{"paper_id":"1f3eeb31-fd15-4b3b-b32d-7e612491f49c","body_text":"Clinical outcomes and mutation analysis of carbon ion radiotherapy for bladder urothelial carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical outcomes and mutation analysis of carbon ion radiotherapy for bladder urothelial carcinoma Qinleng Zhang, Yihe Zhang, Xiaojun Li, Xin Pan, Wenjun Jin, Yuhan Wang, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6725919/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Carbon–ion radiotherapy (CIRT) improves the survival outcomes in patients with tumor recurrence or metastasis. However, there is little data to support the clinical efficacy of CIRT for bladder cancer. Ten patients underwent CIRT between November 2020 and July 2023 (ChiCTR2100043607). A comprehensive evaluation of the tumor control and toxicity of CIRT was performed, and the clinical characteristics associated with CIRT resistance were analyzed. The median age of the patients was 69 years (range, 32–81) at the start of CIRT, and 90% were male. With a median follow-up of 28 months (range, 18.3–35.6), and 2 patients (20%) developed local recurrence and/or distant metastases. Complete clinical response was achieved in 4 patients (40%), whereas partial response was documented in 4 patients (40%). The 1-year overall survival, progression-free survival, and local progression-free survival rates were 100.0%, 80.0%, 90.0%, respectively. The median prescription dose of CIRT was 70 Gy [relative biological effectiveness (RBE)], ranging from 64Gy (RBE) to 74Gy (RBE). One of the cases harbored mutations associated with CIRT resistance, including ARID1A V1982I, CCND1 S43F, and FANCA C1159S mutations. Moreover, individuals carrying FGFR3 R34Q and PIK3CA E545K mutations showed significant improvement. The acute and late toxicities were grade 2 or lower. No grade III or higher toxicity was observed. CIRT provides favourable tumor control and acceptable toxicity in bladder cancer. Further prospective clinical studies are necessary to use predictive and prognostic biomarkers to evaluate the therapeutic efficacy and adverse events of CIRT in patients with bladder urothelial carcinoma. Muscle invasive bladder cancer Carbon ion radiotherapy Bladder preservation Trimodal therapy Next-Generation Sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bladder cancer (BC) is a heterogeneous disease characterized by genomic instability and a relatively high mutation frequency, and is associated with diverse responses to therapeutic schedules and various clinical outcomes [ 1 , 2 ]. Radical cystectomy remains the gold standard for patients with muscle-invasive bladder cancer (MIBC) [ 3 ]. However, radical cystectomy remains a highly complex procedure with considerably frequent complications, a high degree of risk of perioperative mortality, and changes in quality of life [ 4 ]. Since the 1980s, radiotherapy investigations have focused on organ preservation, usually combining limited resection and chemoradiotherapy [ 5 ]. Bladder preservation has become increasingly important as greater attention is focused on the health-related quality of life and avoiding the potential morbidity of extensive surgery while achieving similar cancer outcomes [ 6 ]. The safety and efficacy of bladder preservation therapy have been supported by a series of studies [ 7 , 8 ]. The National Comprehensive Cancer Network (NCCN) [ 9 ] and the European Association of Urology (EAU) [ 10 ] have recommended concurrent chemoradiotherapy as an effective and potentially curative treatment for MIBC. Adjuvant chemotherapy plus radiotherapy has been associated with increased local control and improved locoregional recurrence–free survival [ 11 , 12 ]. Postoperative radiotherapy has been shown to reduce local recurrence and significantly improve disease-free survival (DFS) [ 13 ]. However, photon radiotherapy for the bladder can cause acute bladder and bowel toxicity in most patients and rarely leads to long-term toxicity, with the most severe cases requiring a cystectomy for symptom alleviation [ 14 ]. High linear energy transfer (LET) carbon ion beams are becoming very promising tools for various cancer treatments and are more efficient than conventional low LET gamma or X-rays in killing malignant or radioresistant cells [ 15 ]. The rationale for the use of high-LET radiation for radiotherapy of cancer has been based on the consideration of a relatively high RBE, highly conformal dose distribution and reduced oxygen enhancement ratio (OER), while simultaneously reducing the volume of normal tissue irradiated [ 16 ]. CIRT has low toxicity when it passes through normal tissue in the entrance channel and can produce excellent cell killing in the tumor target region, compared with conventional photon irradiation [ 17 ]. However, the application of CIRT in bladder cancer has rarely been reported. Carbon beams can reduce the dose delivered to the bowel and normal bladder wall, while increasing the dose to bladder tumors. In this regard, CIRT has obvious advantages in terms of radiation physics and radiation biology compared with conventional X-rays. Therefore, CIRT may further optimize the comprehensive treatment regimen, which is characterized by fewer adverse events and enhanced survival with respect to local control and bladder preservation. Locoregional failure in patients with locally advanced bladder cancer after chemoradiotherapy is associated with high morbidity and mortality. Therefore, predictive biomarkers are urgently needed to support clinical decisions in MIBC. This study also aimed to evaluate the clinical outcomes and adverse events associated with CIRT through long-term follow up. Materials and Methods Patient eligibility A prospective clinical study on the clinical efficacy of CIRT for bladder cancer was designed for patients presenting with MIBC, according to the TNM stage published by the International Union Against Cancer in 2017. Pelvic computed tomography (CT) or magnetic resonance imaging (MRI), ultrasound, chest CT, and bone scintigraphy were performed to determine the clinical stage. Renal function was evaluated based on the creatinine clearance. Patients with poor general status (World Health Organization performance status of 3 or poorer) were excluded from this investigation. Written informed consent was obtained from all patients before the initiation of treatment. This study was reviewed and approved by the ethical committee of the Gansu Wuwei Tumor Hospital and registered at https://www.chictr.org.cn/ (Trial registration number: ChiCTR2100043607). All patients signed an informed consent form before the initiation of CIRT. CIRT treatment plan The therapeutic target area of bladder irradiation was presented in Fig. 1 . Treatment planning CT images were merged with dynamic contrast-enhanced CT or MRI and FDG-PET CT images to define the visible tumor as the gross tumor volume (GTV). The clinical target volume (CTV) of whole-bladder irradiation included all areas of gross and potentially microscopic disease, which consisted of the primary site and the whole pelvic node region. GTV with a safety margin of 2–5 mm external expansion formed planning the target volume (PTV1). For patients who received a local CIRT boost after whole-bladder irradiation, the PTV1 was used for the CIRT boost without expansion to the CTV. The internal target volume (ITV) was generated by an external expansion of 3 mm based on the GTV. The planning target volume (PTV) was expanded by a safety margin of 2–3 mm on the ITV with respect to the carbon ion beam dose distribution and patient position movement, and the 3 mm margins were tailored appropriately if the location of the intestinal tract was close to the bladder. The \"galloping scheme\" was used to control the constant bladder volume and position throughout CIRT [ 18 ]. Tumor response and follow-up Clinical responses on the basis of imaging findings were analyzed using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. The effects of the treatment were evaluated in terms of local control and overall survival. Complete response (CR) constituted the eradication of all treated tumor tissue. Partial response (PR) was defined as a > 30% decrease in tumor size, measured at the longest diameter. Stable disease (SD) ranged from a 30% decrease to a 20% increase in size. Progressive disease (PD) indicated a > 20% increase in tumor size during the post-treatment period. Local control was defined as the absence of local tumor regrowth or recurrence in the treatment volume according to physical examination, imaging and cystoscopy. The emergence of a tumor in the irradiated tumor bed was defined as local recurrence, and cystoscopy biopsy was performed immediately. All patients underwent cystoscopy and urine cytology every 3 months in the first year after CIRT therapy, and every 3–6 months for the following 5 years. The imaging follow-up policy, including contrast-enhanced MRI or CT scans of the pelvic, was scheduled six to eight weeks after treatment and afterwards every three months thereafter within the first year after CIRT for a minimum of two years (Fig. 2 ). Current symptoms and toxicities related to treatment, as well as the patients’weight and Karnofsky Performance Scale score, were recorded by a radiation oncologist at each follow-up visit. Next-Generation Sequencing (NGS) Formalin-fixed paraffin-embedded (FFPE) samples were obtained from the patients before CIRT. DNA was extracted from the FFPE samples using a HiPure FFPE DNA kit. The quantity and quality of the extracted DNA were checked using a Qubit fluorimeter and Tapestation 4200. A total of 50 to 200 ng of fragmented DNA was then subjected to library construction, including end-repair dA-tailing and adapter ligation. Ligated library fragments with appropriate adapters were amplified by PCR. The amplified DNA libraries were further checked using a Bioanalyzer 2100 and samples with sufficient yields were proceeded to hybrid capture. Library capture was conducted using the use of biotin-labelled DNA probes. Briefly, the library was hybridized overnight with a predicted NGS panel and paramagnetic beads. The unbound fragments were washed away, and the enriched fragments were amplified by PCR amplification. Similar to library preparation, the purified product was checked on Bioanalyzer 2100 and then loaded into Illumina NovaSeq 6000 for next-generation sequencing (NGS) with paired-end 2 x 150 bp sequencing kits. The data were analyzed using the predicine in-house developed analysis pipeline, which starts with the raw sequencing data (BCL files) and outputs the final mutation calls. Briefly, the pipeline first performs adapter trimming, barcode checking, and correction. The cleaned paired FASTQ files were aligned to the human reference genome build hg19 using the BWA alignment tool. Candidate variants, consisting of point mutations, small insertions and deletions were identified across the targeted regions covered in the panel. Candidate variants with low base quality, mapping scores, and other quality metrics were filtered out. Candidate variants with an allelic frequency of < 1% were excluded. Criteria for toxicity and statistics Toxicity data were recorded