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.

Conventional chemotherapy achieves clinical efficacy not only through its cytotoxic effects but also by reactivating immune surveillance. However, whether chemotherapy can inversely suppress antitumor immunity remains largely unexplored. Here, we integrate cross-species single-cell and spatial transcriptomics to investigate how chemotherapy programs immune cell plasticity. Our findings reveal that chemotherapy-educated liver-resident Kupffer cells (KCs) promote immune evasion and chemoresistance in liver metastases. These reprogrammed KCs, characterized by leptin receptor expression (LEPR+), originate from preexisting KCs and differentiate via STING (Stimulator of interferon genes)-ID1 signaling, driven by paracrine cGAMP (cyclic GMP-AMP) released from chemotherapy-treated tumor cells. Unlike conventional KCs at the tumor periphery, LEPR+ KCs infiltrate tumors and suppress antitumor immunity through MerTK-dependent efferocytosis that eliminates chemotherapy-induced immunogenic cell death (ICD) signals. Targeting LEPR+ KCs enhances tumor immunogenicity and promotes antitumor T cell responses. Together, our study highlights the potential of combining efferocytosis inhibitors with immunotherapy to overcome chemoresistance.

3026 Background: Treatment options in China are limited for patients with late-line, HER2-expressing advanced solid tumors. In Part 1 of DESTINY-PanTumor02, T-DXd showed clinically meaningful antitumor activity in HER2-expressing advanced solid tumors, with the greatest benefit observed in HER2 IHC 3+ tumors. Based in part on these findings, T-DXd has been approved in multiple countries worldwide, including the US, as treatment for patients with unresectable or metastatic HER2-positive (IHC 3+) solid tumors who have received prior treatment and/or have no satisfactory alternative therapies. Following the results from DESTINY-PanTumor02, DESTINY-PanTumor03 is evaluating T-DXd in patients in China with HER2-expressing advanced solid tumors. The primary analysis of Part 1 of DESTINY-PanTumor03 is presented here. Methods: DESTINY-PanTumor03 is an open-label, Phase 2 study (NCT06271837). Part 1 is evaluating T-DXd (5.4 mg/kg IV Q3W) in patients in China with HER2 IHC 3+ (by central testing), locally advanced, unresectable, or metastatic solid tumors (excluding breast and gastric cancers) after ≥1 prior systemic treatment for advanced disease or without treatment options. The primary endpoint is confirmed objective response rate (ORR) by independent central review (ICR) per RECIST 1.1. Secondary endpoints include ORR by investigator assessment (INV) per RECIST 1.1; duration of response (DOR), disease control rate (DCR), and progression-free survival (PFS) by INV and ICR per RECIST 1.1; overall survival (OS); and safety. Results: At primary analysis data cutoff (November 28, 2025), 50 patients with biliary tract (n = 11), colorectal (n = 6), cervical (n = 10), endometrial (n = 7), ovarian (n = 5), non-small cell lung (n = 7), or other cancers (n = 4) had received T-DXd. Median follow-up duration was 9.9 (range 1.1–18.3) months. Median number of prior treatment regimens was 2 (range 1–10). By ICR, ORR (95% CI) was 58.0% (43.2, 71.8), median DOR (95% CI) was 15.4 (12.5, not evaluable [NE]) months, DCR (95% CI) at Week 6 was 88.0% (75.7, 95.5), and median PFS (95% CI) was 15.7 (7.2, NE) months. By INV, ORR (95% CI) was 56.0% (41.3, 70.0). Median OS was not reached. Grade ≥3 drug-related adverse events occurred in 31 (62.0%) patients, and adjudicated drug-related interstitial lung disease / pneumonitis occurred in 4 (8.0%) patients (Grade 2 n = 3 [6.0%], Grade 3 n = 1 [2.0%]). Conclusions: T-DXd demonstrated durable and clinically meaningful antitumor activity in pretreated patients in China with HER2 IHC 3+ advanced solid tumors. Safety was generally consistent with the established T-DXd profile. Results from DESTINY-PanTumor03 Part 1 support T-DXd as a tumor-agnostic treatment for patients in China with HER2 IHC 3+ solid tumors. Clinical trial information: NCT06271837 .

