SHOX2-Driven Lymphatic Metastasis in pT1 Non-Clear Cell Renal Cell Carcinoma: A Biomarker-Guided Risk Stratification and Therapeutic Strategy

SHOX2-Driven Lymphatic Metastasis in pT1 Non-Clear Cell Renal Cell Carcinoma: A Biomarker-Guided Risk Stratification and Therapeutic Strategy | 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 SHOX2-Driven Lymphatic Metastasis in pT1 Non-Clear Cell Renal Cell Carcinoma: A Biomarker-Guided Risk Stratification and Therapeutic Strategy Xu Zhen, Qiang Ma, Wenjin Yang², Hongjun Yin, Rui Si, Lina Dai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6688444/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 Non-clear cell renal cell carcinoma (nccRCC), representing 25–30% of RCC cases, exhibits higher lymph node metastasis (LNM) risk than clear cell RCC (ccRCC). Despite favorable outcomes in early-stage pT1 nccRCC, metastatic potential persists, necessitating improved risk stratification. Methods A single-center retrospective cohort of 528 pT1 RCC patients (96 nccRCC; 432 ccRCC) and TCGA data (n = 355) were analyzed. Differential gene expression (DESeq2, log2FC > 1, FDR < 0.05, p < 0.05 ), survival analyses (Kaplan-Meier/Cox regression), and immunohistochemistry (IHC) validated SHOX2 as a biomarker. Results The LNM rate in pT1 nccRCC was significantly higher than in ccRCC (4.1% vs. 0.4%, p < 0.001 ). SHOX2 was markedly overexpressed in LNM + tumors (log2FC = 6.01, p < 0.01 ) and independently predicted adverse prognosis (HR = 2.896, 95% CI 1.329–6.31, p < 0.01 ). Functional enrichment linked SHOX2 to ECM remodeling (r = 0.401, p < 0.001 ) and EMT activation (r = 0.381, p < 0.001 ). IHC validation demonstrated SHOX2’s predictive value for LNM (AUC = 0.77, 95% CI 0.61–0.93), with optimal cutoff ≥ 3.5 (70% sensitivity, 90% specificity). SHOX2 positivity was enriched in LNM + specimens (70% vs. 10%, p < 0.001 ). Conclusions SHOX2 drives lymphatic dissemination in pT1 nccRCC via ECM-EMT crosstalk, serving as a robust biomarker. A tripartite strategy integrating preoperative mSHOX2 liquid biopsy, image-guided lymph node dissection, and postoperative targeted-immunotherapy (e.g., toripalimab-axitinib) is proposed to optimize outcomes. Multicenter validation is warranted for clinical translation. Non-clear cell renal cell carcinoma SHOX2 Lymph node metastasis Biomarker validation Precision medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Renal cell carcinoma (RCC) accounted for 434,419 new cases and 155,702 deaths globally in 2022[ 1 ]. Histologically, RCC is classified into clear cell RCC (ccRCC) and non-clear cell RCC (nccRCC). As the predominant subtype (70–75% of RCC), ccRCC has been extensively studied through both basic research and large-scale randomized controlled/prospective phase III trials, yielding significant advances in molecular mechanisms and clinical management[ 2 – 4 ]. In contrast, nccRCC—encompassing papillary, chromophobe, and other rare subtypes with marked heterogeneity—constitutes 25–30% of RCC cases[ 5 ]. Notably, no standardized first-line therapy exists for metastatic nccRCC, and current treatment protocols predominantly extrapolate from ccRCC guidelines due to the absence of successful phase III trials[ 6 , 7 ]. Recent imaging advancements have increased the detection of small renal masses (SRMs; ≦4 cm) in nccRCC, accounting for 48–66% of renal tumors[ 8 ].However, despite favorable prognosis in pT1-stage RCC, lifelong surveillance remains necessary due to reported late recurrences[ 9 , 10 ], including rare cases of metastatic SRMs[ 11 ]. Adjuvant therapy demonstrates efficacy for postoperative patients with microscopic residual disease (MRD) undetectable by imaging[ 12 ], The ASCO Annual Meeting has emphasized MRD detection in guiding RCC management. While circulating tumor DNA (ctDNA) analysis shows promise for identifying high-risk patients, its clinical utility in RCC is limited by economic constraints and low detection rates[ 13 ]. Both SRMs and MRD populations exhibit elevated recurrence risks, yet nccRCC surgical strategies largely mirror ccRCC protocols due to insufficient evidence. This study compares clinicopathological features between pT1 nccRCC and ccRCC to elucidate prognostic