Changes in complement C3a in the immunotherapy of advanced clear cell renal cell carcinoma

Introduction

Clear cell renal cell carcinoma (CCRCC) is a subtype of renal cancer, and accounts for about 70–75% of all renal cancers (1). Advanced CCRCC has low sensitivity to chemotherapy, and the treatment options are mainly small molecule tyrosine kinase inhibitors (TKIs), such as sorafenib, axitinib, sunitinib or pazopanib alone. The overall prognosis of patients treated with drugs and immune card control drugs represented by programmed cell death protein 1 (PD-1) antibodies is poor (2). Thus, more potential targets need to be identified.

In recent years, a study has confirmed that the complement system plays an important role in tumor growth and metastasis (3). Complement C3 is the core molecule of complement activation and is mainly synthesized in the liver. A study has found that malignant tumor cells can also synthesize and secrete complement C3 (4). Tumor-derived complement C3 plays an important role in tumor proliferation and metastasis (5). After activation, complement C3 is cleaved into C3a, and C3a has been confirmed to play a role in promoting tumor metastasis in the tumor microenvironment (3).

Through the analysis of data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, several studies have found that CCRCC patients with a high expression of complement C3 have a poor prognosis and short survival time (6,7). Thus, C3 could serve as a potential prognostic factor for CCRCC. However, the changes in C3a, which is an expression product of C3, in renal cancer immunotherapy and its relationship with the curative effect is unclear.

This study sought to detect the expression of C3a in renal cancer tissues and adjacent tissue samples by immunohistochemistry. The level of serum complement C3a in patients with advanced CCRCC before and after immunotherapy was detected by enzyme-linked immunosorbent assay (ELISA), and the relationship between its changes and clinical efficacy was observed to explore the significance of C3a in immunotherapy for advanced CCRCC. We present this article in accordance with the REMARK reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-24-549/rc).

Methods

Data acquisition and the prognostic value of C3

The C3 expression data of CCRCC patients were downloaded from TIMER2.0 database (http://timer.cistrome.org/) (8). The CCRCC data set comprised 72 normal tissue samples and 533 tumor samples. The Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/detail.php?gene=C3) was used to verify the relationship between C3 and overall survival (OS) (9).

Participants

Immunohistochemistry participants

Specimens from 110 CCRCC patients obtained from surgeries conducted at The First People’s Hospital of Yancheng between January 2018 and December 2021 were included in the retrospective study. None of the patients had undergone any anti-tumor treatment prior to surgery.

The study was a retrospective study and conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of The First People’s Hospital of Yancheng (No. 2022-K-121) and informed consent was taken from all the patients.

ELISA experiment participants

A total of 30 patients with advanced CCRCC who received PD-1 antibody therapy between January 2019 and December 2021 were enrolled in the retrospective study.

Inclusion criteria

To be eligible for inclusion in this study, the patients had to meet the following inclusion criteria: (I) have a histologically confirmed diagnosis of CCRCC; (II) have lymph node or organ metastasis with measurable lesions [i.e., a maximum transverse diameter ≥1 cm on computed tomography (CT)]; (III) have received at least four cycles of PD-1 antibody therapy and/or TKI (sunitinib, axitinib, or sorafenib) treatment; (IV) have undergone a clinical efficacy evaluation using the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST1.1); and (V) have a performance status (PS) score ≤2.

Exclusion criteria

Patients were excluded from the study if they met any of the following exclusion criteria: (I) had discontinued treatment due to grade 3–4 immune-related adverse reactions; (II) were aged over 75 years); and/or (III) had multiple primary cancers.

Clinical efficacy

Before treatment and after four cycles (12 weeks) of treatment, CT or magnetic resonance imaging (MRI) examinations were performed to evaluate the treatment efficacy of the patients using the RECIST 1.1. Complete response (CR) was defined as the disappearance of all target lesions with a short-axis lymph node diameter <10 mm. Partial response (PR) was defined as a reduction of ≥30% in the sum of the longest diameters of all target lesions. Progressive disease (PD) was defined as an increase of at least 20% in the sum of the longest diameters of target lesions with an absolute increase of at least 5 mm, and the appearance of one or more new lesions. Stable disease (SD) was defined as tumor shrinkage of less than 30% and an increase of less than 20% between the PR and PD.

Immunohistochemistry staining analysis

The expression of complement C3a was detected using immunohistochemical staining with the LEICA BOND-MAX fully automated immunohistochemistry system (Leica, Germany); normal renal tissue >3 cm from the tumor edge served as the control group. Negative controls were treated with phosphate buffered saline (PBS) instead of the primary antibody. C3a (mouse monoclonal antibody, clone K13/16,1:200) was purchased from Santa Cruz Biotechnology (Shanghai, China). Positive staining of C3a was localized in the cytoplasm and/or cell membrane, appearing as a pale yellow to brownish-brown color. A minimum of 500 tumor cells were randomly counted in each slide, and expression was considered negative if ≤20% of the cells were positive, and positive if >20% of the cells were positive. Two experienced pathologists reviewed the slides to determine the histological grade and clinical stage.

