BACKGROUND:Colorectal cancer (CRC) is characterized by aberrant mucin expression, but the full spectrum of mucin profiles and their clinical relevance remain unclear. Here, we developed a novel mucin-based CRC subtyping using multiple datasets to tailor therapeutic strategies. METHODS:Mucin-related signatures were used to stratify CRC into four novel mucin-based subtypes: metabolism initiated, inflammatory, immune activated, and progressing. Highly accurate predictions were achieved, with under the curve (AUC) values of 0.977 for PC1 and 0.997 for PC2. RESULTS:The novel subtypes demonstrated prognostic potential in predicting CRC patient survival outcomes. We identified interactions between mucin-based subtypes and tumor-associated macrophages (TAMs). Mechanistic research revealed that MUC20 overexpression promoted M2 macrophage-dependent tumor glycolysis and hypoxia. Specifically, M2 phenotype TAMs secreted TNF-α, which enhanced tumor glycolysis and upregulated MUC20 expression in cancer cells. This established a MUC20/glycolysis positive feedback loop that exacerbated hypoxia and aerobic glycolysis in CRC. Additionally, chemotherapy responses in patient organoids were highly matched to these subtypes. Patients in cluster 2 were sensitive to 5-Fu or CPT-11 but potentially resistant to routine FOLFOX treatment. CONCLUSION:We identified a novel four mucin-characterized subtypes, which advance precision medicine by refining chemotherapy regimen selection. Our study provides valuable insights for tailoring therapeutic strategies.

ABSTRACT Organoids are innovative three‐dimensional (3D) cellular constructs, offering a unique platform to replicate the architectural and functional complexity of organs and tissues. In oncology, the tumor microenvironment (TME) dictates tumor evolution and therapeutic resistance. Consequently, therapies targeting TME components have emerged as a burgeoning frontier in cancer treatment. However, accurately recapitulating the dynamic, multicellular crosstalk of TME remains a significant hurdle for clinical translation. This review encapsulates the spectrum of current organoid coculture methodologies, ranging from direct coculture and air–liquid interface to advanced microfluidics and 3D bioprinting. These models not only deepen our understanding of the fundamental mechanisms at play in cancer but also evaluate emerging therapeutic modalities, such as antibody–drug conjugates and immunotherapy. By closely mimicking the in vivo tumor milieu, organoid cocultures enhance our ability to predict therapeutic outcomes and pave the way for the development of precision medicine approaches, thereby propelling forward the frontiers of oncology. This review aims to provide a comprehensive overview of organoid coculture models, spanning from construction methodologies to clinical applications. We envision this work serving as a definitive guide for the field, ultimately accelerating the transition from theoretical research to clinical practice.

3644 Background: In patients with proficient mismatch repair (pMMR) locally advanced rectal cancer (LARC), the integration of PD-1 inhibitor into total neoadjuvant chemoradiotherapy (iTNT) has demonstrated promising potential to enhance complete response (CR) rates and facilitate organ preservation. However, the combined use of radiotherapy and surgical intervention frequently leads to increased functional impairment relative to either modality administered independently. Concurrently, the combination of chemotherapy and PD-1 inhibitor has been shown to promote tumor downstaging, thereby enabling sphincter-preserving surgery while mitigating radiation-induced injury. This trial evaluates oncological efficacy and functional outcomes of two iTNT regimens: CAPOX plus PD-1 inhibitor with or without SCRT in neoadjuvant treatment of pMMR LARC patients. Methods: The TORCH-iTNT trial (NCT06281405) is a prospective, multicenter, randomized phase II study involving 192 patients with pMMR LARC (T3-4/N+M0). Participants were randomized into Group A (6 cycles of CAPOX plus toripalimab) or Group B (SCRT [25Gy/5Fx] followed by 6 cycles of CAPOX plus toripalimab). Patients achieving clinical complete response (cCR) were offered a watch-and-wait (W&W) strategy, while those without cCR were advised to undergo surgery. Group A patients with positive circumferential margins received adjuvant chemoradiotherapy. The primary endpoint was CR rate (pathological complete response [pCR] plus cCR). Secondary endpoints included organ preservation rate, anorectal function, adverse effects, and survival outcomes. Results: By December 31, 2025, 192 patients were enrolled, with 137 completing treatment (Group A: 65; Group B: 72). Baseline characteristics were balanced, with 97.1% (133/137) exhibiting at least one high-risk feature: lower tumor location (≤5cm), cT4, cN2, MRF+, or EMVI+. In Group A, 5 patients achieved cCR and adopted W&W, compared to 22 in Group B. Of the 54 and 49 patients in Group A and B who underwent surgery, pCR was observed in 19 (35.2%) and 22 (44.9%) cases, respectively. Five non-cCR patients in Group A declined surgery or were still undergoing treatment. Disease progression occurred in one patient per group. The overall CR rates were 36.9% (24/65) in Group A and 61.1% (44/72) in Group B. The most frequent grade 3-4 toxicity was thrombocytopenia (Group A: 12.3%; Group B: 12.5%). Conclusions: This study is the first comparative analysis of two iTNT regimens in LARC. Unprecedentedly, neoadjuvant CAPOX plus PD-1 inhibitor achieved a promising CR rate in pMMR LARC, and this CR rate can be further enhanced with the addition of SCRT prior to immunochemotherapy. Further follow-up is necessary to assess long-term efficacy and functional endpoints. Clinical trial information: NCT06281405 .

