Abstract
Background/Aim: Combination therapy with immune checkpoint inhibitors has become the standard first-line treatment for metastatic renal cell carcinoma (mRCC), leading to changes in second-line treatment options, such as nivolumab or tyrosine kinase inhibitors (TKIs). However, very few studies have compared the efficacy of these drugs in patients with mRCC, particularly those with bone metastases (BM), which are associated with a poor prognosis. This study compared the efficacy of nivolumab and TKIs as second-line treatments for mRCC patients with BM and examined the microenvironments of primary tumors and BM lesions. Patients and Methods: This multi-institutional retrospective study included 87 mRCC patients with BM who received either nivolumab or TKIs as second-line treatments. We analyzed tumor-infiltrating immune cells expressing CD8 and CD20, along with PD-L1, HIF2α, c-MET, VEGFR2, and AXL, in primary tumors and BM sites using immunohistochemistry. Results: This analysis indicated that poor-risk classification, as per the International Metastatic RCC Database Consortium criteria (p<0.01), and elevated serum alkaline phosphatase levels (p=0.031) were significantly associated with poor prognosis. No significant difference in overall survival was observed between patients receiving nivolumab and those receiving TKIs. However, the objective response rate of patients with BM lesions was significantly higher when receiving TKIs than when receiving nivolumab (p=0.014). Immunohistochemistry revealed significantly higher VEGFR2 expression in BM lesions than primary tumors. Conclusion: TKIs could be a promising second-line treatment option for mRCC patients with bone-limited metastases.
- Renal cell carcinoma
- bone metastasis
- second-line treatment
- nivolumab
- tyrosine kinase inhibitor
- overall survival
Renal cell carcinoma (RCC) ranks 14th among the most frequently diagnosed cancers worldwide (1). In 2020, an estimated 431,288 new cases of RCC and 179,368 associated deaths were reported. With advances in imaging and inspection technology, the incidence of RCC has been increasing at an annual rate of 1-2% (2). Immune checkpoint inhibitor (ICI) combination therapy, such as ICI plus ICI or ICI plus tyrosine kinase inhibitor (TKI), has emerged as the first-line systemic treatment for metastatic RCC (mRCC). Owing to the refractoriness of mRCC to ICI combination therapy, the majority of patients develop progressive disease requiring subsequent TKIs or nivolumab for management (3). To date, although the comparable efficacies of axitinib and nivolumab as secondary treatments for mRCC have been reported (4), there are limited real-world clinical reports on second-line treatments for RCC.
Among the various types of metastases, RCC is especially susceptible to bone metastasis (BM), which occurs in approximately 30% of the patients with mRCC (5). BM may lead to severe complications, including skeletal-related events, and is a poor prognostic factor for patients with mRCC (6). Negishi et al. reported that the effectiveness of ICIs against BM in mRCC is limited to only 5% (7). However, the METEOR trial reported that cabozantinib, a well-known TKI, led to a better prognosis in mRCC with BM than everolimus after at least one previous TKI treatment (8). However, the efficacy of cabozantinib in treating bone metastatic lesions remains unclear.
Therefore, we aimed to compare the treatment efficacies of second-line TKIs and nivolumab in patients with RCC and BM. Additionally, we evaluated the expression of receptors targeted by cabozantinib, the immune microenvironment, and hypoxic conditions using immunohistochemistry to compare primary tumors and BM lesions.
Patients and Methods
Patient selection. We retrospectively reviewed the clinical data of 721 patients with RCC and analyzed the clinicopathological data of 87 patients with mRCC with BM who initiated second-line treatment with nivolumab (n=29) or TKI (n=58) at Osaka University Hospital, Osaka Police Hospital, Osaka General Medical Center, Osaka Rosai Hospital, and Osaka National Hospital between January 2008 and December 2023. BM was diagnosed using computed tomography with MRI or bone scintigraphy, which was performed as needed (9). The TKIs included were sunitinib, axitinib, cabozantinib, sorafenib, and pazopanib. The dosage and administration intervals followed standard treatment protocols.
The following patient data were collected from the hospital records of the patients: age, sex, histopathology of RCC, International Metastatic Renal Cell Carcinoma Database (IMDC) classification for first-line therapy, previous nephrectomy, local therapy for BM site, location of metastatic sites, number of BMs, first-line therapy regimen, type of second-line regimen, bone modifying agent, serum concentration of alkaline phosphatase (ALP), serum concentration of lactate dehydrogenase, neutrophil-lymphocyte ratio, best overall response rate (ORR) for the second line therapy, second-line therapy cancer-specific survival (CSS), and overall survival (OS).
