Mesenteric benign lymph node enlargement in colorectal cancer: Friend or Foe?

Mesenteric benign lymph node enlargement in colorectal cancer: Friend or Foe? | 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 Mesenteric benign lymph node enlargement in colorectal cancer: Friend or Foe? Junwei Wang, Xiangchao Shi, Limei Guo, Fei Li, Siyi Lu, Bingyan Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3975650/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Benign lymph node enlargement (BLNE) is common in colorectal cancer; however, few studies have investigatedits influence on prognosis, clinicopathological features, and pathogenesis. Methods: Patients who underwent radical surgery for colorectal cancer at Peking University Third Hospital were grouped according to the presence ofBLNE, and their prognosis and clinical characteristics were analysed. The immune microenvironment and genomic characteristics of primary tumors were comprehensively explored in representative patients. Results: Overall,630 AJCC stage I/IIpatients, with 131 in the BLNE group and 499 in the Non-BLNE (NBLNE) group, were included in the study. Patients in the BLNE group exhibited better disease-free survival (DFS) (hazard ratio [HR] 0.44, P = 0.016) and overall survival (OS) (HR 0.46, P = 0.011) . Interestingly, the prognosis of patients without lymph node metastasis wasworse when the number of harvested lymph nodes was more than 15. On computed tomography (CT) images, significant differences in lymph node morphology were found between BLNE and metastatic lymph node enlargement, including lymph node border, shape and enhancement characteristics. Pathologically, compared with the NBLNE group, the BLNE group had more mature tertiary lymphoid structures (66.7% vs. 36.5%, P = 0.002), greater immunoscore (18.8% vs. 2.1%, P = 0.004) in tumor tissue, and more abundant lymph follicles in lymph nodes (13.69 ± 4.89 vs. 5.08 ± 3.87, P < 0.0001). Whole-exon sequencing analysis revealed greater TMB in the BLNE group [ 6.03 (5.59, 7.59) vs. 5.33 (4.62, 6.34), P = 0.025). 11 differentially mutated genes were identified between the BLNE groupand NBLNE group, with MUC12 (81%) as the most common mutated gene in the BLNE group (odds ratio [OR] 0.10, P = 0.0002). Conclusion: BLNE is a positive factor in predicting the prognosis of colorectal cancer, possibly becausepatients with BLNE have a stronger anti-tumorimmune response. Accurate preoperative lymph node staging is critical. colorectal cancer prognosis tumor immune microenvironment benign lymph node enlargement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Colorectal cancer (CRC) is a common malignancy worldwide, ranking second among the most common cancers in women and third among men, and poses a significant public health problem worldwide [ 1 ]. Lymph node metastasis is generally associated with a poorer prognosis. The 5-year overall survival rate for patients with American joint Committee on cancer (AJCC) stage I/II is greater than 85%, whereas that for patients with AJCC stage III with lymph node metastasis is between 67–80% [ 2 ]. According to the Japanese Guidelines for Colorectal Cancer, extensive lymph node dissection should not be performed in patients with T1 and T2 CRC when lymph node metastasis is absent [ 3 ]. Extensive lymph node dissection may result in a greater complication rate; therefore, determining the presence of lymph node metastasis before surgery is important. Currently, diagnosing preoperative lymph node metastasis mainly relies on computed tomography (CT) and magnetic resonance imaging(MRI). Lymph nodes over 8 mm in diameter with enhancement are usually considered metastatic [ 4 ]. However, the sensitivity and specificity of CT for determining lymph node metastasis are only 55% and 74%, respectively [ 5 , 6 ]. In clinical practice, lymph node enlargement on preoperative imaging and postoperative pathology confirming the absence of metastatic cancer cells are common in CRC patients. In this study, lymph nodes with a diameter > 8 mm on preoperative imaging without metastasis on postoperative pathology were termed mesenteric benign lymph node enlargement (BLNE) [ 7 ]. Within lymph nodes, fibroblast reticular cells comprise 20–50% of the non-hematopoietic region, creating a web of interconnected extracellular matrix components that form tubes that rapidly transport soluble antigens and signalling molecules deep into the lymph node parenchyma. When the adaptive immune response begins, the contractile function of FRC actomyosin is inhibited, facilitating rapid lymph node expansion to accommodate the increased number of activated T and B cells [ 8 ]. Previous studies have also shown lymph node follicular hyperplasia in the drainage areas of patients with stage II colon cancer [ 9 ], suggesting an association between non-metastatic enlargement of tumor-draining lymph nodes and the tumor-adaptive immune response. Recently, some studies have reported that BLNE is a favourable prognostic factor for colorectal cancer [ 7 , 10 ], while other studies have proposed hat it is associated with poorer prognosis [ 11 ]. Therefore, the clinical significance of mesenteric BLNE in stage I/II CRC with remains unclear. This study aimed to investigate the prognostic value of mesenteric BLNE in CRC and explore its clinical significance. METHODS Patients Selection This study was approved by the Medical Science Research Ethics Committee of Peking University Third Hospital (IRB00006761-M2020123) and conducted in accordance with the Declaration of Helsinki. Between January 2015 and June 2023, 2,270 patients with CRC underwent surgery at Peking University Third Hospital. The clinical data of these patients were prospectively recorded, and all eligible patients were divided into two groups. The BLNE group comprised those with mesenteric BLNE, while the non-benign lymph node enlargement (NBLNE) group served as the control group, according to the 9th edition of the TNM staging system published by the AJCC. The inclusion criteria were as follows: 1) no regional lymph node metastasis or distant metastasis; 2) stage I and II patients in the AJCC staging system; 3) radical CRC resection achieving R0 status with no positive lymph nodes; and 4) a single malignant lesion confirmed via colonoscopy. Exclusion criteria: 1) underwent emergency surgery or had preoperative complications such as intestinal obstruction or perforation; 2) had suspected distant metastasis on positron emission tomography (PET-CT); 3) received preoperative neoadjuvant therapy; 4) had hereditary CRC such as familial adenomatous polyposis; and 5) had microsatellite instability (MSI) confirmed by pathology. In early CRC, MSI-H status is associated with a favourable prognosis and an enhanced anti-tumor immune response; thus, we excluded this subset of patients to avoid its impact on prognosis [ 12 ]; 6) lacked imaging data; and 7) were lost to follow-up. In the study of the relationship between lymph node dissection and prognosis, we analysed the number of dissected lymph nodes in 1435 of the 2270 patients (excluding patients with preoperative neoadjuvant therapy and incomplete data). Additionally, we analysed the imaging features of all the 290 patients with enlarged lymph nodes seen on preoperative dual-phase CT scanning. The clinicopathological features assessed included sex, age, tumor location, tumor size, T stage, histological type, preoperative neutrophil and lymphocyte counts in peripheral blood, vascular invasion (VI), and perineural invasion (PNI). CT imaging features Lymph nodes in the drainage area of the tumor were mainly observed, and patients were grouped according to preoperative CT findings. Lymph nodes with a maximum diameter > 8 mm on CT images were considered positive. Patients with positive lymph nodes and no metastasis confirmed by pathology were in the BLNE group, those with positive lymph nodes and metastasis confirmed by pathology were in the metastatic lymph node enlargement (MLNE) group, and those with negative lymph nodes and no metastasis confirmed by pathology were in the NBLNE group. In the imaging analysis, the largest or most suspicious metastatic lymph nodes in the mesenteric drainage area in each patient with CRC were selected for further evaluation. Lymph node morphology, margins, and CT values were evaluated using arterial-phase CT images. Heterogeneous enhancement refers to nonuniform enhancement in a whole lymph node. The unclear outer border refers to the lymph node with rough edges, and the irregular form refers to the shape of the lymph node other than the round and oval. The difference in CT values represents the maximum CT value in the lymph node minus the minimum CT value. The necrotic center was defined as a lymph node with significant marginal enhancement and no central enhancement Two physicians who were blinded to the clinical and pathological data independently analysed the CT images. Discrepancies were resolved through discussion until a consensus was reached. Hematoxylin and eosin (HE), immunohistochemical (IHC), and immunofluorescence (IF) staining To investigate the histopathological characteristics of BLNE, we selected 51 CRC patients with BLNE (diameter > 10 mm) matched with 52 CRC patients with lymph node diameters less than 5 mm. HE sections of primary tumor tissue were assessed to evaluate the distribution of tertiary lymphoid structures (TLS), and CD3\CD19 IHC staining was performed on slides of tumor tissues and lymph nodes from 79 patients with available CRC. The slides of tumor tissues were then subjected to CD3/CD8 IF staining [ 13 ] to evaluate their immunoscore [ 14 ]. TLS are organized aggregates of immune cells formed in non-lymphoid tissues [ 15 ]. We used the method of Ding et al. [ 16 ] to classify TLS as follows: 1) no lymphoid aggregates formed by lymphatic follicles, 2) lymphoid follicular structures without germinal centers, and 3) lymphoid follicular structures with germinal centers. Based on the above grouping, two researchers independently evaluated TLS in all samples, and those with at least one lymphoid aggregation but no lymphoid follicular structure were classified as TLS+, those with at least one lymphoid follicle-like structure were classified as TLS++, and samples without TLS were classified as TLS negative (TLS-) for further analysis. Immunoscore were calculated based on an internationally validated consensus [ 14 , 17 , 18 ]. The immunoscore was calculated based on the expression levels of CD3 and CD8 in the tumor core (CT) and invasive margin (IM) regions. The cell density in each region was calculated and converted to percentiles, and the average of the four percentiles was calculated. The immunoscore was categorised into three groups, with mean percentiles of 0–25% (low), 25–75% (intermediate), and 75–100% (high). The expression results for each index in all stained slices were counted or assessed using ImageJ software. Whole-exome sequencing (WES) To investigate the etiology of BLNE, we selected frozen tumor tissues of 51 patients from the tissue sample bank for WES, comprising 24 samples in the BLNE group and 27 samples in the NBLNE group. After assessing the quality by fastp, clean sequence reads were aligned to the GRCh38 human reference genome using the BWA-MEM algorithm [ 19 , 20 ]. SAMtools was used to sort and index the original BAM files, with duplicate reads marked by Picard [ 21 ]. The Genome Analysis Tool Kit pipeline was employed to detect SNP and short indel somatic variants, adhering to the "Best Practices Workflows" outlined at https://gatk.broadinstitute.org/ [ 22 , 23 ]. The clinical impact of the variants was assessed by referencing the [ 24 ], dbSNP [ 25 ], COSMIC [ 26 ], and OncoKB databases [ 27 ]. The splice-site somatic and non-silent exonic variants were also assessed. The somatic variation data were summarized, and the outcomes were