Relationship between fibrosis induced by preoperative chemoradiotherapy and real-time tissue elastography of the internal anal sphincter

Relationship between fibrosis induced by preoperative chemoradiotherapy and real-time tissue elastography of the internal anal sphincter | 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 Relationship between fibrosis induced by preoperative chemoradiotherapy and real-time tissue elastography of the internal anal sphincter Akira Sakamoto, Kazuhito Sasaki, Hiroyuki Abe, Ryosuke Kikuchi, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7503661/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2025 Read the published version in International Journal of Colorectal Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Purpose: Chemoradiotherapy may cause fibrosis of the internal anal sphincter, resulting in sclerosis. However, no report has quantitatively investigated this relationship by using real-time tissue elastography. This study aimed to clarify the relationship between fibrosis and elasticity of the internal anal sphincter in patients undergoing surgery for lower rectal cancer with or without preoperative chemoradiotherapy. Methods: A single-center, prospective cohort study. Eighteen patients with rectal cancer who underwent abdominoperineal resection in a tertiary referral university hospital between May 2019 and May 2022 were included. Real-time tissue elastography was performed before surgery to measure internal anal sphincter hardness as “elasticity” (hardest (0) to softest (255); decreased elasticity indicated sclerotic changes). Internal anal sphincter fibrosis was evaluated using Masson’s trichrome and Elastica van Gieson staining. We evaluated internal anal sphincter elasticity after preoperative chemoradiotherapy and preoperatively in patients who did not undergo preoperative chemoradiotherapy, and analyzed the correlation with the percentage of internal anal sphincter fibrosis in the resected specimens. Results: Of the 18 patients, 10 underwent preoperative chemoradiotherapy. A significantly higher percentage of internal anal sphincter fibrosis was observed in the chemoradiotherapy group. Post-chemoradiotherapy elasticity was significantly lower than pre-chemoradiotherapy elasticity in patients undergoing chemoradiotherapy. The post-chemoradiotherapy elasticity in these patients was also lower than that in patients not undergoing chemoradiotherapy. The analysis of the correlation between internal anal sphincter elasticity and fibrosis showed that elasticity decreased as the percentage of fibrosis increased. Conclusions : Internal anal sphincter elasticity may reflect tissue sclerosis associated with fibrosis caused by chemoradiotherapy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer is the second leading cause of cancer-related deaths worldwide, accounting for 10% of all cancer types [1]. Preoperative chemoradiotherapy (CRT) followed by total mesorectal excision and postoperative adjuvant chemotherapy have become the standard of care for patients with locally advanced rectal cancer [2, 3]. Furthermore, total neoadjuvant therapy, a combination of radiation therapy and chemotherapy, is now presented as an option in the National Comprehensive Cancer Network (NCCN) guidelines [4, 5]. While some reports have indicated that preoperative CRT has an impact on the development of anorectal dysfunction and fecal incontinence after rectal surgery, others have indicated that preoperative CRT has no significant impact on defecation, an issue that is still controversial [6–10]. Previously, we established a method to quantify the hardness of the internal anal sphincter (IAS) as elasticity using real-time tissue elastography (RTE) on endoanal ultrasound (EAUS)[11]. This method has made it possible to objectively quantify the sclerosis of the IAS, especially before and after CRT. By using RTE on IAS, we reported that IAS sclerosis with preoperative CRT was associated with increased maximum rest pressure and worsened Cleveland Clinic Florida Fecal Incontinence Score (CCFIS) [12]. Since tissue sclerosis generally correlates with fibrosis [13, 14], we hypothesized that IAS sclerosis measured by RTE might also be related to IAS fibrosis induced by CRT. In this study, we aimed to clarify the relationship between IAS fibrosis and elasticity as an indicator of IAS sclerosis in patients with lower rectal cancer who underwent abdominoperineal resection (APR) from a histological aspect. Materials and Methods Patients We enrolled 18 patients with locally advanced rectal cancer whose IAS elasticity was measured using EAUS preoperatively and who underwent APR between May 2019 and May 2022 at the Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo. Data regarding patient background, including age, sex, tumor location from the anal verge before CRT, histology, history of anal surgery, clinical T Stage, and clinical N stage, were collected from medical records. For patients who underwent preoperative