PURPOSE:We hypothesized that a dynamic surveillance strategy guided by circulating tumor DNA (ctDNA) methylation would increase the rate of curative-intent therapy for recurrence in patients with nonmetastatic colorectal cancer (CRC) after curative resection. METHODS:The FIND trial (ClinicalTrials.gov identifier: NCT05904665) is a prospective, multicenter, randomized, phase III study. Patients with nonmetastatic CRC were randomly assigned to ctDNA-guided surveillance or standard computed tomography (CT)-based monitoring. In the ctDNA-guided group, a positive ctDNA result triggered immediate CT imaging; if negative, bimonthly CT continued alongside quarterly ctDNA testing. After two consecutive ctDNA-negative results, imaging reverted to standard frequency. The primary end point was the proportion of patients with recurrence receiving curative-intent metastasis-directed therapy. RESULTS:Among 584 eligible patients (289 ctDNA-guided, 295 control) in the modified intention-to-treat population, with a median follow-up of 23.3 months, recurrence rates were similar (18.0% v 18.6%, P = .919). The ctDNA-guided group had a significantly higher rate of curative-intent treatment (48.1% v 23.6%, relative risk 2.03, P = .008). The median time to clinical recurrence was significantly shorter in the ctDNA-guided group than in the control group (9.5 v 13.4 months; P < .001), representing a lead time of 3.9 months. Among recurrences confined to the liver and/or lungs, the ctDNA-guided group showed higher curative resection rates (42.3% v 18.2%, P = .002). These patients had more favorable hepatic metastatic features: fewer lesions (≤3: 75.0% v 28.6%, P = .005), smaller tumor size (≤3 cm: 90.0% v 57.1%, P = .033), and more unilobar disease (80.0% v 28.6%, P = .002). CONCLUSION:ctDNA methylation-guided dynamic surveillance improves the rate of curative-intent therapy for recurrence in patients with initially nonmetastatic CRC through earlier detection of resectable metastases, pending validation of long-term survival benefit in future analyses with mature data.

BACKGROUND:Current therapeutic outcomes for advanced colorectal cancer (CRC) remain suboptimal, and chemotherapy-based regimens continue to be the mainstay of treatment. Circular RNAs (circRNAs) can serve as templates for translating short peptides or proteins, and the resulting products actively regulate malignant tumour progression, making them attractive therapeutic targets. METHODS:We identified the novel protein PVT1-104aa translated from circPVT1, which is generated by back-splicing of the non-coding PVT1 gene. Its expression and clinical significance were examined in CRC clinical specimens and cell lines. Proliferation, metastasis, and tumour growth were assessed by CCK-8, colony formation, transwell, wound healing, and xenograft syngeneic tumour models. Mechanistic studies were performed by immunoprecipitation, ubiquitination assays, and protein half-life analysis. The relationship between PVT1-104aa, c-Myc, and PD-L1 was evaluated by promoter reporter assays, ChIP-qPCR, and immunohistochemistry. The therapeutic efficacy of combining PVT1-104aa inhibition with anti-PD-L1 therapy was tested in vivo. RESULTS:PVT1-104aa was significantly overexpressed in CRC and correlated with poor patient prognosis. Functionally, it drove tumour progression by promoting proliferation and metastasis. Mechanistically, PVT1-104aa enhanced c-Myc phosphorylation at Ser62, disrupted the c-Myc-FBW7 interaction, and thereby inhibited ubiquitin-mediated degradation of c-Myc, as shown by accelerated c-Myc turnover upon PVT1-104aa knockdown. In addition, PVT1-104aa regulated PD-L1 expression through c-Myc. Combining anti-PVT1-104aa with anti-PD-L1 therapy suppressed CRC growth and increased CD4+ and CD8+ T cell infiltration in xenograft syngeneic tumour models and CRC tissues. CONCLUSIONS:Our results uncover a pathogenic role of the PVT1-originated molecular species PVT1-104aa and suggest that targeting this pathway represents a promising therapeutic strategy for CRC treatment. KEY POINTS:Circ-PVT1 is upregulated in CRC patients and encoded a novel protein: PVT1-104aa. PVT1-104aa promotes CRC progression and predicts worse prognosis. PVT1-104aa enhance Myc expression through inhibition of Myc ubiquitination. PVT1-104aa regulate PD-L1 expression of CRC cells and modulate T cells infiltration in vivo.