graphically represented using the maftools R package [ 28 ]. Additionally, the tumor mutation burden (TMB) values were calculated using the "TMB" function from the maftools software package, representing the number of nonsilent mutations per megabase (Mb) in each dataset sample. TMB was compared between our cohort (BLNE, NBLNE) using the Mann–Whitney U test. To detect genes with significantly different mutational frequencies between the analysed datasets, Fisher exact test was employed using the "mafCompare" function. Mutational signature analysis was conducted on the BLNE and NBLNE groups, utilizing COSMIC single-base substitution signatures. Statistical analysis For the comparison of variables, bicategorical variables were assessed using the chi-square (Fisher exact) test, while continuous variables were analysed using the t-test. Continuous variables with normal distribution were presents as mean ± standard deviation ; non-normal variables were reported as median (interquartile range). Univariate and multivariate analyses were performed for each clinicopathological factor; P < 0.05 was considered as a statistically significant result, and Cox multivariate regression was performed for these factors. Tumor prognosis was assessed based on DFS and OS, and survival analysis was conducted using the Kaplan-Meier method. RESULTS BLNE is associated with a better prognosis in patients with AJCC stage I/II CRC . After screening, a total of 630 patients were included in this study. The flowchart is shown in Additional file 1: Figure S1. The clinicopathological features of the 630 patients included in this study are presented in Table 1. Most patients in this group were males (63.3%), with an average age of 65.59 ± 11.31 years, including 157 (24.9%) in the right colon, 237 (37.6%) in the left colon, and 236 (37.5%) in the rectum. According to the subgroup analysis, the incidence of tumors on the right side of the colon was significantly greater in the BLNE group than in the NBLNE group (48.1% vs. 18.8%, P < 0.0001). The T stages of the tumors in the NBLNE group were mainly T2 and T3, whereas those in the BLNE group were mainly T3 ( P < 0.0001). There were significantly more patients with AJCC stage II disease in the BLNE group than in the NBLNE group (85.5% vs. 61.7%, P < 0.0001). Excluding 34 patients with missing tumor size data, 51.1% of tumors in the BLNE group were over 5 cm, compared with 15.5% in the NBLNE group ( P < 0.0001), with a majority of tumors ≤ 3.5 cm (51.8%) Table 1 Clinicopathological features of BLNE and NBLNE groups Characteristics Total ( n = 630) BLNE ( n = 131) NBLNE ( n = 499) P for Difference Age 65.59 ± 11.31 63.61 ± 12.44 66.61 ± 10.95 0.024 Sex Male 399 (63.3) 86 (65.6) 313 (62.7) Female 231 (36.7) 45 (34.4) 163 (37.3) 0.982 T Stage I 73 (11.6) 2 (1.5) 71 (14.2) II 154 (24.4) 19 (14.5) 135 (27.1) III 380 (60.3) 99 (75.6) 281 (56.3) IV 23 (3.7) 11 (8.4) 12 (2.4) < 0.0001 AJCC Stage I 210 (33.3) 19 (14.5) 191 (38.3) II 420 (66.7) 112 (85.5) 308 (61.7) < 0.0001 Cancer site Right 157 (24.9) 63 (48.1) 94 (18.8) Left 237 (37.6) 31 (23.7) 206 (41.3) Rectum 236 (37.5) 37 (28.2) 199 (39.9) 5cm 133 (22.8) 67 (51.1) 72 (15.5) >3.5cm,≤5cm 202 (34.6) 50 (38.2) 152 (32.7) ≤3.5cm 249 (42.6) 14 (10.7) 241 (51.8) < 0.0001 BLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement; AJCC: American Joint Committee on Cancer; NLR: neutrophil-to-lymphocyte ratio; VI: vascular invasion; PNI: perineural invasion We also compared age, sex, the neutrophil-to-lymphocyte ratio (NLR), the VI, and the PNI distribution between the two groups. Patients in the NBLNE group had a greater mean age (66.61 ± 10.95 vs. 63.61 ± 12.44 years, P = 0.024), with no significant differences observed between the two groups in terms of sex, NLR, VI, or PNI ( P = 0.982/0.374/0.538/0.719). During follow-up, 54 patients (8.6%) experienced recurrent CRC and 576 patients (91.3%) had no signs of recurrence, with 4 patients (3.1%) in the BLNE group and 50 patients (10.0%) in the NBLNE group. At the end of the study, 58 patients died (9.2%): 5 in the BLNE group (3.8%) and 53 in the NBLNE group (10.6%). The KM curves of the two groups are shown in Figure 1 (A and B), with statistically significant differences in DFS and OS between the two groups ( P = 0.016 and P = 0.011, respectively). Further subgroup analysis revealed superior DFS and OS in the BLNE group compared with those in the NBLNE group in the right colon (Figure 1C and 1D), whereas no significant difference was observed in the left colon and rectum (Additional file 2: Figure S2), possibly due to the relatively smaller sample sizes.Univariate analysis revealed that age > 60 years (Hazard ratio (HR) 3.30, 95% confidence interval (CI) 1.51-7.25, P = 0.003) and BLNE (HR 3.07, 95% CI 1.23-7.64, P = 0.016) were significantly correlated with OS, whereas the NLR (HR 1.87, 95% CI 1.10-3.21, P = 0.023) and BLNE (HR 3.26, 95% CI 1.18-9.04, P = 0.023) significantly were correlated with DFS. Multivariate regression analysis, incorporating the above variables and tumor T stage, identified tumor T stage (T3, P =0.048; T4, P = 0.040), NLR (HR 1.73, 95% CI 1.01-2.98, P = 0.047), and BLNE (HR 3.85, 95% CI 1.37-10.79, P = 0.010) as independent risk factors affecting DFS. Age > 60 years (HR 2.65, 95% CI 1.20-5.86, P = 0.016) and BLNE (HR 3.03, 95% CI 1.20-7.61, P = 0.019) were independent risk factors for OS (Additional file 3: Table S1). Extensive lymph node dissection is associated with worse prognosis in AJCC stage I/II CRC patients If lymph node metastasis is suspected before surgery, the surgeon will perform more extensive lymph node dissection. To investigate the relationship between lymph node dissection and prognosis, we analysed the number of lymph nodes harvested from 1435 patients with stage I–IV disease, including 888 patients with stage I/II disease and 547 patients with stage III/IV disease (Additional file 1: Figure S1). When the number of harvested lymph nodes was less than 12, no significant difference in DFS or OS was observed compared with patients with more than 12 lymph nodes harvested ( P = 0.765; P = 0.245). Subgroup analysis revealed that DFS and OS remained unaffected when fewer than 12 lymph nodes were removed, regardless of the presence of lymph node metastasis (Additional file 4: Figure S3A–C; Additional file 5: Figure S4A–C) Interestingly, among patients without lymph node metastasis (AJCC I/II), patients with > 15 harvested lymph nodes had worse DFS ( P = 0.041), while no significant difference was observed in the whole cohort or in patients with lymph node metastasis only. However, no significant difference in OS was observed (Additional file 4: Figure S3D–F; Additional file 5: Figure S4D–F). Among the 131 patients with BLNE, only 4 patients experienced relapse, all of them were with > 15 lymph nodes dissected, indicating a trend of worse prognosis in extended lymph node dissection, although the difference was not statistically significant ( P = 0.231) due to the relatively small sample size (Additional file 4: Figure S3). BLNE has specific radiologic features compared with MLNE Among all the 290 patients with lymph nodes > 8 mm in diameter visible on dual-phase abdominal CT before surgery, 165 patients had lymph node metastases (Additional file 1: Figure S1). The lymph node morphology, margin features, and CT values were evaluated on arterial-phase CT images (Figure 2). The patient information is presented in Additional file 6: Table S2. In our cohort, BLNE appeared in 20.8% of AJCC stage I/II CRC patients, and 43.1% of patients with radiologically visible enlarged lymph nodes (Figure 2E). In terms of clinicopathological features, patients in the MLNE group were predominantly females (53.3% vs. 36%, P = 0.003) and exhibited more T4 stages ( P = 0.042), and VI and PNI were more common (48.9% vs. 9.6%, P < 0.0001 and; 28.5% vs. 8.8%, P < 0.0001, respectively); however, the tumor size was smaller in the MLNE group ( P < 0.0001). Regarding the morphological features on CT, the BLNE group showed less heterogeneity enhancement and necrotic centers (16% vs. 69.1%, P < 0.0001; 1.6% vs. 20.6%, P < 0.0001), more clear outer borders and regular lymph node forms (83.2% vs. 55.8%, P < 0.0001; 94.4% vs. 62.3%, P < 0.0001), and lower CT value differences (31.83 ± 10.56 vs. 41.51 ± 14.72, P < 0.0001). B LN E is associated with characteristics of enhanced adaptive immunity , including TLS CT image, HE staining image, and macroscopic photo of BLNE are shown in Figure 3. The number of follicles per 10 mm 2 area of lymph node slide was calculated. The number and density of follicles were significantly greater in the BLNE group than in the NBLNE group (13.69 ± 4.89 vs. 5.08 ± 3.87, P < 0.001). To investigate the cellular composition of the BLNE , we labelled T cells and B cells with CD3 and CD19 . We observed that both the BLNE and NBLNE primarily consisted of lymphoid nodules containing B cells and diffuse lymphoid tissue containing T cells, with no apparent distinction between them (Additional file 7: Figure S5). HE staining of primary tumor tissue revealed the presence of TLS, which was confirmed by IF staining (Figure 3). There were significantly more mature TLSs (TLS++) in the BLNE group than in the NBLNE group (66.7% vs. 36.5%, P = 0.002) (Figure 3I). Of the 79 patients who underwent IF staining with CD3 and CD8 antibodies, 32 were in the BLNE group and 47 were in the NBLNE group. Three regions of CT and IM regions were randomly selected from each section, and the average density of CD3 and CD8 staining in each region was calculated using Image J (Figure 4A). The results revealed more CD3 positive cells in the CT (571.01 ± 368.30 vs. 341.52 ± 228.86, P = 0.002) and IM (794.18 ± 369.86 vs. 633.51 ± 369.86, P = 0.045) regions of the primary tumor tissue in the BLNE group than in the NBLNE group (Figure 4B–C). CD8 expression in the BLNE group was greater in the IM (231.60 ± 219.75 vs. 119.03 ± 124.72, P = 0.011) than in the NBLNE group, but no difference was found in the CT region ( P = 0.09) (Figure 4D–E). Based on the percentiles of the mean densities of CD3 and CD8 in the CT and IM regions, the immunoscore were classified as high, intermediate, or low. The results indicated a greater proportion of high immunoscore in the primary tumor tissue from the BLNE group than in that from the NBLNE group (Figure 4F). BLNE is associated with greater TMB and specific mutation signatures We performed a high-depth WES of 51 CRC tissues (24 with BLNE and 27 with NBLNE). The mean WES coverage was 114×. Sequencing data revealed that the most common variant classification was missense mutation, and the most common variant type was SNP. Among these SNPs, the C>T mutation was dominant (Additional file 8: Figure S6). The mutation landscape plot showed that TTN and MUC12 had the greatest number of mutations in patients with BLNE and NBLNE, respectively (Figure 5A–B). Fisher's exact test was used to compare the differences in mutations between the two groups, and significant differences were observed for 11 genes ( P < 0.01). The differentially mutated genes had a greater mutation frequency in the BLNE group, except for MUC12 (Figure 5C). TMB was also evaluated and compared between the groups. The BLNE group had a significantly greater TMB than the NBLNE group (Figure 5D). In addition to the aforementioned results, tumor mutation signatures were analysed in detail. The mutation characteristics of all patients showed that C>A and C>T were the major components of SBSs (Additional file 9: Figure S7). Non-negative matrix factorization and cosine similarity revealed that the mutation signatures of all sequence data were associated with signatures 1, 6, 10, 15, and 28 in the COSMIC database. Comparative analysis of these five mutation features revealed that signature 1 was more enriched in the BLNE group (Figure 5E). Signature 1 arises