CRT, elasticity was measured before and after preoperative CRT. Of the 18 patients, 10 received preoperative CRT. Preoperative chemoradiotherapy The indication for CRT was primary adenocarcinoma of the lower rectum (cT3-cT4, any N, M0), without distant metastasis, located below the peritoneal reflection (second Houston valve). The staging was based on the American Joint Committee on Cancer staging system, 8th edition [15].CRT consisted of a total dose of 50.4 Gy of radiation using the 4-field box technique and concomitant 5-fluorouracil-based chemotherapy. Surgery was performed 8–10 weeks after the completion of CRT. All patients underwent APR for radical surgery based on total mesorectal excision. Histological staining and image acquisition To assess IAS fibrosis, tissue including the IAS in rectal resection specimens was fixed, embedded in paraffin, cut into 5-μm slices, and stained with Masson's trichrome (MT) and Elastica van Gieson (EVG) stains. In 16 cases, tissue was obtained from the tumor contralateral side, and in 2 cases, tissue was obtained from the non-contralateral side. The degree of fibrosis was observed in both groups, and all images of the MT- and EVG-stained sections were captured with an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan) using a Plan Apochromat 2× objective (NA0.75, BZ-PA02, KEYENCE, Osaka, Japan). The percentage of stained collagen fibers relative to the total IAS area in the sections was defined and calculated as the percentage of fibrosis. Figure 1 presents MT- and EVG-stained images of the IAS and images analyzed to calculate the area of fibrosis. The percentage of fibrosis was calculated by extracting the range of collagen fibers stained blue by MT staining and the range stained reddish brown by EVG staining. Real-time tissue elastography B-mode ultrasound imaging and RTE measurements of the anal canal were performed using a Noblus ultrasound system (Hitachi, Tokyo, Japan) equipped with a rectal probe (EUP-R54AW-19) and 10-MHz transducer. All ultrasound examinations, including RTE, were performed by two different surgeons. All patients were placed in the left lateral recumbent position and a rectal probe was inserted into the anal canal for measurement (Figure 2a). RTE at the IAS of each patient was performed with freehand manual compression in the anterior, left, posterior, and right positions to determine whether the IAS was well delineated. If IAS elasticity could be assessed, the RTE technique was performed according to the procedure previously reported by Fukui et al [11]. IAS elasticity at the contralateral side of the tumor was recorded and evaluated. Elasticity was expressed as a color scale from 0 to 255 and numerical value, with lower values indicating stiffer tissue and higher values indicating softer tissue. Representative images of IAS evaluation in the posterior position are shown in Figure 2b. Pre- and post-CRT IAS elasticity were recorded 1–3 weeks before initiation and 4–6 weeks after the end of CRT, respectively (Online Resource 1). For the group that did not receive CRT, preoperative elasticity was measured and included in the study. The correlation between IAS fibrosis percentage in the resected specimens and RTE of the IAS was examined. We also compared IAS elasticity and fibrosis rates between the two groups based on whether preoperative CRT was performed or not. Statistical analysis Quantitative data are expressed as means ± standard deviations, and scores are expressed as medians and ranges. Comparisons were made using Fisher’s exact test for categorical variables and Mann-Whitney U test for continuous variables. Statistical correlations for IAS elasticity and fibrosis percentage were made using Spearman's rank correlation coefficient. All analyses were performed using JMP Pro 15.0.0 software (SAS Institute Inc., Cary, NC, USA), and p<0.05 was considered significant for all analyses. Results A comparison of the clinicopathological factors between the two groups classified according to the presence or absence of preoperative CRT is shown in Table 1. Ten patients were in the CRT group and eight were in the non-CRT group. There were no significant differences between groups in terms of age, sex, body mass index, distance from the tumor to the anal verge, or clinical stage. In both groups, no patient had a history of anorectal surgery. Regarding IAS fibrosis assessed using both MT and EVG staining, a significantly higher fibrosis was observed in the CRT group than the non-CRT group (MT staining, median: 46% vs. 23%, p<0.01; EVG staining: 45% vs. 24%, p<0.01; Figure 3a, b). Comparing pre- and post-CRT elasticity in the CRT group and preoperative elasticity in the non-CRT group, post-CRT elasticity in the CRT group was significantly lower than pre-CRT (median: 54.4 vs. 96.5, p<0.01), which was also lower than that in patients who did not undergo CRT (median: 54.4 vs. 119.2, p<0.01; Figure 4). There was no significant difference between the pre-CRT elasticity of the CRT