Trans-anal Multipoint Full-layer Puncture Biopsy Guides Organ Preservation and Improves Prognostic Stratification in Locally Advanced Rectal Cancer After Neoadjuvant Therapy

Trans-anal Multipoint Full-layer Puncture Biopsy Guides Organ Preservation and Improves Prognostic Stratification in Locally Advanced Rectal Cancer After Neoadjuvant Therapy | 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 Trans-anal Multipoint Full-layer Puncture Biopsy Guides Organ Preservation and Improves Prognostic Stratification in Locally Advanced Rectal Cancer After Neoadjuvant Therapy Chunxiang Ye, Zhiwei Zhai, Hao Qu, Jiagang Han, Congqing Jiang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9320255/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Aim To evaluate the value of trans-anal multipoint full-layer puncture biopsy (TMFP) in guiding organ preservation strategies and prognostic stratification for locally advanced rectal cancer (LARC) after neoadjuvant therapy. Method This prospective multicenter study was conducted across four tertiary centers in China. Sixty-one LARC patients after neoadjuvant chemoradiotherapy (± immunotherapy) who underwent in vivo TMFP were enrolled. Primary outcomes were TMFP-guided changes in treatment strategy (Watch-and-Wait, W&W, vs. surgery) and overall survival (OS). Results Among 61 patients, 20 achieved clinical complete response (cCR) and 41 were non-cCR. TMFP results significantly altered treatment decisions in non-cCR patients, with 48.8% (20/41) being TMFP-negative; half of these (10/20) successfully adopted a W&W strategy. The postoperative pCR rate in non-cCR but TMFP-negative patients who underwent surgery was 70% (7/10). TMFP demonstrated superior prognostic stratification compared to cCR: the OS of TMFP-negative patients was significantly better than that of TMFP-positive patients (P = 0.04), whereas no significant difference was found between cCR and non-cCR groups (P = 0.58). Among TMFP-negative patients, no survival difference was observed between the W&W and surgery groups (P = 0.40). The procedure was well-tolerated with a low complication rate. Conclusions TMFP serves as a pivotal clinical decision-making tool that safely expands organ preservation opportunities to a significant subset of non-cCR patients and provides powerful prognostic stratification, outperforming traditional cCR assessment. Integrating TMFP into the post-neoadjuvant workflow optimizes the balance between oncological safety and functional preservation. Locally advanced rectal cancer Neoadjuvant therapy Trans-anal multipoint full-layer puncture biopsy Watch-and-wait Organ preservation Figures Figure 1 Figure 2 Introduction The management of locally advanced rectal cancer (LARC) has evolved towards a more personalized approach, with organ preservation becoming a key goal for patients achieving a complete response to neoadjuvant therapy 1 , 2 . The "watch-and-wait" (W&W) strategy, initially proposed for patients with a clinical complete response (cCR), can avoid the morbidity associated with radical surgery and preserve quality of life 3 , 4 . Currently, patient selection for W&W relies primarily on the assessment of clinical complete response (cCR) using a combination of digital rectal examination (DRE), endoscopy, and magnetic resonance imaging (MRI) 1 , 5 . However, these modalities are hampered by inherent limitations. DRE is highly subjective 6 , endoscopy and mucosal biopsies are limited to evaluating the superficial layers of the bowel wall 1 , 7 , 8 , and MRI struggles to differentiate between post-treatment fibrosis and residual tumor cells 9 . Consequently, a significant proportion of patients with a pathological complete response (pCR) are misclassified as non-cCR and thus may undergo unnecessary surgery 10 . Conversely, some patients managed with W&W based on cCR experience local regrowth, underscoring the limitations of current assessment methods 11 . To bridge this gap between clinical assessment and pathological truth, our group developed the trans-anal multipoint full-layer puncture biopsy (TMFP). This technique involves obtaining multiple full-thickness tissue cores from the tumor bed under direct vision, providing a much more comprehensive pathological sampling than standard forceps biopsies. Previously, our team has reported the high diagnostic accuracy of TMFP for predicting pCR, establishing it as a superior pathological assessment tool compared to standard cCR criteria 12 . While establishing diagnostic accuracy is a crucial first step, it begs a more profound clinical question: How does the integration of this precise diagnostic tool into the clinical decision-making workflow ultimately influence patient management and long-term outcomes? Specifically, can TMFP identify a subgroup of patients traditionally deemed as non-cCR who can safely forego radical surgery and pursue organ preservation? Furthermore, does the pathological information provided by TMFP offer superior prognostic stratification compared to clinical assessment alone? Therefore, this prospective multicenter study was designed not as another diagnostic accuracy study, but to evaluate the clinical utility of TMFP. We focus specifically on its role in guiding therapeutic decisions—particularly in expanding organ preservation opportunities to non-cCR patients—and its power in predicting long-term survival in LARC patients following neoadjuvant therapy. This cohort study has been reported in line with the STROCSS guidelines 13 . Materials and methods Study Design and Participants This was a prospective, multicenter, non-randomized cohort study conducted across four tertiary hospitals in China, namely Beijing Chaoyang Hospital, Capital Medical University (Center 1, n = 34), Beijing Friendship Hospital, Capital Medical University (Center 2, n = 5), Qilu Hospital of Shandong University (Center 3, n = 17), and Zhongnan Hospital of Wuhan University (Center 4, n = 5). The study specifically enrolled a consecutive cohort of patients who underwent in vivo TMFP, reflecting its application in the real-world clinical decision-making context. Between 2021 and 2025, 61 patients with LARC who completed neoadjuvant chemoradiotherapy (nCRT) with or without immunotherapy were included. The inclusion criteria and exclusion criteria were the same as those described previously 12 . This study was approved by the institutional review boards of four hospitals, and all patients provided written informed consent. Neoadjuvant Therapy and Clinical Assessment Neoadjuvant therapy consisted of long-course radiotherapy (45-50.4 Gy in 25–28 fractions) with concurrent capecitabine-based chemotherapy. A subset of patients received additional immune checkpoint inhibitors as part of clinical trials or off-label use. Clinical response was assessed 8–12 weeks after radiotherapy completion using DRE, endoscopy, and pelvic MRI. cCR was defined according to the Chinese Watch and Wait Database (CW&WD) criteria 14 . Due to the small