ELISA experiment

Before treatment and after four cycles (12 weeks) of treatment, 5 mL of peripheral blood was collected from each patient, and the serum complement C3a concentration was measured using the ELISA method. The steps were carried out according to the instructions of the complement C3a assay kit (catalog number JEH-141, Anhui Qiaoyi Biotechnology Co., Ltd., Anhui, China).

Long-term therapeutic effects

Patients were followed up for the first time after four cycles of treatment, and every 3 months thereafter until December 2022. Adverse reactions and survival status during the follow-up period were recorded. Progression-free survival (PFS) was defined as the time from patient enrollment to disease progression (PD) or death.

Statistical methods

The data analysis and graphing were performed using SPSS 22.0 and GraphPad Prism 6.0. The continuous data are presented as the mean ± standard deviation, and the categorical data are presented as the count and percentage. The between-group comparisons were performed using the Chi-squared test. Survival curves were generated using the Kaplan-Meier method, and differences between the groups were analyzed using the log-rank test. A P value <0.05 was considered statistically significant.

Results

Differential expression of complement C3 and its influence on the prognosis of CCRCC patients

The analysis of TIMER2.0 data showed that C3 expression was heterogeneous across cancer patients, but was generally higher in the CCRRC tissues, including the CCRRC tissues, than the normal tissues. The survival curves plotted based on the OS data from the GEPIA database indicated that C3 significantly affected the prognosis of CCRCC patients; that is, the high expression of C3 increased the risk of a poor prognosis (Figure 1).

Figure 1 Expression levels and prognostic value of C3 in CCRCC. (A) Expression levels of C3 in CCRCC from TIMER2.0 database; (B) survival curves of C3 in CCRCC from the GEPIA database. Dotted lines indicate 95% confidence intervals. *, P<0.05; **, P<0.01; ***, P<0.001. TPM, transcripts per million; HR, hazard ratio; CCRCC, clear cell renal cell carcinoma; GEPIA, Gene Expression Profiling Interactive Analysis.

Complement C3a was significantly upregulated in the tissues of CCRCC patients

Among the 110 patients, 76 showed positive expression of C3a in renal cancer tissues, with some expression also observed in the adjacent non-cancerous renal tubules (Figure 2). The positive expression rates of C3a in the adjacent non-cancerous tissues and renal cancer tissues were 76.36% (84/110) and 69.09% (76/110), respectively, with no statistically significant difference (P=0.22).

Figure 2 Expression of complement C3a in renal cell carcinoma and adjacent non-cancerous renal tubules (immunohistochemistry staining). (A,C) Renal cell carcinoma; (B,D) adjacent non-cancerous renal tubules. Magnification: (A,B) 10×20; (C,D) 10×40.

The analysis of the clinical and pathological factors revealed no differences in C3a expression in the tumor tissues in terms of gender, age, location, or histological grade. However, when comparing the different tumor sizes and stages, a significantly higher expression rate of C3a was observed in tumors with a maximum diameter >3.5 cm compared to those with a maximum diameter ≤3.5 cm (P=0.02). Additionally, the expression of C3a in tumor tissues of patients with the stage II TNM classification was significantly higher than that in those with the stage I TNM classification (P=0.005), and the difference was statistically significant (Table 1).

Relationship between serum complement C3a and the clinical response of patients

A total of 30 patients with advanced CCRCC were included in this study, of whom 20 were male and 10 were female. Among the patients, 16 were aged over 60 years, and 14 patients were aged 60 years or below. There were 11 cases with metastasis in ≤2 organ sites and 19 cases with metastasis in >2 organ sites. The PS scores were 0, 1, and 2 in 9, 11, and 10 cases, respectively. There were no differences in complement C3a levels based on age and gender. However, patients with metastasis in >2 organ sites and higher PS scores exhibited higher concentrations of complement C3a, and the differences were statistically significant (Table 2).

All the patients were evaluated for treatment response after four cycles of immunotherapy at week 12. Among them, 12 patients achieved disease remission, including one case of complete remission (CR) and 11 cases of partial remission (PR), while 7 cases achieved SD, and 11 cases showed PD. In these patients, the serum complement C3a overall level before treatment was 2,222.2±250.3 pg/mL, but after treatment, it decreased to 1,787.2±784.1 pg/mL. The pre-treatment serum complement C3a concentration for patients achieving CR + PR + SD was 2,278.1±271.6 pg/mL, which decreased to 1,356.2±647.8 pg/mL after treatment, showing a statistically significant difference (P=0.005). Interestingly, the expression level of C3a was significantly reduced in patients with CR + PR and stabilized in patients with SD. The pre-treatment complement C3a level for patients with PD was 2,125.6±180.9 pg/mL, which increased significantly to 2,531.7±254.6 pg/mL after treatment, and the difference was statistically significant (P<0.001) (Figure 3).

Figure 3 Changes in serum concentration of complement C3a after treatment in renal clear cell carcinoma. **, P<0.01; ***, P<0.001; NS, P≥0.05. CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease.