The therapeutic efficacy of second-line treatment was assessed every 2-3 months using computed tomography according to the Response Evaluation Criteria in Solid Tumors version 1.1 and MD Anderson (MDA) criteria (10). The best response of each patient during treatment was determined and classified as complete response, partial response, stable disease, or progressive disease. This study was approved by the Institutional Review Board of the Osaka University Hospital (# 018-0003). All procedures were conducted in accordance with the Declaration of Helsinki, and informed consent was obtained from all participants.
Immunohistochemistry and quantification. The primary RCC tissue resected after total nephrectomy was stored as formalin-fixed paraffin-embedded tissue blocks. Resected RCC tissue from the BM site in the same patients was subjected to tris-ethylenediami-netetraacetic acid treatment until tissue softening was observed. This was followed by paraffin fixation. Immunohistochemical staining was performed on 4-μm-thick paraffin-embedded tissue sections from primary RCC and BM tissues. The tissue sections were deparaffinized using xylene and a graded series of ethanol solutions and stained with eosin. Other sections were treated with EDTA buffer (pH 9.0) and activated by warming at 125°C for 30 s using a Pascal pressure chamber (S2800, DAKO, Carpinteria, CA, USA) for antigen activation treatment. Endogenous peroxidase activity was blocked by incubating the sections with 0.3% hydrogen peroxide for 5 min, followed by overnight incubation with primary antibodies against CD8 (1:100; ab17147, Abcam, Cambridge, UK) as a marker for cytotoxic T-lymphocytes, CD20 (1:800; ab9475, Abcam) as a marker for B-cells, PD-L1 (clone 28-8, 1:200; ab205921, Abcam), HIF2α (1:200; ab243861, Abcam), c-MET (1:100; ab51067, Abcam), VEGFR2 (1:100; ab115805, Abcam), and AXL (1:2,000; ab219651, Abcam) at 4°C. The EnVision + system horseradish peroxidase-labeled polyclonal anti-rabbit and anti-mouse antibody (DAKO) was used, and staining was performed using 3,3′Diaminobenzidine substrate (MK210, TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Finally, the sections were counterstained with hematoxylin, dehydrated using a graded series of ethanol concentrations, and cleared with xylene.
The CD8+ and CD20+ cell counts within the tumor were quantified separately. Three distinct random regions featuring the highest abundance of positive cells were identified using a ×40 objective lens, and the average count was used for statistical analysis (11). The membrane PD-L1 expression in the tumor cells was assessed at three random locations using a ×40 objective lens, and the average count was used for statistical analysis (12). HIF2α was assessed for positivity exclusively within the nuclei of tumor cells (13). The ratios of c-MET, VEGFR2, and AXL expression in the membrane and cytoplasm of tumor cells were determined (14-16). HIF2α, c-MET, VEGFR2, and AXL were evaluated and analyzed in three random fields of view using a ×40 objective lens. The number of positive cells and their levels of expression were automatically determined using a microscope (BZ-X710; KEYENCE, Osaka, Japan).
Statistical analysis. Clinical characteristics were compared using the Mann–Whitney U-test. Fisher’s exact test was used to analyze the therapeutic effects of the ICIs and TKIs. Univariate and multivariate logistic regression analyses were performed to assess the relative contributions to CSS and OS. CSS and OS were estimated using the Kaplan–Meier method and Cox proportional hazard regression. All p-values were two-sided, and differences were considered statistically significant at p<0.05. Data were analyzed using JMP Pro (v.17.0.0; SAS Institute, Cary, NC, USA).
Results
Patient characteristics. This study included a total of 721 patients with RCC, and the clinicopathological characteristics and outcomes of the 87 patients with mRCC and BM included in the study are summarized in Table I. The median age of the patients in the nivolumab and TKI groups was 66 years (range=29-87 years) and 69 years (range=33-84 years), respectively. The most prevalent histological type was clear cell carcinoma, observed in 25 (86%) and 47 (81%) patients treated with nivolumab and TKI, respectively. The nivolumab group predominantly comprised patients with poor IMDC risk (52.0%), whereas 76.0% of patients treated with a TKI were classified as having favorable and intermediate risk. The most commonly prescribed TKI was axitinib (n=35, 60%), followed by cabozantinib (n=14, 24%).
Patient characteristics.
