Significance of the FGFR3 mutation in Chinese patients with bladder cancer

Introduction

Bladder cancer (BC) is the tenth most common malignancy in the world, with 570,000 new cases and 210,000 reported globally in 2020, placing a massive burden on the healthcare system (1). In terms of the pathological type, >90% of BCs are urothelial cell carcinomas, and about 5% are squamous cell carcinomas, while adenocarcinomas are rare (2). In the clinic, about 75% of newly diagnosed patients have non-muscle invasive BC. The common treatments for non-muscle invasive BC include transurethral resection, followed by intravesical chemotherapy or Bacillus Calmette Guerin immunotherapy. After treatment, the long-term survival rate of patients is reasonable, but the recurrence rate is high, and 10–15% of patients show disease progression. Muscle invasive BC, which is highly malignant, prone to lymph node metastasis, and has a low 5-year survival rate, accounts for about 25% of BC cases (3-5). The main treatment for muscle invasive BC rely on radical cystectomy and lymph node dissection, different urinary diversion performed according to the situation. In addition, local radiotherapy or systemic adjuvant chemotherapy can be used if local progression or distant metastasis occurs. In recent years, the efficacy of surgical treatment, immunotherapy, and combined chemoradiotherapy in the treatment of BC has progressed (6-8). However, the prognosis of invasive and metastatic BC patients has not been significantly improved due to limited understanding of the biological mechanisms of disease recurrence and progression. Thus, an in-depth exploration of the genes related to malignant tumor progression would help to better predict the progression of BC patients and provide new ideas and directions for diagnosis and treatment (9). Some FGFR inhibitors are becoming new treatment options for metastatic urothelial cancer (10).

FGFR3 is a tyrosine-protein kinase family and transmembrane tyrosine kinase receptor with autophosphorylation activity. After binding to fibroblast growth factor, it can activate various downstream signaling pathways, which mainly include the PI3K/AKT and Ras/Raf/MAPK signaling pathways (11-13). Some centers in Europe have studied the mutation frequency and mutation site of FGFR3 gene in bladder cancer (14,15). Several studies have shown that the FGFR3 mutation is significantly associated with low-grade and low-stage BC and is associated with low-grade and low-stage papillary BC (16). van Rhijn et al. found an association between mutations and low-grade BC (17). Hernández et al. found that the A393E mutation has a high frequency in bladder tumors with low malignant potential (15). Rizzo et al.’s study found that 150 of the 358 patients with upper urinary tract urothelial cancer (UTUC) had FGFR3 mutations, and patients with FGFR3 mutations had a longer overall survival than wild-type patients who received radical surgery (18). In addition, FGFR3 mutations were more frequent in low-grade UTUCs with early-stage disease (18).

This study analyzed the detection results of gene mutations in the tissue samples of BC patients and explored the relationship between FGFR3 and the prognosis and recurrence of BC. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-23-247/rc).

Methods

This study is a retrospective study of bladder cancer patients treated at Zhongshan Hospital Xiamen University between January 2017 and December 2019. These patients have all received multi-gene panel targeted next-generation sequencing. We used the results of gene mutations in tissue samples of BC patients to explore the relationship between FGFR3 and BC prognosis and recurrence.

Clinical samples

A total of 82 Chinese patients from the Zhongshan Hospital Xiamen University were enrolled in this study. These patients contain both non-muscle-invasive bladder cancer patients and muscle-invasive bladder cancer patients, and include all clinical stages (Table 1). Of these patients, 34 underwent radical cystectomy (RC), and 48 underwent transurethral resection with intravesical therapy. The tumor samples were obtained from the BC surgery, and the tissue was paraffin-embedded after the surgery. All the samples were checked for quality control and met the requirements for gene sequencing. This study was approved by the Ethics Committee of Zhongshan Hospital Xiamen University (No. 2022-181). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Written informed consent was obtained from all the study participants.

NGS and annotation

Genomic DNA was extracted from the formalin-fixed, paraffin-embedded tissue sections. Sonication was then performed to obtain 200-bp DNA fragments. The Roche SeqCap EZ Exome V3 (Roche NimbleGen Inc., Basel, Switzerland) and TruePrep DNA Library Prep Kit (version 2) were used for Illumina (#TD501, Vazyme, Nanjing, China) sequencing to capture the target DNA fragments. Multi-gene panel targeted next-generation sequencing (NGS) (Tongshu BioTech, Shanghai, China) was performed using the Illumina HiSeq machine to generate paired-end sequence data. Subsequently, the received sequencing results were compared using BWA software. Corrections were compared using the Genome Analysis Toolkit, and CNVKIT was used to detect copy number variations in the sequencing data. Finally, somatic mutations were converted into MAF format and visualized using R maftools (https://bioconductor.org/packages/release/bioc/vignettes/maftools/inst/doc/maftools.html).