The efficacy of immune checkpoint blockade (ICB) therapy in colorectal cancer (CRC) remains limited. Thus, elucidating the molecular mechanisms underlying tumor immune evasion and identifying novel predictive biomarkers are critical for improving immunotherapy outcomes in CRC patients. Here, we report that ubiquitin-specific peptidase 21 (USP21) is overexpressed in tumor tissues from CRC patients who fail to respond to immunotherapy and that its expression is correlated with CD8+ T-cell exclusion and impaired antitumor immunity. To functionally validate these clinical observations, we employed syngeneic mouse models of both microsatellite instability-high (MSI-H) and microsatellite stable (MSS) CRC. Genetic ablation of Usp21 or treatment with the USP21 inhibitor BAY-805 significantly enhanced tumor-reactive CD8+ T-cell responses, suppressed tumor progression, and synergized with anti-PD-1 therapy. Mechanistically, USP21 stabilized β-catenin by removing K48-linked ubiquitin chains, enabling its nuclear translocation and binding to the ATF3 promoter to upregulate ATF3 expression. ATF3 subsequently mediated transcriptional repression of the chemokine CCL4, thereby limiting CD8⁺ T-cell trafficking and function via the CCL4-CCR5 axis. Importantly, the therapeutic potential of targeting USP21 to enhance anti-PD-1 efficacy was further validated in huCD34+ humanized mice. Collectively, our findings identify USP21 as a pivotal regulator of immune evasion, and inhibiting USP21 represents a promising adjuvant strategy to increase the efficacy of ICB in CRC.

BackgroundThe optimal choice of chemotherapy regimen for resectable colorectal liver metastases (CRLM) is still undetermined. The study aimed to estimate the impact of perioperative or adjuvant chemotherapy on disease-free survival (DFS), and explore the underlying mechanisms which influence chemotherapy efficacy.MethodsEight hundred twenty-two patients undergoing curative resection of CRLM were retrospectively collected from May 2018 to December 2023. Treatment effects between perioperative and adjuvant chemotherapy were compared in full subgroup analyses by Kaplan–Meier and Cox proportional hazards methods. Single-cell RNA sequencing and tumor derived organoids were used to explore the molecular mechanisms.ResultsAfter propensity score matching, 630 patients were enrolled with ratio 1:1 in adjuvant and perioperative chemotherapy group. The median DFS in adjuvant and perioperative group was comparable with 28 and 32.5 months, respectively (HR = 0.99, P = 0.945). Perioperative chemotherapy was associated with improved DFS (25 vs. 13 months, P = 0.039) in patients with a clinical risk score (CRS) of 5. Notably, within the high-risk CRS 4-5 population receiving perioperative chemotherapy, patients with chronic hepatitis B (CHB) had significantly worse DFS (HR = 4.31, 95% CI 1.93–9.64; P < 0.001). Single-cell analyses revealed TSPAN8+ stem-like epithelial cells were enriched in sample with CHB. TSPAN8 knockdown sensitized colorectal cancer cells to 5-fluorouracil, and anti-TSPAN8 antibody showed synergistic efficacy with 5-fluorouracil in patient-derived liver metastasis organoids. In addition, the enhanced SPP1-CD44 signaling between TSPAN8+ epithelial cells and SPP1+ macrophages also contributed to 5-fluorouracil resistance.ConclusionsHigher CRS identify a subgroup of CRLM patients who may derive greater benefit from perioperative chemotherapy. HBV-related serological profiles is a potential predictor of chemotherapy resistance. The exploratory findings warrants further validation.