group and preoperative elasticity of the non-CRT group (96.5 vs. 119.2, p=0.18). Notably, the inverse correlation between IAS elasticity and fibrosis was confirmed in all preoperative cases, which showed that the percentage of fibrosis in IAS increased as the tissue of IAS became more sclerotic. This trend was confirmed by MT and EVG staining (MT, r= –0.69, p<0.01; EVG, r= –0.76, p<0.01; Figure 5a, b). Discussion In the present study, the CRT group showed a significantly higher percentage of fibrosis and significantly lower IAS elasticity than the non-CRT group. Additionally, a decrease in IAS elasticity was strongly correlated with tissue fibrosis. RTE was originally developed and clinically applied to diagnose breast cancer based on tissue hardness, but has also been applied to diagnose the extent of liver cirrhosis because progressive fibrosis is a pathological feature of normal tissue exposed to radiation [16–18]. However, no previous studies by other researchers have focused on the RTE of IAS, and to the best of our knowledge, this is the first study to quantify IAS sclerosis using RTE and to confirm its strong correlation with IAS fibrosis. In general, adverse events from radiation therapy occur at the start of irradiation and increase in a time-dependent manner, and RT-induced fibrosis is a well-known side effect that can arise in some patients[19, 20]. In a previous report on the histological changes caused by preoperative CRT in the IAS, Da Silva et al. showed that fibrosis of the IAS was significantly increased in patients who received preoperative CRT compared to those who did not[21]. They further reported that this histological change in the sphincter muscle may affect the stiffness and tone of the IAS, which may cause increased faecal incontinence [21]. Koushi et al. conducted a retrospective study on 95 patients with lower rectal cancer who underwent preoperative CRT, neoadjuvant chemotherapy (NAC), or surgery alone. This previous report also concluded that peritoneal fibrosis was more pronounced in the preoperative CRT group than in the NAC or surgery alone groups [22]. We examined the effect of CRT on the IAS by using two different stains to assess the percentage of IAS fibrosis, mainly on the contralateral side of the tumor, and found increased IAS fibrosis. The results of our study indicate a decrease in elasticity, also known as tissue sclerosis, in patients who received preoperative CRT, which is a new finding. The strong correlation between increased fibrosis and sclerosis of IAS shown in this study indicates that a decrease in IAS elasticity may suggest an increase in local fibrosis due to preoperative CRT. Radiotherapy provides tremendous benefit to patients, but causes long-term undesirable side effects. Because radiation-induced fibrosis is an irreversible condition, treatment primarily focuses on managing symptoms, and there is currently no effective method to achieve complete remission [23, 24]. While radiation dose and the amount of tissue exposed are strongly suspected to be risk factors for radiation-induced fibrosis, individual genetic predisposition has also been shown to contribute to its development [25, 26].How variations in radiation dose owing to tumor location and individual radiation sensitivity affect the elasticity of the IAS and fibrosis require further investigation. This study has some limitations. First, it was conducted at a single institution, and the number of cases was small. Second, this study focused only on collagen fibers; further investigation is needed on factors, such as fibroblasts, that may be involved in the increase in fibrosis. Third, since excised specimens were used for analysis, the timing of IAS elasticity measurement and the rate of fibrosis in the IAS might have been different, potentially introducing error in the relationship between the two. In conclusion, IAS elasticity may reflect tissue stiffness associated with fibrosis caused by CRT. Declarations Funding/Support: No funding. Conflicts of interest : The authors declare no competing interests. Ethics approval: The study protocol was approved by The University of Tokyo Ethics Committee (No. 10046-(5) and No. 3252-(16)). Consent: Written informed consent was obtained from all patients. Data, Material and/or Code availability ： The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Author contributions: All authors (Akira Sakamoto, Kazuhito Sasaki, Hiroyuki Abe, Ryosuke Kikuchi, Hiroaki Nozawa, Koji Murono, Shigenobu Emoto, Kensuke Kaneko, Yuichiro Yokoyama, Yuzo Nagai, Shinya Abe, Takahide Shinagawa, Yuichi Tachikawa, Satoshi Okada, Tetsuo Ushiku, Soichiro Ishihara) have met all three of the following criteria: a. 