3534 Background: Maintenance therapy with capecitabine (Cap) is widely recommended to unresectable mCRC patients (pts). Fruquintinib (Fru) is a highly selective inhibitor of VEGFR1/2/3. We sought to compare the therapeutic potential of Fru plus Cap versus Cap as maintenance therapy for mCRC. Methods: Eligible pts with histologically confirmed mCRC who achieved disease control (including CR/PR and SD) after at least six cycles of first-line standard chemotherapy were included in the study. During phase Ib, pts received Fru (4 mg, p.o. qd, 3 weeks on/1 week off, q4w) plus Cap (850 mg/m 2 , p.o. bid, d1-7 and d15-21, q4w). In phase II, pts were randomized in a 1:1 ratio to receive either Fru (RP2D, 3mg, 3 weeks on/1 week off) plus Cap or Cap alone. The primary endpoint was progression-free survival (PFS). Secondary endpoints included objective response rate (ORR), disease control rate (DCR) and safety. Results: As of November 15, 2025, 113 eligible patients were enrolled, including 6 in phase Ib and 107 in phase II. In this presentation, we report only the updated data from Phase II. 107 were randomly assigned to receive either fruquintinib plus capecitabine (Fru+Cap; n=52) or capecitabine alone (Cap; n=55). Baseline characteristics included median age: 61 years (range, 30-78) vs. 60 years (range, 31-80), male: 51.9% vs. 58.2%, left-sided colorectal primary tumors: 55.8% vs. 61.8% and RAS-mutant tumors: 53.8% vs. 52.7%. As first-line maintenance therapy, the median PFS1 (defined as time from randomization to disease progression or death) in the per-protocol population was 6.03 months with Fru+Cap versus 3.30 months with Cap (p=0.003). Furthermore, the Fru+Cap arm demonstrated significantly prolonged PFS2 (defined as the time from first receipt of first-line treatment to disease progression or death): 12.1 vs. 9.7 months, p=0.006. Consistent improvements were observed in objective response rate (ORR: 14.6% vs. 7.0%) and disease control rate (DCR: 75.6% vs. 51.2%). Grade ≥3 treatment-emergent adverse events (TEAEs) in Fru+Cap arm included hypertension (5.77%), palmar-plantar erythrodysesthesia syndrome (1.9%) and albuminuria (1.9%). Diarrhea (3.64%), palmar-plantar erythrodysesthesia syndrome (1.8%) and oral mucositis (1.8%) were the most frequently reported grade ≥3 TEAEs in Cap arm. Conclusions: PFS was significantly prolonged and ORR was improved in mCRC pts treated with Fru plus Cap as first-line maintenance treatment. The safety profile of the combination regimen was manageable and aligned with the expected toxicity patterns of the individual agents. Clinical trial information: NCT05451719 .

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.

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.

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.

Ultra-low-pass whole-genome sequencing (ULP-WGS) of cell-free DNA (cfDNA) offers a cost-efficient strategy for cancer detection, but its clinical application is limited by extreme data sparsity and poor model generalization. We developed Fragmentia-AI™ WGS, a mutation-calling-independent framework that uses a transformer-based multiple-instance learning architecture with sequential fine-tuning across tumor fraction (TF) strata to extract latent cancer-associated signals from ULP-WGS data. Model performance was evaluated in multiple independent cohorts, including a pan-cancer test set covering 17 cancer types, an external public dataset generated on a different sequencing platform, and a technical variability cohort with heterogeneous pre-analytical and experimental conditions. Clinical relevance was assessed by correlating model predictions with progression-free survival (PFS) in patients with advanced non-small cell lung cancer receiving chemoimmunotherapy. Sequential fine-tuning across TF strata significantly improved performance in low-TF samples, achieving a 35.6