3634 Background: The prognosis for locally advanced colon cancer (LACC) with bulky nodal disease and/or clinically T4 stage remains poor, with high rates of recurrence and metastasis. Immunotherapy has become the standard treatment for deficient mismatch repair or microsatellite instability-high (dMMR/MSI-H) colon cancer. However, there has been little progress for the majority of cases, which was mismatch-repair proficient or microsatellite stable (pMMR/MSS). For locally advanced colon cancer with T4b stage or bulky lymph nodes, neoadjuvant chemotherapy is currently widely used. Whether combining it with immunotherapy and radiotherapy can improve efficacy is worthy of exploration. TORCH-C is the first study to evaluate this combination. Methods: TORCH-C is a prospective, multicenter, randomized phase 2 study that enrolled patients from April 3, 2023, through September 9, 2025. A total of 120 high-risk LACC (T4/bulky N+M0，pMMR/MSS) patients were 1:1 randomized to either an immunotherapy combined with SCRT and CAPOX chemotherapy group (group A) or a chemotherapy alone group (group B). Patients in the group B received 4 cycles of CAPOX (oxaliplatin 130 mg/m 2 intravenously day 1 and capecitabine 1000 mg/m 2 orally days 1-14). Patients in the group A received SCRT (25Gy in 5 fraction) and 4 cycles of the PD-1 inhibitor (serplulimab, 300mg intravenously day 1) combined with CAPOX. The radiotherapy target volume includes only the primary colon tumor and enlarged lymph nodes, without elective nodal irradiation. After neoadjuvant therapy, patients will be evaluated for radical colon resection. All patients will receive adjuvant chemotherapy of four cycles of CAPOX. The primary outcome was pathological complete response (pCR) rate. The secondary outcomes included tumor downstaging, R0 resection, 3-year disease free survival (DFS), 3-year overall survival (OS), 3-year local recurrence-free survival and treatment-related toxicity. Results: 120 patients have been enrolled and randomized. 107 patients were included in the mITT analysis (54 in group A and 53 in group B). 103 have received surgery (50 in the chemotherapy group, 53 in the radiotherapy group). The pCR rate was 10% (5/50) in chemotherapy group (group B) and 45.3% (24/53) in the radiotherapy group (group A) ( P <0.05). The most common grade 3-4 adverse event (AE) among patients was thrombocytopenia, (22.2%, 12/54) in the group A and (18.9%, 10/53) in the group B. Conclusions: The combination of PD-1 inhibitor, SCRT, and chemotherapy shows promising efficacy and significantly improved pCR rates in patients with MSS/pMMR high-risk LACC. This regimen may provide a new therapeutic option to achieve R0 resection and improve long-term survival. Clinical trial information: NCT05732493 .

BACKGROUND:BRAFV600E-mutant colorectal cancer (CRC) represents an aggressive molecular subtype characterized by poor prognosis and resistance to standard therapies. Predictive models to guide treatment selection and overcome drug resistance are urgently needed. METHODS:We established a biobank of patient-derived organoids (PDOs) and matched xenografts (PDOXs) from BRAFV600E-mutant CRC, capturing inter- and intratumoral heterogeneity. PDOs were characterized by histopathology, whole-exome sequencing, bulk and single-cell RNA sequencing, and drug sensitivity testing to chemotherapies and targeted agents. Immunogenicity was assessed via PDO-T cell co-culture and humanized xenograft models. RESULTS:PDOs faithfully preserved parental tumor histology, mismatch repair status, mutational landscapes, and copy number alterations (91.6% concordance in driver genes). Single-cell transcriptomics confirmed high fidelity between PDOs and original tumors. Drug sensitivity assays revealed marked interpatient heterogeneity and accurately recapitulated clinical responses to FOLFIRI, FOLFOXIRI, and BRAF/EGFR inhibitor combinations. Notably, RNF43-mutant PDOs exhibited significantly enhanced sensitivity to encorafenib plus cetuximab. Integrative transcriptomic analysis (TCGA and PDO cohorts) identified that RNF43 mutations are associated with upregulation of E2F, G2M, interferon-α/γ, and inflammatory pathways, along with elevated MHC-I expression. In co-culture with CD8⁺ T cells, RNF43-mutant PDOs triggered stronger T-cell activation, enhanced cytotoxicity, and increased tumor cell apoptosis. In humanized PDOX models, RNF43-mutant tumors showed suppressed growth and heightened CD8⁺ T-cell infiltration compared to wild-type controls. CONCLUSIONS:This functional precision oncology platform integrates multi-omics and immune co-culture to uncover RNF43 mutation as a dual biomarker for targeted therapy sensitivity and tumor immunogenicity in BRAFV600E-mutant CRC. Our findings provide mechanistic rationale for combining BRAF/EGFR inhibition with immunotherapy to overcome drug resistance and improve outcomes in this aggressive subtype.