from an endogenous mutational process initiated by the spontaneous deamination of 5-methylcytosine. Discussion In this study, we analysed the clinicopathological features of 630 patients with BLNE and performed HE staining, IHC, IF staining and WES to explore BLNE occurrence in the tumor immune microenvironment and at the genomic level. This is the first study to evaluate the relationships among BLNE, the tumor immune microenvironment, and genomic features in patients with CRC. We observed that patients with BLNE were younger, with a predominantly right colon location, T3 stage and larger tumors. Lymph node metastasis in the tumor drainage area impacts both postoperative adjuvant therapy and preoperative treatment strategies. The Japanese CRC guidelines recommend D2 lymph node dissection for stage T1 and stage T2 patients without lymph node metastasis [ 3 ]. A smaller lymph node dissection area often results in fewer surgical complications [ 29 – 32 ]. Additionally, precursor-exhausted cells (Tpex) expressing the transcription factor TCF-1, which is essential for maintaining antigen-specific CD8 + T cell responses, proliferate mainly in tumor-draining lymph nodes [ 33 ]. Therefore, as a local hub of anti-tumor immunoity [ 34 ], whether lymph nodes should be extensively dissected warrants consideration. Thus, we analysed the effect of the number of lymph node dissections on patient prognosis of patients. Although fewer than 12 lymph nodes dissection is recommended as a risk factor for postoperative chemotherapy in stage II CRC patients [ 35 ], in our study, fewer than 12 lymph nodes dissection did not affect the long-term prognosis of AJCC I-II patients. Interestingly, the prognosis was worse when the number of lymph nodes was more than 15 in patients without lymph node metastasis, possibly because of destruction of the anti-tumor adaptive immune response. Therefore, accurate BLNE identification before surgery might affect surgical decision-making. Preoperative diagnosis of lymph node status in colon cancer remains a significant challenge, mainly relying on imaging methods such as enhanced CT, MRI, and PET-CT. By combining features of morphology and signal features instead of size criteria [ 36 ], MRI can achieve 80–85% sensitivity and 97–98% specificity [ 37 , 38 ], while the accuracy of CT, the most common test for colon cancer, is only 59–71% [ 39 ]. Although recently artificial intelligence [ 40 ], liquid biopsy [ 41 ], and various clinicopathological factors [ 42 ], including tumor budding, submucosal infiltration depth, vascular nerve invasion, and histological grade, have been used to predict lymph node metastasis, surgeons rely mainly on morphological features on imaging to diagnose lymph node metastasis. Thus, we compared the differences in CT images between the BLNE and MLNE groups. The results showed that MLNE had more unclear outer border, heterogeneity enhancement, and irregular form, especially the necrotic centre, an important indicator of lymph node metastasis, which can help surgeons to identify lymph node metastasis more intuitively, necessitating further radiomic studies. We speculated that the BLNE is correlated with the adaptive immune response of tumors. When antigens enter the lymph nodes through the afferent lymphatic vessels, fibroblast reticular cells proliferate, and the number of activated T and B cells increases rapidly, leading to rapid expansion of the lymph nodes [ 8 ]. HE and IHC staining of lymph nodes revealed morphological similarities between BLNE and NBLNE. However, there were significant differences in the number and density of lymphatic follicles, confirming that BLNE was the consequence of activated B and T cell proliferation. Our study revealed superior OS ( P = 0.011) and DFS ( P = 0.016) in the BLNE group than in the NBLNE group. Whether this survival advantage was due to the activation of the immune system was unclear; therefore, we performed an immunoscore analysis of the primary tumor tissue. The results revealed significantly more CD3 + T cells in the tumor core ( P = 0.002) and IM ( P = 0.045) in the BLNE group than in the NBLNE group and significantly more CD8 + T cells in the IM ( P = 0.011) than in the NBLNE group. BLNE was closely related to a greater immunoscore. The prognostic value of CD8 + T cells for DFS and OS has been demonstrated in 17 solid tumors, and among different immune cell subtypes, CD8 + T cells have the most positive impact on cancer survival [ 43 ]. Immunoscore plays a prognostic role in CRC, surpassing classical TNM stages in predicting DFS and OS [ 14 , 44 , 45 ]. In addition, more mature TLS were found in tumor tissues from the BLNE group. TLS has structural and functional characteristics similar to those of lymph nodes and is considered the first site at which T cells may be activated to form an anti-tumour immune response [ 15 , 46 ]. These results suggest that the positive effect of BLNE on prognosis may be related to the activation of anti-tumor immunity. Although the NLR in the peripheral blood was a predictor of CRC prognosis ( P = 0.047), no significant differences were observed between the BLNE and NBLNE groups. Therefore, we conclude that the effect of BLNE on tumor immunity is mainly associated with the local tumor microenvironment and is not influenced by circulating immune cells. TMB is an effective tool for predicting immune checkpoint blockade response in patients. Somatic mutations in tumor DNA may produce antigens that are recognized by the immune system. Although not all mutations produce neoantigens, somatic mutations in a tumor correspond to increased recognition of neoantigens [ 47 ]. In our study, the TMB was significantly greater in the BLNE group than in the NBLNE group ( P = 0.025), which may be responsible for the stronger immune response in patients in the BLNE group. Additionally, we identified 11 mutated genes with significant differences between the two groups (MUC12, PLCH1, LRP1B, OBSL1, FHOD3, MCM3AP, MYH9, NBPF1, NLRP8, PCDHA1, and PTGIR). Except for MUC12, which mainly occurred in the NBLNE group, the rest mainly occurred in the BLNE group. MUC12 encodes a complete membrane glycoprotein, a member of the mucin family, and is more highly expressed in normal colon tissue than in colon cancer. Matsuyama et al. reported that MUC12 is an independent predictor of prognosis in stage II and III CRC,patients and that poor prognosis is associated with low MUC12 expression [ 48 ], consistent with our finding of a high MUC12 mutation rate in the NBLNE group. We must admit that our paper has some limitations. First of all, our data were from a single center, and lacked the validation from multiple centers, however, the relatively larger cohort size makes our results reliable. Second, our study focused mainly on lymphocytes in the tumor microenvironment, and other immune cell types such as neutrophils and macrophages also need further study. Moreover, our study is limited to exon sequencing, which needs to be analysed in combination with transcriptome sequencing data, and further functional testing is necessary to verify the identified mutated genes. Our study confirmed the positive impact of BLNE on CRC prognosis and delineated its clinicpathological features. Additionally, our investigation emphasized the significance of accurately identifying BLNE preoperatively and highlighted its morphological characteristics on CT scans for surgeons. Subsequent pathological staining revealed a potential association between BLNE and an enhanced immune response against tumor cells. Supported by WES data, the identification of differentially mutated genes potentially linked to BLNE offers avenues for further exploration. Conclusions In conclusion, BLNE is a positive prognostic factor for colorectal cancer, possibly because BLNE patients have a stronger anti-tumor immune response. In patients without lymph node metastasis, excessive lymph node dissection can lead to a worse prognosis, and accurate preoperative lymph node staging is critical. Abbreviations BLNE: Benign lymph node enlargement; NBLNE: Non-Benign lymph node enlargement; DFS: Disease-free survival; OS: Overall survival; HR: Hazard ratio; OR: Odds ratio; CRC: Colorectal cancer; CT: computed tomography; MRI: magnetic resonance imaging; AJCC: American joint Committee on cancer; MSI: Microsatellite instability; VI: vascular invasion; PNI: perineural invasion; MLNE: metastatic lymph node enlargement; HE: Hematoxylin and eosin; IHC: Immunohistochemical; IF: Immunofluorescence TLS: tertiary lymphoid structures; CT: tumor core; IM: invasive margin; WES: Whole-exome sequencing; TMB: Tumor mutation burden; NLR: neutrophil-to-lymphocyte ratio; CI: confidence interval; Declarations Acknowledgements During the research process, the technical support and help from the Center of Basic Medical Research, Institute of Medical Innovation and Research are indispensable, and we would like to express our sincere thanks to the staff of the center. Authors' contributions Ideas and research design: ZX, WJW, FW; Data collection: LXY, LSY; Image reading: WBY, LF; Analyze and interpret data: WJW, SXC, CX; Manuscript: WJW, SXC, GLM. All authors read and approved the final manuscript. Funding Xin Zhou was supported by Beijing Nova Program (20220484153 and 20230484485)；Wei Fu was was supported by grants from the Beijing Natural Science Foundation（L234001） Availability of data and materials All relevant data are available from the corresponding authors for research use only. Ethics approval and consent to participate This study was approved by the Medical Science Research Ethics Committee of Peking University Third Hospital (IRB00006761-M2020123) and conducted in accordance with the Declaration of Helsinki. Informed consent from all patients prior to obtaining tumor tissue. Consent for publication Not required. Competing interests The authors declare that they have no competing interests. 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Supplementary Files Additionalfile1FigureS1.jpg Additional file 1: Figure S1 .jpg Flow chart for selecting patients with colorectal cancer Additionalfile2FigureS2.jpg Additional file 2: Figure S2 .jpg Subgroup analysis of BLNE association with DFS and OS (A-B) In left-sided colon cancer, DFS and OS were not significantly different between BLNE and NBLNE; (C-D) In rectal cancer, DFS and OS were not significantly different between BLNE and NBLNE DFS: disease-free survival; OS: overall survival; BLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement Additionalfile3TableS1.docx Additional file 3: TableS1 .doc Prognostic factors for DFS and OS DFS: disease-free survival; OS: overall survival; NLR: neutrophil-to-lymphocyte ratio; VI: vascular invasion; PNI: perineural invasion; NBLNE: non-benign lymph node enlargement Additionalfile4FigureS3.jpg Additional file 4: Figure S3 .jpg Relationship between the number of lymph node dissection and DFS in patients with different AJCC stages (A-C) DFS was not affected by lymph node dissection with fewer than 12 nodes; (D-F) In stage I/II patients, lymph node dissection greater than 15 was associated with worse DFS; (H) Relationship between the number of dissected lymph nodes and DFS in BLNE group BLNE: benign lymph node enlargement; DFS: disease-free survival AJCC: American Joint Committee on Cancer Additionalfile5FigureS4.jpg Additional file 5: Figure S4 .jpg Relationship between the number of lymph node dissection and OS in patients with different AJCC stages (A-C) OS was not affected by lymph node dissection with fewer than 12 nodes; (D-F) In stage I/II patients, lymph node dissection greater than 15 was associated with worse OS; (H) Relationship between the number of dissected lymph nodes and OS in BLNE group BLNE: benign lymph node enlargement; OS: overall survival; AJCC: American Joint Committee on Cancer Additionalfile6TableS2.docx