Relationship between serum complement C3a expression level and PFS in CCRCC patients

Patients were initially followed up after four cycles of treatment, and subsequent follow-up visits were conducted every 3 months until December 2022. The results demonstrated that patients with a decrease in complement C3a levels had a significantly prolonged PFS compared to those with no decrease or an increase in complement C3a levels [11.2±4.7 vs. 4.3±2.4 months, HR (95% CI): 0.31 (0.13–0.71), P<0.001] (Figure 4).

Figure 4 Progression-free survival in patients with rising and declining serum complement C3a levels after treatment (P<0.001). HR, hazard ratio; CI, confidence interval.

Discussion

CCRCC is one of the most common tumors in the urinary system. Early-stage CCRCC has a favorable prognosis and can be treated with surgical resection. However, even after surgical resection, approximately 40–50% of CCRCC patients experience recurrence and metastasis (10). Metastatic CCRCC (mCCRCC) is resistant to conventional chemotherapy and has a poor prognosis. Targeted therapies (TKI and mTOR inhibitors) and immune checkpoint inhibitors (ICIs) are the main treatment options for mCCRCC (11). However, the PFS with immune and targeted therapies is typically limited to about 12–24 months, necessitating the active exploration of new treatment approaches and medications.

The complement system is an important component of the tumor immune microenvironment (12-15). It comprises a group of enzymatically active proteins present in the serum, tissue fluids, and cell membrane surfaces of humans and animals. The complement system includes components of the innate immune system, complement regulatory proteins, and complement receptors. The complement system is involved in the initial stages of non-specific and adaptive immune responses and plays a regulatory role in humoral and cellular immune reactions (16).

In a study conducted in 2020 (17), the researchers analyzed 72 normal samples and 539 tumor samples obtained from TCGA database, as well as 23 normal samples and 32 tumor samples from the GEO database. The findings revealed a higher expression of complement C3 in CCRCC, and a correlation between higher C3 expression and poorer survival outcomes. Similarly, in a study by Quan et al. in 2021 (6), which used data from the GEO database to analyze differential gene expression in advanced CCRCC and normal individuals, it was also observed that the expression of complement C3 was elevated in advanced CCRCC, and was associated with a worse prognosis. These results are consistent with those of our database review. However, there is a lack of a sample analysis of the C3a in the database, which needs to be further supplemented.

In our present investigation, we employed immunohistochemical methods and found that complement C3a was upregulated in partial CCRCC, with some expression also observed in the adjacent non-cancerous renal tubules. We observed that C3a was significantly more highly expressed in tumors with a maximum diameter >3.5 cm than those with a maximum diameter of ≤3.5 cm (P=0.02). Further, C3a was significantly more highly expressed in the TNM stage II tumors than stage I tumors (P=0.005). Thus, the results suggest that increased C3a expression is associated with a larger tumor size and more advanced TNM staging.

A study has suggested that the complement system, when activated, exerts anti-tumor effects through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, inhibiting tumor growth (16). However, recent research has highlighted the significant role of the complement system in tumorigenesis and tumor progression. Malignant tumor cells can secrete complement C3, which is subsequently cleaved to form complement C3a (18). It has been shown that C3a promotes tumor growth and metastasis, and this effect has been validated in various cancers, such as lung, colon, gastric, and cutaneous squamous cell carcinoma (19-21).

We employed ELISA to detect the expression of serum complement C3a in patients with advanced CCRCC. We observed higher levels of complement C3a in patients with metastasis in more than two organs and higher PS scores. This could be attributed to the fact that patients with late-stage cancer often display higher PS scores, which in turn correlates with increased C3a expression. Following treatment, the patients with a PR or CR showed a decrease in complement C3a levels, and patients with SD showed a stability in complement C3a levels, while patients with PD exhibited a significant increase in C3a levels. This indicates a correlation between C3a concentration and changes in patient condition. However, it remains to be further investigated whether the alterations in C3a levels are a direct consequence of tumor lysis or if there are other indirect mechanisms that implicate immune functions. Moreover, our study found that patients with a decrease in C3a had significantly longer PFS compared to those with an increase in C3a. Therefore, C3a could serve as an important prognostic indicator for disease outcome and a predictive marker for treatment efficacy.

Conclusions

In conclusion, the expression of C3a in advanced CCRCC is associated with the tumor burden, with a higher expression observed in later-stage tumors. The changes in complement C3a during immunotherapy are related to treatment efficacy, with better outcomes and longer PFS observed in patients with a decrease in C3a levels. However, it is important to note that our study had a small sample size and a relatively short follow-up period. Future research should aim to increase the sample size and extend the duration of the follow-up period to further validate these findings.

Acknowledgments

Funding: This work was supported by Health Commission project of Jiangsu (No. Z2021041), Health Commission project of Yancheng (No. YK2018017), Jiangsu Province Traditional Chinese Medicine Science and Technology Development Program Young Talent Project (No. QN202327), and Yancheng Municipal Health Commission Medical Research Key Project (No. YK2023002).

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-24-549/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-549/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-549/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-549/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was a retrospective study and conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of The First People’s Hospital of Yancheng (No. 2022-K-121) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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