as carefully as possible by inquiring about genitourinary and gastrointestinal symptoms and performing blood and urine tests at each follow-up visit. Within 3 months of the initiation of CIRT, the acute toxicity was graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 of the National Cancer Institute. The late toxicity in normal tissue was graded according to the European Organization for Research and Treatment of Cancer (EORTC) and Radiation Therapy Oncology Group (RTOG) guidelines, starting 3 months after CIRT. The overall survival (OS), progression-free survival (PFS), and local progression-free survival (LPFS) rates were estimated from the initiation of CIRT using the Kaplan– Meier method. Results Patient characteristics Between November 2020 and July 2023, 10 patients who were continuously diagnosed with transitional cell carcinoma of the urinary bladder were considered satisfactory in terms of patient eligibility criteria and consented to undergo CIRT. The baseline demographic data and tumor characteristics of these patients are summarized in Table 1 . The patient population consisted of 9 males and 1 female, and 6 (60%) patients had multiple tumors. The median age of the patients was 69 years (range, 32–81), and 7 (70%) patients were received transurethral resection of bladder tumor (TURBT) before CIRT. The disease stage in these patients was Stage II in 5 patients, III in 3 patients, and IV in 2 patients. According to histopathological diagnosis after cystoscopy biopsy or TURBT, high-grade urothelial carcinoma and low-grade urothelial carcinoma account for 80% and 20% respectively. Among this group of 10 patients, the reasons for the use of CIRT were newly diagnosed bladder cancer on histopathology of the resected specimen in 5 (50%), and clinically evident tumor recurrence with previous intervention on follow-up examination in 5 (50%). Table 1 Demographic information and tumour characteristics of patients treated with CIRT. Abbreviation: CR, complete response; PR, partial response; PD, progressive disease. Patient characteristics (N = 10) Gender Male 9 (90%) Female 1 (10%) Median age, y (range) 69 (32–81) T category, n (%) T2 7 (70%) T3 1 (10%) T4 2 (20%) Pathological type, n (%) Low grade 2 (20%) High grade 8 (80%) Tumor number, n (%) Solitary 4 (40%) Multiple 6 (60%) Median follow-up, month (range) 24 (14–32) Recurrent site, n (%) Bladder 2 (20%) Distant metastases site, n (%) Lung 2 (20%) Bone 1 (10%) Overall tumor response rate complete response 4 (40%) partial response 4 (40%) progressive disease 2 (20%) Antitumour effect Among the 10 patients, the 1-year overall survival, 1-year progression-free survival, and 1-year local progression-free survival rates were 100.0%, 80.0%, 90.0%, respectively (Fig. 3 ). At the last follow-up, the overall tumor response rate was 80% (8 of 10), which was a complete response in 4 (40%) patients and a partial response in 4 (40%) patients (Table 1 ). The overall control rates were presented in Table 2 . The overall control rate for low grade was 100% (2 of 2), which was better than the 75% (6 of 8) for high grade. The overall control rates were 100% (4 of 4) and 66.7% (4 of 6) for patients with solitary tumor patients and multiple tumor patients, respectively. The overall tumor control rates for stages II, III, and IV were 80% (4 of 5), 66.7% (2 of 3), and 100.0% (2 of 2), respectively. Table 2 The overall tumour control rate of bladder cancer patients receiving CIRT. Abbreviation: CIRT, Carbon–ion radiotherapy; RBE, relative biological effectiveness. Overall tumour control well-controlled poor-controlled T category, n (%) T2 6 (85.7%) 1 (14.3%) T3 1 (100.0%) 0 (0.0%) T4 1 (50.0%) 1 (50.0%) Pathological type, n (%) Low grade 2 (100.0%) 0 (0.0%) High grade 6 (75.0%) 2 (25.0%) Tumor number, n (%) Solitary 4 (100.0%) 0 (0.0%) Multiple 4 (66.7%) 2 (33.3%) Prescription dose, Gy(RBE), n (%) 64 1 (100.0%) 0 (0.0%) 68 3 (75.0%) 1 (25.0%) 72 3 (100.0%) 0 (0.0%) 74 1 (50.0%) 1 (50.0%) TNM category, n (%) II 4 (80.0%) 1 (20.0%) III 2 (66.7%) 1 (33.3%) IV 2 (100.0%) 0 (0.0%) Filure therapy During the median follow-up period of 28.0 months (range, 18.3–35.6 months), 2 (20%) distant metastases developed, combined with 1 (10%) patient of regional recurrences in the bladder. Distant metastasis occurred in the lung of 1 patient and in the bone of 1 patient during the 1-year follow-up period. The 2 patients with distant metastasis had high-grade urothelial carcinoma before CIRT, including 1 patient with recurrent tumors after TURBT with GC chemotherapy. Moreover, there were 2 patients with distant metastasis had multiple tumors before CIRT. One patient experienced symptoms of bone pain, which was considered a metastatic lesion by bone scan despite the primary lesion being well controlled. In addition, one patient had lung metastasis during the 1-year follow-up period. None of the patients with local recurrence underwent radical cystectomy because the conservative therapy may be the best choice for patients with recurrence and distant metastasis. Recurrence was not detected in the pelvic lymphatic drainage area. Recommended dose The median total dose of CIRT was 70 Gy (RBE). No Grade 3 or worse adverse effects occurred at any dose level from 64 to 74 Gy (RBE). These results confirmed the safety of the therapeutic doses. The overall tumor control rates for 64, 68, 72, and 74 Gy (RBE) were 100.0%, 75.0%, 100.0%, and 50%, respectively (Table 2 ). Although the local control rate was also 100% with 74 Gy (RBE) (Supplementary data 1), this dose was not considered adequate as the recommended dose because of the occurrence of radiation cystitis at high doses. The recommended dose was actually determined as the minimum dose that led to the greatest local control rate. Therefore, 72 Gy (RBE) was used as the recommended dose based on the tumor control rates. Toxicity There were no treatment-related deaths. None of the patients died within 1 year of starting treatment. The acute and late toxicities that occurred after CIRT are summarized in Table 3 and Table 4 , respectively. All patients completed the scheduled treatment course without interruption of treatment, and no dose-limiting toxicity (DLT) was observed. Within 3 months after the start of treatment (acute toxicity), grade 1 toxicity and grade 2 toxicity in the bladder were observed in 5 (50%) and 2 (20%) of the 10 patients, respectively. Within 3–12 months after the start of treatment (late toxicity), grade 1 toxicity and grade 2 toxicity in the bladder were observed in 1 (10%) and 2 (20%) of the 10 patients, respectively. No other serious adverse events were observed. Table 3 Therapy-related acute toxicity was graded according to the CTCAE version 5.0. Abbreviation: CTCAE, Common Terminology Criteria for Adverse Events. Total dose, Gy (RBE) Genitourinary Gastrointestinal Skin G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 64 1 0 0 0 0 0 0 0 0 0 0 0 68 4 0 0 0 0 0 0 0 0 0 0 0 72 0 1 0 0 0 1 0 0 0 0 0 0 74 0 1 0 0 0 0 0 0 0 0 0 0 Table 4 The late toxicity in normal tissue was graded according to the EORTC and RTOG. Total dose, Gy (RBE) Genitourinary Gastrointestinal G1 G2 G3 G4 G1 G2 G3 G4 64 0 0 0 0 0 0 0 0 68 1 2 0 0 0 0 0 0 72 0 0 0 0 0 0 0 0 74 0 0 0 0 0 0 0 0 Abbreviation: EORTC, European Organization for Research and Treatment of Cancer; RTOG, Radiation Therapy Oncology Group. Mutation analyses To determine the mutation profile characteristics of radioresistant bladder cancer, we performed targeted next generation sequencing of pretreatment tumor samples obtained from 3 patients with CIRT. Among the 3 patients, one developed distant metastasis post-CIRT. The status of whole exome regions of 103 genes and specific exon regions associated with 49 gene variations in bladder cancer was evaluated via gene panel-based sequencing. To assess whether molecular pathological characteristics of tumors affects the CIRT sensitivity, we compared the effects of CIRT treatment in both high-grade and low-grade urothelial carcinoma of the bladder. The multifocal high-grade urothelial carcinoma of the bladder carrying the ARID1A V1982I, CCND1 S43F, and FANCA C1159S mutations were resistant for CIRT (Fig. 4 A, Supplementary table 1 , 2). As anticipated, we also found that the low-grade urothelial carcinoma of the bladder carrying CHEK2 R474H, FGFR3 R34Q, and PDCD1LG2 A73T mutations were sensitive to CIRT (Fig. 4 B, Supplementary table 1 , 2). However, next-generation sequencing revealed that PIK3CA E545K, POLE A503D, POLE E491K, ROS1 P2329T, ROS1 V1831I, and TP53 H214R mutations were sensitive in high-grade MIBC accompanied by squamous differentiation (Supplementary table 1 , 2). Discussion Radical cystectomy with pelvic lymph node dissection remains the gold-standard therapy for patients with MIBC as well as for those with high-risk NMIBC, preceded by cisplatin-based neoadjuvant chemotherapy [ 19 ]. Bladder preservation therapy is an alternative to radical cystectomy for the management of locally advanced bladder cancer. The two largest phase 3, randomized controlled trials of bladder preservation showed benefits in terms of locoregional recurrence free survival (the BC2001 trial [ 20 ]) and overall survival (BCON [ 21 ]) with the use of chemotherapy or hypoxia-modifying therapy concurrent with radiotherapy. However, the treatment outcomes of radiotherapy are unfavourable in the absence of chemotherapy: the 2-year locoregional disease–free survival and 5-year overall survival rates of radiotherapy alone are 54% and 35%, respectively [ 22 ]. In particular, the safety and efficacy of CIRT have been confirmed in recent decades in prospective and large multicenter Japanese studies. Several clinical studies have shown that CIRT provides high local tumor control and confers a great overall survival benefit in many cancer types [ 15 ]. However, there have been few reports on the safety and clinical control of CIRT in patients with bladder cancer. Protocol ChiCTR2100043607 is the first prospective clinical trial of CIRT for bladder cancer. We described the prospective cohort outcomes of 10 patients with MIBC who received CIRT treatment. Our data show that an excellent cancer control rate can be achieved after CIRT, with a 1-year local progression-free survival rate of 90% in all 10 patients. The overall control rate for patients with multiple tumors was 66.7%, which was inferior to the 100% for patients with a solitary tumor. Jenkins et al [ 23 ]. advocated the use of radical radiotherapy with the appropriate use of salvage cystectomy for patients whose tumors were not fully responsive to radiotherapy. Patients who underwent salvage cystectomy had a 5-year survival of 47%, whereas the 65 patients who did not undergo cystectomy had a 5-year survival of 3%. Most importantly, the addition of CIRT was associated with a statistically significant improvement in patients with urothelial carcinoma. CIRT offers robust results in terms of survival for patients with MIBC. The present study adds to the evidence by providing the results of a single-center survey, presenting the largest group of patients treated with this approach reported thus far. In this treatment modality, the tumor response is evaluated through cystoscopic