Additional file 6: Table S2 .doc Clinicopathological features of BLNE and MLNE groups VI: vascular invasion; PNI: perineural invasion; BLNE: benign lymph node enlargement; MLNE: Metastatic lymph node enlargement Additionalfile7FigureS5.jpg Additional file 7: Figure S5 .jpg BLNE and NBLNE in IHC images (A-B) IHC labels CD19 and CD3 in BLNE; (C-D) IHC labels CD19 and CD3 in NBLNE BLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement; IHC: immunohistochemistry Additionalfile8FigureS6.jpg Additional file 8: Figure S6 .jpg Summary plots of the variants found in CRC CRC: colorectal cancer; SNV: single nucleotide variants Additionalfile9FigureS7.jpg Additional file 9: Figure S7 .jpg Somatic mutation signatures comparison between BLNE and NBLNE samples BLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-3975650","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274185115,"identity":"95c5e9d1-44b9-486a-8e3b-08c2ef2b5215","order_by":0,"name":"Junwei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACNv7GhgMfeGzsGOeff/ggoaKGsBY+icMHH86QSUtmnsHDbPDgzDHCWuQY0pKNeWwOM7bP4GGTfNjCTITDGM6YSfPkMDPzzu49VpHYwMbA396dgF8Lc4+Z5JwzbHySc86l3UjcIcMgcebsBoK2SLzt4WE2bEgwu5F4ho3BQCKXkJYcMwnefxKM+w8kmBUktjEToyUt2ZCHx4CxcUaOGQNxWsCBzJOQzNhzLFki4cwxHoJ+ke8HR+V/O8b25oMff1TUyPG39+LXggF4SFM+CkbBKBgFowArAAAmFEv/t8qNbQAAAABJRU5ErkJggg==","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":true,"prefix":"","firstName":"Junwei","middleName":"","lastName":"Wang","suffix":""},{"id":274185116,"identity":"c47768e6-6689-4b2b-8aa0-3ad700058efe","order_by":1,"name":"Xiangchao Shi","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiangchao","middleName":"","lastName":"Shi","suffix":""},{"id":274185117,"identity":"1aaebed4-b05f-436b-adad-6225619639d7","order_by":2,"name":"Limei Guo","email":"","orcid":"","institution":"Department of Pathology, School of Basic Medical Sciences, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Limei","middleName":"","lastName":"Guo","suffix":""},{"id":274185118,"identity":"27a5589f-dcc1-4673-a08a-9cf4ec76d45d","order_by":3,"name":"Fei Li","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Li","suffix":""},{"id":274185119,"identity":"d1f81651-a05e-4c8b-bff9-9a9e36d98226","order_by":4,"name":"Siyi Lu","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Siyi","middleName":"","lastName":"Lu","suffix":""},{"id":274185120,"identity":"9bba72fa-f95b-43fe-80fd-6465051c63f6","order_by":5,"name":"Bingyan Wang","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bingyan","middleName":"","lastName":"Wang","suffix":""},{"id":274185121,"identity":"9e80c31a-02f3-4519-9848-2ea5446d8589","order_by":6,"name":"Xinyi Lin","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Lin","suffix":""},{"id":274185122,"identity":"98543678-62e9-4563-910b-f3c57fe5b033","order_by":7,"name":"Xin Chen","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Chen","suffix":""},{"id":274185124,"identity":"7122096a-7509-4d18-9e10-555fb7b9423c","order_by":8,"name":"Wei Fu","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Fu","suffix":""},{"id":274185126,"identity":"4db8034f-9c48-435e-9092-904fec6534d7","order_by":9,"name":"Xin Zhou","email":"","orcid":"","institution":"Department of General Surgery, Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-02-21 13:32:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3975650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3975650/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51563080,"identity":"8d35f614-9f56-45c0-a64c-a24ef35bdfa2","added_by":"auto","created_at":"2024-02-23 18:44:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":612970,"visible":true,"origin":"","legend":"\u003cp\u003ePatients in the BLNE group had a better long-term prognosis. (A,B) Patients in the BLNE group had better DFS and OS compared to the NBLNE group. (C,D) In subgroup analysis, patients with right-sided colon cancer had better DFS and OS in the BLNE group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/2d4cb984fe1d659fa68bd693.png"},{"id":51563989,"identity":"83bc1af5-7a94-49d6-bbe9-7f59b7821112","added_by":"auto","created_at":"2024-02-23 18:52:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":371565,"visible":true,"origin":"","legend":"\u003cp\u003eCT features and distribution of enlarged lymph nodes. (A)Heterogeneity enhancement and unclear outer border; (B) Heterogeneity enhancement; (C) Heterogeneity enhancement and irregular form; (D) Necrotic center; (E) The proportion of lymph node enlargement.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/95e004d1d02e851b8e77bf97.png"},{"id":51563090,"identity":"a7da4ff7-14a4-42ee-908f-74dc7ab8f921","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1928075,"visible":true,"origin":"","legend":"\u003cp\u003ePathological features of BLNE and corresponding primary tumor tissue. (A) BLNE on CT image; (B) Macroscopic photo of BLNE; (C) Lymph nodes in the BLNE group had more abundant follicles than those in the NBLNE group; (D-F) HE image of BLNE, NBLNE and MLNE; (G) HE image of TLS in tumor tissue; (H) IF image of TLS in tumor tissue, blue represents DAPI, green represents CD3, and red represents CD8; (I) Mature TLS in the BLNE group were significantly more than those in the NBLNE group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/17f5da0171de72acb9eeaeff.png"},{"id":51563086,"identity":"ecbdb055-ee47-4aa1-9313-840e2cfe166a","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1063839,"visible":true,"origin":"","legend":"\u003cp\u003eThe immunoscore of primary tumor tissue in BLNE and NBLNE group.\u003c/p\u003e\n\u003cp\u003e(A) CT and IM in IF staining image, DAPI in blue, CD3 in green, and CD8 in red; (B,C) The expression of CD3 in BLNE group was more abundant in the CD and IM regions; (D,E) The expression of CD8 in BLNE group was more abundant in IM region; (F) The BLNE group had greater immunoscore. CT: tumor core; IM: Invasive margin\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/f50cfcbdf3ba60d3d2e482a2.png"},{"id":51563990,"identity":"901a0bbf-9b3b-4e53-82df-9129be83e1da","added_by":"auto","created_at":"2024-02-23 18:52:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":806668,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in mutated genes and TMB between the BLNE and NBLNE groups. (A-B) Somatic mutation landscape of colorectal cancer; (C) Forest map of mutation differences between the two groups; (D) The TMB of the BLNE group is greater than that of the NBLNE group, TMB is compared in lg (mut / Mb) ; (E) Somatic mutation signatures comparison between BLNE and NBLNE groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/b80f116d8ff835b8268fa78c.png"},{"id":51654084,"identity":"71e7a698-82f0-4bb2-98b0-6b00a2c23fa1","added_by":"auto","created_at":"2024-02-26 16:23:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4498728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/06c1a51e-df6a-4660-b536-5b4a109fcc04.pdf"},{"id":51563081,"identity":"cccd72c4-2774-4f31-8923-fc9002e8784f","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1763925,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1: Figure S1 .jpg\u003c/p\u003e\n\u003cp\u003eFlow chart for selecting patients with colorectal cancer\u003c/p\u003e","description":"","filename":"Additionalfile1FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/aa15aeca15875497c1acddb2.jpg"},{"id":51563083,"identity":"2e3fbe36-b09f-4f67-a7a2-43d4ea66c562","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":256335,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2: Figure S2 .jpg\u003c/p\u003e\n\u003cp\u003eSubgroup analysis of BLNE association with DFS and OS\u003c/p\u003e\n\u003cp\u003e(A-B) In left-sided colon cancer, DFS and OS were not significantly different between BLNE and NBLNE; (C-D) In rectal cancer, DFS and OS were not significantly different between BLNE and NBLNE\u003c/p\u003e\n\u003cp\u003eDFS: disease-free survival; OS: overall survival; BLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement\u003c/p\u003e","description":"","filename":"Additionalfile2FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/7b8ce2b85415a92414b60056.jpg"},{"id":51563089,"identity":"a32240c6-5160-46cc-a7ed-5daa68f9bfdd","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16008,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 3: TableS1 .doc\u003c/p\u003e\n\u003cp\u003ePrognostic factors for DFS and OS\u003c/p\u003e\n\u003cp\u003eDFS: disease-free survival; OS: overall survival; NLR: neutrophil-to-lymphocyte ratio; VI: vascular invasion; PNI: perineural invasion; NBLNE: non-benign lymph node enlargement\u003c/p\u003e","description":"","filename":"Additionalfile3TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/6576946af3400560a3e6cef2.docx"},{"id":51563085,"identity":"245498fc-d513-4c75-a0ec-9017f73cb55f","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":251898,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 4: Figure S3 .jpg\u003c/p\u003e\n\u003cp\u003eRelationship between the number of lymph node dissection and DFS in patients with different AJCC stages\u003c/p\u003e\n\u003cp\u003e(A-C) DFS was not affected by lymph node dissection with fewer than 12 nodes; (D-F) In stage I/II patients, lymph node dissection greater than 15 was associated with worse DFS; (H) Relationship between the number of dissected lymph nodes and DFS in BLNE group\u003c/p\u003e\n\u003cp\u003eBLNE: benign lymph node enlargement; DFS: disease-free survival AJCC: American Joint Committee on Cancer\u003c/p\u003e","description":"","filename":"Additionalfile4FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/7ba58289257127d2cec0c39f.jpg"},{"id":51563991,"identity":"f5d3cbe2-e190-45b5-8e62-09e257c33464","added_by":"auto","created_at":"2024-02-23 18:52:09","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":253872,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 5: Figure S4 .jpg\u003c/p\u003e\n\u003cp\u003eRelationship between the number of lymph node dissection and OS in patients with different AJCC stages\u003c/p\u003e\n\u003cp\u003e(A-C) OS was not affected by lymph node dissection with fewer than 12 nodes; (D-F) In stage I/II patients, lymph node dissection greater than 15 was associated with worse OS; (H) Relationship between the number of dissected lymph nodes and OS in BLNE group\u003c/p\u003e\n\u003cp\u003eBLNE: benign lymph node enlargement; OS: overall survival; AJCC: American Joint Committee on Cancer\u003c/p\u003e","description":"","filename":"Additionalfile5FigureS4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/1ef8d5a612eab9b541ecab11.jpg"},{"id":51563093,"identity":"4e2f0ed9-ac3a-46b8-8833-f9a8fec1c7f7","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15204,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 6: Table S2 .doc\u003c/p\u003e\n\u003cp\u003eClinicopathological features of BLNE and MLNE groups\u003c/p\u003e\n\u003cp\u003eVI: vascular invasion; PNI: perineural invasion; BLNE: benign lymph node enlargement; MLNE: Metastatic lymph node enlargement\u003c/p\u003e","description":"","filename":"Additionalfile6TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/3fc65555192e8c770cd86997.docx"},{"id":51563091,"identity":"ebd894a9-6998-423e-bd65-235edded7dbf","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":490151,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 7: Figure S5 .jpg\u003c/p\u003e\n\u003cp\u003eBLNE and NBLNE in IHC images\u003c/p\u003e\n\u003cp\u003e(A-B) IHC labels CD19 and CD3 in BLNE; (C-D) IHC labels CD19 and CD3 in NBLNE\u003c/p\u003e\n\u003cp\u003eBLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement; IHC: immunohistochemistry\u003c/p\u003e","description":"","filename":"Additionalfile7FigureS5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/946d2de13eddb5fd349339fc.jpg"},{"id":51563094,"identity":"c9f35ea4-b0d3-4786-85a9-ee2d2dfcae7d","added_by":"auto","created_at":"2024-02-23 18:44:10","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":105289,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 8: Figure S6 .jpg\u003c/p\u003e\n\u003cp\u003eSummary plots of the variants found in CRC\u003c/p\u003e\n\u003cp\u003eCRC: colorectal cancer; SNV: single nucleotide variants\u003c/p\u003e","description":"","filename":"Additionalfile8FigureS6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/b11349d80219149271b835be.jpg"},{"id":51563088,"identity":"10f9c9ae-251c-430e-9ca7-6d1f7721040e","added_by":"auto","created_at":"2024-02-23 18:44:09","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":79123,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 9: Figure S7 .jpg\u003c/p\u003e\n\u003cp\u003eSomatic mutation signatures comparison between BLNE and NBLNE samples\u003c/p\u003e\n\u003cp\u003eBLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement\u003c/p\u003e","description":"","filename":"Additionalfile9FigureS7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3975650/v1/9cf54156aae1c4cdb6924ecd.