Background:Palliative chemotherapy is the standard first treatment for patients with ≥10 liver metastases, irrespective of resectability. This study aimed to assess the clinical benefits of hepatic resection and identify optimal treatments for colorectal cancer patients with ≥10 liver-limited metastases (LLMs). Methods:A retrospective study of 373 patients with unresectable colorectal cancer and ≥10 liver metastases. Patients were grouped into hepatic surgery ± radiofrequency ablation (RFA) + systemic therapy, RFA + systemic therapy, and systemic therapy alone. Kaplan-Meier and Cox proportional hazards methods were used to analyze overall survival (OS) and progression-free survival (PFS). A nomogram was developed and validated. Results:Three-year survival rates were 39.2%, 18.6%, and 8.2% for surgery ± RFA + systemic, RFA + systemic, and systemic only groups, respectively. Significant survival differences were noted in both KRAS/NRAS/BRAF wildtype and mutated cohorts. For wildtype LLMs, OS and PFS were 39.3 and 13.8 months for surgery ± RFA + systemic, compared to 23.9 and 9.9 months for RFA + systemic. The nomogram (C-index =0.709) identified independent prognostic factors for OS. Conclusions:Hepatectomy, with or without RFA, is associated with significantly improved survival in selected patients with ≥10 liver metastases. Further prospective studies are needed to confirm these findings.

Characteristics of Patients Developing Tumor Recurrence

Duration of cCR in each individual patient who adopted WW

Representativeness of Study Participants

Colorectal liver metastasis (CRLM) is one of the leading death causes among colorectal cancer (CRC) patients, yet its underlying molecular events remain poorly understood, particularly at the proteomic and phosphoproteomic levels. A proteogenomic analysis combining genomics, transcriptomics, proteomics, and phosphoproteomics is performed on 102 samples from 34 treatment-naïve CRLM patients, including primary CRC, adjacent normal colorectal, and matched liver metastasis tissues. CRC cell lines, organoids, mouse models, and an independent patient cohort are used to validate the findings. Proteomics and phosphoproteomics show profoundly dysregulated pathways in liver metastasis tissues, notably disruptions in carbon metabolism. Functional validation using CRC organoids and mouse models demonstrates that the one-carbon metabolism enzyme SHMT1 promotes CRC tumorigenesis and metastasis via formate-mediated AMPK inhibition, whereas PIM kinase-dependent NDRG1 Ser330 phosphorylation exacerbates liver metastasis by promoting ubiquitin-dependent degradation of NDRG1. Unsupervised clustering identifies two proteomic subtypes of liver metastasis samples with distinct clinical outcomes: a poor-prognosis C1 (metabolism) subtype and a better-prognosis C2 (RNA function) subtype. Considering expression frequency, specificity, and functional relevance, FTCD, GPD1, SOD2, and EIF4B Ser422 phosphorylation are further identified and validated as subtype prognostic biomarkers. This study provides critical insights into the molecular mechanisms underlying CRLM and offers resources for high-risk metastatic CRC.

Postoperative complications according to Clavien- Dindo classification

Genetic mutation detection for colorectal cancer (CRC) is crucial for precision diagnosis and treatment, yet current methods often suffer from challenges such as low sensitivity, time consumption, and high costs. In our preliminary bioinformatic analysis of 751 CRC cases from The Cancer Genome Atlas and 131 Chinese patient samples, APC, TP53, and KRAS were identified as the most frequently mutated genes. Among them, KRAS missense mutations emerged as key diagnostic biomarkers. In this study, we applied a fluorescence-based long block displacement amplification (LBDA) sensing method for the rapid, high-throughput, and cost-effective detection of KRAS genetic mutations. In the LBDA system, SYBR Green dye binds to the amplified double-stranded DNA, generating a fluorescence signal that directly reflects the abundance of mutant types (MTs). This real-time signal output enables the enrichment and sensitive detection of MTs, establishing LBDA as an efficient biosensing platform for KRAS genotyping. Using this technique, a detection limit of 0.08% variant allele frequency was achieved with 20 ng of synthetic DNA input. To evaluate clinical performance, the LBDA method was applied to 118 tissue samples from 59 CRC patients, including tumor and matched peritumoral tissues. For 59 CRC tumor samples, LBDA successfully identified KRAS mutations in 37.29% of cases, closely matching results (42.37%) obtained by next-generation sequencing and achieving 88% sensitivity and 100% specificity. In conclusion, this study presents a rapid and cost-effective mutation detection method based on optical biosensing, offering strong potential for advancing personalized CRC diagnosis and treatment.