Total neoadjuvant therapy (TNT) combined with immunotherapy (iTNT) has shown promising results for locally advanced rectal cancer (LARC) in microsatellite stable (MSS) patients. However, traditional clinical approaches often fail to accurately forecast clinical outcomes, and reliable methods to predict treatment response and recurrence risk are essential for optimizing treatment strategies and maximizing organ preservation. We analyzed clinical and genetic data from 63 patients from the prospectively registered TORCH clinical trial (NCT04518280) who received iTNT from May 1, 2021, to September 15, 2022. Baseline tissue mutations, magnetic resonance tumor regression grade (mrTRG), and circulating tumor DNA (ctDNA) positivity at different time points were collected and examined for their association with complete response (CR) and recurrence. Integrated models incorporating these features were developed to assess their accuracy in distinguishing CR from non-CR patients. Prognostic features were analyzed using Cox proportional hazards regression and Kaplan-Meier analyses. Baseline SMAD4 mutation was significantly associated with poor response to iTNT (P = 0.007), while post-treatment mrTRG (P < 0.0001), tumor mutational burden (TMB) (P = 0.03) and latest ctDNA status (P = 0.03) also correlated with CR rates. Integration of post-treatment mrTRG, SMAD4 mutation, TMB, and latest ctDNA status significantly improved CR discrimination accuracy compared with the mrTRG-only model (area under the curve (AUC) 0.92 vs. 0.75, P = 0.0098). Early integrated models incorporating mid-iTNT mrTRG and these genetic features showed better discriminative performance than the mrTRG-only model (AUC 0.89 vs. 0.76, P = 0.047). Furthermore, baseline SMAD4 mutations and ctDNA positivity at later time points correlated with an increased risk of disease progression, including local recurrence and distant metastasis. Our findings highlight the discriminative power of combining genetic features, particularly SMAD4 mutation and ctDNA, with clinical assessments like post-treatment mrTRG, for improving CR identification and early recurrence risk stratification over mrTRG alone in iTNT-treated LARC. These models could serve as complementary risk stratification tools to guide personalized treatment strategies, enabling early identification of patients requiring intensified therapy or nonoperative management. https://ClinicalTrials.gov identifier NCT04518280; Registration date August 15, 2020.

Colorectal cancer liver metastasis (CRLM) and chemotherapy resistance remain major clinical challenges, with the underlying molecular mechanisms yet to be fully elucidated. In this study, based on analyses of five initial clinical cohorts from FUSCC, the E3 ubiquitin ligase COP1 was identified as a critical driver of CRLM and resistance to oxaliplatin-based chemotherapy. Using an organoid biobank derived from paired primary colorectal tumors and liver metastases, integrated multi-omics analyses (WES, bulk RNA-seq, scRNA-seq) of patient-derived organoids (PDOs) from CRLM revealed significantly elevated COP1 expression in liver metastases compared to primary tumors. High COP1 levels were associated with poor prognosis, increased liver metastatic burden, and resistance to oxaliplatin-based chemotherapy. In vitro and in vivo functional experiments demonstrated that COP1 facilitates CRLM progression by ubiquitinating and degrading LUZP1, thereby releasing DAPK3 from LUZP1-mediated suppression. This process leads to enhanced MYL9 phosphorylation and activation of epithelial-mesenchymal transition (EMT) as well as the JAK2-STAT3-CCND2 signaling axis—pathways crucial for liver metastasis and resistance to oxaliplatin-based chemotherapy. These findings establish the COP1-LUZP1-MYL9 axis as a therapeutic target for CRLM and oxaliplatin-based chemoresistance. Clinically, COP1 expression profiling in PDOs from postoperative specimens enables a precision strategy for managing oxaliplatin-based chemoresistance, especially in the context of FOLFOX.