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mesenteric benign lymph node enlargement in colorectal cancer: Friend or Foe?","fulltext":[{"header":"Background","content":"\u003cp\u003eColorectal cancer (CRC) is a common malignancy worldwide, ranking second among the most common cancers in women and third among men, and poses a significant public health problem worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Lymph node metastasis is generally associated with a poorer prognosis. The 5-year overall survival rate for patients with American joint Committee on cancer (AJCC) stage I/II is greater than 85%, whereas that for patients with AJCC stage III with lymph node metastasis is between 67\u0026ndash;80% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the Japanese Guidelines for Colorectal Cancer, extensive lymph node dissection should not be performed in patients with T1 and T2 CRC when lymph node metastasis is absent [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Extensive lymph node dissection may result in a greater complication rate; therefore, determining the presence of lymph node metastasis before surgery is important.\u003c/p\u003e \u003cp\u003eCurrently, diagnosing preoperative lymph node metastasis mainly relies on computed tomography (CT) and magnetic resonance imaging(MRI). Lymph nodes over 8 mm in diameter with enhancement are usually considered metastatic [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the sensitivity and specificity of CT for determining lymph node metastasis are only 55% and 74%, respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In clinical practice, lymph node enlargement on preoperative imaging and postoperative pathology confirming the absence of metastatic cancer cells are common in CRC patients. In this study, lymph nodes with a diameter\u0026thinsp;\u0026gt;\u0026thinsp;8 mm on preoperative imaging without metastasis on postoperative pathology were termed mesenteric benign lymph node enlargement (BLNE) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Within lymph nodes, fibroblast reticular cells comprise 20\u0026ndash;50% of the non-hematopoietic region, creating a web of interconnected extracellular matrix components that form tubes that rapidly transport soluble antigens and signalling molecules deep into the lymph node parenchyma. When the adaptive immune response begins, the contractile function of FRC actomyosin is inhibited, facilitating rapid lymph node expansion to accommodate the increased number of activated T and B cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have also shown lymph node follicular hyperplasia in the drainage areas of patients with stage II colon cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], suggesting an association between non-metastatic enlargement of tumor-draining lymph nodes and the tumor-adaptive immune response. Recently, some studies have reported that BLNE is a favourable prognostic factor for colorectal cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while other studies have proposed hat it is associated with poorer prognosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, the clinical significance of mesenteric BLNE in stage I/II CRC with remains unclear. This study aimed to investigate the prognostic value of mesenteric BLNE in CRC and explore its clinical significance.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients Selection\u003c/h2\u003e \u003cp\u003e This study was approved by the Medical Science Research Ethics Committee of Peking University Third Hospital (IRB00006761-M2020123) and conducted in accordance with the Declaration of Helsinki. Between January 2015 and June 2023, 2,270 patients with CRC underwent surgery at Peking University Third Hospital. The clinical data of these patients were prospectively recorded, and all eligible patients were divided into two groups. The BLNE group comprised those with mesenteric BLNE, while the non-benign lymph node enlargement (NBLNE) group served as the control group, according to the 9th edition of the TNM staging system published by the AJCC. The inclusion criteria were as follows: 1) no regional lymph node metastasis or distant metastasis; 2) stage I and II patients in the AJCC staging system; 3) radical CRC resection achieving R0 status with no positive lymph nodes; and 4) a single malignant lesion confirmed via colonoscopy. Exclusion criteria: 1) underwent emergency surgery or had preoperative complications such as intestinal obstruction or perforation; 2) had suspected distant metastasis on positron emission tomography (PET-CT); 3) received preoperative neoadjuvant therapy; 4) had hereditary CRC such as familial adenomatous polyposis; and 5) had microsatellite instability (MSI) confirmed by pathology. In early CRC, MSI-H status is associated with a favourable prognosis and an enhanced anti-tumor immune response; thus, we excluded this subset of patients to avoid its impact on prognosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; 6) lacked imaging data; and 7) were lost to follow-up.\u003c/p\u003e \u003cp\u003eIn the study of the relationship between lymph node dissection and prognosis, we analysed the number of dissected lymph nodes in 1435 of the 2270 patients (excluding patients with preoperative neoadjuvant therapy and incomplete data). Additionally, we analysed the imaging features of all the 290 patients with enlarged lymph nodes seen on preoperative dual-phase CT scanning.\u003c/p\u003e \u003cp\u003eThe clinicopathological features assessed included sex, age, tumor location, tumor size, T stage, histological type, preoperative neutrophil and lymphocyte counts in peripheral blood, vascular invasion (VI), and perineural invasion (PNI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCT imaging features\u003c/h2\u003e \u003cp\u003eLymph nodes in the drainage area of the tumor were mainly observed, and patients were grouped according to preoperative CT findings. Lymph nodes with a maximum diameter\u0026thinsp;\u0026gt;\u0026thinsp;8 mm on CT images were considered positive. Patients with positive lymph nodes and no metastasis confirmed by pathology were in the BLNE group, those with positive lymph nodes and metastasis confirmed by pathology were in the metastatic lymph node enlargement (MLNE) group, and those with negative lymph nodes and no metastasis confirmed by pathology were in the NBLNE group.\u003c/p\u003e \u003cp\u003eIn the imaging analysis, the largest or most suspicious metastatic lymph nodes in the mesenteric drainage area in each patient with CRC were selected for further evaluation. Lymph node morphology, margins, and CT values were evaluated using arterial-phase CT images. Heterogeneous enhancement refers to nonuniform enhancement in a whole lymph node. The unclear outer border refers to the lymph node with rough edges, and the irregular form refers to the shape of the lymph node other than the round and oval. The difference in CT values represents the maximum CT value in the lymph node minus the minimum CT value. The necrotic center was defined as a lymph node with significant marginal enhancement and no central enhancement\u003c/p\u003e \u003cp\u003eTwo physicians who were blinded to the clinical and pathological data independently analysed the CT images. Discrepancies were resolved through discussion until a consensus was reached.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (HE), immunohistochemical (IHC), and immunofluorescence (IF) staining\u003c/h2\u003e \u003cp\u003eTo investigate the histopathological characteristics of BLNE, we selected 51 CRC patients with BLNE (diameter\u0026thinsp;\u0026gt;\u0026thinsp;10 mm) matched with 52 CRC patients with lymph node diameters less than 5 mm. HE sections of primary tumor tissue were assessed to evaluate the distribution of tertiary lymphoid structures (TLS), and CD3\\CD19 IHC staining was performed on slides of tumor tissues and lymph nodes from 79 patients with available CRC. The slides of tumor tissues were then subjected to CD3/CD8 IF staining [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] to evaluate their immunoscore [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTLS are organized aggregates of immune cells formed in non-lymphoid tissues [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We used the method of Ding et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] to classify TLS as follows: 1) no lymphoid aggregates formed by lymphatic follicles, 2) lymphoid follicular structures without germinal centers, and 3) lymphoid follicular structures with germinal centers. Based on the above grouping, two researchers independently evaluated TLS in all samples, and those with at least one lymphoid aggregation but no lymphoid follicular structure were classified as TLS+, those with at least one lymphoid follicle-like structure were classified as TLS++, and samples without TLS were classified as TLS negative (TLS-) for further analysis.\u003c/p\u003e \u003cp\u003eImmunoscore were calculated based on an internationally validated consensus [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The immunoscore was calculated based on the expression levels of CD3 and CD8 in the tumor core (CT) and invasive margin (IM) regions. The cell density in each region was calculated and converted to percentiles, and the average of the four percentiles was calculated. The immunoscore was categorised into three groups, with mean percentiles of 0\u0026ndash;25% (low), 25\u0026ndash;75% (intermediate), and 75\u0026ndash;100% (high). The expression results for each index in all stained slices were counted or assessed using ImageJ software.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eWhole-exome sequencing (WES)\u003c/h2\u003e \u003cp\u003eTo investigate the etiology of BLNE, we selected frozen tumor tissues of 51 patients from the tissue sample bank for WES, comprising 24 samples in the BLNE group and 27 samples in the NBLNE group. After assessing the quality by fastp, clean sequence reads were aligned to the GRCh38 human reference genome using the BWA-MEM algorithm [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. SAMtools was used to sort and index the original BAM files, with duplicate reads marked by Picard [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The Genome Analysis Tool Kit pipeline was employed to detect SNP and short indel somatic variants, adhering to the \"Best Practices Workflows\" outlined at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gatk.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://gatk.