biopsy and urine cytology after CIRT, with a median dose of 64–74 Gy (RBE), and concurrent chemotherapy or immunotherapy of several courses. In this clinical trial, the overall tumor response rate was 80%, and the complete response and partial response rates were 40% and 40% patients, respectively. However, the overall control rates were 75.0% and 100.0% for high-grade and low-grade patients, respectively. Thrasher et al [ 24 ] identified several clinical variables as predictors of cancer-specific survival following radical cystectomy and reported that clinical T stage and tumor grade were the best predictors of survival. Chahal et al [ 25 ] reported that the clinical T stage was an independent predictor of survival. Concurrent chemoradiotherapy can improve the efficacy of radiotherapy at the primary tumor site and effectively control microscopic metastases. The toxicity data obtained in this study are also encouraging, especially in terms of long-term side effects. We have demonstrated that there was no acute or late genitourinary toxicity of grade 3 or higher. Acute genitourinary toxicity is an accepted toxicity in all pelvic chemoradiotherapy regimens for both this and other disease subsites. Moreover, the incidence of long-term complications and late toxicities in both the urinary and intestinal was low. Furthermore, no patients in our study underwent a cystectomy or urinary diversion for complications. After normal tissue responses were compared and observed, the dose was gradually increased to 64–74 Gy (RBE) over 4 weeks, which might be equivalent to or slightly higher than that of photon radiotherapy [ 25 ]. The aim of radiotherapy is not only to provide oncological cure but also to preserve bladder function. Higher radiation doses have been reported to improve local control at the expense of increased toxicity. The low incidence of toxicity and complications could be attributed to the excellent dose distribution of the carbon ion beams. Further complications should be limited so that the requirement for cystectomy, both salvage and palliative is minimized. The recommended dose was actually determined as the minimum dose that led to the greatest local control rate and the least adverse events. According to the clinical trial ChiCTR2100043607, 72 Gy (RBE) was used as the recommended dose. Bladder cancer is considered a rapidly proliferating cancer, with a high α/β ratio of 10 Gy in the linear quadratic model [ 26 ]. In external radiotherapy for MIBC, a concomitant boost technique coupled with a bladder tumor boost with shortening of the overall treatment time provides a high probability of local control with acceptable toxicity [ 27 ]. Despite the short follow-up time, we have confirmed for the first time in a prospective cohort that CIRT is a promising treatment for bladder cancer because of its encouraging outcomes and safety. Furthermore, local recurrence revealed tumors outside of the primary tumor sites. This finding suggests that continued dose escalation to the primary tumor site may not lead to improved local control. Our study revealed that the dose of whole bladder carbon ion irradiation is low, but it can cause more obvious cystitis and fail to prevent recurrence. Therefore, whole bladder radiotherapy will not be continued in subsequent treatment, thereby reducing the incidence of cystitis and improving the quality of life of patients to a greater extent. The major challenges of radiotherapy for bladder cancer are controlling bladder capacity and minimizing bladder motion and reliably explaining the related intestinal and rectal motion. Our research innovatively adopts the \"Galloping Scheme\" to solve the problem of bladder capacity and position changes during radiotherapy for bladder cancer, and to ensure the accuracy of radiotherapy. If these obstacles can be further overcome, organ-preserving and effective combined-modality treatment for selected patients with MIBC as an alternative to radical surgery should be possible in the near future. Radiation resistance has become the major obstacle to reducing tumor recurrence and improving prognosis in the treatment of MIBC. In the present study, we observed that carrying FGFR3 R34Q mutation was sensitive to CIRT in a low-grade urothelial carcinoma of the bladder. Darwis et al [ 28 ]. reported the exon sequence differences of 409 cancer-related genes in the CIRT recurrent tumors compared with the treatment-naïve tumors in the same patient and demonstrated that CIRT-resistant tumors had a significantly higher prevalence of somatic mutations in FGFR3 and FGFR4. In that study, the authors revealed that the pan-FGFR inhibitor LY2874455 enhanced CIRT sensitivity by occupying the ATP-binding pocket in the kinase domains to suppress FGFR signalling. Fisher et al [ 29 ]. reported that a significant tumor growth delay was observed when AZD4547 was combined with radiation compared with radiation or AZD4547 alone in a FGFR-expressing HNSCC cell line xenograft and PDX. The molecular mechanisms underlying the effects in particular types of FGFR3 mutations on MIBC radiotherapy sensitivity remain unclear and require further research. The current study further identified a wide range of both actionable and nonactionable alterations in oncogenes, tumor suppressors, chromatin remodeling and DNA repair genes. One of the ARID1A V1982I gene alteration had been considered an adverse prognostic factor for CIRT in current research on MIBC. ARID1A abnormalities lead to the transcriptional activation of oncogenic driver genes, double-stranded DNA damage repair, tumorigenesis and tumor progression [ 30 ]. Loss of ARID1A resulted in an increased expression and secretion of prolyl 4-hydroxylase beta in lung cancer cells, which induced the activation of lung fibroblasts [ 31 ]. P4HB-activated lung fibroblasts promoted the proliferation, invasion, and chemoresistance in lung cancer cells. However, neutralizing P4HB hampered the tumor growth and increased cisplatin cytotoxic efficacy in PDX models. Integrating tumor molecular genetics and tumor microenvironment can help us better understand the molecular mechanisms of CIRT resistance and enhance radiation sensitivity. Targeted region sequencing results revealed the CHEK2 R474H mutations in low-grade tumors was sensitive to CIRT. Andreas et al [ 32 ]. demonstrated that patients who received postoperative radiotherapy following breast-conservative surgery and who carried the CHEK2 mutation had a less favourable outcome in terms of local recurrence, distant metastasis-free survival and overall survival at 7 years. De Bock et al [ 33 ]. reported that patients with the CHEK2 variant had a more unfavourable prognosis regarding the occurrence of contralateral breast cancer, and disease-free survival. Compared with our analysis, our data also indicate that the clinical relevance of CHEK2 gene mutations may not be restricted to the 1100delC allele but may extend to other mutations conferring CHEK2 dysfunction, such as the R474H substitution. To our knowledge, this is the first report on the clinical efficacy of associated CIRT in a consecutive series of unselected patients with bladder cancer. Although our study has substantial strengths, its potential limitations need to be considered. Our study reflects the experience of a single institution, and the relatively short follow-up period. Bladder cancer has distinct molecular subtypes with multiple pathogenic pathways [ 34 ]. Comprehensive genomic analyses of bladder cancer provide detailed molecular characterization of genetic and epigenetic alterations, and facilitate the understanding of the potential impact on diagnosis, treatment selection and follow-up plans. Although we performed a small number of genetic analyses on our CIRT patient cohort, we could not determine the definite mechanism of CIRT resistance. However, if the genetic changes associated with unfavourable prognosis for patients in the article are also independently confirmed in other larger cohorts of CIRT patients, particular mutation status may become a pivotal determinant when considering treatment selection, intensive observation and recurrence prevention on the basis of the intrinsic biology of the tumor. Consequently, our observations warrant further consideration and validation in a larger sample size, which may provide more clinical evidence to clarify the resistance mechanism of MIBC to CIRT. Despite these limitations, these results provide good evidence that CIRT is feasible and well tolerated for MIBC. These regimens have many clinical and practical advantages for patients. Given that there is no internationally agreed upon standard CIRT regimen for MIBC and bladder preservation strategies are increasingly being used in the oncological community. It is a necessary to conduct prospective trials to explore the application of CIRT in locally advanced or metastatic disease in order to establish safe and effective treatment schedules. Therefore, CIRT is a topic of the prospective randomized controlled trials for bladder preservation that should be conducted in the future. Declarations Author contribution Qinleng Zhang and Yihe Zhang contributed equally conceived research ideas, designed analysis methodology, and collected and sorted out raw data. Xiaojun Li, Xin Pan, Yuling Yang and Xin Wang wrote the initial draft. Xiaofeng Shi, Wenjun Jin and Yuhan Wang prepared the figure. Pengfei Sun, Zhilong Dong, Yizhao Zhang and Chengzong Zhao contributed to the analysis of the results. Yancheng Ye, Yanshan Zhang and Zhiping Wang contributed equally supervised the research and provided financial support for the project. All authors contributed to the article and approved the submitted version. Funding This work was supported by the Natural Science Foundation of Gansu Province under Grant No.23JRRH0006. Data availability This study was a systematic review and analysis of existing clinical studies and thus no new data are available for sharing. Ethics declarations Institution and ethics approval and informed consent This study was reviewed and approved by the ethical committee of the Gansu Wuwei Tumor Hospital (approval no. 2020-Ethical Review-12) and registered at https://www.chictr.org.cn/ (Trial registration number: ChiCTR2100043607). All patients signed an informed consent form before the initiation of CIRT. Conflict of interest The authors have no competing interests to declare that are relevant to the content of this article. 