147 Background: The prognosis for locally advanced colon cancer (LACC) with bulky nodal disease and/or clinically T4 stage remains poor, with high rate of recurrence and metastasis. Although neoadjuvant chemotherapy is recommended, tumor regression is often limited. Hypofractionated radiotherapy has demonstrated enhanced synergistic effects when combined with PD-1 inhibitors and chemotherapy in rectal cancer. TORCH-C is the first prospective study evaluating neoadjuvant short course radiotherapy (SCRT) combined with chemotherapy and a PD-1 inhibitor in patients with microsatellite stable or mismatch-repair proficient (MSS/pMMR) LACC. Methods: This is a prospective, multicenter, randomized phase II trial (NCT05732493). A total of 120 LACC (T4/bulky N+M0，pMMR/MSS) patients will be randomized to either a chemotherapy group or a radiotherapy group. The chemotherapy group receives 4 cycles of CAPOX. The radiotherapy group receives SCRT（25Gy in 5 fraction) and 4 cycles of the PD-1 inhibitor (serplulimab) combined with CAPOX. The radiotherapy target volume includes only the primary colon tumor and enlarged lymph nodes, without elective nodal irradiation. After neoadjuvant therapy, patients will be evaluated for radical colon resection. The primary endpoint is pathological complete response (pCR) rate. The secondary endpoints include tumor downstaging, R0 resection, 3-year disease free survival (DFS), 3-year overall survival (OS) , 3-year local recurrence-free survival and treatment-related toxicity. Results: As of August 31, 2025, 120 patients have been enrolled and randomized. Of these, 79 patients have completed neoadjuvant treatment and surgery (43 in the chemotherapy group, 36 in the radiotherapy group). All 79 patients achieved radical resection, with R0 resection rates of 95.3% (41/43) in the chemotherapy group (95.3%, 41/43), and 97.2% (35/36) in the radiotherapy group. The pCR rate (TRG 0) was 11.6% (5/43) in chemotherapy group and 47.2% (17/36) in the radiotherapy group. The most common grade 3-4 adverse event (AE) among patients completing neoadjuvant treatment was thrombocytopenia (15.3%, 15/98), including 5 cases of grade 4 thrombocytopenia. Conclusions: The combination of PD-1 inhibitor, SCRT, and chemotherapy shows promising efficacy and may significantly improve pCR rates in patients with MSS/pMMR high-risk LACC. This regimen may provide a new therapeutic option to achieve R0 resection and improve long-term survival. Clinical trial information: NCT05732493 .

Serum bile acids (BAs) emerge as risk factors for cancer, but their roles in colorectal cancer (CRC) remain unclear. We show that glycocholic acid (GCA), a primary BA, is elevated in the serum of CRC patients. In a mouse CRC model, GCA promotes tumor programmed death-ligand 1 (PD-L1) expression in tumors, suppressing CD8⁺ T cell-mediated antitumor immunity and facilitating tumor growth. Mechanistically, GCA inhibits the BA receptor farnesoid X receptor (FXR), a transcriptional repressor for SRY-box transcription factor 14 (SOX14). Loss of FXR repression upregulates SOX14-mediated expression of zinc finger DHHC-type palmitoyl transferase 9 (DHHC9), thereby reducing PD-L1 palmitoylation and stabilization. Silencing SOX14 or DHHC9, or activating FXR, synergizes with anti-PD-1 therapy, reducing tumor growth in GCA-treated mice. These findings uncover a mechanism that GCA remodels the tumor microenvironment to mediate CRC resistance to immunotherapy, highlighting therapeutic opportunities targeting the FXR-PD-L1 axis in CRC patients with elevated serum GCA.