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The clinical impact of the variants was assessed by referencing the [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], dbSNP [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], COSMIC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and OncoKB databases [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The splice-site somatic and non-silent exonic variants were also assessed. The somatic variation data were summarized, and the outcomes were graphically represented using the maftools R package [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, the tumor mutation burden (TMB) values were calculated using the \"TMB\" function from the maftools software package, representing the number of nonsilent mutations per megabase (Mb) in each dataset sample. TMB was compared between our cohort (BLNE, NBLNE) using the Mann\u0026ndash;Whitney U test. To detect genes with significantly different mutational frequencies between the analysed datasets, Fisher exact test was employed using the \"mafCompare\" function. Mutational signature analysis was conducted on the BLNE and NBLNE groups, utilizing COSMIC single-base substitution signatures.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor the comparison of variables, bicategorical variables were assessed using the chi-square (Fisher exact) test, while continuous variables were analysed using the t-test. Continuous variables with normal distribution were presents as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation ; non-normal variables were reported as median (interquartile range). Univariate and multivariate analyses were performed for each clinicopathological factor; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as a statistically significant result, and Cox multivariate regression was performed for these factors. Tumor prognosis was assessed based on DFS and OS, and survival analysis was conducted using the Kaplan-Meier method.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eBLNE is associated with\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;a\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;better prognosis in patients with\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAJCC stage I/II CRC\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAfter screening, a total of 630 patients were included in this study. The flowchart is shown in Additional file 1: Figure S1. The clinicopathological features of the 630 patients included in this study are presented in Table 1. Most patients in this group were males (63.3%), with an average age of 65.59 \u0026plusmn; 11.31 years, including 157 (24.9%) in the right colon, 237 (37.6%) in the left colon, and 236 (37.5%) in the rectum. According to the subgroup analysis, the incidence of tumors on the right side of the colon was significantly greater in the BLNE group than in the NBLNE group (48.1% vs. 18.8%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). The T stages of the tumors in the NBLNE group were mainly T2 and T3, whereas those in the BLNE group were mainly T3 ( \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). There were significantly more patients with AJCC stage II disease in the BLNE group than in the NBLNE group (85.5% vs. 61.7%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). Excluding 34 patients with missing tumor size data, 51.1% of tumors in the BLNE group were over 5 cm, compared with 15.5% in the NBLNE group ( \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), with a majority of tumors \u0026le; 3.5 cm (51.8%)\u003c/p\u003e\n\u003cp\u003eTable 1 Clinicopathological features of BLNE and NBLNE groups\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"709\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003eTotal (\u003cem\u003en\u003c/em\u003e = 630)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003eBLNE (\u003cem\u003en\u003c/em\u003e = 131)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003eNBLNE (\u003cem\u003en\u003c/em\u003e = 499)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e for Difference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e65.59\u0026nbsp;\u0026plusmn;\u0026nbsp;11.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e63.61\u0026nbsp;\u0026plusmn;\u0026nbsp;12.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e66.61\u0026nbsp;\u0026plusmn;\u0026nbsp;10.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e399 (63.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e86 (65.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e313 (62.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e231 (36.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e45 (34.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e163 (37.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e0.982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eT Stage\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e73 (11.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e2 (1.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e71 (14.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e154 (24.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e19 (14.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e135 (27.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e380 (60.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e99 (75.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e281 (56.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e23 (3.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e11 (8.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e12 (2.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eAJCC Stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e210 (33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e19 (14.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e191 (38.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e420 (66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e112 (85.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e308 (61.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eCancer site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eRight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e157 (24.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e63 (48.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e94 (18.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eLeft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e237 (37.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e31 (23.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e206 (41.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eRectum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e236 (37.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e37 (28.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e199 (39.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eVI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e67 (10.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e12 (9.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e55 (11.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003ePNI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e58 (9.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e11 (8.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e47 (9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e0.719\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eNLR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e2.90 (\u0026plusmn;2.12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e3.05 (\u0026plusmn;1.79)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e2.86 (\u0026plusmn;2.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e0.374\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003eTumor Size\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026gt;5cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e133 (22.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e67 (51.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e72 (15.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026gt;3.5cm,\u0026le;5cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e202 (34.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e50 (38.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e152 (32.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.408450704225352%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le;3.5cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e249 (42.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.183098591549296%\" valign=\"top\"\u003e\n \u003cp\u003e14 (10.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.014084507042252%\" valign=\"top\"\u003e\n \u003cp\u003e241 (51.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.211267605633804%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBLNE: benign lymph node enlargement; NBLNE: non-benign lymph node enlargement; AJCC: American Joint Committee on Cancer; NLR: neutrophil-to-lymphocyte ratio; VI: vascular invasion; PNI: perineural invasion\u003c/p\u003e\n\u003cp\u003eWe also compared age, sex, the neutrophil-to-lymphocyte ratio (NLR), the VI, and the PNI distribution between the two groups. Patients in the NBLNE group had a greater mean age (66.61 \u0026plusmn; 10.95 vs. 63.61 \u0026plusmn; 12.44 years, \u003cem\u003eP\u003c/em\u003e = 0.024), with no significant differences observed between the two groups in terms of sex, NLR, VI, or PNI ( \u003cem\u003eP\u003c/em\u003e = 0.982/0.374/0.538/0.719).\u003c/p\u003e\n\u003cp\u003eDuring follow-up, 54 patients (8.6%) experienced recurrent CRC and 576 patients (91.3%) had no signs of recurrence, with 4 patients (3.1%) in the BLNE group and 50 patients (10.0%) in the NBLNE group. At the end of the study, 58 patients died (9.2%): 5 in the BLNE group (3.8%) and 53 in the NBLNE group (10.6%). The KM curves of the two groups are shown in Figure 1 (A and B), with statistically significant differences in DFS and OS between the two groups ( \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.016 and \u003cem\u003eP\u0026nbsp;\u003c/em\u003e=\u0026nbsp;0.011, respectively).\u003c/p\u003e\n\u003cp\u003eFurther subgroup analysis revealed superior DFS and OS in the BLNE group compared with those in the NBLNE group in the right colon (Figure 1C and 1D), whereas no significant difference was observed in the left colon and rectum (Additional file 2: Figure S2), possibly due to the relatively smaller sample sizes.Univariate analysis revealed that age \u0026gt; 60 years (Hazard ratio (HR) 3.30, 95% confidence interval (CI) 1.51-7.25, \u003cem\u003eP\u003c/em\u003e = 0.003) and BLNE (HR 3.07, 95% CI 1.23-7.64, \u003cem\u003eP\u003c/em\u003e = 0.016) were significantly correlated with OS, whereas the NLR (HR 1.87, 95% CI 1.10-3.21, \u003cem\u003eP\u003c/em\u003e = 0.023) and BLNE (HR 3.26, 95% CI 1.18-9.04, \u003cem\u003eP\u003c/em\u003e = 0.023) significantly were correlated with DFS. Multivariate regression analysis, incorporating the above variables and tumor T stage, identified tumor T stage (T3, \u003cem\u003eP\u003c/em\u003e =0.048; T4, \u003cem\u003eP\u003c/em\u003e = 0.040), NLR (HR 1.73, 95% CI 1.01-2.98, \u003cem\u003eP\u003c/em\u003e = 0.047), and BLNE (HR 3.85, 95% CI 1.37-10.79, \u003cem\u003eP\u003c/em\u003e = 0.010) as independent risk factors affecting DFS. Age \u0026gt; 60 years (HR 2.65, 95% CI 1.20-5.86, \u003cem\u003eP\u003c/em\u003e = 0.016) and BLNE (HR 3.03, 95% CI 1.20-7.61, \u003cem\u003eP\u003c/em\u003e = 0.019) were independent risk factors for OS (Additional file 3: Table S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtensive\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;lymph node dissection is associated with\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eworse prognosis in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAJCC stage I/II\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCRC patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIf lymph node metastasis is\u0026nbsp;suspected\u0026nbsp;before surgery, the surgeon will\u0026nbsp;perform\u0026nbsp;more extensive lymph node dissection. To investigate the relationship between lymph node dissection and prognosis, we\u0026nbsp;analysed\u0026nbsp;the number of lymph nodes harvested\u0026nbsp;from\u0026nbsp;1435 patients\u0026nbsp;with\u0026nbsp;stage I\u0026ndash;IV disease, including 888 patients\u0026nbsp;with\u0026nbsp;stage I/II disease and 547 patients\u0026nbsp;with\u0026nbsp;stage III/IV\u0026nbsp;disease (Additional file 1:\u0026nbsp;Figure S1).