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Supplementary Files Table1.docx FigS1.tif Cite Share Download PDF Status: Published Journal Publication published 08 Nov, 2025 Read the published version in International Journal of Colorectal Disease → Version 1 posted Editorial decision: Revision requested 13 Oct, 2025 Reviews received at journal 12 Oct, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviews received at journal 14 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers invited by journal 05 Sep, 2025 Editor assigned by journal 31 Aug, 2025 Submission checks completed at journal 31 Aug, 2025 First submitted to journal 31 Aug, 2025 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. 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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-7503661","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":514770378,"identity":"06e76be2-af60-4450-87cd-da129b149c97","order_by":0,"name":"Akira Sakamoto","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Sakamoto","suffix":""},{"id":514770379,"identity":"101f731d-f341-4eb3-9a71-374e47d7d52c","order_by":1,"name":"Kazuhito 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06:52:12","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":836976,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/de31f10742bd999f2ca8308b.tif"},{"id":91816621,"identity":"6d924cdc-f82a-4e5d-bfd4-ede3d08faf5f","added_by":"auto","created_at":"2025-09-22 06:52:14","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67295,"visible":true,"origin":"","legend":"","description":"","filename":"e50d389349b44ef39cda2207b6a7617d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/2f8707b9a85e264d561e3580.xml"},{"id":91486108,"identity":"1883c5e5-ccc4-4cdd-ab9f-abd785f7fa29","added_by":"auto","created_at":"2025-09-17 04:55:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16637996,"visible":true,"origin":"","legend":"\u003cp\u003eCalculation of fibrosis percentage\u003c/p\u003e\n\u003cp\u003e(a) Overall view of Masson’s trichrome (MT) and (b) Elastica van Gieson (EVG) staining of the internal anal sphincter (IAS) in the rectal resection specimen. (c) Extraction of IAS areas in the visible range for MT staining and (d) EVG staining. (e) In the analyzed images, the blue areas indicate the extent of collagen fibers for MT staining and (f) EVG staining.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/bfff9aec7ba8908cdda9ac5c.png"},{"id":91487528,"identity":"70eff218-9793-4bfd-b2de-947290421b21","added_by":"auto","created_at":"2025-09-17 05:03:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57023439,"visible":true,"origin":"","legend":"\u003cp\u003eInternal anal sphincter evaluation\u003c/p\u003e\n\u003cp\u003e(a) Schematic representation of the anal canal and the level of real-time tissue elastography examination. (b)\u003cstrong\u003e \u003c/strong\u003eReal-time tissue elastography was performed at the level of the anal canal between the puborectalis muscle and subcutaneous external anal sphincter. Endoanal ultrasonography demonstrating the internal (IAS; white arrows) and external (EAS; yellow dotted arrows) anal sphincters. A region of interest was established in the IAS and elasticity was measured.\u003c/p\u003e","description":"","filename":"Fig02.png","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/d996de17b8314bf9e6d3abf1.png"},{"id":91486105,"identity":"f343d473-d56f-44dd-8180-bfb2af4ebd6e","added_by":"auto","created_at":"2025-09-17 04:55:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4527535,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the fibrosis area percentage of the internal anal sphincter between the chemoradiotherapy (CRT) and non-CRT groups\u003c/p\u003e\n\u003cp\u003eFibrosis area percentage of the internal anal sphincter measured using (a) Masson’s trichrome and (b) Elastica van Gieson staining.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/4a16b954ff4fd60041657364.png"},{"id":91486104,"identity":"fa19e9be-fb34-4644-8a36-3f9ab5d666b5","added_by":"auto","created_at":"2025-09-17 04:55:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1611771,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of internal anal sphincter elasticity between the chemoradiotherapy (CRT; before and after CRT) and non-CRT groups\u003c/p\u003e","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/9d6c80b240bfc3b7ccf925c6.png"},{"id":91487520,"identity":"fa4edecd-f826-4635-b48a-df1a287bd084","added_by":"auto","created_at":"2025-09-17 05:03:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3574413,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between internal anal sphincter (IAS) elasticity and fibrosis area percentage of the IAS\u003c/p\u003e\n\u003cp\u003e(a) Fibrosis area percentage of the IAS measured using Masson’s trichrome and (b) Elastica van Gieson staining.