%; n = 14) died during the 2-year observational period. Out of the given clinicopathological characteristics, median age, the histological type of breast cancer, and the scheduled line of therapy significantly predicted OS. Menopausal state, metastatic site, and the number of metastases were not significantly linked with OS in our study. Respective results from Kaplan-Meier analyses are illustrated in online supplementary Figure 2.
Median age and the histological type of the primary tumor were significantly associated with both GPC4 values and OS. Multivariable Cox regression analyses were performed to evaluate the impact of these probably confounding factors on the association between GPC4 and OS. Respective HRs and 95% confidence intervals of continuous GPC4 are displayed in Figure 2b. The association between plasma GPC4 and OS remained significant fitting the regression model either with age or the histological breast cancer type. It should be noted that adjustment for several putative cofounders in multivariable Cox regression analysis requires larger sample sizes to avoid overfitting the regression model . In fact, the association between GPC4 and OS exceeded the predefined significance level by including all three variables in the regression model. Similarly, the associations of age and histological tumor type with OS were no more significant in the three-variable model (p = 0.112 and p = 0.758, respectively). In an exploratory analysis, further adjustments for menopausal state, scheduled line of therapy, metastatic site, and the number of metastases in addition to age and the histological type of breast cancer were performed, revealing that elevated plasma GPC4 still increased mortality risk in the fully adjusted regression model (HR = 2.12).
This study is the first to examine the association between plasma GPC4 levels and outcome in breast cancer patients. We identified circulating GPC4 as a significant predictor of OS in patients with ER+/HER2− MBC treated with endocrine therapy in combination with CDK4/6 inhibitors. The reported results are in line with a previous study from our research group including patients with metastatic colorectal cancer  and demonstrating that shed GPC4 is significantly linked to the prognosis of those patients.
Only a few studies have investigated the role of GPC4 in breast cancer. Munir et al.  found that GPC4 expression was downregulated in metastatic breast tumors compared to nonmetastatic tumors. In vitro studies using metastatic and non-MBC cells confirmed these results. The authors further showed that over-expressing GPC4 in metastatic cells decreased cell migration/invasion and cell proliferation and concluded that GPC4 might be a tumor suppressor in breast carcinomas. Consistently, a study by Grillo et al.  demonstrated that for overall unclassified breast cancer, patients with high expression levels of membrane-bound GPC4 showed a more prolonged relapse-free survival. However, subgroup analysis concerning ER status showed that increased GPC4 expression did not significantly predict survival in patients with ER+ breast cancer and was even a risk factor for reduced relapse-free survival in ER-negative breast cancer patients. The authors, therefore, suggested GPC4 to be a breast cancer subtype-specific biomarker.
GPC4 and other glypicans control the wingless/int-1 (Wnt) signaling pathway and other oncogenic pathways [22‒24]. That said, it remains unclear if GPC4 expression, which is potentially involved in the regulation of signaling pathways within the tumor microenvironment, is proportional to the concentration of free GPC4 ectodomain in plasma which is linked to the outcome of cancer patients. The picture gets even more complex, as membrane-bound GPC4 enhances Wnt signaling, but GPC4 secreted into the extracellular environment acts as a competitive inhibitor of the Wnt signaling pathway . Therefore, functional studies are needed to understand the biological role of GPC4 in breast cancer.
Our study suggests that GPC4 levels are linked to age, menopausal state, metastatic site, and tumor histopathology. With regard to the latter, plasma GPC4 levels were highly and significantly increased in patients with ILC compared to NST breast cancer patients. Although ILC remains an understudied entity, long-term survival and clinical outcomes in patients with ILC seem to be worse than in stage- and grade-matched patients with IDC . In our study, both ILC and GPC4 were associated with a worse prognosis, but our study’s multivariable Cox regression analysis revealed that the significant association between GPC4 and OS was independent of the histologic type of tumor. Across histologies, differences in metastatic patterns , genomic profiles [28, 29], and other clinicopathological factors [30, 31] have been reported, which might have contributed to the varying GPC4 levels between NST and ILC.
Previous studies showed that increased circulating GPC4 levels were associated with various metabolic disorders linked to insulin resistance, including obesity, elevated systolic blood pressure, nonalcoholic fatty liver disease, chronic kidney disease, and cardiovascular disease [15, 16, 32‒37]. The prevalence of comorbid conditions is high among patients with cancer and increases with age . In fact, plasma GPC4 levels significantly correlated with advanced age in the present study. Therefore, the putative presence of comorbidities might have led to elevated GPC4 levels and exposed patients to an increased mortality risk a priory. Consequently, it could be argued that increased levels of GPC4 represent a marker of compromised organ function more broadly rather than being a specific tumor marker. However, although the aptitude of shed GPC4 as a tumor marker has to be clarified further, our study is the first to suggest that plasma circulating GPC4 represents a valuable prognostic biomarker in patients with ER+/HER2− MBC starting treatment with CDK4/6 inhibitors combined with endocrine therapy.
The present study has several limitations. First, our study is of limited sample size. That said, a power calculation demonstrated that our study was sufficiently powered to prove the observed significant association between plasma GPC4 and survival in breast cancer patients. However, GPC4 levels have been associated with various morbidities and, therefore, may not be specific to the malignant progression of a particular cancer type. The observational design of our study prevents causal conclusions regarding relationships between GPC4 and study parameters. Therefore, the molecular backgrounds behind our findings need to be addressed in future studies.
In conclusion, we report for the first time that plasma GPC4 levels significantly predict 24-month OS in ER+/HER2− MBC patients. In addition, plasma GPC4 was significantly linked to various clinicopathological characteristics of our MBC patients. Due to these promising results and the limited knowledge, so far, of circulating GPC4 and its role in cancer prognosis, the molecule deserves further investigation.
The authors sincerely thank the assistance of Dr. Kathrin Geiger and Stella Gaenger (both “Vorarlberg Institute for Vascular Investigation and Treatment,” Dornbirn, Austria) in laboratory work.
Statement of Ethics
This study was conducted as a prospective analysis of patient data and was approved by the Ethics Commission of the Landesärztekammer Baden-Württemberg, Germany (F-2017-46; October 11, 2017). Written informed consent was obtained from all patients for participation in this study. This research project complied with the guidelines for human studies and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.
Conflict of Interest Statement
Thomas Decker received honoraria from iOMEDICO and advisory board honoraria from Novartis and Roche. Tobias Dechow received honoraria from AstraZeneca, Boehringer Ingelheim, iOMEDICO, Merck, and Sanofi. The sponsors had no involvement in study design, in the collection, analysis, and interpretation of data, in the writing of the manuscript, or in the decision to submit the paper for publication. The remaining authors have no conflicts of interest to declare.
This present study was partly financed by the European Regional Development Fund through the INTERREG V program “Alpenrhein-Bodensee-Hochrein,” project number: ABH055.
Conception and design, statistical analysis, and drafting of the manuscript: Thomas Decker and Axel Mündlein. Patients’ recruitment, sample, and data collection: Thomas Decker and Tobias Dechow. Sample analysis: Christine Heinzle, Eva Maria Brandtner, and Axel Muendlein. Data analysis and interpretation: Thomas Decker, Tobias Dechow, Heinz Drexel, Andreas Leiherer, and Christine Heinzle. All the authors participated in the critical revision and validation of the final manuscript.
Data Availability Statement
All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author.