\u003c/p\u003e\n\u003cp\u003eWhen the number of harvested lymph nodes was less than 12, no significant difference in DFS or OS was observed compared with patients with more than 12 lymph nodes harvested ( \u003cem\u003eP\u003c/em\u003e = 0.765; \u003cem\u003eP\u003c/em\u003e = 0.245). Subgroup analysis revealed that DFS and OS remained unaffected when fewer than 12 lymph nodes were removed, regardless of the presence of lymph node metastasis (Additional file 4: Figure S3A\u0026ndash;C; Additional file 5: Figure S4A\u0026ndash;C)\u003c/p\u003e\n\u003cp\u003eInterestingly, among patients without lymph node metastasis (AJCC I/II), patients with \u0026gt; 15 harvested lymph nodes had worse DFS ( \u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e= 0.041), while no significant difference was observed in the whole cohort or in patients with lymph node metastasis only. However, no significant difference in OS was observed (Additional file 4: Figure S3D\u0026ndash;F; Additional file 5: Figure S4D\u0026ndash;F). Among the 131 patients with BLNE, only 4 patients experienced relapse, all of them were with \u0026gt; 15 lymph nodes dissected, indicating a trend of worse prognosis in extended lymph node dissection, although the difference was not statistically significant ( \u003cem\u003eP\u003c/em\u003e = 0.231) due to the relatively small sample size (Additional file 4: Figure S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBLNE has specific radiologic features compared with MLNE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong\u0026nbsp;all\u0026nbsp;the\u0026nbsp;290 patients with lymph nodes \u0026gt; 8\u0026nbsp;mm in diameter visible on\u0026nbsp;dual-phase\u0026nbsp;abdominal CT before surgery, 165 patients had lymph node metastases (Additional file 1:\u0026nbsp;Figure S1).\u0026nbsp;The lymph node morphology, margin features,\u0026nbsp;and CT values were evaluated\u0026nbsp;on\u0026nbsp;arterial-phase\u0026nbsp;CT\u0026nbsp;images\u0026nbsp;(Figure 2).\u0026nbsp;The patient information is presented in Additional file\u0026nbsp;6:\u0026nbsp;Table\u0026nbsp;S2.\u0026nbsp;In\u0026nbsp;our\u0026nbsp;cohort, BLNE appeared in\u0026nbsp;20.8% of AJCC stage I/II CRC patients, and 43.1% of patients\u0026nbsp;with\u0026nbsp;radiologically visible enlarged lymph nodes\u0026nbsp;(Figure 2E).\u003c/p\u003e\n\u003cp\u003eIn terms of clinicopathological features, patients in the MLNE group were predominantly females (53.3% vs. 36%, \u003cem\u003eP\u003c/em\u003e = 0.003) and exhibited more T4 stages ( \u003cem\u003eP\u003c/em\u003e = 0.042), and VI and PNI were more common (48.9% vs. 9.6%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 and; 28.5% vs. 8.8%, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001, respectively); however, the tumor size was smaller in the MLNE group ( \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003eRegarding the morphological features on CT, the BLNE group showed less heterogeneity enhancement and necrotic centers (16% vs. 69.1%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; 1.6% vs. 20.6%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), more clear outer borders and regular lymph node forms (83.2% vs. 55.8%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; 94.4% vs. 62.3%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), and lower CT value differences (31.83 \u0026plusmn; 10.56 vs. 41.51 \u0026plusmn; 14.72, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003eLN\u003c/strong\u003e\u003cstrong\u003eE is\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eassociated\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewith\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003echaracteristics of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eenhanced adaptive immunity\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;including TLS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT\u0026nbsp;image, HE\u0026nbsp;staining\u0026nbsp;image,\u0026nbsp;and\u0026nbsp;macroscopic photo\u0026nbsp;of\u0026nbsp;BLNE\u0026nbsp;are shown in Figure\u0026nbsp;3.\u0026nbsp;The number of follicles per\u0026nbsp;10 mm\u003csup\u003e2\u003c/sup\u003e area of lymph node slide was calculated. The number and density of follicles were significantly greater in the BLNE group than in the NBLNE group (13.69 \u0026plusmn; 4.89 vs. 5.08 \u0026plusmn; 3.87, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.001).\u0026nbsp;To investigate\u0026nbsp;the cellular\u0026nbsp;composition\u0026nbsp;of the\u0026nbsp;BLNE\u0026nbsp;, we\u0026nbsp;labelled\u0026nbsp;T\u0026nbsp;cells and\u0026nbsp;B\u0026nbsp;cells\u0026nbsp;with\u0026nbsp;CD3 and CD19\u0026nbsp;. We observed\u0026nbsp;that\u0026nbsp;both\u0026nbsp;the\u0026nbsp;BLNE and NBLNE\u0026nbsp;primarily consisted\u0026nbsp;of lymphoid nodules\u0026nbsp;containing\u0026nbsp;B cells and diffuse lymphoid tissue\u0026nbsp;containing\u0026nbsp;T cells,\u0026nbsp;with no apparent distinction\u0026nbsp;between them\u0026nbsp;(Additional file\u0026nbsp;7: Figure S5).\u003c/p\u003e\n\u003cp\u003eHE staining of primary tumor tissue revealed the presence of TLS, which was confirmed by IF staining (Figure 3). There were significantly more mature TLSs (TLS++) in the BLNE group than in the NBLNE group (66.7% vs. 36.5%, \u003cem\u003eP\u003c/em\u003e = 0.002) (Figure 3I).\u003c/p\u003e\n\u003cp\u003eOf the 79 patients who underwent IF staining with CD3 and CD8 antibodies, 32 were in the BLNE group and 47 were in the NBLNE group. Three regions of CT and IM regions were randomly selected from each section, and the average density of CD3 and CD8 staining in each region was calculated using Image J (Figure 4A). The results revealed more CD3 positive cells in the CT (571.01 \u0026plusmn; 368.30 vs. 341.52 \u0026plusmn; 228.86, \u003cem\u003eP\u003c/em\u003e = 0.002) and IM (794.18 \u0026plusmn; 369.86 vs. 633.51 \u0026plusmn; 369.86, \u003cem\u003eP\u003c/em\u003e = 0.045) regions of the primary tumor tissue in the BLNE group than in the NBLNE group (Figure 4B\u0026ndash;C). CD8 expression in the BLNE group was greater in the IM (231.60 \u0026plusmn; 219.75 vs. 119.03 \u0026plusmn; 124.72, \u003cem\u003eP\u003c/em\u003e = 0.011) than in the NBLNE group, but no difference was found in the CT region ( \u003cem\u003eP\u003c/em\u003e = 0.09) (Figure 4D\u0026ndash;E). Based on the percentiles of the mean densities of CD3 and CD8 in the CT and IM regions, the immunoscore were classified as high, intermediate, or low. The results indicated a greater proportion of high immunoscore in the primary tumor tissue from the BLNE group than in that from the NBLNE group (Figure 4F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBLNE is associated with greater TMB and specific mutation signatures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed a high-depth WES of 51 CRC tissues (24 with BLNE and 27 with NBLNE). The mean WES coverage was 114\u0026times;. Sequencing data revealed that the most common variant classification was missense mutation, and the most common variant type was SNP. Among these SNPs, the C\u0026gt;T mutation was dominant (Additional file 8: Figure S6). The mutation landscape plot showed that TTN and MUC12 had the greatest number of mutations in patients with BLNE and NBLNE, respectively (Figure 5A\u0026ndash;B). Fisher\u0026apos;s exact test was used to compare the differences in mutations between the two groups, and significant differences were observed for 11 genes ( \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). The differentially mutated genes had a greater mutation frequency in the BLNE group, except for MUC12 (Figure 5C). TMB was also evaluated and compared between the groups. The BLNE group had a significantly greater TMB than the NBLNE group (Figure 5D). In addition to the aforementioned results, tumor mutation signatures were analysed in detail. The mutation characteristics of all patients showed that C\u0026gt;A and C\u0026gt;T were the major components of SBSs (Additional file 9: Figure S7). Non-negative matrix factorization and cosine similarity revealed that the mutation signatures of all sequence data were associated with signatures 1, 6, 10, 15, and 28 in the COSMIC database. Comparative analysis of these five mutation features revealed that signature 1 was more enriched in the BLNE group (Figure 5E). Signature 1 arises from an endogenous mutational process initiated by the spontaneous deamination of 5-methylcytosine.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we analysed the clinicopathological features of 630 patients with BLNE and performed HE staining, IHC, IF staining and WES to explore BLNE occurrence in the tumor immune microenvironment and at the genomic level. This is the first study to evaluate the relationships among BLNE, the tumor immune microenvironment, and genomic features in patients with CRC. We observed that patients with BLNE were younger, with a predominantly right colon location, T3 stage and larger tumors.\u003c/p\u003e \u003cp\u003eLymph node metastasis in the tumor drainage area impacts both postoperative adjuvant therapy and preoperative treatment strategies. The Japanese CRC guidelines recommend D2 lymph node dissection for stage T1 and stage T2 patients without lymph node metastasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A smaller lymph node dissection area often results in fewer surgical complications [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, precursor-exhausted cells (Tpex) expressing the transcription factor TCF-1, which is essential for maintaining antigen-specific CD8\u0026thinsp;+\u0026thinsp;T cell responses, proliferate mainly in tumor-draining lymph nodes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, as a local hub of anti-tumor immunoity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], whether lymph nodes should be extensively dissected warrants consideration. Thus, we analysed the effect of the number of lymph node dissections on patient prognosis of patients. Although fewer than 12 lymph nodes dissection is recommended as a risk factor for postoperative chemotherapy in stage II CRC patients [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], in our study, fewer than 12 lymph nodes dissection did not affect the long-term prognosis of AJCC I-II patients. Interestingly, the prognosis was worse when the number of lymph nodes was more than 15 in patients without lymph node metastasis, possibly because of destruction of the anti-tumor adaptive immune response. Therefore, accurate BLNE identification before surgery might affect surgical decision-making.\u003c/p\u003e \u003cp\u003ePreoperative diagnosis of lymph node status in colon cancer remains a significant challenge, mainly relying on imaging methods such as enhanced CT, MRI, and PET-CT. By combining features of morphology and signal features instead of size criteria [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], MRI can achieve 80\u0026ndash;85% sensitivity and 97\u0026ndash;98% specificity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], while the accuracy of CT, the most common test for colon cancer, is only 59\u0026ndash;71% [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Although recently artificial intelligence [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], liquid biopsy [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and various clinicopathological factors [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], including tumor budding, submucosal infiltration depth, vascular nerve invasion, and histological grade, have been used to predict lymph node metastasis, surgeons rely mainly on morphological features on imaging to diagnose lymph node metastasis. Thus, we compared the differences in CT images between the BLNE and MLNE groups. The results showed that MLNE had more unclear outer border, heterogeneity enhancement, and irregular form, especially the necrotic centre, an important indicator of lymph node metastasis, which can help surgeons to identify lymph node metastasis more intuitively, necessitating further radiomic studies.