Statistical analysis

The results are presented as the mean ± standard deviation, and the Mann-Whitney U test, Wilcoxon signed-rank test, and a Spearman correlation analysis were used in this study. Relapse-free survival (RFS) was determined using the Kaplan-Meier method. A 2-tailed P value of <0.05 was considered statistically significant in this study. Multivariate analysis was done using Cox regression analysis after adjusting for other prognostic factors (P value <0.05 was considered significant). All the statistical analyses were conducted using IBM SPSS Statistics V.25.0, R V.3.5.1, and GraphPad Prism Software V.8.0.

Results

Patient characteristics

A total of 82 BC patients who met the inclusion criteria were enrolled in this study. The available characteristics of the patients are set out in Table 1 and Table S1. All the participants were aged between 36 and 88 years (median age: 69 years), and 13 (15.85%) of the patients were female and 69 (84.15%) were male. Among the 82 patients, 41 (50.00%), 4 (4.88%), 12 (14.63%), 20 (24.39%), and 5 (6.10%) patients had clinical stages 0a, I, II, III, and IV, respectively. Among the patients, 33 (40.24%) were smokers/ex-smokers and 49 (59.76%) were non-smokers. Surgery was performed in all patients.

The mutational profile of the BC patients

Multi-gene panel targeted NGS was performed on samples from the 82 BC patients to detect somatic mutations (available online: https://cdn.amegroups.cn/static/public/10.21037tau-23-247-1.xlsx). The average coverage depth for the samples was ×2,000. We then analyzed a total of 82 mutation profiles using NGS detection. The mutational spectra revealed that C > T was the most common base substitution, and SNP and DEL were the common variant types in our cohort (Figure 1A,1B). The variant classification revealed that that the missense mutation was the most common type of mutation, followed by the nonsense nutation, frame-shift deletion, in-frame deletion, frame-shift insertion, and in-frame insertion (Figure 1C). As Figure 1D shows, the top 10 mutant genes were ROS1 (37%), PIK3CA (35%), FGFR3 (34%), BRAF (34%), ERBB2 (32%), ALK (27%), RET (27%), NTRK1 (24%), MET (23%), and EGFR (18%).

Figure 1 Mutation spectrum of the BC patients. (A) Variant classification summary; (B) number of variants; (C) SNV class; (D) mutation spectrum of the BC samples. SNV, single nucleotide variant; BC, bladder cancer; TNP, triple nucleotide polymorphism; SNP, single nucleotide polymorphism; INS, insertion; DNP, double nucleotide polymorphism; DEL, deletion.

Correlations between the FGFR3 mutations and patients’ clinical characteristic

The mutation frequency of FGFR3 in male patients (22/69, 31.88%) was similar to that in female patients (5/13, 38.46%). Patients under the median age (12/40, 30.0%), and elderly patients (≥ median age) (15/42, 35.71%) had similar mutation frequencies. In terms of the clinical stage, FGFR3 mutations were detected more frequently in stage 0a and I (non-muscle-invasive bladder cancer) patients (35.56%, 16/45) than stage II, III, and IV (muscle-invasive bladder cancer) patients (29.73%, 11/37). The proportions were similar in patients with a history of smoking and non-smokers (15/49 smokers, 30.61%; non-smokers 12/33, 36.36%). The top 3 altered types were p.Ser249Cys, p.Tyr375Cys, and p.Arg248Cys (Figure 2A,2B).

Figure 2 FGFR3 mutations in BC patients. (A) The pie charts display the distribution of frequencies and positions of the FGFR3 mutations in BC patients. (B) FGFR3 mutation status of BC patients. FGFR3, fibroblast growth factor receptor 3; BC, bladder cancer.