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Eur Urol 85:17–31. https://doi.org/10.1016/j.eururo.2023.08.016 Caffo O, Thompson C, De Santis M, Kragelj B, Hamstra DA, Azria D et al (2016) Concurrent gemcitabine and radiotherapy for the treatment of muscle-invasive bladder cancer: a pooled individual data analysis of eight phase i–II trials. Radiother Oncol 121:193–198. https://doi.org/10.1016/j.radonc.2016.09.006 Zaghloul MS, Christodouleas JP, Smith A, Abdallah A, William H, Khaled HM et al (2018) Adjuvant sandwich chemotherapy plus radiotherapy vs adjuvant chemotherapy alone for locally advanced bladder cancer after radical cystectomy. JAMA Surg 153:e174591. https://doi.org/10.1001/jamasurg.2017.4591 Zaghloul MS, Awwad HK, Akoush HH, Omar S, Soliman O, El AI (1992) Postoperative radiotherapy of carcinoma in bilharzial bladder: improved disease free survival through improving local control. Int J Radiat Oncol Biol Phys 23:511–517. https://doi.org/10.1016/0360-3016(92)90005-3 Qiao B, Kerr M, Groselj B, Teo MTW, Knowles MA, Bristow RG et al (2013) Imatinib radiosensitizes bladder cancer by targeting homologous recombination. Cancer Res 73:1611–1620. https://doi.org/10.1158/0008-5472.CAN-12-1170 Kamada T, Tsujii H, Blakely EA, Debus J, De Neve W, Durante M et al (2015) Carbon ion radiotherapy in japan: an assessment of 20 years of clinical experience. Lancet Oncol 16:e93–e100. https://doi.org/10.1016/S1470-2045(14)70412-7 Frese MCPD, Yu VKMS, Stewart RDPD, Carlson DJPD (2012) A mechanism-based approach to predict the relative biological effectiveness of protons and carbon ions in radiation therapy. Int J Radiat Oncol Biol Phys 83:442–450. https://doi.org/10.1016/j.ijrobp.2011.06.1983 Durante M, Debus J, Loeffler JS (2021) Physics and biomedical challenges of cancer therapy with accelerated heavy ions. Nat Rev Phys 3:777–790. https://doi.org/10.1038/s42254-021-00368-5 Zhang Y, Li X, Zhang Y, Hu T, Chen W, Pan X et al (2021) Carbon ion radiotherapy for bladder cancer: a case report. World J Clin Cases 9:7833–7839. https://doi.org/10.12998/wjcc.v9.i26.7833 Lopez-Beltran A, Cookson MS, Guercio BJ, Cheng L (2024) Advances in diagnosis and treatment of bladder cancer. BMJ (Online) 384:e076743. https://doi.org/10.1136/bmj-2023-076743 Hall E, Hussain SA, Porta N, Lewis R, Crundwell M, Jenkins P et al (2022) Chemoradiotherapy in muscle-invasive bladder cancer: 10-yr follow-up of the phase 3 randomised controlled BC2001 trial. Eur Urol 82:273–279. https://doi.org/https://doi.org/10.1016/j.eururo.2022.04.017 Hoskin PJ, Rojas AM, Bentzen SM, Saunders MI (2010) Radiotherapy with concurrent carbogen and nicotinamide in bladder carcinoma. J Clin Oncol 28:4912–4918. https://doi.org/10.1200/JCO.2010.28.4950 James ND, Hussain SA, Hall E, Jenkins P, Tremlett J, Rawlings C et al (2012) Radiotherapy with or without chemotherapy in muscle-invasive bladder cancer. N Engl J Med 366:1477–1488. https://doi.org/10.1056/NEJMoa1106106 Jenkins BJ, Caulfield MJ, Fowler CG, Badenoch DF, Tiptaft RC, Paris AM et al (1988) Reappraisal of the role of radical radiotherapy and salvage cystectomy in the treatment of invasive (t2/t3) bladder cancer. Br J Urol 62:343–346. https://doi.org/10.1111/j.1464-410x.1988.tb04362.x Thrasher JB, Frazier HA, Robertson JE, Dodge RK, Paulson DF (1994) Clinical variables which serve as predictors of cancer-specific survival among patients treated with radical cystectomy for transitional cell carcinoma of the bladder and prostate. Cancer 73:1708–1715. https://doi.org/10.1002/1097-0142(19940315)73:6<1708::AID-CNCR2820730626>3.0.CO;2-J Chahal R, Sundaram SK, Iddenden R, Forman DF, Weston PMT, Harrison SCW (2003) A study of the morbidity, mortality and long-term survival following radical cystectomy and radical radiotherapy in the treatment of invasive bladder cancer in yorkshire. Eur Urol 43:246–257. https://doi.org/10.1016/S0302-2838(02)00581-X Pos FJ, Hart G, Schneider C, Sminia P (2006) Radical radiotherapy for invasive bladder cancer: what dose and fractionation schedule to choose? Int J Radiation Oncology*Biology*Physics 64:1168–1173. https://doi.org/10.1016/j.ijrobp.2005.09.023 Pos FJ, van Tienhoven G, Hulshof MCCM, Koedooder K, González González D (2003) Concomitant boost radiotherapy for muscle invasive bladder cancer. Radiother Oncol 68:75–80. https://doi.org/https://doi.org/10.1016/S0167-8140(03)00019-7 Darwis NDM, Nachankar A, Sasaki Y, Matsui T, Noda S, Murata K et al (2019) FGFR signaling as a candidate therapeutic target for cancers resistant to carbon ion radiotherapy. Int J Mol Sci 20:4563. https://doi.org/10.3390/ijms20184563 Fisher MM, Senthilkumar G, Hu R, Goldstein S, Ong IM, Miller MC et al (2020) Fibroblast growth factor receptors as targets for radiosensitization in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys 107:793–803. https://doi.org/10.1016/j.ijrobp.2020.03.040 Jana S, Brahma S, Arora S, Wladyka CL, Hoang P, Blinka S et al (2023) Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression. Cancer Cell 41:853–870. https://doi.org/https://doi.org/10.1016/j.ccell.2023.03.021 Huang R, Wu D, Zhang K, Hu G, Liu Y, Jiang Y et al (2024) ARID1a loss inducesp4HB to activate fibroblasts to support lung cancer cell growth, invasion, and chemoresistance. Cancer Sci 115:439–451. https://doi.org/10.1111/cas.16052 Meyer A, Dörk T, Sohn C, Karstens JH, Bremer M (2007) Breast cancer in patients carrying a germ-line CHEK2 mutation: outcome after breast conserving surgery and adjuvant radiotherapy. Radiother Oncol 82:349–353. https://doi.org/10.1016/j.radonc.2006.12.002 de Bock GH, Schutte M, Krol-Warmerdam EMM, Seynaeve C, Blom J, Brekelmans CTM et al (2004) Tumour characteristics and prognosis of breast cancer patients carrying the germline CHEK2*1100delc variant. J Med Genet 41:731–735. https://doi.org/10.1136/jmg.2004.019737 Lopez-Beltran A, Cookson MS, Guercio BJ, Cheng L (2024) Advances in diagnosis and treatment of bladder cancer. https://doi.org/10.1136/bmj-2023-076743 . BMJ:e076743 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.xlsx SupplementaryTable2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6725919\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":481769435,\"identity\":\"48221ffa-30cd-4e26-831c-7fd186d24aa3\",\"order_by\":0,\"name\":\"Qinleng Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Lanzhou University Second Hospital, Gansu Province Clinical Research Center for urinary system 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Ye\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Radiotherapy, Lanzhou University Second Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yancheng\",\"middleName\":\"\",\"lastName\":\"Ye\",\"suffix\":\"\"},{\"id\":481769449,\"identity\":\"5e6b6491-a4f5-4d63-af2b-d2fbf9e1afde\",\"order_by\":14,\"name\":\"Yanshan Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Radiation Oncology, Heavy Ion Center, Gansu Wuwei Tumour Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yanshan\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":481769450,\"identity\":\"5e2731fe-8cda-40cc-998e-85f90b7c303b\",\"order_by\":15,\"name\":\"Zhiping Wang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYPACCwYG9sbGhx9I0CLBwMBzuNlYgjQtEultAjzEqDU4fvbwy587JOwNbj5sA+q0k9NtIKTlTF6aheQZCWaD24ltDwoYko3NDhDQYnYgx8zAsE2CDail3UCC4UDiNoJazr8xM0hsk+AxuHkQSBKl5UaO8QOgYgmDG4xEarG/8caMsfGMhIHkmURgIBsQ4RfJ/hzjjz932NjzHT/+8OGHCjs5glqAgE2CsQHGNiCsHASYPyC0jIJRMApGwSjAAgCzdENoyAsAKAAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Lanzhou University Second Hospital, Gansu Province Clinical Research Center for urinary system disease\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Zhiping\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-05-22 14:23:19\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6725919/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6725919/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":86421936,\"identity\":\"f7de616d-679e-41ea-a956-fb35ce8f51ab\",\"added_by\":\"auto\",\"created_at\":\"2025-07-10 12:54:00\",\"extension\":\"jpeg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":183787,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIsodose distribution of the carbon ion beam in a patient with high-grade bladder urothelial carcinoma.\\u003c/p\\u003e\\n\\u003cp\\u003eIsodose distributions of the carbon ion beam in the transverse (A), coronal (B), and sagittal (C) at the entrance of the left ureter bladder. The target area was irradiated with a total dose of 72 Gy (RBE)/18 Fx.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/c8db586cd7151a3e9fbac183.jpeg\"},{\"id\":86420438,\"identity\":\"a477c4db-4631-4b5c-b12c-acec0f6d5ad0\",\"added_by\":\"auto\",\"created_at\":\"2025-07-10 12:46:00\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":83137,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePelvic MRI images throughout the disease course after CIRT treatment.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) T2-weighted image before CIRT; (B) T2-weighted images for 1 day after CIRT; (C) T2-weighted images for 11 months after CIRT.\\u003c/p\\u003e\\n\\u003cp\\u003eAbbreviation: CIRT, Carbon–ion radiotherapy.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/57cd79c3f5ea44a7011dbf71.jpeg\"},{\"id\":86420436,\"identity\":\"57019cf0-7185-4b9c-a4e5-2d2b5d2221ad\",\"added_by\":\"auto\",\"created_at\":\"2025-07-10 12:46:00\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":37923,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eKaplan–Meier estimates of OS, PFS, and LPFS for all 10 patients in one year.\\u003c/p\\u003e\\n\\u003cp\\u003eAbbreviations: OS, overall survival; PFS, progression-free survival; LPFS, local progression-free survival.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/a480e36c2ef5daeb413033f5.png\"},{\"id\":86421937,\"identity\":\"0c0ce598-4278-414d-97b8-dbb76efe5a3c\",\"added_by\":\"auto\",\"created_at\":\"2025-07-10 12:54:00\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":215313,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSubtypes of germline variants identified by NGS before receiving CIRT treatment.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) One patient with CIRT resistance carried ARID1A V1982I mutation; (B) One patient with CIRT sensitivity carried FGFR3 R34Q mutation.\\u003c/p\\u003e\\n\\u003cp\\u003eAbbreviation: CIRT, Carbon–ion radiotherapy.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/e964114fdbfd70747a992b7d.png\"},{\"id\":86816069,\"identity\":\"0b5e0276-5ae8-4909-b978-be82e47b75ee\",\"added_by\":\"auto\",\"created_at\":\"2025-07-16 00:31:20\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1218231,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/7ac2c655-26e0-44a5-9412-482a41cab02c.pdf\"},{\"id\":86424002,\"identity\":\"fbd4882d-94d4-4fb8-baff-bd2938d13fe2\",\"added_by\":\"auto\",\"created_at\":\"2025-07-10 13:10:00\",\"extension\":\"xlsx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":12118,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable1.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/97f61c718ceae7168bf9ad90.xlsx\"},{\"id\":86424003,\"identity\":\"79b74569-727f-456d-958e-e93e9e214366\",\"added_by\":\"auto\",\"created_at\":\"2025-07-10 13:10:01\",\"extension\":\"xlsx\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":13877,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable2.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6725919/v1/1008cb7017710551327b30a9.