\u003c/p\u003e","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/9384d4fb44772e069ae7e81a.png"},{"id":95564230,"identity":"dad7ad8a-ba15-4c1f-976f-a43cb114379b","added_by":"auto","created_at":"2025-11-10 16:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":65618857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/74b4fe52-0cb7-448b-bebf-639594ff5c99.pdf"},{"id":91486100,"identity":"7ef95d28-56b1-42ae-a27e-39f31cdfdff1","added_by":"auto","created_at":"2025-09-17 04:55:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18553,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/c323b24a7c4a4e391e51a21d.docx"},{"id":91487515,"identity":"3506d85d-daf9-4e29-bcac-4b0b665744b8","added_by":"auto","created_at":"2025-09-17 05:03:29","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":325939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7503661/v1/d0395f1d40088b5f76847c7e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Relationship between fibrosis induced by preoperative chemoradiotherapy and real-time tissue elastography of the internal anal sphincter","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer is the second leading cause of cancer-related deaths worldwide, accounting for 10% of all cancer types [1]. Preoperative chemoradiotherapy (CRT) followed by total mesorectal excision and postoperative adjuvant chemotherapy have become the standard of care for patients with locally advanced rectal cancer [2, 3]. Furthermore, total neoadjuvant therapy, a combination of radiation therapy and chemotherapy, is now presented as an option in the National Comprehensive Cancer Network (NCCN) guidelines [4, 5].\u003c/p\u003e\n\u003cp\u003eWhile some reports have indicated that preoperative CRT has an impact on the development of anorectal dysfunction and fecal incontinence after rectal surgery, others have indicated that preoperative CRT has no significant impact on defecation, an issue that is still controversial [6–10].\u003c/p\u003e\n\u003cp\u003ePreviously, we established a method to quantify the hardness of the internal anal sphincter (IAS) as elasticity using real-time tissue elastography (RTE) on endoanal ultrasound (EAUS)[11]. This method has made it possible to objectively quantify the sclerosis of the IAS, especially before and after CRT. By using RTE on IAS, we reported that IAS sclerosis with preoperative CRT was associated with increased maximum rest pressure and worsened Cleveland Clinic Florida Fecal Incontinence Score (CCFIS) [12]. Since tissue sclerosis generally correlates with fibrosis [13, 14], we hypothesized that IAS sclerosis measured by RTE might also be related to IAS fibrosis induced by CRT. In this study, we aimed to clarify the relationship between IAS fibrosis and elasticity as an indicator of IAS sclerosis in patients with lower rectal cancer who underwent abdominoperineal resection (APR) from a histological aspect.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003ePatients\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe enrolled 18 patients with locally advanced rectal cancer whose IAS elasticity was measured using EAUS preoperatively and who underwent APR between May 2019 and May 2022 at the Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo. Data regarding patient background, including age, sex, tumor location from the anal verge before CRT, histology, history of anal surgery, clinical T Stage, and clinical N stage, were collected from medical records. For patients who underwent preoperative CRT, elasticity was measured before and after preoperative CRT. Of the 18 patients, 10 received preoperative CRT.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePreoperative chemoradiotherapy\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe indication for CRT was primary adenocarcinoma of the lower rectum (cT3-cT4, any N, M0), without distant metastasis, located below the peritoneal reflection (second Houston valve). The staging was based on the American Joint Committee on Cancer staging system, 8th edition [15].CRT consisted of a total dose of 50.4 Gy of radiation using the 4-field box technique and concomitant 5-fluorouracil-based chemotherapy. Surgery was performed 8–10 weeks after the completion of CRT. All patients underwent APR for radical surgery based on total mesorectal excision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHistological staining and image acquisition\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess IAS fibrosis, tissue including the IAS in rectal resection specimens was fixed, embedded in paraffin, cut into 5-μm slices, and stained with Masson's trichrome (MT) and Elastica van Gieson (EVG) stains. In 16 cases, tissue was obtained from the tumor contralateral side, and in 2 cases, tissue was obtained from the non-contralateral side.\u003c/p\u003e\n\u003cp\u003eThe degree of fibrosis was observed in both groups, and all images of the MT- and EVG-stained sections were captured with an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan) using a Plan Apochromat 2× objective (NA0.75, BZ-PA02, KEYENCE, Osaka, Japan). The percentage of stained collagen fibers relative to the total IAS area in the sections was defined and calculated as the percentage of fibrosis. Figure 1 presents MT- and EVG-stained images of the IAS and images analyzed to calculate the area of fibrosis. The percentage of fibrosis was calculated by extracting the range of collagen fibers stained blue by MT staining and the range stained reddish brown by EVG staining.