Characteristics of Patients Developing Tumor Recurrence

e15637 Background: Thrombocytopenia is one of the main adverse effects of oxaliplatin-based chemotherapy, probably due to the hepatic sinusoidal obstruction syndrome and splenomegaly. The integration of PD-1 inhibitors with oxaliplatin-based total neoadjuvant therapy (TNT) has achieved remarkable tumor regression but increased the incidence and severity of thrombocytopenia in patients with locally advanced rectal cancer (LARC) in our previous TORCH trial. Herein, we aimed to compare the thrombocytopenia in LARC patients receiving TNT with and without immunotherapy, and explore the correlation among liver injury, splenomegaly and thrombocytopenia. Methods: We conducted a retrospective study involving 181 LARC patients who received either Ox-based TNT (N = 44, long-course chemoradiotherapy [LCCRT] followed by oxaliplatin-based chemotherapy), Iri-based TNT (N = 41, LCCRT followed by irinotecan-based chemotherapy), or iTNT (N = 96, short-course radiotherapy and 6 cycles of CAPOX and PD-1 inhibitor from TORCH trial [NCT04518280]) at the Fudan University Shanghai Cancer Center. Blood laboratory tests were extracted before, during and after TNT/iTNT. Splenic and hepatic volumes were measured using automated techniques based on CT, MRI, or PET-CT at baseline and at the end of treatment. Thrombocytopenia was graded according to the CTCAE5.0 criteria. Splenomegaly was defined as an increase in spleen volume of more than 30% from baseline. Results: Among the 181 patients, 118 (65.2%) developed thrombocytopenia, and 69 (38.1%) developed splenomegaly. The iTNT group exhibited significantly higher rates of thrombocytopenia (89.6%) and splenomegaly (61.5%) compared to Ox-based TNT group(thrombocytopenia50.0%, splenomegaly13.6%) and Iri-based TNT group(thrombocytopenia61.0%, splenomegaly9.8%). Platelet count was negatively correlated with the increase in AST level (P < 0.001) and spleen volume (P < 0.001). In the multivariate analysis of the entire cohort, immunotherapy(P = 0.002), splenomegaly(P = 0.003) and older age(P = 0.004) were identified as independent predictive factors for G2-4 thrombocytopenia. Additionally, immunotherapy(P < 0.001), direct bilirubin(P = 0.031), hepatomegaly(P = 0.032) and AST(P = 0.032)were identified as independent predictive factors for splenomegaly. In patients receiving iTNT, splenomegaly (P = 0.009) and older age (P = 0.033) were still the major predictive factors for G2-4 thrombocytopenia. Conclusions: In LARC patients, the addition of PD-1 inhibitors to TNT lead to more severe thrombocytopenia compared to traditional TNT, probably due to increased liver injury and splenomegaly.

BACKGROUND:The optimal regimen and chemotherapy intensity are still under investigation for neoadjuvant treatment of locally advanced rectal cancer (LARC). The CinClare trial has demonstrated improved pathologic complete response (pCR) with the addition of irinotecan to neoadjuvant chemoradiotherapy (CRT) guided by uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) genotype in LARC. Here, we report the 5-year follow-up outcomes of the CinClare study. METHODS:From November 2015 to December 2017, this randomized, open-label, multicenter, phase III trial enrolled 360 patients with LARC and assigned them in a 1:1 ratio to CapIriRT (radiation with capecitabine combined with irinotecan followed by irinotecan and capecitabine) or CapRT (radiation with concurrent capecitabine followed by oxaliplatin and capecitabine). Irinotecan dosing was guided by UGT1A1 genotype (80 mg/m2 for *1/*1 and 65 mg/m2 for *1/*28). The endpoints, including local control (LC), distant metastasis-free survival (DMFS), disease-free survival (DFS), and overall survival (OS), were analyzed using the log-rank test, Cox proportional hazards regression and restricted mean survival time (RMST) test at the data cut-off date of June 2023. RESULTS:With a median follow-up of 60 months, the CapIriRT group showed numerically higher 5-year LC (95.6% vs. 93.9%), 5-year DMFS (83.9% vs. 77.9%), 5-year DFS (77.7% vs. 70.6%), and 5-year OS rates (82.9% vs. 76.1%) than the CapRT group. Further RMST test also showed a statistically significant difference in DFS (P < 0.05) and a borderline difference in OS (P = 0.050). Among the UGT1A1 *1/*1 population, the CapIriRT group had significantly improved 5-year rates of DMFS, DFS, and OS (all P < 0.05). Patients achieving pCR also had significantly longer DFS and OS compared to non-pCR patients (P < 0.05). CONCLUSIONS:The addition of irinotecan guided by UGT1A1 genotype to a standard capecitabine-based scheme brings clinical benefits with improved LC, DMFS, DFS, and OS. Patients with the UGT1A1 *1/*1 genotype derived notable benefit from irinotecan, with improved survival outcomes. Achievement of pCR is crucial as it is associated with improved long-term survival. These findings support the integration of genomic testing into clinical practice to achieve a personalized irinotecan dosing regimen, which can optimize efficacy and safety. TRIAL REGISTRATION:ClinicalTrials.gov (NCT02605265).