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Clinical outcomes and mutation analysis of carbon ion radiotherapy for bladder urothelial carcinoma\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eBladder cancer (BC) is a heterogeneous disease characterized by genomic instability and a relatively high mutation frequency, and is associated with diverse responses to therapeutic schedules and various clinical outcomes [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Radical cystectomy remains the gold standard for patients with muscle-invasive bladder cancer (MIBC) [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. However, radical cystectomy remains a highly complex procedure with considerably frequent complications, a high degree of risk of perioperative mortality, and changes in quality of life [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Since the 1980s, radiotherapy investigations have focused on organ preservation, usually combining limited resection and chemoradiotherapy [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Bladder preservation has become increasingly important as greater attention is focused on the health-related quality of life and avoiding the potential morbidity of extensive surgery while achieving similar cancer outcomes [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. The safety and efficacy of bladder preservation therapy have been supported by a series of studies [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. The National Comprehensive Cancer Network (NCCN) [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] and the European Association of Urology (EAU) [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e] have recommended concurrent chemoradiotherapy as an effective and potentially curative treatment for MIBC.\\u003c/p\\u003e\\u003cp\\u003eAdjuvant chemotherapy plus radiotherapy has been associated with increased local control and improved locoregional recurrence\\u0026ndash;free survival [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Postoperative radiotherapy has been shown to reduce local recurrence and significantly improve disease-free survival (DFS) [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. However, photon radiotherapy for the bladder can cause acute bladder and bowel toxicity in most patients and rarely leads to long-term toxicity, with the most severe cases requiring a cystectomy for symptom alleviation [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. High linear energy transfer (LET) carbon ion beams are becoming very promising tools for various cancer treatments and are more efficient than conventional low LET gamma or X-rays in killing malignant or radioresistant cells [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. The rationale for the use of high-LET radiation for radiotherapy of cancer has been based on the consideration of a relatively high RBE, highly conformal dose distribution and reduced oxygen enhancement ratio (OER), while simultaneously reducing the volume of normal tissue irradiated [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. CIRT has low toxicity when it passes through normal tissue in the entrance channel and can produce excellent cell killing in the tumor target region, compared with conventional photon irradiation [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eHowever, the application of CIRT in bladder cancer has rarely been reported. Carbon beams can reduce the dose delivered to the bowel and normal bladder wall, while increasing the dose to bladder tumors. In this regard, CIRT has obvious advantages in terms of radiation physics and radiation biology compared with conventional X-rays. Therefore, CIRT may further optimize the comprehensive treatment regimen, which is characterized by fewer adverse events and enhanced survival with respect to local control and bladder preservation. Locoregional failure in patients with locally advanced bladder cancer after chemoradiotherapy is associated with high morbidity and mortality. Therefore, predictive biomarkers are urgently needed to support clinical decisions in MIBC. This study also aimed to evaluate the clinical outcomes and adverse events associated with CIRT through long-term follow up.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003ePatient eligibility\\u003c/p\\u003e\\u003cp\\u003e A prospective clinical study on the clinical efficacy of CIRT for bladder cancer was designed for patients presenting with MIBC, according to the TNM stage published by the International Union Against Cancer in 2017. Pelvic computed tomography (CT) or magnetic resonance imaging (MRI), ultrasound, chest CT, and bone scintigraphy were performed to determine the clinical stage. Renal function was evaluated based on the creatinine clearance. Patients with poor general status (World Health Organization performance status of 3 or poorer) were excluded from this investigation. Written informed consent was obtained from all patients before the initiation of treatment. This study was reviewed and approved by the ethical committee of the Gansu Wuwei Tumor Hospital and registered at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.chictr.org.cn/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.chictr.org.cn/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e (Trial registration number: ChiCTR2100043607). All patients signed an informed consent form before the initiation of CIRT.\\u003c/p\\u003e\\u003cp\\u003eCIRT treatment plan\\u003c/p\\u003e\\u003cp\\u003eThe therapeutic target area of bladder irradiation was presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. Treatment planning CT images were merged with dynamic contrast-enhanced CT or MRI and FDG-PET CT images to define the visible tumor as the gross tumor volume (GTV). The clinical target volume (CTV) of whole-bladder irradiation included all areas of gross and potentially microscopic disease, which consisted of the primary site and the whole pelvic node region. GTV with a safety margin of 2\\u0026ndash;5 mm external expansion formed planning the target volume (PTV1). For patients who received a local CIRT boost after whole-bladder irradiation, the PTV1 was used for the CIRT boost without expansion to the CTV. The internal target volume (ITV) was generated by an external expansion of 3 mm based on the GTV. The planning target volume (PTV) was expanded by a safety margin of 2\\u0026ndash;3 mm on the ITV with respect to the carbon ion beam dose distribution and patient position movement, and the 3 mm margins were tailored appropriately if the location of the intestinal tract was close to the bladder. The \\\"galloping scheme\\\" was used to control the constant bladder volume and position throughout CIRT [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eTumor response and follow-up\\u003c/p\\u003e\\u003cp\\u003eClinical responses on the basis of imaging findings were analyzed using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. The effects of the treatment were evaluated in terms of local control and overall survival. Complete response (CR) constituted the eradication of all treated tumor tissue. Partial response (PR) was defined as a\\u0026thinsp;\\u0026gt;\\u0026thinsp;30% decrease in tumor size, measured at the longest diameter. Stable disease (SD) ranged from a 30% decrease to a 20% increase in size. Progressive disease (PD) indicated a\\u0026thinsp;\\u0026gt;\\u0026thinsp;20% increase in tumor size during the post-treatment period. Local control was defined as the absence of local tumor regrowth or recurrence in the treatment volume according to physical examination, imaging and cystoscopy. The emergence of a tumor in the irradiated tumor bed was defined as local recurrence, and cystoscopy biopsy was performed immediately.\\u003c/p\\u003e\\u003cp\\u003eAll patients underwent cystoscopy and urine cytology every 3 months in the first year after CIRT therapy, and every 3\\u0026ndash;6 months for the following 5 years. The imaging follow-up policy, including contrast-enhanced MRI or CT scans of the pelvic, was scheduled six to eight weeks after treatment and afterwards every three months thereafter within the first year after CIRT for a minimum of two years (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Current symptoms and toxicities related to treatment, as well as the patients\\u0026rsquo;weight and Karnofsky Performance Scale score, were recorded by a radiation oncologist at each follow-up visit.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eNext-Generation Sequencing (NGS)\\u003c/p\\u003e\\u003cp\\u003eFormalin-fixed paraffin-embedded (FFPE) samples were obtained from the patients before CIRT. DNA was extracted from the FFPE samples using a HiPure FFPE DNA kit. The quantity and quality of the extracted DNA were checked using a Qubit fluorimeter and Tapestation 4200. A total of 50 to 200 ng of fragmented DNA was then subjected to library construction, including end-repair dA-tailing and adapter ligation. Ligated library fragments with appropriate adapters were amplified by PCR. The amplified DNA libraries were further checked using a Bioanalyzer 2100 and samples with sufficient yields were proceeded to hybrid capture.\\u003c/p\\u003e\\u003cp\\u003eLibrary capture was conducted using the use of biotin-labelled DNA probes. Briefly, the library was hybridized overnight with a predicted NGS panel and paramagnetic beads. The unbound fragments were washed away, and the enriched fragments were amplified by PCR amplification. Similar to library preparation, the purified product was checked on Bioanalyzer 2100 and then loaded into Illumina NovaSeq 6000 for next-generation sequencing (NGS) with paired-end 2 x 150 bp sequencing kits.\\u003c/p\\u003e\\u003cp\\u003eThe data were analyzed using the predicine in-house developed analysis pipeline, which starts with the raw sequencing data (BCL files) and outputs the final mutation calls. Briefly, the pipeline first performs adapter trimming, barcode checking, and correction. The cleaned paired FASTQ files were aligned to the human reference genome build hg19 using the BWA alignment tool. Candidate variants, consisting of point mutations, small insertions and deletions were identified across the targeted regions covered in the panel. Candidate variants with low base quality, mapping scores, and other quality metrics were filtered out. Candidate variants with an allelic frequency of \\u0026lt;\\u0026thinsp;1% were excluded.\\u003c/p\\u003e\\u003cp\\u003eCriteria for toxicity and statistics\\u003c/p\\u003e\\u003cp\\u003eToxicity data were recorded as carefully as possible by inquiring about genitourinary and gastrointestinal symptoms and performing blood and urine tests at each follow-up visit. Within 3 months of the initiation of CIRT, the acute toxicity was graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 of the National Cancer Institute. The late toxicity in normal tissue was graded according to the European Organization for Research and Treatment of Cancer (EORTC) and Radiation Therapy Oncology Group (RTOG) guidelines, starting 3 months after CIRT. The overall survival (OS), progression-free survival (PFS), and local progression-free survival (LPFS) rates were estimated from the initiation of CIRT using the Kaplan\\u0026ndash; Meier method.