disparities, preliminarily explore molecular mechanisms, and propose optimized perioperative strategies for early nccRCC with high lymph node metastasis (LNM) risk. Our objectives are to: (1) address the lack of prognostic biomarkers in nccRCC; (2) develop novel therapeutic approaches to mitigate metastasis risk; and (3) improve survival outcomes for high-risk nccRCC populations. Study Design and Ethics This single-center retrospective cohort study received approval from Nanjing Gulou Hospital Ethics Committee. Inclusion criteria: pathologically confirmed pT1 RCC (AJCC 8th edition) treated with partial nephrectomy, complete clinical data, and endpoint events defined as metastasis, recurrence, or cancer-specific death. Patients receiving neoadjuvant therapy were excluded. The final cohort comprised 528 RCC cases (96 nccRCC; 432 ccRCC), all treated via robotic-assisted surgery. From TCGA database ( https://portal.gdc.cancer.gov ), we obtained STAR-counts data and clinical information for nccRCC subtypes (KICH and KIRP). After data cleaning (excluding incomplete records), 355 cases were stratified into LN+ (n = 20) 、LN- (n = 89) and LNx(n = 246) groups. Statistical Analysis Survival analysis (Kaplan-Meier method), multivariate Cox regression, and Spearman correlation were performed using SPSS 26.0 and R 4.2.2. TCGA differential gene expression analysis employed DESeq2 (threshold: |log2FC|>1, p < 0.05 ). Gene set variation analysis (GSVA) utilized the ssGSEA algorithm (method='ssgsea'). Statistical significance was set at p < 0.05 . Immunohistochemistry (IHC) Twenty paraffin-embedded specimens from robotic partial nephrectomy patients (2016–2024) with pT1 nccRCC and LND (10 LN+, 10 LN-) were analyzed. SHOX2 staining used Proteintech polyclonal antibody (Cat No.16366-1-AP; optimized dilution 1:1500). Standardized protocols included: Deparaffinization : Xylene (2×20 min) → ethanol series (100%→60%);Antigen retrieval : TE buffer (pH 9.0) microwaved 10 min;Primary antibody incubation: 1.5 h at RT; Signal detection: HRP-conjugated secondary antibody (30 min) → DAB (2–5 min); Counterstaining: Hematoxylin (3 min) → ethanol dehydration → xylene clearing. Two pathologists independently scored specimens using semi-quantitative criteria : Intensity Score (IS): 0 (none), 1 (weak), 2 (moderate), 3 (strong);Proportion Score (PS): 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), 4 (76–100%). Total Score (TS): IS + PS (range 0–7). Inter-rater reliability met acceptable standards ( Kappa > 0.6 ). Results Clinical Characteristics Both groups exhibited negative surgical margins without renal vein/perirenal fat invasion. Propensity score matching was deemed unnecessary (SMD < 0.2; Fig. 1A). Among 528 participants, 96 (18.2%) had non-clear cell renal carcinoma, while 432 (81.8%) had clear cell renal carcinoma. The non-clear cell group had a lower mean age (52.58 ± 14.42 years vs 55.01 ± 13.23 years; p = 0.111 ) and similar BMI (24.95 ± 2.82 vs 24.86 ± 3.54; p = 0.821 ). Tumor size showed no difference between groups (3.50 ± 1.44 cm vs 3.71 ± 1.27 cm; p = 0.169 ). Gender distribution was comparable (34.38% male vs 38.19% male; p = 0.484 ). The non-clear cell group had higher rates of metastasis or death (6.25% vs 0.69%; p < 0.001 ), lymph node metastasis (5.21% vs 0.46%; p = 0.001 ), and high-grade Fuhrman classification (31.25% vs 21.99%; p = 0.054 ). No differences were observed in renal capsule penetration (20.83% vs 24.36%; p = 0.462 )(Fig. 1B). Survival Analysis (dup: abstract ?) The nccRCC cohort demonstrated significantly lower 5-year DFS (93.2% vs 99.2%; log-rank p < 0.001 ), indicating intrinsic biological aggressiveness (Fig. 2A). Lymph Node Metastasis Prognostication Univariate Cox regression identified LNM as the strongest predictor of poor outcomes in nccRCC (HR = 38.63, 95% CI 6.92-215.73, p < 0.001 ). This association persisted in multivariate analysis adjusting for Fuhrman grade and renal capsule involvement (adjusted HR = 36.44, 95% CI 4.67-284.47, p < 0.001 ) (Fig. 3). Differential Gene Expression Volcano plot analysis revealed SHOX2 as the most significantly upregulated gene in LN + versus LN- groups (log2FC = 6.01, p < 0.01 ; Fig. 2B). Survival Analysis Using TCGA database, 