Clinical outcomes of patients treated with second-line nivolumab or tyrosine kinase inhibitors. Overall, the median follow-up durations of patients treated with nivolumab and TKIs were 7.0 months (range=1-73 months) and 11.5 months (range=1-93 months) months, respectively. There were no significant differences between the CSS and OS of the patients treated with nivolumab and TKI (p=0.76 and p=0.55, respectively) (Figure 1, Table II, Table III). Multivariate analyses revealed that poor risk based on the IMDC classification and high serum ALP concentration (≥288 U/l) were significantly associated with reduced OS [hazard ratio (HR)=3.24, 95%CI=1.84-5.71, p<0.01; HR=1.78, 95%CI=1.05-3.01, p<0.01, Table III]. Accordingly, OS was significantly shorter for patients with poor risk based on the IMDC classification and high serum ALP concentration (≥288 U/l) (HR=3.58, 95%CI=2.08-6.17, p<0.01; HR=2.01, 95%CI=1.21-3.35, p<0.01, Figure 2).
Kaplan–Meier curves of cancer-specific survival (A) and overall survival (B) for patients with metastatic renal cell carcinoma after second-line treatment with immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs).
Univariate and multivariate analyses of CSS after second-line therapy.
Univariate and multivariate analyses of OS after the introduction of second-line therapy.
Kaplan–Meier curves of cancer-specific survival and overall survival for patients with metastatic renal cell carcinoma stratified according to International Metastatic Database Consortium (IMDC) risk classification (A, C) and alkaline phosphatase (ALP) concentration (B, D).
To exclude the influence of radiation therapy on the BM site, we examined 72 patients with untreated BM sites (Table IV). A comparison of the outcomes of second-line treatment for BM according to the MDA criteria indicated that the ORR was significantly higher for the TKI group (29.2%) than for the nivolumab group (4.2%) (p=0.014) (Figure 3, Table V). This indicated that TKIs may be more effective than nivolumab for treatment-naïve BM.
Background of patients with locally untreated bone metastasis.
Bone metastasis is shown for a 66-year-old male with metastatic clear cell renal cell carcinoma whose treatment was initiated with ipilimumab plus nivolumab; however, was switched to cabozantinib after 13 months. (A) Computed tomography (CT) images just before induction of cabozantinib. (B) CT images after 11 months of cabozantinib treatment show that according to RECIST criteria, the metastatic lesion in the right iliac bone is assessed as SD (stable disease). However, according to MDA criteria, the appearance of sclerotic bone lesions indicates PR (partial response). The white arrow indicates the development of a partial sclerotic fill-in of lytic lesions.
Overall response rate of second-line treatment to locally untreated BM according to the MDA criteria and RECIST 1.1.
Significant difference between the tumor microenvironments of primary tumors and bone metastasis. We investigated the presence of tumor-infiltrated immune cells and several molecules targeted by TKIs in the tumor microenvironment of primary tumors and BM sites. Representative immuno-histochemistry results are shown in Figure 4 and Figure 5. Consequently, we observed a significant increase in VEGFR2-positive cells in the BM lesions (p=0.048). In addition, we confirmed that the CD8+ T-cell and CD20+ B-cell counts were higher in primary tumors (p=0.075 and p=0.10, respectively; Figure 6). Collectively, these results indicate that primary tumors and BM have different tumor microenvironments, affecting the efficacy of TKIs and nivolumab therapies.
Immunohistochemical staining results for a primary site of clear cell renal cell carcinoma. Scale bar represents 200 μm.
Immunohistochemical staining results for a bone metastatic lesion of clear cell renal cell carcinoma. Scale bar represents 200 μm.
Comparison of expression levels of CD8, CD20, PD-L1, HIF2α, c-MET, VEGFR2, and AXL in the primary tumor and bone metastatic lesions of clear cell renal cell carcinoma.
Discussion
The guidelines of the European Association of Urology recommend ICI combination therapy as the first-line treatment for mRCC (17). There is currently no established drug selection for the second-line treatment of mRCC; however, several studies have suggested that second-line treatment should generally comprise nivolumab monotherapy or TKI therapy (17). On the contrary, the effectiveness of TKI and ICI greatly depends on the tumor microenvironment, which varies significantly due to several patient factors including tumor heterogeneity (18). For RCC, the characteristics of metastatic tumor cells and the surrounding microenvironment may not always match those of primary tumors (19). Therefore, a comparative analysis of the molecular biomarker expression profiles of primary and metastatic specimens, such as the bone, should be performed (20).