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003ePatient characteristics\\u003c/p\\u003e\\u003cp\\u003eBetween November 2020 and July 2023, 10 patients who were continuously diagnosed with transitional cell carcinoma of the urinary bladder were considered satisfactory in terms of patient eligibility criteria and consented to undergo CIRT. The baseline demographic data and tumor characteristics of these patients are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. The patient population consisted of 9 males and 1 female, and 6 (60%) patients had multiple tumors. The median age of the patients was 69 years (range, 32\\u0026ndash;81), and 7 (70%) patients were received transurethral resection of bladder tumor (TURBT) before CIRT. The disease stage in these patients was Stage II in 5 patients, III in 3 patients, and IV in 2 patients. According to histopathological diagnosis after cystoscopy biopsy or TURBT, high-grade urothelial carcinoma and low-grade urothelial carcinoma account for 80% and 20% respectively. Among this group of 10 patients, the reasons for the use of CIRT were newly diagnosed bladder cancer on histopathology of the resected specimen in 5 (50%), and clinically evident tumor recurrence with previous intervention on follow-up examination in 5 (50%).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eDemographic information and tumour characteristics of patients treated with CIRT. Abbreviation: CR, complete response; PR, partial response; PD, progressive disease.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"2\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePatient characteristics (N\\u0026thinsp;=\\u0026thinsp;10)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGender\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMale\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e9 (90%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eFemale\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (10%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMedian age, y (range)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e69 (32\\u0026ndash;81)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT category, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e7 (70%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT3\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (10%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (20%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePathological type, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eLow grade\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (20%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eHigh grade\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e8 (80%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTumor number, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eSolitary\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4 (40%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMultiple\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e6 (60%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMedian follow-up, month (range)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e24 (14\\u0026ndash;32)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eRecurrent site, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBladder\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (20%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eDistant metastases site, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eLung\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (20%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBone\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (10%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOverall tumor response rate\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ecomplete response\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4 (40%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003epartial response\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4 (40%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eprogressive disease\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (20%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eAntitumour effect\\u003c/p\\u003e\\u003cp\\u003eAmong the 10 patients, the 1-year overall survival, 1-year progression-free survival, and 1-year local progression-free survival rates were 100.0%, 80.0%, 90.0%, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). At the last follow-up, the overall tumor response rate was 80% (8 of 10), which was a complete response in 4 (40%) patients and a partial response in 4 (40%) patients (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The overall control rates were presented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The overall control rate for low grade was 100% (2 of 2), which was better than the 75% (6 of 8) for high grade. The overall control rates were 100% (4 of 4) and 66.7% (4 of 6) for patients with solitary tumor patients and multiple tumor patients, respectively. The overall tumor control rates for stages II, III, and IV were 80% (4 of 5), 66.7% (2 of 3), and 100.0% (2 of 2), respectively.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eThe overall tumour control rate of bladder cancer patients receiving CIRT. Abbreviation: CIRT, Carbon\\u0026ndash;ion radiotherapy; RBE, relative biological effectiveness.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"3\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOverall tumour control\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003ewell-controlled\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003epoor-controlled\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT category, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e6 (85.7%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (14.3%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT3\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (100.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0 (0.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eT4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (50.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (50.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePathological type, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eLow grade\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (100.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0 (0.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eHigh grade\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e6 (75.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2 (25.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTumor number, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eSolitary\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4 (100.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0 (0.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMultiple\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4 (66.7%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2 (33.3%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePrescription dose, Gy(RBE), n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e64\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (100.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0 (0.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e68\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e3 (75.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (25.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e72\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e3 (100.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0 (0.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e74\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 (50.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (50.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTNM category, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eII\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4 (80.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (20.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eIII\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (66.7%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (33.3%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eIV\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2 (100.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0 (0.0%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eFilure therapy\\u003c/p\\u003e\\u003cp\\u003eDuring the median follow-up period of 28.0 months (range, 18.3\\u0026ndash;35.6 months), 2 (20%) distant metastases developed, combined with 1 (10%) patient of regional recurrences in the bladder. Distant metastasis occurred in the lung of 1 patient and in the bone of 1 patient during the 1-year follow-up period. The 2 patients with distant metastasis had high-grade urothelial carcinoma before CIRT, including 1 patient with recurrent tumors after TURBT with GC chemotherapy. Moreover, there were 2 patients with distant metastasis had multiple tumors before CIRT. One patient experienced symptoms of bone pain, which was considered a metastatic lesion by bone scan despite the primary lesion being well controlled. In addition, one patient had lung metastasis during the 1-year follow-up period. None of the patients with local recurrence underwent radical cystectomy because the conservative therapy may be the best choice for patients with recurrence and distant metastasis. Recurrence was not detected in the pelvic lymphatic drainage area.\\u003c/p\\u003e\\u003cp\\u003eRecommended dose\\u003c/p\\u003e\\u003cp\\u003eThe median total dose of CIRT was 70 Gy (RBE). No Grade 3 or worse adverse effects occurred at any dose level from 64 to 74 Gy (RBE). These results confirmed the safety of the therapeutic doses. The overall tumor control rates for 64, 68, 72, and 74 Gy (RBE) were 100.0%, 75.0%, 100.0%, and 50%, respectively (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Although the local control rate was also 100% with 74 Gy (RBE) (Supplementary data 1), this dose was not considered adequate as the recommended dose because of the occurrence of radiation cystitis at high doses. The recommended dose was actually determined as the minimum dose that led to the greatest local control rate. Therefore, 72 Gy (RBE) was used as the recommended dose based on the tumor control rates.\\u003c/p\\u003e\\u003cp\\u003eToxicity\\u003c/p\\u003e\\u003cp\\u003eThere were no treatment-related deaths. None of the patients died within 1 year of starting treatment. The acute and late toxicities that occurred after CIRT are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, respectively. All patients completed the scheduled treatment course without interruption of treatment, and no dose-limiting toxicity (DLT) was observed. Within 3 months after the start of treatment (acute toxicity), grade 1 toxicity and grade 2 toxicity in the bladder were observed in 5 (50%) and 2 (20%) of the 10 patients, respectively. Within 3\\u0026ndash;12 months after the start of treatment (late toxicity), grade 1 toxicity and grade 2 toxicity in the bladder were observed in 1 (10%) and 2 (20%) of the 10 patients, respectively. No other serious adverse events were observed.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eTherapy-related acute toxicity was graded according to the CTCAE version 5.0. Abbreviation: CTCAE, Common Terminology Criteria for Adverse Events.