Patients with metastatic colorectal cancer (mCRC) can achieve no evidence of disease (NED) after curative treatment. However, the survival benefit of adjuvant chemotherapy (ACT) for these patients remains uncertain. Circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection has shown to help predict recurrence risk in CRC. Recent retrospective studies have indicated that ACT offers no clinical benefit in surgically resected mCRC with negative ctDNA. In this study, we conducted a prospective, interventiaonal clinical trial (NCT05635630) to evaluate the efficacy of ctDNA-guided ACT in patients with mCRC who achieved NED. Eligible mCRC patients who achieved NED after curative treatment were enrolled. Surgical tissues or puncture tissues were sequenced by whole-exon sequencing, and up to 50 highly ranked patient-specific variants were selected for the personalized panel design. Post curative treatment blood samples was sequenced by the personalized panel to determine the MRD status. Patients with positive MRD post curative treatment were offered investigator-determined ACT, while those with negative MRD were under surveillance. During surveillance, blood samples were collected every 3 months and patients whose MRD turned positive would receive intensive image surveillance every 2 months. A total of 104 eligible patients were included in the current analysis, among them 7 patients did not receive assigned treatment strictly adhered to the study protocol and were excluded from the per protocol set (PPS). In the PPS, as date cutoff of October 2024, the median follow-up was 14 months with 58 patients (58/97, 59.8%) who experienced recurrence. MRD was detected in 53 patients (54.6%, 53/97) post curative treatment. The median recurrence-free survival (RFS) was 8.5 months and not reached for patients with positive or negative MRD post curative treatment, respectively (HR: 4.28, 95% CI: 2.36-7.77; P<0.001). Six patients with negative MRD received ACT and had non-superior RFS than those who did not receive ACT (HR: 1.60, 95% CI: 0.46-5.58; P = 0.46). During surveillance, the median RFS was 6.1 months, 11.8 months, 16.8 months and not reached in patients with persistently positive ctDNA, converted positive ctDNA, converted negative ctDNA, and persistently negative ctDNA, respectively (log-rank P for trend <0.0001). All recurrent patients had at least one positive ctDNA detection before imaging confirmed recurrence, and the median lead time interval of ctDNA positivity to radiographic recurrence was 7.8 months (range, 0-20.2 months). Our results demonstrated the feasibility of using ctDNA to guide ACT in mCRC patients with NED. Future randomized clinical trials are needed to further demonstrate the utility of ctDNA-guided ACT in mCRC patients with NED. The follow-up is still on-going. Yaqi Li, Chengcheng Li, Peng Cui, Xiang Hu, Fangqi Liu, Wenhua Li, Guoqiang Wang, Kunli Zhao, Qi Pan, Jing Wang, Bing Li, Shangli Cai, Junjie Peng. Circulating tumor DNA-based molecular residual disease guides treatment in metastatic colorectal cancer patients with no evidence of disease: An open-label, prospective, interventional phase II study [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 4567.