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eReal-time tissue elastography\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eB-mode ultrasound imaging and RTE measurements of the anal canal were performed using a Noblus ultrasound system (Hitachi, Tokyo, Japan) equipped with a rectal probe (EUP-R54AW-19) and 10-MHz transducer. All ultrasound examinations, including RTE, were performed by two different surgeons. All patients were placed in the left lateral recumbent position and a rectal probe was inserted into the anal canal for measurement (Figure 2a). RTE at the IAS of each patient was performed with freehand manual compression in the anterior, left, posterior, and right positions to determine whether the IAS was well delineated. If IAS elasticity could be assessed, the RTE technique was performed according to the procedure previously reported by Fukui et al [11]. IAS elasticity at the contralateral side of the tumor was recorded and evaluated. Elasticity was expressed as a color scale from 0 to 255 and numerical value, with lower values indicating stiffer tissue and higher values indicating softer tissue. Representative images of IAS evaluation in the posterior position are shown in Figure 2b. Pre- and post-CRT IAS elasticity were recorded 1–3 weeks before initiation and 4–6 weeks after the end of CRT, respectively (Online Resource 1). For the group that did not receive CRT, preoperative elasticity was measured and included in the study. The correlation between IAS fibrosis percentage in the resected specimens and RTE of the IAS was examined. We also compared IAS elasticity and fibrosis rates between the two groups based on whether preoperative CRT was performed or not.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative data are expressed as means ± standard deviations, and scores are expressed as medians and ranges. Comparisons were made using Fisher’s exact test for categorical variables and Mann-Whitney U test for continuous variables. Statistical correlations for IAS elasticity and fibrosis percentage were made using Spearman's rank correlation coefficient. All analyses were performed using JMP Pro 15.0.0 software (SAS Institute Inc., Cary, NC, USA), and p\u0026lt;0.05 was considered significant for all analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA comparison of the clinicopathological factors between the two groups classified according to the presence or absence of preoperative CRT is shown in Table 1. Ten patients were in the CRT group and eight were in the non-CRT group. There were no significant differences between groups in terms of age, sex, body mass index, distance from the tumor to the anal verge, or clinical stage. In both groups, no patient had a history of anorectal surgery.\u003c/p\u003e\n\u003cp\u003eRegarding IAS fibrosis assessed using both MT and EVG staining, a significantly higher fibrosis was observed in the CRT group than the non-CRT group (MT staining, median: 46% vs. 23%, p\u0026lt;0.01; EVG staining: 45% vs. 24%, p\u0026lt;0.01; Figure 3a, b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComparing pre- and post-CRT elasticity in the CRT group and preoperative elasticity in the non-CRT group, post-CRT elasticity in the CRT group was significantly lower than pre-CRT (median: 54.4 vs. 96.5, p\u0026lt;0.01), which was also lower than that in patients who did not undergo CRT (median: 54.4 vs. 119.2, p\u0026lt;0.01; Figure 4). There was no significant difference between the pre-CRT elasticity of the CRT group and preoperative elasticity of the non-CRT group (96.5 vs. 119.2, p=0.18).\u003c/p\u003e\n\u003cp\u003eNotably, the inverse correlation between IAS elasticity and fibrosis was confirmed in all preoperative cases, which showed that the percentage of fibrosis in IAS increased as the tissue of IAS became more sclerotic. This trend was confirmed by MT and EVG staining (MT, r= –0.69, p\u0026lt;0.01; EVG, r= –0.76, p\u0026lt;0.01; Figure 5a, b).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, the CRT group showed a significantly higher percentage of fibrosis and significantly lower IAS elasticity than the non-CRT group. Additionally, a decrease in IAS elasticity was strongly correlated with tissue fibrosis. RTE was originally developed and clinically applied to diagnose breast cancer based on tissue hardness, but has also been applied to diagnose the extent of liver cirrhosis because progressive fibrosis is a pathological feature of normal tissue exposed to radiation [16–18]. However, no previous studies by other researchers have focused on the RTE of IAS, and to the best of our knowledge, this is the first study to quantify IAS sclerosis using RTE and to confirm its strong correlation with IAS fibrosis.