\u003c/p\u003e \u003cp\u003eWe speculated that the BLNE is correlated with the adaptive immune response of tumors. When antigens enter the lymph nodes through the afferent lymphatic vessels, fibroblast reticular cells proliferate, and the number of activated T and B cells increases rapidly, leading to rapid expansion of the lymph nodes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. HE and IHC staining of lymph nodes revealed morphological similarities between BLNE and NBLNE. However, there were significant differences in the number and density of lymphatic follicles, confirming that BLNE was the consequence of activated B and T cell proliferation. Our study revealed superior OS ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011) and DFS ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016) in the BLNE group than in the NBLNE group. Whether this survival advantage was due to the activation of the immune system was unclear; therefore, we performed an immunoscore analysis of the primary tumor tissue. The results revealed significantly more CD3\u003csup\u003e+\u003c/sup\u003e T cells in the tumor core ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and IM ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045) in the BLNE group than in the NBLNE group and significantly more CD8\u003csup\u003e+\u003c/sup\u003e T cells in the IM ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011) than in the NBLNE group. BLNE was closely related to a greater immunoscore. The prognostic value of CD8\u003csup\u003e+\u003c/sup\u003e T cells for DFS and OS has been demonstrated in 17 solid tumors, and among different immune cell subtypes, CD8\u003csup\u003e+\u003c/sup\u003e T cells have the most positive impact on cancer survival [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Immunoscore plays a prognostic role in CRC, surpassing classical TNM stages in predicting DFS and OS [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, more mature TLS were found in tumor tissues from the BLNE group. TLS has structural and functional characteristics similar to those of lymph nodes and is considered the first site at which T cells may be activated to form an anti-tumour immune response [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These results suggest that the positive effect of BLNE on prognosis may be related to the activation of anti-tumor immunity. Although the NLR in the peripheral blood was a predictor of CRC prognosis ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047), no significant differences were observed between the BLNE and NBLNE groups. Therefore, we conclude that the effect of BLNE on tumor immunity is mainly associated with the local tumor microenvironment and is not influenced by circulating immune cells.\u003c/p\u003e \u003cp\u003eTMB is an effective tool for predicting immune checkpoint blockade response in patients. Somatic mutations in tumor DNA may produce antigens that are recognized by the immune system. Although not all mutations produce neoantigens, somatic mutations in a tumor correspond to increased recognition of neoantigens [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our study, the TMB was significantly greater in the BLNE group than in the NBLNE group ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025), which may be responsible for the stronger immune response in patients in the BLNE group. Additionally, we identified 11 mutated genes with significant differences between the two groups (MUC12, PLCH1, LRP1B, OBSL1, FHOD3, MCM3AP, MYH9, NBPF1, NLRP8, PCDHA1, and PTGIR). Except for MUC12, which mainly occurred in the NBLNE group, the rest mainly occurred in the BLNE group. MUC12 encodes a complete membrane glycoprotein, a member of the mucin family, and is more highly expressed in normal colon tissue than in colon cancer. Matsuyama et al. reported that MUC12 is an independent predictor of prognosis in stage II and III CRC,patients and that poor prognosis is associated with low MUC12 expression [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], consistent with our finding of a high MUC12 mutation rate in the NBLNE group.\u003c/p\u003e \u003cp\u003eWe must admit that our paper has some limitations. First of all, our data were from a single center, and lacked the validation from multiple centers, however, the relatively larger cohort size makes our results reliable. Second, our study focused mainly on lymphocytes in the tumor microenvironment, and other immune cell types such as neutrophils and macrophages also need further study. Moreover, our study is limited to exon sequencing, which needs to be analysed in combination with transcriptome sequencing data, and further functional testing is necessary to verify the identified mutated genes.\u003c/p\u003e \u003cp\u003eOur study confirmed the positive impact of BLNE on CRC prognosis and delineated its clinicpathological features. Additionally, our investigation emphasized the significance of accurately identifying BLNE preoperatively and highlighted its morphological characteristics on CT scans for surgeons. Subsequent pathological staining revealed a potential association between BLNE and an enhanced immune response against tumor cells. Supported by WES data, the identification of differentially mutated genes potentially linked to BLNE offers avenues for further exploration.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, BLNE is a positive prognostic factor for colorectal cancer, possibly because BLNE patients have a stronger anti-tumor immune response. In patients without lymph node metastasis, excessive lymph node dissection can lead to a worse prognosis, and accurate preoperative lymph node staging is critical.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBLNE: Benign lymph node enlargement; NBLNE: Non-Benign lymph node enlargement; DFS: Disease-free survival; OS: Overall survival; HR: Hazard ratio; OR: Odds ratio; CRC: Colorectal cancer; CT: computed tomography; MRI: magnetic resonance imaging; AJCC: American joint Committee on cancer; MSI: Microsatellite instability; VI: vascular invasion; PNI: perineural invasion; MLNE: metastatic lymph node enlargement; HE: Hematoxylin and eosin; IHC: Immunohistochemical; IF: Immunofluorescence TLS: tertiary lymphoid structures; CT: tumor core; IM: invasive margin; WES: Whole-exome sequencing; TMB: Tumor mutation burden; NLR: neutrophil-to-lymphocyte ratio; CI: confidence interval;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the research process, the technical support and help from the Center of Basic Medical Research, Institute of Medical Innovation and Research are indispensable, and we would like to express our sincere thanks to the staff of the center.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdeas and research design: ZX, WJW, FW; Data collection: LXY, LSY; Image reading: WBY, LF; Analyze and interpret data: WJW, SXC, CX; Manuscript: WJW, SXC, GLM. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXin Zhou was supported by Beijing Nova Program (20220484153 and 20230484485)；Wei Fu was was supported by grants from the Beijing Natural Science Foundation（L234001）\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are available from the corresponding authors for research use only.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Science Research Ethics Committee of Peking University Third Hospital (IRB00006761-M2020123) and conducted in accordance with the Declaration of Helsinki. Informed consent from all patients prior to obtaining tumor tissue. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of General Surgery, Peking University Third Hospital; \u003csup\u003e2\u003c/sup\u003ePeking university third hospital cancer center; \u003csup\u003e3\u003c/sup\u003eDepartment of Pathology, School of Basic Medical Sciences, Peking University Third Hospital, Beijing 100191, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDekker E, Tanis PJ, Vleugels JLA, et al. 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Int J Cancer. 2010;127:2292\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"colorectal cancer, prognosis, tumor immune microenvironment, benign lymph node enlargement","lastPublishedDoi":"10.21203/rs.3.rs-3975650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3975650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eBenign lymph node enlargement (BLNE) is common in colorectal cancer; however, few studies have investigatedits influence on prognosis, clinicopathological features, and pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003ePatients who underwent radical surgery for colorectal cancer at Peking University Third Hospital were grouped according to the presence ofBLNE, and their prognosis and clinical characteristics were analysed. The immune microenvironment and genomic characteristics of primary tumors were comprehensively explored in representative patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Overall,630 AJCC stage I/IIpatients, with 131 in the BLNE group and 499 in the Non-BLNE (NBLNE) group, were included in the study. Patients in the BLNE group exhibited better disease-free survival (DFS) (hazard ratio [HR] 0.44, \u003cem\u003eP\u003c/em\u003e = 0.016) and overall survival (OS) (HR 0.46, \u003cem\u003eP\u003c/em\u003e = 0.011) . Interestingly, the prognosis of patients without lymph node metastasis wasworse when the number of harvested lymph nodes was more than 15. On computed tomography (CT) images, significant differences in lymph node morphology were found between BLNE and metastatic lymph node enlargement, including lymph node border, shape and enhancement characteristics. Pathologically, compared with the NBLNE group, the BLNE group had more mature tertiary lymphoid structures (66.7% vs. 36.5%, \u003cem\u003eP\u003c/em\u003e = 0.002), greater immunoscore (18.8% vs. 2.1%, \u003cem\u003eP\u003c/em\u003e = 0.004) in tumor tissue, and more abundant lymph follicles in lymph nodes (13.69 ± 4.89 vs. 5.08 ± 3.87, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). Whole-exon sequencing analysis revealed greater TMB in the BLNE group [ 6.03 (5.59, 7.59) vs. 5.33 (4.62, 6.34), \u003cem\u003eP\u003c/em\u003e = 0.025). 11 differentially mutated genes were identified between the BLNE groupand NBLNE group, with MUC12 (81%) as the most common mutated gene in the BLNE group (odds ratio [OR] 0.10, \u003cem\u003eP\u003c/em\u003e = 0.0002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eBLNE is a positive factor in predicting the prognosis of colorectal cancer, possibly becausepatients with BLNE have a stronger anti-tumorimmune response. Accurate preoperative lymph node staging is critical.\u003c/p\u003e","manuscriptTitle":"Mesenteric benign lymph node enlargement in colorectal cancer: Friend or Foe?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 18:44:04","doi":"10.21203/rs.3.rs-3975650/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"411473a2-9dc2-4281-9de1-7bf4db51aaaa","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-26T16:23:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-23 18:44:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3975650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3975650","identity":"rs-3975650","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}