Correlations between the FGFR3 mutations and patient outcomes

Of the 82 patients, 78 (95.12%; 78/82) had recurrence-free survival data, of whom 34 (41.46%; 34/82) underwent RC and 48 (58.54%; 48/82) underwent transurethral resection with intravesical therapy. We also analyzed the association between FGFR3 mutations and post-treatment RFS. After treatment, no significant difference was observed in the RFS of the FGFR3 mutation patients and the non-mutation patients [P=0.36; hazard ratio (HR) =1.59, 95% confidence interval (CI): 0.74 to 3.38] (Figure 3A). Similarly, there was no RFS difference between the FGFR3 mutation patients and non-mutation patients in the RC cohort (P=0.91; HR =1.44, 95% CI: 0.28 to 7.31) (Figure 3B). Intriguingly, transurethral resection with intravesical therapy appeared to be more effective in patients with FGFR3 mutations (P=0.15; HR =1.87, 95% CI: 0.79 to 4.42) (Figure 3C). In the multivariable analysis, the RFS is worse with age (P=0.016), other factors were no statistical difference (Table S2).

Figure 3 Kaplan-Meier survival curves for the BC patients. (A) RFS of the BC patients (FGFR3 mutations vs. non-mutations); (B) RFS of the BC patients with radical cystectomy (FGFR3 mutations vs. non-mutations); (C) RFS of the BC patients with transurethral resection with intravesical therapy (FGFR3 mutations vs. non-mutations). RFS, relapse-free survival; BC, bladder cancer; FGFR3, fibroblast growth factor receptor 3; WT, wild type.

Discussion

BC is one of the most common malignant tumors of the urinary system. According to global tumor epidemiological statistics, there are about 573,278 new cases worldwide every year (1). The occurrence of BC seriously affects the survival of patients. The typical clinical treatment methods mainly include surgery, chemotherapy, radiation therapy, and biological therapy (6). There are many clinical treatment methods for BC; however, various side effects, such as bladder irritation, occur. In addition, BC has a high recurrence rate, and once it recurs, it is often accompanied by metastasis, and tumor invasiveness and malignancy are also high (19). Thus, research on the prevention and treatment of BC urgently needs to be conducted.

Cappellen et al. were the first to report the FGFR3 mutation in BC (20). Of the 26 BC patients examined by Cappellen et al., 9 had the FGFR3 mutation, and the mutation sites were R248C, S249C, G372C, and K652E. All of these mutations are consistent with germline mutations. Additional mutation sites in BC have also been found in skeletal dysplasia (21). However, the frequency and distribution of FGFR3 mutations differ. Codons 248, 249, and 375 are the most common mutations in BC. Among these mutations, codon 249 mutations account for 70% (14). The mutation frequency of the FGFR3 in BC is about 17.86%, and the FGFR3 mutation is associated with low-grade and low-stage BC and the generation of cysteine residues in the first part of the extracellular domain of the transmembrane region, such as S249C, Y375C, R248C, and G372C (22). This study found that FGFR3 was more frequent in stage 0a and I patients than stage II, III, and IV patients. The top 3 altered types of FGFR3 were p.Ser249Cys, p.Tyr375Cys, and p.Arg248Cys.

Several studies have shown that FGFR3 expression is abnormal in BC patients. Matsumoto et al. reported that 49% of BCs expressed FGFR3 protein at moderate to high levels, but found no correlation between stage and grade (23). In another study, FGFR3 overexpression was associated with low-stage and low-grade tumors (24). Compared to the normal ureter and bladder control group, the FGFR3 protein was overexpressed in the tumor group. Increased FGFR3 protein expression was associated with FGFR3-mutated BCs, and non-invasive BCs had higher mutation frequencies than invasive BCs (16). A higher proportion of FGFR3 protein overexpression was found in invasive tumors without mutations, it may be that the tumor FGFR3 signaling pathway without mutation promotes phenotypic transformation (12).

With the continuous progress of molecular biology research, tumor-targeted therapy has reached a new stage of development. Targeted therapy aims to interfere with tumor-specific signaling pathways, and the targets currently being researched include human epidermal growth factor receptor 2 (HER2), fibroblast EGFR, and immune checkpoints (9). Some targeted therapies have entered clinical trials; for example, clinical trials are being conducted on the use of the EGFR inhibitor gefitinib combined with chemotherapy as a first-line treatment for metastatic BC patients, and on the use of trastuzumab combined with chemotherapy as a treatment for HER2-positive metastatic BC patients (25). Currently, many EGF/EGFR signaling pathway inhibitors are being used in the clinic to treat lung cancer, breast cancer, and other related tumors, which have achieved sound curative effects (26). However, the activation of the FGF/FGFR signaling pathway was observed in many cases of later recurrence (27). The recurrence or metastasis of tumors is closely related to the stemness of the tumor (28), which proves that the FGF/FGFR signaling pathway has a function independent of the EGF/EGFR signaling pathway in tumors and that the function is related to the stemness of the tumor.