\u003c/p\u003e\n\u003cp\u003eIn general, adverse events from radiation therapy occur at the start of irradiation and increase in a time-dependent manner, and RT-induced fibrosis is a well-known side effect that can arise in some patients[19, 20]. In a previous report on the histological changes caused by preoperative CRT in the IAS, Da Silva et al. showed that fibrosis of the IAS was significantly increased in patients who received preoperative CRT compared to those who did not[21]. They further reported that this histological change in the sphincter muscle may affect the stiffness and tone of the IAS, which may cause increased faecal incontinence [21]. Koushi et al. conducted a retrospective study on 95 patients with lower rectal cancer who underwent preoperative CRT, neoadjuvant chemotherapy (NAC), or surgery alone. This previous report also concluded that peritoneal fibrosis was more pronounced in the preoperative CRT group than in the NAC or surgery alone groups [22]. We examined the effect of CRT on the IAS by using two different stains to assess the percentage of IAS fibrosis, mainly on the contralateral side of the tumor, and found increased IAS fibrosis.\u003c/p\u003e\n\u003cp\u003eThe results of our study indicate a decrease in elasticity, also known as tissue sclerosis, in patients who received preoperative CRT, which is a new finding. The strong correlation between increased fibrosis and sclerosis of IAS shown in this study indicates that a decrease in IAS elasticity may suggest an increase in local fibrosis due to preoperative CRT. Radiotherapy provides tremendous benefit to patients, but causes long-term undesirable side effects. Because radiation-induced fibrosis is an irreversible condition, treatment primarily focuses on managing symptoms, and there is currently no effective method to achieve complete remission [23, 24]. While radiation dose and the amount of tissue exposed are strongly suspected to be risk factors for radiation-induced fibrosis, individual genetic predisposition has also been shown to contribute to its development [25, 26].How variations in radiation dose owing to tumor location and individual radiation sensitivity affect the elasticity of the IAS and fibrosis require further investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study has some limitations. First, it was conducted at a single institution, and the number of cases was small. Second, this study focused only on collagen fibers; further investigation is needed on factors, such as fibroblasts, that may be involved in the increase in fibrosis. Third, since excised specimens were used for analysis, the timing of IAS elasticity measurement and the rate of fibrosis in the IAS might have been different, potentially introducing error in the relationship between the two.\u003c/p\u003e\n\u003cp\u003eIn conclusion, IAS elasticity may reflect tissue stiffness associated with fibrosis caused by CRT.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding/Support:\u003c/strong\u003e No funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThe study protocol was approved by The University of Tokyo Ethics Committee (No. 10046-(5) and No. 3252-(16)).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent:\u003c/strong\u003e Written informed consent was obtained from all patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData, Material and/or Code availability\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e All authors (Akira Sakamoto, Kazuhito Sasaki, Hiroyuki Abe, Ryosuke Kikuchi, Hiroaki Nozawa, Koji Murono, Shigenobu Emoto, Kensuke Kaneko, Yuichiro Yokoyama, Yuzo Nagai, Shinya Abe, Takahide Shinagawa, Yuichi Tachikawa, Satoshi Okada, Tetsuo Ushiku, Soichiro Ishihara) have met all three of the following criteria: \u003cstrong\u003ea.\u003c/strong\u003e Substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; \u003cstrong\u003eb.\u0026nbsp;\u003c/strong\u003eDrafting the article or revising it critically for important intellectual content; \u003cstrong\u003ec.\u003c/strong\u003e Final approval of the version to be published.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fedewa SA, et al (2017) Colorectal cancer statistics, 2017. 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Dis Colon Rectum 46:1492–1497. https://doi.org/10.1007/s10350-004-6800-1\u003c/li\u003e\n\u003cli\u003eKoushi K, Nishizawa Y, Kojima M, et al (2016) Erratum to: Association between pathologic features of peripheral nerves and postoperative anal function after neoadjuvant therapy for low rectal cancer. Int J Colorectal Dis 31:1853. https://doi.org/10.1007/s00384-016-2676-5. Erratum in: (2016). Int J Colorectal Dis 31:1853. https://doi.org/10.1007/s00384-016-2676-5\u003c/li\u003e\n\u003cli\u003eBourgeois JF, Gourgou S, Kramar A, Lagarde JM, Guillot B (2008) A randomized, prospective study using the LPG technique in treating radiation-induced skin fibrosis: clinical and profilometric analysis. Skin Res Technol 14:71–76. https://doi.org/10.1111/j.1600-0846.2007.00263.x\u003c/li\u003e\n\u003cli\u003eHartl DM, Cohen M, Juliéron M, Marandas P, Janot F, Bourhis J (2008) Botulinum toxin for radiation-induced facial pain and trismus. 