Background:Microsatellite stability (MSS) colorectal cancers (CRCs) have a limited response to immune checkpoint inhibitors (ICIs) compared to microsatellite instability-high (MSI-H) CRCs. Nevertheless, previous studies have shown that some MSS CRCs are sensitive to ICIs, although established criteria for treatment justification are still lacking. Objective:This study aimed to test the tumor-infiltrating lymphocyte (TIL) features of MSS and develop a novel computational tool for the similarity prediction between MSS and MSI-H status in patients with CRC based on multiple factors. Methods:We collected and analyzed data from 188 patients with CRC, including MSI status, immune cell distributions, clinical features, and gene mutations, using statistical methods and Cox regression. An ensemble machine learning-based MSI-H score was developed using stacked extreme gradient boosting classifiers to quantify the similarity of patient data to MSI-H data based on immune cell distributions, clinical features, and gene mutations. The model was robust and could address missing input data for immune cell distributions and gene mutations. Results:The scorer performed well (mean Cohen κ of 0.40, SD 0.05, over 10 random seeds) in identifying MSI-H-like MSS samples with TIL distributions similar to genuine MSI-H CRCs. No significant difference was observed between the TIL features of MSI-H-like MSS CRCs and MSI-H CRCs. The disparity between MSI-H-like MSS CRCs and MSS CRCs potentially lies in the T regulatory cells (P=.09) and macrophage (P=.16) populations within the tumor stromal region. Conclusions:Some patients with MSS CRC presented similar immune cell distributions with high immunoactivity compared to patients with MSI-H CRC. The MSI-H score serves as a metric to quantify the similarity of MSS CRCs to MSI-H CRCs and presents a promising avenue for more personalized and effective cancer immunotherapy treatment, offering a clinical reference for potential ICI targets in MSS CRCs.

Objective·To comprehensively evaluate the efficiency of different kits and methods for DNA and RNA extraction from blood samples.Methods·A total of 145 blood samples were collected, including those from patients with Alzheimer's disease (20 cases), fibrosis (5 cases), colorectal cancer (108 cases), and healthy individuals (12 cases). A column-based kit (Kit A) and a nucleic acid extraction instrument were used to extract genomic DNA (gDNA) from leukocytes in the blood. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in plasma were extracted using five different kits (Kit B‒F), which employed either column-based (Kit B, E) or magnetic bead-based methods (Kit C, D, F). The extraction process of Kit B was optimized by increasing the plasma sample volume and extending the elution incubation time. Furthermore, this protocol was applied to extracting cfDNA from plasma samples of 100 colorectal cancer patients. Quantitative real-time PCR (qPCR) was used to quantify the extracted DNA and RNA, and the molecular yields were compared to evaluate the extraction efficiency. A comprehensive assessment was conducted, considering factors such as cost and operation time.Results·In gDNA extraction, although the the operation time was shortened by using the nucleic acid extraction instrument, the median number of DNA molecules extracted using Kit A (column-based method) was 25.36-fold higher than that obtained with the instrument (P<0.05). For cfDNA extraction, while the overall efficiency of the three kits (Kit B‒D) was similar, Kit B (column-based method) showed superior performance in low-concentration samples, with average DNA yields 4.24-fold and 1.18-fold higher than those of Kit D and Kit C (both magnetic bead-based). Optimization of Kit B's extraction protocol further improved cfDNA yield. When comparing three samples, the cfDNA yields from larger plasma input volumes was 3.98-fold, 2.38-fold, and 3.82-fold higher than those from smaller input volumes, respectively. The results of cfDNA extraction from 100 colorectal cancer patients indicated that this extraction protocol reliably extracted sufficient amounts of cfDNA from clinical samples. For cfRNA extraction, Kit E (column-based method) was widely recommended due to its high efficiency, convenience, and cost-effectiveness. The median RNA content extracted using Kit E was 5.01-fold higher than that of Kit F (magnetic bead-based method). Lastly, a comparison of the copy numbers of cfDNA and cfRNA in plasma revealed that the average copy number of cfRNA per milliliter of plasma was 27.65-fold higher than that of cfDNA.Conclusion·Kit A, Kit B, and Kit E show outstanding performance in leukocyte gDNA extraction, plasma cfDNA extraction, and plasma cfRNA extraction, respectively. However, although Kit E has advantages in extraction efficiency and cost, its safety requires further evaluation.