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"13\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c10\\\" colnum=\\\"10\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c11\\\" colnum=\\\"11\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c12\\\" colnum=\\\"12\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c13\\\" colnum=\\\"13\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eTotal dose,\\u003c/p\\u003e\\u003cp\\u003eGy 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align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c11\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c12\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c13\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e68\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c11\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c12\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c13\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e72\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c11\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c12\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c13\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e74\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c11\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c12\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c13\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eThe late toxicity in normal tissue was graded according to the EORTC and RTOG.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"9\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eTotal dose,\\u003c/p\\u003e\\u003cp\\u003eGy (RBE)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c5\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eGenitourinary\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c9\\\" namest=\\\"c6\\\"\\u003e\\u003cp\\u003eGastrointestinal\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eG1\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eG2\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eG3\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eG4\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eG1\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eG2\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003eG3\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003eG4\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e64\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e68\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e72\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e74\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003ctfoot\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"9\\\"\\u003eAbbreviation: EORTC, European Organization for Research and Treatment of Cancer; RTOG, Radiation Therapy Oncology Group.\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tfoot\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eMutation analyses\\u003c/p\\u003e\\u003cp\\u003eTo determine the mutation profile characteristics of radioresistant bladder cancer, we performed targeted next generation sequencing of pretreatment tumor samples obtained from 3 patients with CIRT. Among the 3 patients, one developed distant metastasis post-CIRT. The status of whole exome regions of 103 genes and specific exon regions associated with 49 gene variations in bladder cancer was evaluated via gene panel-based sequencing.\\u003c/p\\u003e\\u003cp\\u003eTo assess whether molecular pathological characteristics of tumors affects the CIRT sensitivity, we compared the effects of CIRT treatment in both high-grade and low-grade urothelial carcinoma of the bladder. The multifocal high-grade urothelial carcinoma of the bladder carrying the ARID1A V1982I, CCND1 S43F, and FANCA C1159S mutations were resistant for CIRT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, Supplementary table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, 2). As anticipated, we also found that the low-grade urothelial carcinoma of the bladder carrying CHEK2 R474H, FGFR3 R34Q, and PDCD1LG2 A73T mutations were sensitive to CIRT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB, Supplementary table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, 2). However, next-generation sequencing revealed that PIK3CA E545K, POLE A503D, POLE E491K, ROS1 P2329T, ROS1 V1831I, and TP53 H214R mutations were sensitive in high-grade MIBC accompanied by squamous differentiation (Supplementary table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, 2).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eRadical cystectomy with pelvic lymph node dissection remains the gold-standard therapy for patients with MIBC as well as for those with high-risk NMIBC, preceded by cisplatin-based neoadjuvant chemotherapy [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Bladder preservation therapy is an alternative to radical cystectomy for the management of locally advanced bladder cancer. The two largest phase 3, randomized controlled trials of bladder preservation showed benefits in terms of locoregional recurrence free survival (the BC2001 trial [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]) and overall survival (BCON [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]) with the use of chemotherapy or hypoxia-modifying therapy concurrent with radiotherapy. However, the treatment outcomes of radiotherapy are unfavourable in the absence of chemotherapy: the 2-year locoregional disease\\u0026ndash;free survival and 5-year overall survival rates of radiotherapy alone are 54% and 35%, respectively [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. In particular, the safety and efficacy of CIRT have been confirmed in recent decades in prospective and large multicenter Japanese studies. Several clinical studies have shown that CIRT provides high local tumor control and confers a great overall survival benefit in many cancer types [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. However, there have been few reports on the safety and clinical control of CIRT in patients with bladder cancer.\\u003c/p\\u003e\\u003cp\\u003eProtocol ChiCTR2100043607 is the first prospective clinical trial of CIRT for bladder cancer. We described the prospective cohort outcomes of 10 patients with MIBC who received CIRT treatment. Our data show that an excellent cancer control rate can be achieved after CIRT, with a 1-year local progression-free survival rate of 90% in all 10 patients. The overall control rate for patients with multiple tumors was 66.7%, which was inferior to the 100% for patients with a solitary tumor. Jenkins et al [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. advocated the use of radical radiotherapy with the appropriate use of salvage cystectomy for patients whose tumors were not fully responsive to radiotherapy. Patients who underwent salvage cystectomy had a 5-year survival of 47%, whereas the 65 patients who did not undergo cystectomy had a 5-year survival of 3%. Most importantly, the addition of CIRT was associated with a statistically significant improvement in patients with urothelial carcinoma.\\u003c/p\\u003e\\u003cp\\u003eCIRT offers robust results in terms of survival for patients with MIBC. The present study adds to the evidence by providing the results of a single-center survey, presenting the largest group of patients treated with this approach reported thus far. In this treatment modality, the tumor response is evaluated through cystoscopic biopsy and urine cytology after CIRT, with a median dose of 64\\u0026ndash;74 Gy (RBE), and concurrent chemotherapy or immunotherapy of several courses. In this clinical trial, the overall tumor response rate was 80%, and the complete response and partial response rates were 40% and 40% patients, respectively. However, the overall control rates were 75.0% and 100.0% for high-grade and low-grade patients, respectively. Thrasher et al [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] identified several clinical variables as predictors of cancer-specific survival following radical cystectomy and reported that clinical T stage and tumor grade were the best predictors of survival. Chahal et al [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e] reported that the clinical T stage was an independent predictor of survival. Concurrent chemoradiotherapy can improve the efficacy of radiotherapy at the primary tumor site and effectively control microscopic metastases.\\u003c/p\\u003e\\u003cp\\u003eThe toxicity data obtained in this study are also encouraging, especially in terms of long-term side effects. We have demonstrated that there was no acute or late genitourinary toxicity of grade 3 or higher. Acute genitourinary toxicity is an accepted toxicity in all pelvic chemoradiotherapy regimens for both this and other disease subsites. Moreover, the incidence of long-term complications and late toxicities in both the urinary and intestinal was low. Furthermore, no patients in our study underwent a cystectomy or urinary diversion for complications. After normal tissue responses were compared and observed, the dose was gradually increased to 64\\u0026ndash;74 Gy (RBE) over 4 weeks, which might be equivalent to or slightly higher than that of photon radiotherapy [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. The aim of radiotherapy is not only to provide oncological cure but also to preserve bladder function. Higher radiation doses have been reported to improve local control at the expense of increased toxicity. The low incidence of toxicity and complications could be attributed to the excellent dose distribution of the carbon ion beams. Further complications should be limited so that the requirement for cystectomy, both salvage and palliative is minimized. The recommended dose was actually determined as the minimum dose that led to the greatest local control rate and the least adverse events. According to the clinical trial ChiCTR2100043607, 72 Gy (RBE) was used as the recommended dose.\\u003c/p\\u003e\\u003cp\\u003eBladder cancer is considered a rapidly proliferating cancer, with a high α/β ratio of 10 Gy in the linear quadratic model [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. In external radiotherapy for MIBC, a concomitant boost technique coupled with a bladder tumor boost with shortening of the overall treatment time provides a high probability of local control with acceptable toxicity [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Despite the short follow-up time, we have confirmed for the first time in a prospective cohort that CIRT is a promising treatment for bladder cancer because of its encouraging outcomes and safety. Furthermore, local recurrence revealed tumors outside of the primary tumor sites. This finding suggests that continued dose escalation to the primary tumor site may not lead to improved local control. Our study revealed that the dose of whole bladder carbon ion irradiation is low, but it can cause more obvious cystitis and fail to prevent recurrence. Therefore, whole bladder radiotherapy will not be continued in subsequent treatment, thereby reducing the incidence of cystitis and improving the quality of life of patients to a greater extent. The major challenges of radiotherapy for bladder cancer are controlling bladder capacity and minimizing bladder motion and reliably explaining the related intestinal and rectal motion. Our research innovatively adopts the \\\"Galloping Scheme\\\" to solve the problem of bladder capacity and position changes during radiotherapy for bladder cancer, and to ensure the accuracy of radiotherapy. If these obstacles can be further overcome, organ-preserving and effective combined-modality treatment for selected patients with MIBC as an alternative to radical surgery should be possible in the near future.