The bone matrix is inherently rich in growth factors, making it a favorable and fertile environment for metastatic tumors, irrespective of the primary tumor type. BM is considered a poor prognostic factor for patients with mRCC (21). In the microenvironment of BM lesions, regulatory T-cells and myeloid-derived suppressor cells are abundant, suppressing tumor immunity (22, 23), which may lead to poor prognosis for patients with mRCC and BM. Moreover, in RCC, inactivation of the von Hippel-Lindau tumor suppressor gene is common and leads to an increase in hypoxia-inducible factor-alpha, resulting in the over-expression of vascular endothelial growth factor (VEGF) (24). This growth factor promotes tumor angiogenesis and plays a crucial role in the invasion and metastasis of RCC. On the contrary, tyrosine kinase is an important enzyme in intracellular signal transduction, and binding of VEGF to the VEGF receptor (VEGFR) increases receptor tyrosine kinase activity, initiating intracellular signal transduction. Therefore, we aimed to compare the efficacies of nivolumab and TKIs as second-line treatments for patients with mRCC with BM, elucidating the differences between the tumor microenvironments of primary and BM lesions.
In this study, we present two novel findings. First, we found that TKIs were more effective as second-line treatments for BM lesions in RCC than nivolumab monotherapy, as evidenced by the higher ORR at the BM sites (Table V). Previous studies have demonstrated significant differences in the treatment efficacy of ICI across metastatic lesions (25). In a large-scale tumor-agnostic study, BM lesions were associated with resistance to ICI (26). In patients with mRCC treated with second-line nivolumab, treatment efficacy varies significantly with the metastatic site, with bone metastases exhibiting a particularly low ORR of 5%, compared with 36% for lung and 50% for liver metastases (7). In patients with mRCC treated with nivolumab as second-line therapy or beyond, BM, rather than lung or lymph node metastases, was associated with poor progression-free survival (PFS) and OS (27). In the present study, an elevated serum ALP concentration, which reflects the overall deterioration of systemic conditions such as BM volume, was an independent poor prognostic factor for second-line treatment of mRCC with BM (Table III). Therefore, TKIs are considered a promising option for patients with mRCC with BM and without other visceral metastases.
Second, VEGFR2 expression was higher in BM lesions than in primary tumors, and BM lesions exhibited a trend of decreased CD8+ tumor-infiltrating T-lymphocytes and tumor-infiltrating CD20+ B-cells (Figure 6), which may contribute to the lower ORR of nivolumab than of TKI for BM lesions. Tumor-infiltrating B-lymphocytes in mRCC might recruit and activate CD8+ tumor-infiltrating T-lymphocytes, thereby enhancing antitumor effects, and this process is involved in the formation of tertiary lymphoid structures (28, 29). TKIs are also considered more likely to be effective for cases of RCC with high expression of VEGFR2 (30). In this study, we described a significant increase in VEGFR2- positive cells in BM sites, demonstrating the potential benefits of TKI in patients with mRCC with BM. Specifically, considering that cabozantinib exhibited the strongest inhibition toward VEGFR2 among several types of TKIs, cabozantinib may likely contribute to the enhanced antitumor activity in BM (31).
Study limitations. First, this was a retrospective study with few cases. While it was a multicenter study including five hospitals, the number of patients with mRCC with BM receiving second-line treatment was limited, and even fewer underwent surgery for BM lesions. The prior therapy does not affect the response to nivolumab (32), but a study design with a certain first line therapy is needed. Therefore, future studies with larger sample sizes and more well-organized designs are warranted. Second, we used the MDA criteria and Response Evaluation Criteria in Solid Tumors to evaluate the treatment efficacy for BM lesions, but there is currently no consensus on the best response evaluation system (33). Notwithstanding, several studies have used the MDA criteria to evaluate BM lesions (34). ICI combination therapy for mRCC is safe for elderly patients (35) and more effective than TKI alone (36), but the efficacy for BM is an issue for further investigation.
In this study, we revealed that TKIs may have higher efficacy than nivolumab against BM lesions, and poor IMDC risk classification and elevated serum ALP concentrations are associated with a poorer prognosis in patients with mRCC and BM receiving second-line therapy. Overall, TKIs may be more effective than nivolumab in treating patients with mRCC with metastases limited to the bone.
Footnotes
Authors’ Contributions
G.Y. contributed to data collection and analysis, table and figure preparation, reference collection, and manuscript writing. G.Y. and T.K. contributed to data collection and analysis, and supervised all activities. A.Y., M.T., Y.H., L.Y., S.N., Y.O., T.O., T.U., A.Y., Y.I., T.H., Y.Y., K.H., A.K., T.T., K.N., S.T., M.T., and N.N. supervised all the activities. The first draft of the manuscript was prepared by G. Y. and T. K. All Authors have read and approved the published version of the manuscript.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
Funding
This study was supported by JSPS KAKENHI (grant number: 21K09396).
- Received December 20, 2024.
- Revision received January 7, 2025.
- Accepted January 8, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