sample size, for the convenience of statistical analysis, and considering the significant differences between near-cCR patients and cCR patients in terms of local recurrence rate and 5-year organ preservation rate 15 , 16 , near-cCR patients were classified as non-cCR for statistical analysis in this paper. TMFP Procedure and Pathological Evaluation The in vivo TMFP procedure was performed as previously described [11]. Briefly, under anesthesia and with adequate bowel preparation, a 16G core biopsy needle was used to obtain full-thickness tissue samples from at least 13 predetermined sites around the original tumor bed or scar under direct vision. The puncture angle was 60°-70° with a depth of 0.8-1.0 cm. Hemostasis was achieved by direct pressure or electrocautery. A result was considered TMFP-positive if any specimen contained viable tumor cells; otherwise, it was TMFP-negative. To ensure procedural and data consistency across centers, a standardized protocol was implemented. All surgeons underwent centralized TMFP training and submitted procedural videos for approval to minimize inter-operator variability. For the intervention itself, all TMFP and surgical specimens were independently reviewed by two blinded pathologists, with consensus resolution for discrepancies. Treatment Decision and Follow-up Following the TMFP procedure, a comprehensive treatment recommendation was made based on a synthesis of clinical assessment (cCR status) and TMFP results. Patients were thoroughly counseled, and the final choice between radical surgery (Total meso-rectal excision, TME) or a W&W strategy was made jointly by the physician and patient. Patients opting for W&W underwent intensified follow-up with DRE, endoscopy, MRI, and serum carcinoembryonic antigen (CEA) testing every 3–6 months. Overall survival (OS) was defined as the time from the date of TMFP to death from any cause or last follow-up. Statistical Analysis Statistical analyses were performed using SPSS 26.0 (IBM Corporation, Chicago, IL, USA). Categorical variables were described as frequencies and percentages. The Kaplan-Meier method was used to generate survival curves, and differences were compared using the log-rank test. A two-sided P-value < 0.05 was considered statistically significant. Results Patient Characteristics and TMFP-guided Treatment Pathways A total of 61 patients with a mean age of 59.8 years were included. All had mid-low stage III rectal cancer and received nCRT, with 8.2% (5/61) receiving additional immunotherapy. The clinicopathological characteristics are summarized in Table 1 . Table 1 Clinicopathological characteristics of the patients Characteristics Number(%) Gender Male 42(68.85%) Female 19(31.15%) Age <60 26(42.6%) ≥60 35(57.4%) Distance from the lower edge of the tumor to the anal verge <5cm 35(57.4%) ≥5cm 26(42.6%) cT stage cT1-2 9(14.8%) cT3 41(67.2%) cT4 11(18.0%) cN stage cN0 15(24.6%) cN1-2 46(75.4%) Interval between radiotherapy and biopsy (weeks) <8 9(14.8%) ≥8 52(85.2%) Immune Checkpoint Inhibitors (ICIs)‌‌ Yes 5(8.2%) No 56(91.8%) ycT stage cT0-2 16(26.2%) cT3 13(21.3%) cT4 3(4.9%) Positive 19(31.1%) NA 10(16.4%) ycN stage cN0 30(49.2%) cN1-2 9(14.7%) Positive 17(27.9%) NA 5(8.2%) ccR Yes 19(31.1%) No 42(68.9%) TMFP Positive 22(36.1%) Negative 39(63.9%) pCR Yes 13(35.1%) No 24(64.9%) Of the 61 patients, 20 (32.8%) were assessed as cCR, and 41 (67.2%) as non-cCR. In the cCR group (n = 20), TMFP was negative in 17 patients (85.0%). The majority of these (10/17, 58.8%) opted for W&W, while 7 patients (41.2%) chose surgery. TMFP was positive in 3 cCR patients (15%), 2 of whom underwent surgery. In the non-cCR group (n = 41), TMFP had a profound impact. Nearly half (20/41, 48.8%) were TMFP-negative. Of these, 10 patients (50%) chose W&W, while the other 10 (50%) proceeded to surgery. The remaining 21 non-cCR patients were TMFP-positive, and the vast majority (18/21, 85.7%) underwent radical surgery. Oncological Safety of Organ Preservation Guided by Clinical and TMFP Assessment We first sought to validate the oncological safety of the W&W strategy in established patient groups. With a median follow-up of 28.9 months, analysis of the 20 patients who achieved a clinical complete response (cCR) confirmed that the choice of treatment strategy—W&W versus surgery—did not lead to a significant difference in overall survival (P = 0.59, Fig. 1 A). This finding reinforces the safety of the current cCR-based paradigm for organ preservation. We then evaluated the safety of W&W in the broader cohort of patients identified as TMFP-negative, irrespective of their initial cCR status. Reassuringly, among all 37 TMFP-negative patients, there was no significant difference in OS between those who opted for W&W and those who underwent radical surgery (P = 0.40, Fig. 1 B). This pivotal finding establishes TMFP-negativity as a robust and safe criterion for offering organ preservation. Expanding the Boundaries: W&W in Non-cCR Patients with Negative TMFP The critical question was whether this safety could be extended to patients traditionally excluded from organ preservation. We therefore focused specifically on the subgroup of non-cCR patients who were TMFP-negative (n = 20). Strikingly, in this cohort, there was no significant difference in overall survival between the 10 patients who chose a W&W strategy and the 10 who underwent immediate surgery (P > 0.99, Fig. 1 C). The outcomes within this subgroup were particularly informative. Among the 10 patients who chose surgery, the postoperative pCR rate was 70% (7/10), providing strong pathological corroboration that TMFP successfully identifies a large subset of non-cCR patients with an excellent pathological response. Conversely, the 10 patients who chose W&W had a 100% 2-year survival rate with no deaths, no distant metastasis and no tumor regrowth during a mean follow-up of 32.9 months. This collective evidence robustly demonstrates that for non-cCR patients, a negative TMFP result can identify a subgroup for whom organ preservation is a safe and viable alternative to radical surgery. Superior Prognostic Stratification of TMFP Compared to cCR We further investigated the comparative prognostic value of TMFP and standard cCR assessment. Initial analysis showed that the 5-year OS was not significantly different between patients classified as cCR and those as non-cCR (P = 0.58, Fig. 2 A), highlighting the limited prognostic power of clinical assessment alone. In stark contrast, TMFP status provided powerful prognostic stratification across the entire cohort. Patients with a negative TMFP result had a significantly better OS than those with a positive TMFP (P = 0.04, Fig. 2 B). The prognostic dominance of TMFP was further cemented when we compared extreme groups. We found no significant survival difference between patients in the most favorable group by traditional standards [cCR(+)TMFP(-), n = 16] and those in a less favorable group [non-cCR(+)TMFP(+), n = 18] (P = 0.40, Fig. 2 