178 nccRCC patients with high SHOX2 expression and 177 patients with low SHOX2 expression were stratified. Patients in the high-expression group exhibited significantly shorter survival time compared to the low-expression group (HR = 2.896, 95% CI 1.329–6.31, p < 0.01 ). Samples were dichotomized into high- and low-expression groups based on median gene expression. Survival differences between groups were assessed using the log-rank test in Kaplan-Meier analysis. The Kaplan-Meier curves, analyzed by log-rank test and univariate Cox regression, demonstrated statistically significant prognostic relevance of SHOX2 (log-rank p < 0.05), with higher expression correlating with poorer outcomes. When sorted by ascending SHOX2 expression (Fig. 4A), the intermediate scatterplot revealed a left-to-right trend of increasing mortality and decreasing survival time. The heatmap (Fig. 4B) illustrates gene expression patterns, confirming SHOX2 as a risk factor. Time-dependent ROC analysis showed AUC values of 0.809 (95% CI 0.7-0.918) at 1 year, 0.707 (95% CI 0.605–0.809) at 3 years, and 0.658 (95% CI 0.544–0.773) at 5 years (Fig. 4C). Pathway Correlation ssGSEA revealed positive correlations between SHOX2 and Extracellular matrix (ECM) (r = 0.401, p < 0.001 )/Epithelial-mesenchymal transition (EMT) pathways (r = 0.381, p < 0.001 ; Fig. 5). IHC Validation ROC analysis demonstrated SHOX2's predictive value for LNM (AUC = 0.77, 95% CI 0.54–0.99). At optimal cutoff (TS ≧ 3.5; Youden index: sensitivity 70.0%, specificity 90.0%), SHOX2 positivity was observed in 7/10 LN + versus 1/10 LN- cases ( p < 0.01 ; Fig. 6B-C). Key Findings This study first reports a 4.1% incidence of LNM in Chinese patients with pT1 nccRCC, significantly higher than in ccRCC counterparts (0.4%; p < 0.001 ). This observation aligns with prior studies demonstrating elevated pathological LNM rates in nccRCC versus ccRCC[ 14 ], potentially attributable to the predilection of nccRCC for lymphatic dissemination [ 15 ]. Although early-stage pT1 nccRCC generally exhibits favorable postoperative outcomes, the presence of metastatic risk persists despite low pathological stage. Both univariate and multivariate Cox proportional hazards analyses identified LNM as an independent risk factor for adverse prognosis in nccRCC. Recent advancements in systemic therapies—including molecular targeted agents and immune checkpoint inhibitors (ICIs)—have significantly improved survival outcomes in ccRCC through combination regimens [ 16 , 17 ],However, the management of nccRCC remains extrapolated from ccRCC protocols due to insufficient randomized controlled trial data. nccRCC exhibits distinct immune evasion mechanisms: VEGF-targeted inhibition upregulates PD-L1 expression on lymphocytes, while dual pathway blockade enhances lymphocyte infiltration and activity via lymphotoxin beta receptor (LTBR) signaling[ 18 ].The 2022 EAU guidelines recommend cabozantinib over sunitinib for papillary RCC based on prolonged progression-free survival [ 9 ].Although ICI-targeted combinations demonstrate superior survival in ccRCC [ 17 ], their efficacy in nccRCC requires validation through dedicated trials. Lymph node dissection (LND) remains crucial for high-risk RCC management[ 19 ], with studies demonstrating survival benefits in locally advanced high-risk RCC (median overall survival 43.10 vs 40.52 months, p < 0.01)[ 20 ]. Nevertheless, LND carries significant risks: Suzuki et al. reported a 9.7% incidence of chylous leakage associated with LND[ 21 ]. 99mTc-sulfur colloid lymphoscintigraphy provides high diagnostic accuracy for nodal staging (sensitivity 89%, specificity 93% [ 22 ]), utilizing SPECT imaging to track radiotracer transport through lymphatic endothelial cells. However, its clinical application requires judicious consideration of radiation exposure and cost-effectiveness. The dual challenges of selecting appropriate candidates for lymphoscintigraphy and determining optimal LND timing underscore the need for improved risk stratification tools." While early surgical intervention achieves favorable outcomes in most pT1 nccRCC cases, a subset with high-risk features demonstrates significantly greater LNM propensity compared to ccRCC (4.1% vs 0.4%, p < 0.001 ). The presence of MRD in these patients may drive subclinical progression, as current therapeutic strategies fail to adequately address high-risk nodal disease. Our findings position SHOX2 as a central mediator of lymphatic metastasis in nccRCC, extending its known pan-cancer oncogenic roles (e.g., WASF3 activation in breast cancer[ 23 ]; STAT3 signaling in gastric cancer[ 24 ]; Hepatocellular carcinoma: Associates with early recurrence[ 25 ]; Lung cancer: Validated as a prognostic biomarker in NSCLC[ 26 – 28 ]) to RCC pathogenesis. Specifically, the strong correlation between SHOX2 and EMT/ECM pathways (r = 0.381/0.401, p < 0.001 ) suggests conserved mechanisms across malignancies. Our TCGA analysis of nccRCC (n = 355) identified SHOX2 as the most differentially expressed gene between LN + and LN- groups (log2FC = 6.01, FDR = 0.003). Elevated SHOX2 expression correlated with reduced survival (HR = 2.896, 95% CI 1.329–6.31, p < 0.01 ), suggesting its prognostic utility in nccRCC. In bladder cancer models, SHOX2 overexpression induces EMT, enhancing migratory capacity[ 29 ].Although direct evidence in RCC remains limited, these findings suggest SHOX2 promotes lymphatic metastasis through conserved mechanisms involving ECM degradation and EMT activation. Methylation of SHOX2 (mSHOX2) occurs at cytosine-phosphate-guanine (CpG) islands within the promoter region. A single-center prospective study demonstrated that mSHOX2 serves as a molecular biomarker for predicting poor prognosis in RCC patients at high risk of metastasis. Multivariable Cox analysis confirmed mSHOX2 as an independent risk factor for recurrence, with preoperative mSHOX2 levels significantly elevated in RCC patients with unfavorable outcomes. Furthermore, mSHOX2 in ctDNA showed significant correlation with lymph node metastasis in RCC[ 30 ].Hypomethylation of mSHOX2 may contribute to upregulated SHOX2 mRNA expression in tumor tissues[ 31 ].The U.S. Food and Drug Administration has approved an mSHOX2 detection kit for lung cancer diagnosis[ 32 ], suggesting its potential utility in perioperative therapeutic decision-making for high-risk nccRCC patients. These findings collectively support mSHOX2 as a biomarker for identifying nccRCC patients at high risk of lymph node metastasis. IHC analysis of 20 pT1 nccRCC specimens from patients undergoing lymph node dissection revealed significantly higher SHOX2 positivity in the LN + group compared to the LN- group (70% vs. 10%, p < 0.01 ). This indicates SHOX2's potential as a prognostic marker for screening MRD in high-risk nccRCC patients, enabling early personalized interventions to prevent adverse outcomes. Based on existing evidence, we propose the following clinical pathway :Preoperative screening: Detect mSHOX2 levels to identify high-risk populations for lymph node metastasis. For patients with elevated mSHOX2 levels or suspicious lymph nodes on contrast-enhanced CT/MRI, perform 99mTc-sulfur colloid lymphoscintigraphy to confirm nodal status; Intraoperative management: Implement LND for confirmed high-risk cases, balancing therapeutic benefits against surgical risks; Postoperative surveillance: Quantify SHOX2 expression in resected specimens. For patients with SHOX2-positive IHC scores (TS ≧ 3.5), initiate contrast-enhanced abdominal CT every 6 months and consider early targeted-immunotherapy combinations.The recently reported IUNU-RC-102 trial (ASCO-GU 2025) further supports the feasibility of adjuvant toripalimab-axitinib in high-risk nccRCC, demonstrating preliminary efficacy in reducing recurrence rates. Our SHOX2-driven stratification strategy could synergize with such regimens to optimize patient selection. Clinical Translational Significance In accordance with the EAU guidelines, we have developed a perioperative optimization strategy (Fig. 7) comprising sequential phases: Preoperative assessment preoperative mSHOX2 testing to identify high-risk populations for lymph node metastasis, followed by 99mTc-sulfur colloid lymphoscintigraphy in mSHOX2-elevated cases to confirm nodal status; Intraoperative decision-making:meticulous lymph node evaluation guides selective LND in high-risk patients, balanced against overtreatment