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Int J Radiat Oncol Biol Phys 64:776–783. https://doi.org/10.1016/j.ijrobp.2005.09.014\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-colorectal-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijcd","sideBox":"Learn more about [International Journal of Colorectal Disease](http://link.springer.com/journal/384)","snPcode":"384","submissionUrl":"https://submission.nature.com/new-submission/384/3","title":"International Journal of Colorectal Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7503661/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7503661/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003ePurpose: \u003c/em\u003eChemoradiotherapy may cause fibrosis of the internal anal sphincter, resulting in sclerosis. However, no report has quantitatively investigated this relationship by using real-time tissue elastography. This study aimed to clarify the relationship between fibrosis and elasticity of the internal anal sphincter in patients undergoing surgery for lower rectal cancer with or without preoperative chemoradiotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethods: \u003c/em\u003eA single-center, prospective cohort study.\u003cem\u003e \u003c/em\u003eEighteen patients with rectal cancer who underwent abdominoperineal resection in a tertiary referral university hospital between May 2019 and May 2022 were included. Real-time tissue elastography was performed before surgery to measure internal anal sphincter hardness as “elasticity” (hardest (0) to softest (255); decreased elasticity indicated sclerotic changes). Internal anal sphincter fibrosis was evaluated using Masson’s trichrome and Elastica van Gieson staining. We evaluated internal anal sphincter elasticity after preoperative chemoradiotherapy and preoperatively in patients who did not undergo preoperative chemoradiotherapy, and analyzed the correlation with the percentage of internal anal sphincter fibrosis in the resected specimens.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults:\u003c/em\u003e Of the 18 patients, 10 underwent preoperative chemoradiotherapy. A significantly higher percentage of internal anal sphincter fibrosis was observed in the chemoradiotherapy group. Post-chemoradiotherapy elasticity was significantly lower than pre-chemoradiotherapy elasticity in patients undergoing chemoradiotherapy. The post-chemoradiotherapy elasticity in these patients was also lower than that in patients not undergoing chemoradiotherapy. The analysis of the correlation between internal anal sphincter elasticity and fibrosis showed that elasticity decreased as the percentage of fibrosis increased.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusions\u003c/em\u003e: Internal anal sphincter elasticity may reflect tissue sclerosis associated with fibrosis caused by chemoradiotherapy.\u003c/p\u003e","manuscriptTitle":"Relationship between fibrosis induced by preoperative chemoradiotherapy and real-time tissue elastography of the internal anal sphincter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 04:55:24","doi":"10.21203/rs.3.rs-7503661/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T12:10:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-12T12:51:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140020905266522615709732062588729418592","date":"2025-09-18T04:32:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T08:39:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291253480668995384730294914888103584589","date":"2025-09-10T00:24:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T17:26:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T03:52:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-01T03:52:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Colorectal Disease","date":"2025-09-01T02:51:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-colorectal-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijcd","sideBox":"Learn more about [International Journal of Colorectal Disease](http://link.springer.com/journal/384)","snPcode":"384","submissionUrl":"https://submission.nature.com/new-submission/384/3","title":"International Journal of Colorectal Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9a88f77a-cfdc-457d-8e8f-a3942c523bed","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:04:49+00:00","versionOfRecord":{"articleIdentity":"rs-7503661","link":"https://doi.org/10.1007/s00384-025-05026-1","journal":{"identity":"international-journal-of-colorectal-disease","isVorOnly":false,"title":"International Journal of Colorectal Disease"},"publishedOn":"2025-11-08 15:57:42","publishedOnDateReadable":"November 8th, 2025"},"versionCreatedAt":"2025-09-17 04:55:24","video":"","vorDoi":"10.1007/s00384-025-05026-1","vorDoiUrl":"https://doi.org/10.1007/s00384-025-05026-1","workflowStages":[]},"version":"v1","identity":"rs-7503661","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7503661","identity":"rs-7503661","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}