C). This counterintuitive result indicates that the inherent inaccuracy of cCR assessment introduces significant confounding when combined with the precise pathological information from TMFP. TMFP as a Dynamic Guide for Conversion Therapy The study also provided insights into the dynamic use of TMFP. Two initially TMFP-positive patients (one cCR and one non-cCR) received consolidation chemotherapy. Repeat TMFP after chemotherapy showed conversion to negativity, and both patients subsequently qualified for and maintained disease-free survival under W&W. This highlights TMFP's potential role in monitoring response to additional therapy and guiding "conversion" to organ preservation. Procedure Safety The safety profile and functional impact of the TMFP procedure were rigorously evaluated. The procedure was well-tolerated across all patients. Five patients (8.2%) experienced intraoperative bleeding that was readily controlled—four via simple compression and one requiring suture ligation. No major procedural complications, such as perforation, or injuries to adjacent genitourinary structures, were recorded. Furthermore, no anesthesia-related adverse events occurred. Discussion This prospective multicenter study demonstrates that TMFP transcends its established role as a diagnostic tool to become a pivotal asset in clinical decision-making and prognostic stratification for LARC after neoadjuvant therapy. Our principal finding is that TMFP can safely expand the boundaries of organ preservation by identifying a substantial subgroup of non-cCR patients—comprising nearly half of this population in our cohort—who have a high probability of pCR and can achieve excellent survival outcomes with a W&W strategy. This moves the clinical application of TMFP beyond mere pathological confirmation into the realm of strategic therapeutic guidance. The current paradigm for organ preservation heavily relies on the cCR status, which is limited by subjectivity and imperfect accuracy 1 , 6 , 7 . Our data robustly challenge the long-standing dogma that "non-cCR necessitates surgery." We found that 48.8% of non-cCR patients were TMFP-negative, and importantly, those who opted for W&W had survival outcomes equivalent to their surgical counterparts within the TMFP-negative group. The high pCR rate (70%) in non-cCR/TMFP-negative patients who underwent surgery provides strong pathological corroboration for this strategy. This finding is pivotal, as it directly addresses a major source of clinical dilemma and potential overtreatment. It introduces a new, pathology-guided paradigm of "Selective Watch-and-Wait," which promises to significantly increase organ preservation rates without compromising oncological safety. This approach effectively de-escalates treatment for a large subset of patients who would have been previously committed to radical surgery based on clinical assessment alone. A second major contribution of our study is the demonstration of TMFP's superior prognostic power. While cCR status failed to stratify patient survival in our cohort, a finding consistent with previous studies 17 , 18 , TMFP results clearly distinguished between patients with favorable (TMFP-negative) and unfavorable (TMFP-positive) long-term outcomes. This suggests that the residual tumor burden detected by TMFP is a more biologically relevant marker of underlying disease aggressiveness and treatment resistance than the macroscopic and radiological features defining cCR. The fact that the integration of cCR status fails to enhance, and may even diminish, the clear prognostic stratification achieved by TMFP alone, underscoring TMFP's primacy as an independent prognostic biomarker, and TMFP might capture the essential prognostic information. Therefore, TMFP serves as a valuable standalone biomarker for risk-adapted management post-neoadjuvant therapy, potentially identifying TMFP-positive patients who might benefit from more intensive surveillance or adjuvant therapy, even after surgery. The dynamic application of TMFP, as seen in patients who converted from positive to negative after consolidation therapy, opens another exciting avenue. It positions TMFP not just as a static assessment tool, but as a dynamic monitoring platform to guide "conversion therapy." This capability is particularly relevant in the era of total neoadjuvant therapy (TNT) and novel agents, where the goal is to maximize response 19 . TMFP can provide an objective pathological endpoint to assess the efficacy of consolidation or intensification regimens, potentially enabling more patients to achieve organ preservation through response-adapted treatment strategies 20 . This aligns with the concept of "adaptive treatment strategies" in rectal cancer, moving towards a more personalized and iterative treatment mode 19 . When compared to other organ preservation (OP) strategies, TMFP demonstrates unique clinical advantages. In contrast to local excision, this technique not only causes minimal trauma, but more importantly, preserves intact anatomical structures. It avoids the technical challenges and surgical risks associated with post-excision scarring during subsequent salvage total meso-rectal excision (TME) surgery 21 , while offering superior anal sphincter preservation. Notably, local excision may compromise fecal continence, particularly in patients with low-lying tumors requiring extensive resection 22 ,23 . Additionally, TMFP's repeatable nature enables dynamic monitoring of treatment response, establishing a practical foundation for implementing the "extended waiting period" therapeutic concept 24,25 . Our study has limitations, including its non-randomized design and modest sample size, which preclude definitive conclusions regarding long-term oncological equivalence. TMFP cannot assess meso-rectal lymph node status, a known factor in prognosis, and sampling error, though reduced by multiple cores, remains a possibility. Future studies with larger cohorts and longer follow-up are warranted to validate our findings. Conclusions This study establishes that the value of TMFP extends far beyond diagnosing pCR. It is a powerful tool that directly influences and improves clinical practice by enabling safer organ preservation in a broader patient population and by providing critical, independent prognostic information. By objectively identifying both candidates for treatment de-escalation and patients with higher-risk disease, integrating TMFP into the post-neoadjuvant evaluation represents a significant step forward in the personalized, function-preserving management of rectal cancer. Declarations Author contributions Ye Chunxiang: Investigation; Methodology; writing original draft; data curation; writing–review and editing; investigation. Zhai Zhiwei: Methodology; data curation. Qu Hao : Methodology; data curation. Han jiagang: Methodology; data curation. Jiang Cong Qing: Investigation; methodology; writing–review and editing. Yong Dai: Investigation; methodology; writing–review and editing. Hong Wei Yao: Investigation; methodology; writing–review and editing. Zhen Jun Wang: Study design; Investigation; methodology; writing–review and editing. Funding None. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics statement The study was retrospectively registered (NCT04743102, BIOPSY-01 study). Ethical approval was granted by the Institutional Review Board of Beijing Chaoyang Hospital, Capital Medical University (2021-ke-6). 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 May, 2026 Reviews received at journal 02 May, 2026 Reviews received at journal 30 Apr, 2026 Reviewers agreed at journal 12 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers invited by journal 08 Apr, 2026 Editor assigned by journal 08 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 04 Apr, 2026 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. 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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-9320255","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622992398,"identity":"ad11d341-6c56-442f-ae15-3fae63849aa8","order_by":0,"name":"Chunxiang Ye","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunxiang","middleName":"","lastName":"Ye","suffix":""},{"id":622992399,"identity":"991ec645-c051-479b-9007-b105ead86f18","order_by":1,"name":"Zhiwei Zhai","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Zhai","suffix":""},{"id":622992400,"identity":"828a74e5-1623-4f51-ab36-e22981c08ffa","order_by":2,"name":"Hao Qu","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Qu","suffix":""},{"id":622992401,"identity":"6f2873c7-2980-4dbb-b484-17c2e3ec49c5","order_by":3,"name":"Jiagang Han","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiagang","middleName":"","lastName":"Han","suffix":""},{"id":622992402,"identity":"91a340c1-2541-4f7f-8e39-4340813590b0","order_by":4,"name":"Congqing Jiang","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Congqing","middleName":"","lastName":"Jiang","suffix":""},{"id":622992403,"identity":"f759e308-a7fc-4449-9379-91aca457d6a9","order_by":5,"name":"Yong Dai","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Dai","suffix":""},{"id":622992404,"identity":"371b0e9b-2e6c-4de6-8ce8-22c36153f837","order_by":6,"name":"Hongwei Yao","email":"","orcid":"","institution":"Beijing Friendship Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Yao","suffix":""},{"id":622992405,"identity":"9690e9e8-97b1-47c2-b0d6-32ed98a7c6c0","order_by":7,"name":"Zhenjun Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDCCAwxsQBKI2RsbH34gTQvP4WZjCRK0AIFEepsADzE6+G4ff/bg4w6+xO2SD9sYJBjs5HQbCGiRPJeQbjjzDFviztmJbQ8KGJKNzQ4Q0GJwhuGYNG8bW+KG24ntBhIMBxK3EdbC2Cb9F6Tl5sE2CR7itDCzSTOCtNxgJFKL5Bk2NsneNjbjDWcSgYFsQIRf+M6wP5P42XZMdsPx4w8ffqiwkyOoBQqOwdxJnHIQqCFe6SgYBaNgFIw8AAAmqkTdtAbZEwAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":true,"prefix":"","firstName":"Zhenjun","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-04-04 11:55:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9320255/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9320255/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107245500,"identity":"fe75e4ef-656f-4ee7-bb3e-912187bac04b","added_by":"auto","created_at":"2026-04-19 08:05:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOncological Safety of Organ Preservation Across Differently Selected Patient Groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Overall survival (OS) among patients with clinical complete response (cCR) choosing Watch-and-Wait (W\u0026amp;W) (n=10) vs. Surgery (n=8) (P=0.62).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eOS among all TMFP-negative patients choosing W\u0026amp;W (n=19) vs. Surgery (n=17) (P=0.39).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e OS among non-cCR patients with negative TMFP results choosing W\u0026amp;W (n=10) vs. Surgery (n=10) (P \u0026gt; 0.99).\u003c/p\u003e\n\u003cp\u003eSurvival curves were generated using the Kaplan-Meier method, and the log-rank test was used for comparisons.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9320255/v1/2cb8cf4cc3069a76633f22c4.png"},{"id":107484338,"identity":"39ac769f-669e-4117-a526-388908caa243","added_by":"auto","created_at":"2026-04-22 02:31:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":176529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrognostic Stratification Value of cCR Status versus TMFP Status.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eOverall \u003cstrong\u003esurvival\u003c/strong\u003e (OS) comparison between cCR (n=18) and non-cCR (n=41) groups (P=0.56).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eOS comparison between TMFP-negative (n=36) and TMFP-positive (n=23) patients (P=0.04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eOS comparison between cCR(+)TMFP(-) (n=15) and non-cCR(+)TMFP(+) (n=18) patients (P=0.42).\u003c/p\u003e\n\u003cp\u003eSurvival curves were generated using the Kaplan-Meier method, and the log-rank test was used for comparisons.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9320255/v1/ddd3cb94f32547e850b7cd22.png"},{"id":108005876,"identity":"c3537414-2fd2-498c-9d48-ec025fab5f62","added_by":"auto","created_at":"2026-04-28 12:49:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":380408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9320255/v1/931c325b-7d2d-4740-8b92-c4bfa329d177.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Trans-anal Multipoint Full-layer Puncture Biopsy Guides Organ Preservation and Improves Prognostic Stratification in Locally Advanced Rectal Cancer After Neoadjuvant Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe management of locally advanced rectal cancer (LARC) has evolved towards a more personalized approach, with organ preservation becoming a key goal for patients achieving a complete response to neoadjuvant therapy\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The \"watch-and-wait\" (W\u0026amp;W) strategy, initially proposed for patients with a clinical complete response (cCR), can avoid the morbidity associated with radical surgery and preserve quality of life\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, patient selection for W\u0026amp;W relies primarily on the assessment of clinical complete response (cCR) using a combination of digital rectal examination (DRE), endoscopy, and magnetic resonance imaging (MRI)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, these modalities are hampered by inherent limitations. DRE is highly subjective\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, endoscopy and mucosal biopsies are limited to evaluating the superficial layers of the bowel wall\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and MRI struggles to differentiate between post-treatment fibrosis and residual tumor cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Consequently, a significant proportion of patients with a pathological complete response (pCR) are misclassified as non-cCR