FGFR belongs to receptor tyrosine kinase (RTK), and its direct downstream channels include MAPK, PI3K/AKT, ERK, and cross-talk with WNT and other signaling pathways (29). Abnormalities in these pathways can cause tumor occurrence and deterioration, including epithelial-mesenchymal transition, increased tumor cell stemness, and related metabolic changes (29). As the MAPK pathway downstream of RTK is closely related to tumor proliferation and survival, many drugs targeting RTK are currently being used for the clinical treatment of tumors. The inhibition of RTK can effectively kill tumors that depend on the MAPK pathway (30).

Based on these findings, researchers have begun to focus on the FGF/FGFR signaling pathway, and its inhibitors have also entered the clinical testing stage. This research may lead to new treatment options for tumors. FGFR3 is a class of targeted drugs that is being studied and has excellent prospects. Preliminary clinical studies have shown that the short-hairpin RNA-mediated knockout of FGFR3 in FGFR3b mutant cell lines inhibits proliferation, supports independent growth, and reduces colony formation (31). Tumor cells rely solely on FGFR3 mutants for growth to maintain the neoplastic phenotype, which may be a drug-targeted therapy with minimal systemic toxicity (32).

FGFR-targeted inhibitors have also entered an era of rapid development. Some FGFR-targeted inhibitors (such as erdafitinib) have been approved by FDA for clinical use, and many FGFR-targeted inhibitors are in preclinical or clinical trials (33). FGFR targeting is expected to improve the prognosis of tumor patients. Recently, at the American Society of Clinical Oncology (ASCO) 2023 Congress, data from the THOR-2 cohort 3 were reported, showing that erdafitinib was effective in the treatment of adult patients with intermediate-risk non-muscle-invasive bladder cancer carrying FGFR mutations. Some pathogenic mechanisms of FGFR have been confirmed in preclinical models. However, they still need to be verified in clinical trials, and there are more pathogenic mechanisms of FGFR to be discovered.

Nevertheless, this study also has some limitations. Even though we used 82 samples for analysis, the number of BC samples still needs to be increased, which can lead to potential errors/biases. In addition, multiple centers and more extensive sample size data are still required to support it. A clinical study of patients with metastatic urothelial carcinoma treated with the FGFR inhibitor erdafitinib showed higher response rates in patients who had previously received cancer immunotherapy. Bladder cancer patients with FGFR3 mutations were significantly associated with a lower tumor mutation burden (TMB). Future combination therapy strategies may show more responsiveness in the TMB-high tumor patient population (34). The data in this study are unsuitable for analyzing TMB, and the relationship between TMB and FGFR3 mutations still needs to be explored, which is also a limitation of this study.

Conclusions

This study conducted NGS and found that ROS1, PIK3CA, FGFR3, BRAF, ERBB2, and other genes were frequently mutated in BC patients. FGFR3 has a higher mutation frequency in non-muscle-invasive bladder cancer patients. The primary mutation types of FGFR3 were p.Ser249Cys, p.Tyr375Cys, and p.Arg248Cy. It appears that patients with FGFR3 mutation are more likely to benefit from anti-FGFR3 therapy. In addition, we hope that our findings will enable clinical individualization strategies for BC patients to be optimized.

Acknowledgments

The authors are grateful to Shanghai Tongshu Biotechnology Co., Ltd. for its technical support.

Funding: This work was supported by the Project of Science and Technology Bureau of Xiamen (Nos. 3502Z20184033 and 3502Z20194022), the Natural Science Foundation of Fujian Province (No. 2020J011213), and the Young/Middle-aged Talent Cultivation Project that was funded by the Fujian Provincial Health and Family Planning Commission and Xiamen Health and Family Planning Commission (No. 2021GGB028).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-23-247/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-247/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-247/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-23-247/coif). All authors report the technical support from Shanghai Tongshu Biotechnology Co., Ltd. The authors have no other 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. This study was approved by the Ethics Committee of Zhongshan Hospital Xiamen University (No. 2022-181). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Written informed consent was obtained from all the study participants.

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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(English Language Editor: L. Huleatt)