\\u003c/p\\u003e\\u003cp\\u003eRadiation resistance has become the major obstacle to reducing tumor recurrence and improving prognosis in the treatment of MIBC. In the present study, we observed that carrying FGFR3 R34Q mutation was sensitive to CIRT in a low-grade urothelial carcinoma of the bladder. Darwis et al [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. reported the exon sequence differences of 409 cancer-related genes in the CIRT recurrent tumors compared with the treatment-na\\u0026iuml;ve tumors in the same patient and demonstrated that CIRT-resistant tumors had a significantly higher prevalence of somatic mutations in FGFR3 and FGFR4. In that study, the authors revealed that the pan-FGFR inhibitor LY2874455 enhanced CIRT sensitivity by occupying the ATP-binding pocket in the kinase domains to suppress FGFR signalling. Fisher et al [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. reported that a significant tumor growth delay was observed when AZD4547 was combined with radiation compared with radiation or AZD4547 alone in a FGFR-expressing HNSCC cell line xenograft and PDX. The molecular mechanisms underlying the effects in particular types of FGFR3 mutations on MIBC radiotherapy sensitivity remain unclear and require further research.\\u003c/p\\u003e\\u003cp\\u003eThe current study further identified a wide range of both actionable and nonactionable alterations in oncogenes, tumor suppressors, chromatin remodeling and DNA repair genes. One of the ARID1A V1982I gene alteration had been considered an adverse prognostic factor for CIRT in current research on MIBC. ARID1A abnormalities lead to the transcriptional activation of oncogenic driver genes, double-stranded DNA damage repair, tumorigenesis and tumor progression [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Loss of ARID1A resulted in an increased expression and secretion of prolyl 4-hydroxylase beta in lung cancer cells, which induced the activation of lung fibroblasts [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. P4HB-activated lung fibroblasts promoted the proliferation, invasion, and chemoresistance in lung cancer cells. However, neutralizing P4HB hampered the tumor growth and increased cisplatin cytotoxic efficacy in PDX models. Integrating tumor molecular genetics and tumor microenvironment can help us better understand the molecular mechanisms of CIRT resistance and enhance radiation sensitivity.\\u003c/p\\u003e\\u003cp\\u003eTargeted region sequencing results revealed the CHEK2 R474H mutations in low-grade tumors was sensitive to CIRT. Andreas et al [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. demonstrated that patients who received postoperative radiotherapy following breast-conservative surgery and who carried the CHEK2 mutation had a less favourable outcome in terms of local recurrence, distant metastasis-free survival and overall survival at 7 years. De Bock et al [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. reported that patients with the CHEK2 variant had a more unfavourable prognosis regarding the occurrence of contralateral breast cancer, and disease-free survival. Compared with our analysis, our data also indicate that the clinical relevance of CHEK2 gene mutations may not be restricted to the 1100delC allele but may extend to other mutations conferring CHEK2 dysfunction, such as the R474H substitution.\\u003c/p\\u003e\\u003cp\\u003eTo our knowledge, this is the first report on the clinical efficacy of associated CIRT in a consecutive series of unselected patients with bladder cancer. Although our study has substantial strengths, its potential limitations need to be considered. Our study reflects the experience of a single institution, and the relatively short follow-up period. Bladder cancer has distinct molecular subtypes with multiple pathogenic pathways [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Comprehensive genomic analyses of bladder cancer provide detailed molecular characterization of genetic and epigenetic alterations, and facilitate the understanding of the potential impact on diagnosis, treatment selection and follow-up plans. Although we performed a small number of genetic analyses on our CIRT patient cohort, we could not determine the definite mechanism of CIRT resistance. However, if the genetic changes associated with unfavourable prognosis for patients in the article are also independently confirmed in other larger cohorts of CIRT patients, particular mutation status may become a pivotal determinant when considering treatment selection, intensive observation and recurrence prevention on the basis of the intrinsic biology of the tumor. Consequently, our observations warrant further consideration and validation in a larger sample size, which may provide more clinical evidence to clarify the resistance mechanism of MIBC to CIRT.\\u003c/p\\u003e\\u003cp\\u003eDespite these limitations, these results provide good evidence that CIRT is feasible and well tolerated for MIBC. These regimens have many clinical and practical advantages for patients. Given that there is no internationally agreed upon standard CIRT regimen for MIBC and bladder preservation strategies are increasingly being used in the oncological community. It is a necessary to conduct prospective trials to explore the application of CIRT in locally advanced or metastatic disease in order to establish safe and effective treatment schedules. Therefore, CIRT is a topic of the prospective randomized controlled trials for bladder preservation that should be conducted in the future.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u003c/strong\\u003e Qinleng Zhang and Yihe Zhang contributed equally conceived research ideas, designed analysis methodology, and collected and sorted out raw data. Xiaojun Li, Xin Pan, Yuling Yang and Xin Wang wrote the initial draft. Xiaofeng Shi, Wenjun Jin and Yuhan Wang prepared the figure. Pengfei Sun, Zhilong Dong, Yizhao Zhang and Chengzong Zhao contributed to the analysis of the results. Yancheng Ye, Yanshan Zhang and Zhiping Wang contributed equally supervised the research and provided financial support for the project. All authors contributed to the article and approved the submitted version.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e This work was supported by the Natural Science Foundation of Gansu Province under Grant No.23JRRH0006.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e This study was a systematic review and analysis of existing clinical studies and thus no new data are available for sharing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInstitution and ethics approval and informed consent\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was reviewed and approved by the ethical committee of the Gansu Wuwei Tumor Hospital (approval no. 2020-Ethical Review-12) and registered at https://www.chictr.org.cn/ (Trial registration number: ChiCTR2100043607). All patients signed an informed consent form before the initiation of CIRT.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e The authors have no competing interests to declare that are relevant to the content of this article.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eGerlinger M, Catto JW, Orntoft TF, Real FX, Zwarthoff EC, Swanton C (2015) Intratumour heterogeneity in urologic cancers: from molecular evidence to clinical implications. 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J Med Genet 41:731\\u0026ndash;735. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1136/jmg.2004.019737\\u003c/span\\u003e\\u003cspan address=\\\"10.1136/jmg.2004.019737\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLopez-Beltran A, Cookson MS, Guercio BJ, Cheng L (2024) Advances in diagnosis and treatment of bladder cancer. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1136/bmj-2023-076743\\u003c/span\\u003e\\u003cspan address=\\\"10.1136/bmj-2023-076743\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. BMJ:e076743\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Muscle invasive bladder cancer, Carbon ion radiotherapy, Bladder preservation, Trimodal therapy, Next-Generation Sequencing\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6725919/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6725919/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eCarbon\\u0026ndash;ion radiotherapy (CIRT) improves the survival outcomes in patients with tumor recurrence or metastasis. However, there is little data to support the clinical efficacy of CIRT for bladder cancer. Ten patients underwent CIRT between November 2020 and July 2023 (ChiCTR2100043607). A comprehensive evaluation of the tumor control and toxicity of CIRT was performed, and the clinical characteristics associated with CIRT resistance were analyzed. The median age of the patients was 69 years (range, 32\\u0026ndash;81) at the start of CIRT, and 90% were male. With a median follow-up of 28 months (range, 18.3\\u0026ndash;35.6), and 2 patients (20%) developed local recurrence and/or distant metastases. Complete clinical response was achieved in 4 patients (40%), whereas partial response was documented in 4 patients (40%). The 1-year overall survival, progression-free survival, and local progression-free survival rates were 100.0%, 80.0%, 90.0%, respectively. The median prescription dose of CIRT was 70 Gy [relative biological effectiveness (RBE)], ranging from 64Gy (RBE) to 74Gy (RBE). One of the cases harbored mutations associated with CIRT resistance, including ARID1A V1982I, CCND1 S43F, and FANCA C1159S mutations. Moreover, individuals carrying FGFR3 R34Q and PIK3CA E545K mutations showed significant improvement. The acute and late toxicities were grade 2 or lower. No grade III or higher toxicity was observed. CIRT provides favourable tumor control and acceptable toxicity in bladder cancer. Further prospective clinical studies are necessary to use predictive and prognostic biomarkers to evaluate the therapeutic efficacy and adverse events of CIRT in patients with bladder urothelial carcinoma.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Clinical outcomes and mutation analysis of carbon ion radiotherapy for bladder urothelial carcinoma\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-07-10 12:45:56\",\"doi\":\"10.21203/rs.3.rs-6725919/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f888e94a-fe8e-4d32-94fe-878274db3754\",\"owner\":[],\"postedDate\":\"July 10th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-07-16T00:23:11+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-07-10 12:45:56\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6725919\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6725919\",\"identity\":\"rs-6725919\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}