and thus may undergo unnecessary surgery\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Conversely, some patients managed with W\u0026amp;W based on cCR experience local regrowth, underscoring the limitations of current assessment methods\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo bridge this gap between clinical assessment and pathological truth, our group developed the trans-anal multipoint full-layer puncture biopsy (TMFP). This technique involves obtaining multiple full-thickness tissue cores from the tumor bed under direct vision, providing a much more comprehensive pathological sampling than standard forceps biopsies. Previously, our team has reported the high diagnostic accuracy of TMFP for predicting pCR, establishing it as a superior pathological assessment tool compared to standard cCR criteria\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile establishing diagnostic accuracy is a crucial first step, it begs a more profound clinical question: How does the integration of this precise diagnostic tool into the clinical decision-making workflow ultimately influence patient management and long-term outcomes? Specifically, can TMFP identify a subgroup of patients traditionally deemed as non-cCR who can safely forego radical surgery and pursue organ preservation? Furthermore, does the pathological information provided by TMFP offer superior prognostic stratification compared to clinical assessment alone?\u003c/p\u003e \u003cp\u003eTherefore, this prospective multicenter study was designed not as another diagnostic accuracy study, but to evaluate the clinical utility of TMFP. We focus specifically on its role in guiding therapeutic decisions\u0026mdash;particularly in expanding organ preservation opportunities to non-cCR patients\u0026mdash;and its power in predicting long-term survival in LARC patients following neoadjuvant therapy. This cohort study has been reported in line with the STROCSS guidelines\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Participants\u003c/h2\u003e \u003cp\u003eThis was a prospective, multicenter, non-randomized cohort study conducted across four tertiary hospitals in China, namely Beijing Chaoyang Hospital, Capital Medical University (Center 1, n\u0026thinsp;=\u0026thinsp;34), Beijing Friendship Hospital, Capital Medical University (Center 2, n\u0026thinsp;=\u0026thinsp;5), Qilu Hospital of Shandong University (Center 3, n\u0026thinsp;=\u0026thinsp;17), and Zhongnan Hospital of Wuhan University (Center 4, n\u0026thinsp;=\u0026thinsp;5). The study specifically enrolled a consecutive cohort of patients who underwent \u003cem\u003ein vivo\u003c/em\u003e TMFP, reflecting its application in the real-world clinical decision-making context. Between 2021 and 2025, 61 patients with LARC who completed neoadjuvant chemoradiotherapy (nCRT) with or without immunotherapy were included. The inclusion criteria and exclusion criteria were the same as those described previously\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This study was approved by the institutional review boards of four hospitals, and all patients provided written informed consent.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeoadjuvant Therapy and Clinical Assessment\u003c/h3\u003e\n\u003cp\u003eNeoadjuvant therapy consisted of long-course radiotherapy (45-50.4 Gy in 25\u0026ndash;28 fractions) with concurrent capecitabine-based chemotherapy. A subset of patients received additional immune checkpoint inhibitors as part of clinical trials or off-label use. Clinical response was assessed 8\u0026ndash;12 weeks after radiotherapy completion using DRE, endoscopy, and pelvic MRI. cCR was defined according to the Chinese Watch and Wait Database (CW\u0026amp;WD) criteria\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Due to the small sample size, for the convenience of statistical analysis, and considering the significant differences between near-cCR patients and cCR patients in terms of local recurrence rate and 5-year organ preservation rate\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, near-cCR patients were classified as non-cCR for statistical analysis in this paper.\u003c/p\u003e\n\u003ch3\u003eTMFP Procedure and Pathological Evaluation\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e TMFP procedure was performed as previously described [11]. Briefly, under anesthesia and with adequate bowel preparation, a 16G core biopsy needle was used to obtain full-thickness tissue samples from at least 13 predetermined sites around the original tumor bed or scar under direct vision. The puncture angle was 60\u0026deg;-70\u0026deg; with a depth of 0.8-1.0 cm. Hemostasis was achieved by direct pressure or electrocautery. A result was considered TMFP-positive if any specimen contained viable tumor cells; otherwise, it was TMFP-negative. To ensure procedural and data consistency across centers, a standardized protocol was implemented. All surgeons underwent centralized TMFP training and submitted procedural videos for approval to minimize inter-operator variability. For the intervention itself, all TMFP and surgical specimens were independently reviewed by two blinded pathologists, with consensus resolution for discrepancies.\u003c/p\u003e\n\u003ch3\u003eTreatment Decision and Follow-up\u003c/h3\u003e\n\u003cp\u003eFollowing the TMFP procedure, a comprehensive treatment recommendation was made based on a synthesis of clinical assessment (cCR status) and TMFP results. Patients were thoroughly counseled, and the final choice between radical surgery (Total meso-rectal excision, TME) or a W\u0026amp;W strategy was made jointly by the physician and patient. Patients opting for W\u0026amp;W underwent intensified follow-up with DRE, endoscopy, MRI, and serum carcinoembryonic antigen (CEA) testing every 3\u0026ndash;6 months. Overall survival (OS) was defined as the time from the date of TMFP to death from any cause or last follow-up.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS 26.0 (IBM Corporation, Chicago, IL, USA). Categorical variables were described as frequencies and percentages. The Kaplan-Meier method was used to generate survival curves, and differences were compared using the log-rank test. A two-sided P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePatient Characteristics and TMFP-guided Treatment Pathways\u003c/h2\u003e \u003cp\u003eA total of 61 patients with a mean age of 59.8 years were included. All had mid-low stage III rectal cancer and received nCRT, with 8.2% (5/61) receiving additional immunotherapy. The clinicopathological characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eClinicopathological characteristics of the patients\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber(%)\u003c/p\u003e \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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42(68.85%)\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19(31.15%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\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\u003e\u0026lt;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26(42.