PURPOSE:EGFR inhibition, combined with chemotherapy, forms a mainstay of treatment of first-line RAS wild-type (RASwt) metastatic CRC (mCRC). We compared the anti-EGFR antibody, A140, with cetuximab (both combined with chemotherapy) for RASwt mCRC. METHODS:In this phase 3, randomized, double-blind, multi-center equivalence trial in China, patients were randomized (1:1) to oxaliplatin, 5-fluorouracil, and leucovorin (modified [m] FOLFOX6), plus either A140 or cetuximab for ≤ 16 weeks until disease progression or unacceptable toxicity. After the 16-week treatment period, patients who investigators deemed could benefit received A140 plus mFOLFOX6. Eligible patients were 18-75 years old with histologically confirmed RASwt mCRC and an Eastern Cooperative Oncology Group (ECOG) score of 0-1. Primary endpoint was objective response rate (ORR [RECIST v1.1]), per independent review committee, over the 16 weeks. A140 was considered equivalent if the 90 % CIs for the ORR ratio were between 0.83 and 1.20. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, immunogenicity, and pharmacokinetics. RESULTS:Overall, 688 patients were randomized to A140 (n = 341) or cetuximab (n = 347). As of 22-Mar-2023, the ORR was 71.0 % (A140) and 77.5 % (cetuximab); the ORR ratio was 0.93 (90 % CI, 0.87-0.99). With a median follow-up of 19.6 months, no notable differences in PFS, OS, or duration of response were observed between arms. Safety profiles and immunogenicity were similar between arms and no new safety signals were observed. CONCLUSION:A140 plus mFOLFOX6 is equivalent to cetuximab plus mFOLFOX6 for RASwt mCRC, with no appreciable differences in safety profiles. A140 plus mFOLFOX6 may provide a new treatment option for patients with RASwt mCRC. TRIAL REGISTRATION:NCT04835142.

Total neoadjuvant therapy (TNT) combined with immunotherapy (iTNT) has shown promising results for locally advanced rectal cancer (LARC), especially in proficient mismatch repair (pMMR)/microsatellite stable (MSS) patients. However, traditional clinical approaches often fail to accurately forecast clinical outcomes, and reliable methods to predict treatment response and recurrence risk are essential for optimizing treatment strategies and maximizing organ preservation. We analyzed clinical and genetic data from 63 patients in the TORCH clinical trial who received iTNT. Baseline tissue mutations, magnetic resonance tumor regression grade (mrTRG), and circulating tumor DNA (ctDNA) positivity at different time points were examined for their association with complete response (CR) and recurrence. Predictive models integrating these features were developed to assess their accuracy in distinguishing CR from non-CR patients. Prognostic features were analyzed using COX regression and Kaplan-Meier analyses. Baseline SMAD4 mutation was significantly associated with poor response to iTNT (P=0.007), while post-treatment mrTRG (P<0.0001), tumor mutational burden (TMB) (P=0.03), and latest ctDNA status (P=0.03) also correlated with CR rates. Integration of post-treatment mrTRG, SMAD4 mutation, TMB, and latest ctDNA status significantly improved CR prediction accuracy compared with mrTRG-only model (area under the curve (AUC) 0.92 vs 0.75, P=0.0098). Early prediction models incorporating middle-iTNT mrTRG and these genetic features showed superior accuracy to predict CR than mrTRG-only model (AUC 0.89 vs 0.76, P=0.047). Furthermore, ctDNA positivity at later time points and SMAD4 mutations correlated with worse disease progression, including local recurrence and distant metastases. Our findings highlight the predictive power of combining genetic and clinical features, particularly ctDNA and SMAD4mutation, for improving CR prediction and early recurrence risk stratification in iTNT-treated LARC. These models could guide more personalized treatment strategies, enabling early identification of patients requiring intensified therapy or nonoperative management. Yaqi Wang, Yang Xu, Fan Xia, Juefeng Wan, Lijun Shen, Yan Wang, Hui Zhang, Hairong Bao, Haimeng Tang, Qiuxiang Ou, Hua Bao, Yang Shao, Sanjun Cai, Xinxiang Li, Zhen Zhang. Characterization and estimation of treatment response of immunotherapy-based total neoadjuvant therapy (iTNT) for microsatellite stable (MSS) locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 722.