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35(57.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance from the lower edge of the tumor to the anal verge\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\u003e\u0026lt;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35(57.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26(42.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecT stage\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\u003ecT1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9(14.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41(67.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11(18.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecN stage\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\u003ecN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15(24.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecN1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46(75.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInterval between radiotherapy and biopsy (weeks)\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\u003e\u0026lt;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9(14.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52(85.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmune Checkpoint Inhibitors (ICIs)\u0026zwnj;\u0026zwnj;\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\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5(8.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56(91.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eycT stage\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\u003ecT0-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16(26.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13(21.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3(4.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19(31.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10(16.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eycN stage\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\u003ecN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30(49.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecN1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9(14.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17(27.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5(8.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eccR\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\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19(31.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42(68.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMFP\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\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22(36.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39(63.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epCR\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\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13(35.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24(64.9%)\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\u003eOf the 61 patients, 20 (32.8%) were assessed as cCR, and 41 (67.2%) as non-cCR. In the cCR group (n\u0026thinsp;=\u0026thinsp;20), TMFP was negative in 17 patients (85.0%). The majority of these (10/17, 58.8%) opted for W\u0026amp;W, while 7 patients (41.2%) chose surgery. TMFP was positive in 3 cCR patients (15%), 2 of whom underwent surgery.\u003c/p\u003e \u003cp\u003eIn the non-cCR group (n\u0026thinsp;=\u0026thinsp;41), TMFP had a profound impact. Nearly half (20/41, 48.8%) were TMFP-negative. Of these, 10 patients (50%) chose W\u0026amp;W, while the other 10 (50%) proceeded to surgery. The remaining 21 non-cCR patients were TMFP-positive, and the vast majority (18/21, 85.7%) underwent radical surgery.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOncological Safety of Organ Preservation Guided by Clinical and TMFP Assessment\u003c/h3\u003e\n\u003cp\u003eWe first sought to validate the oncological safety of the W\u0026amp;W strategy in established patient groups. With a median follow-up of 28.9 months, analysis of the 20 patients who achieved a clinical complete response (cCR) confirmed that the choice of treatment strategy\u0026mdash;W\u0026amp;W versus surgery\u0026mdash;did not lead to a significant difference in overall survival (P\u0026thinsp;=\u0026thinsp;0.59, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This finding reinforces the safety of the current cCR-based paradigm for organ preservation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then evaluated the safety of W\u0026amp;W in the broader cohort of patients identified as TMFP-negative, irrespective of their initial cCR status. Reassuringly, among all 37 TMFP-negative patients, there was no significant difference in OS between those who opted for W\u0026amp;W and those who underwent radical surgery (P\u0026thinsp;=\u0026thinsp;0.40, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This pivotal finding establishes TMFP-negativity as a robust and safe criterion for offering organ preservation.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExpanding the Boundaries: W\u0026amp;W in Non-cCR Patients with Negative TMFP\u003c/h2\u003e \u003cp\u003eThe critical question was whether this safety could be extended to patients traditionally excluded from organ preservation. We therefore focused specifically on the subgroup of non-cCR patients who were TMFP-negative (n\u0026thinsp;=\u0026thinsp;20). Strikingly, in this cohort, there was no significant difference in overall survival between the 10 patients who chose a W\u0026amp;W strategy and the 10 who underwent immediate surgery (P\u0026thinsp;\u0026gt;\u0026thinsp;0.99, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The outcomes within this subgroup were particularly informative. Among the 10 patients who chose surgery, the postoperative pCR rate was 70% (7/10), providing strong pathological corroboration that TMFP successfully identifies a large subset of non-cCR patients with an excellent pathological response. Conversely, the 10 patients who chose W\u0026amp;W had a 100% 2-year survival rate with no deaths, no distant metastasis and no tumor regrowth during a mean follow-up of 32.9 months. This collective evidence robustly demonstrates that for non-cCR patients, a negative TMFP result can identify a subgroup for whom organ preservation is a safe and viable alternative to radical surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSuperior Prognostic Stratification of TMFP Compared to cCR\u003c/h2\u003e \u003cp\u003eWe further investigated the comparative prognostic value of TMFP and standard cCR assessment. Initial analysis showed that the 5-year OS was not significantly different between patients classified as cCR and those as non-cCR (P\u0026thinsp;=\u0026thinsp;0.58, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), highlighting the limited prognostic power of clinical assessment alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn stark contrast, TMFP status provided powerful prognostic stratification across the entire cohort. Patients with a negative TMFP result had a significantly better OS than those with a positive TMFP (P\u0026thinsp;=\u0026thinsp;0.04, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe prognostic dominance of TMFP was further cemented when we compared extreme groups. We found no significant survival difference between patients in the most favorable group by traditional standards [cCR(+)TMFP(-), n\u0026thinsp;=\u0026thinsp;16] and those in a less favorable group [non-cCR(+)TMFP(+), n\u0026thinsp;=\u0026thinsp;18] (P\u0026thinsp;=\u0026thinsp;0.40, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This counterintuitive result indicates that the inherent inaccuracy of cCR assessment introduces significant confounding when combined with the precise pathological information from TMFP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTMFP as a Dynamic Guide for Conversion Therapy\u003c/h2\u003e \u003cp\u003eThe study also provided insights into the dynamic use of TMFP. Two initially TMFP-positive patients (one cCR and one non-cCR) received consolidation chemotherapy. Repeat TMFP after chemotherapy showed conversion to negativity, and both patients subsequently qualified for and maintained disease-free survival under W\u0026amp;W. This highlights TMFP's potential role in monitoring response to additional therapy and guiding \"conversion\" to organ preservation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProcedure Safety\u003c/h2\u003e \u003cp\u003eThe safety profile and functional impact of the TMFP procedure were rigorously evaluated. The procedure was well-tolerated across all patients. Five patients (8.2%) experienced intraoperative bleeding that was readily controlled\u0026mdash;four via simple compression and one requiring suture ligation. No major procedural complications, such as perforation, or injuries to adjacent genitourinary structures, were recorded. Furthermore, no anesthesia-related adverse events occurred.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis prospective multicenter study demonstrates that TMFP transcends its established role as a diagnostic tool to become a pivotal asset in clinical decision-making and prognostic stratification for LARC after neoadjuvant therapy. Our principal finding is that TMFP can safely expand the boundaries of organ preservation by identifying a substantial subgroup of non-cCR patients\u0026mdash;comprising nearly half of this population in our cohort\u0026mdash;who have a high probability of pCR and can achieve excellent survival outcomes with a W\u0026amp;W strategy. This moves the clinical application of TMFP beyond mere pathological confirmation into the realm of strategic therapeutic guidance.\u003c/p\u003e \u003cp\u003eThe current paradigm for organ preservation heavily relies on the cCR status, which is limited by subjectivity and imperfect accuracy\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Our data robustly challenge the long-standing dogma that \"non-cCR necessitates surgery.\" We found that 48.8% of non-cCR patients were TMFP-negative, and importantly, those who opted for W\u0026amp;W had survival outcomes equivalent to their surgical counterparts within the TMFP-negative group. The high pCR rate (70%) in non-cCR/TMFP-negative patients who underwent surgery provides strong pathological corroboration for this strategy. This finding is pivotal, as it directly addresses a major source of clinical dilemma and potential overtreatment. It introduces a new, pathology-guided paradigm of \"Selective Watch-and-Wait,\" which promises to significantly increase organ preservation rates without compromising oncological safety. This approach effectively de-escalates treatment for a large subset of patients who would have been previously committed to radical surgery based on clinical assessment alone.\u003c/p\u003e \u003cp\u003eA second major contribution of our study is the demonstration of TMFP's superior prognostic power. While cCR status failed to stratify patient survival in our cohort, a finding consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, TMFP results clearly distinguished between patients with favorable (TMFP-negative) and unfavorable (TMFP-positive) long-term outcomes. This suggests that the residual tumor burden detected by TMFP is a more biologically relevant marker of underlying disease aggressiveness and treatment resistance than the macroscopic and radiological features defining cCR. The fact that the integration of cCR status fails to enhance, and may even diminish, the clear prognostic stratification achieved by TMFP alone, underscoring TMFP's primacy as an independent prognostic biomarker, and TMFP might capture the essential prognostic information. Therefore, TMFP serves as a valuable standalone biomarker for risk-adapted management post-neoadjuvant therapy, potentially identifying TMFP-positive patients who might benefit from more intensive surveillance or adjuvant therapy, even after surgery.\u003c/p\u003e \u003cp\u003eThe dynamic application of TMFP, as seen in patients who converted from positive to negative after consolidation therapy, opens another exciting avenue. It positions TMFP not just as a static assessment tool, but as a dynamic monitoring platform to guide \"conversion therapy.\" This capability is particularly relevant in the era of total neoadjuvant therapy (TNT) and novel agents, where the goal is to maximize response\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. TMFP can provide an objective pathological endpoint to assess the efficacy of consolidation or intensification regimens, potentially enabling more patients to achieve organ preservation through response-adapted treatment strategies\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This aligns with the concept of \"adaptive treatment strategies\" in rectal cancer, moving towards a more personalized and iterative treatment mode\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen compared to other organ preservation (OP) strategies, TMFP demonstrates unique clinical advantages. In contrast to local excision, this technique not only causes minimal trauma, but more importantly, preserves intact anatomical structures. It avoids the technical challenges and surgical risks associated with post-excision scarring during subsequent salvage total meso-rectal excision (TME) surgery\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, while offering superior anal sphincter preservation. Notably, local excision may compromise fecal continence, particularly in patients with low-lying tumors requiring extensive resection\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,23\u003c/sup\u003e. Additionally, TMFP's repeatable nature enables dynamic monitoring of treatment response, establishing a practical foundation for implementing the \"extended waiting period\" therapeutic concept\u003csup\u003e24,25\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study has limitations, including its non-randomized design and modest sample size, which preclude definitive conclusions regarding long-term oncological equivalence. TMFP cannot assess meso-rectal lymph node status, a known factor in prognosis, and sampling error, though reduced by multiple cores, remains a possibility. Future studies with larger cohorts and longer follow-up are warranted to validate our findings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study establishes that the value of TMFP extends far beyond diagnosing pCR. It is a powerful tool that directly influences and improves clinical practice by enabling safer organ preservation in a broader patient population and by providing critical, independent prognostic information. By objectively identifying both candidates for treatment de-escalation and patients with higher-risk disease, integrating TMFP into the post-neoadjuvant evaluation represents a significant step forward in the personalized, function-preserving management of rectal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYe Chunxiang:\u003c/strong\u003e Investigation; Methodology; writing original draft; data curation; writing\u0026ndash;review and editing; investigation. \u003cstrong\u003eZhai Zhiwei:\u003c/strong\u003e Methodology; data curation. \u003cstrong\u003eQu Hao\u003c/strong\u003e: Methodology; data curation. \u003cstrong\u003eHan jiagang:\u0026nbsp;\u003c/strong\u003eMethodology; data curation. \u003cstrong\u003eJiang Cong Qing:\u003c/strong\u003e Investigation; methodology; writing\u0026ndash;review and editing. \u003cstrong\u003eYong Dai:\u0026nbsp;\u003c/strong\u003eInvestigation; methodology; writing\u0026ndash;review and editing. \u003cstrong\u003eHong Wei Yao:\u0026nbsp;\u003c/strong\u003eInvestigation; methodology; writing\u0026ndash;review and editing. \u003cstrong\u003eZhen Jun Wang:\u003c/strong\u003e Study design; Investigation; methodology; writing\u0026ndash;review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was retrospectively registered (NCT04743102, BIOPSY-01 study). Ethical approval was granted by the Institutional Review Board of Beijing Chaoyang Hospital, Capital Medical University (2021-ke-6). All patients gave informed consent for the use of their data in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMaas M, Lambregts DM, Nelemans PJ et al (2015) Assessment of Clinical Complete Response After Chemoradiation for Rectal Cancer with Digital Rectal Examination, Endoscopy, and MRI: Selection for Organ-Saving Treatment. 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Front Oncol 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2020.01280\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2020.01280\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Locally advanced rectal cancer, Neoadjuvant therapy, Trans-anal multipoint full-layer puncture biopsy, Watch-and-wait, Organ preservation","lastPublishedDoi":"10.21203/rs.3.rs-9320255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9320255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eTo evaluate the value of trans-anal multipoint full-layer puncture biopsy (TMFP) in guiding organ preservation strategies and prognostic stratification for locally advanced rectal cancer (LARC) after neoadjuvant therapy.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eThis prospective multicenter study was conducted across four tertiary centers in China. Sixty-one LARC patients after neoadjuvant chemoradiotherapy (\u0026plusmn;\u0026thinsp;immunotherapy) who underwent \u003cem\u003ein vivo\u003c/em\u003e TMFP were enrolled. Primary outcomes were TMFP-guided changes in treatment strategy (Watch-and-Wait, W\u0026amp;W, vs. surgery) and overall survival (OS).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong 61 patients, 20 achieved clinical complete response (cCR) and 41 were non-cCR. TMFP results significantly altered treatment decisions in non-cCR patients, with 48.8% (20/41) being TMFP-negative; half of these (10/20) successfully adopted a W\u0026amp;W strategy. The postoperative pCR rate in non-cCR but TMFP-negative patients who underwent surgery was 70% (7/10). TMFP demonstrated superior prognostic stratification compared to cCR: the OS of TMFP-negative patients was significantly better than that of TMFP-positive patients (P\u0026thinsp;=\u0026thinsp;0.04), whereas no significant difference was found between cCR and non-cCR groups (P\u0026thinsp;=\u0026thinsp;0.58). Among TMFP-negative patients, no survival difference was observed between the W\u0026amp;W and surgery groups (P\u0026thinsp;=\u0026thinsp;0.40). The procedure was well-tolerated with a low complication rate.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTMFP serves as a pivotal clinical decision-making tool that safely expands organ preservation opportunities to a significant subset of non-cCR patients and provides powerful prognostic stratification, outperforming traditional cCR assessment. Integrating TMFP into the post-neoadjuvant workflow optimizes the balance between oncological safety and functional preservation.\u003c/p\u003e","manuscriptTitle":"Trans-anal Multipoint Full-layer Puncture Biopsy Guides Organ Preservation and Improves Prognostic Stratification in Locally Advanced Rectal Cancer After Neoadjuvant Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-19 08:05:32","doi":"10.21203/rs.3.rs-9320255/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-03T06:59:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T19:01:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T02:28:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255879993270842490379541647209275345970","date":"2026-04-13T02:39:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65117593644946541203587211112856512366","date":"2026-04-10T17:52:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T17:24:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T05:47:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T05:47:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Colorectal Disease","date":"2026-04-04T11:48:47+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":"40df8174-53e5-4589-90d2-ee4fd73fbd8e","owner":[],"postedDate":"April 19th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-03T06:59:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T19:01:34+00:00","index":65,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T02:28:16+00:00","index":64,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T05:38:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-19 08:05:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9320255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9320255","identity":"rs-9320255","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}