risks; Postoperative surveillance Postoperative management integrates IHC quantification of tumor SHOX2 expression with periodic mSHOX2 monitoring, prompting six-monthly contrast-enhanced abdominal CT for early micrometastasis detection. Adjuvant therapy Emerging evidence supporting targeted-immunotherapy combinations (e.g., toripalimab-axitinib) in high-risk cohorts warrants exploration of adjuvant regimens for SHOX2-overexpressing subgroups. Innovations and Limitations This study provides the first Asian population-derived framework that synergizes molecular biomarkers (SHOX2/mSHOX2) with clinical stratification, addressing the historical overreliance on Eurocentric data in nccRCC management. By establishing actionable protocols for pT1 nccRCC—a clinical scenario lacking consensus guidelines—we bridge a critical knowledge gap. The retrospective single-center design inherently limits causal inference, necessitating validation through multicenter prospective cohorts to mitigate selection bias and enhance generalizability. Conclusion Early surgical intervention for pT1 nccRCC is associated with a significantly elevated lymph node metastasis risk compared to ccRCC, mechanistically linked to SHOX2-mediated ECM remodeling and EMT. A precision medicine approach combining liquid biopsy (mSHOX2-guided lymphoscintigraphy), histopathological profiling (SHOX2 IHC scoring), and postoperative biomarker surveillance is proposed to optimize therapeutic decision-making. Validation through collaborative multicenter studies remains imperative to translate this biomarker-driven strategy into clinical practice. Abbreviations nccRCC Non-clear cell renal cell carcinoma LNM lymph node metastasis ccRCC Clear cell renal cell carcinoma HR Hazards Ratio CI Confidence Interval IHC Immunohistochemistry RCC Renal cell carcinoma SRMs Small renal masses MRD Microscopic residual disease ctDNA Circulating tumor DNA LNM Lymph node metastasis GSVA Gene set variation analysis IS Intensity Score PS Proportion Score TS Total Score SMD Standardized Mean Difference DEGs Differentially Expressed Genes t t-test χ 2 Chi-square test SD Standard deviation LN+ Lymph node-positive LN− Lymph node-negative ECM Extracellular matrix EMT Epithelial-mesenchymal transition ICIs Immune checkpoint inhibitors LTBR lymphotoxin beta receptor LND Lymph node dissection NSCLC Nonsmall-cell lung cancer Declarations Acknowledgements Not applicable. Funding This work was supported by grants from the General Program of National Natural Science Foundation of China (Grant No. 82172777) and Jiangsu Provincial Medical Key Discipline (Laboratory) Cultivation Unit (JSDW202221). Author information Author notes Xu Zhen contributed to this work. Authors and Affiliations ¹Department of Urology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, 321 Zhongshan Road, Nanjing 210008, China Xu Zhen¹, Qiang Ma 1 , Changwei Ji 1 ²Department of Urology, Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing 210008, China Wenjin Yang², Hongjun Yin 2 , Rui Si²,Lina Dai 2 ,Hongqian Guo 2 Contributions Study design: Z.X; Experimental operation: Z.X. and J.CW.; Data acquisition: Z.X.,D.LN and Y.WJ.; Data analysis:Z.X.,Y.HJ and S.R.; Research writing and revision of the manuscript: Z.X.,G.HQ.and M.Q.; Study supervision: J.CW. Corresponding authors Correspondence to Changwei Ji. Data availability The datasets used or analyzed during the study are available from the corresponding author on reasonable request. Ethics declarations Ethics approval and consent to participate This retrospective study was approved by and the Ethics Committee of the Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School. All methods were carried out in accordance with relevant guidelines and regulation. Informed consent was obtained from of all subjects and/or their legal guardian(s). Consent for publication All authors agree to publish. Competing interests The authors declare no competing interests. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. Motzer RJ, Powles T, Burotto M, Escudier B, Bourlon MT, Shah AY, Suarez C, Hamzaj A, Porta C, Hocking CM, et al. 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Buttner T, Zarbl R, Krausewitz P, Strieth S, Kristiansen G, Eckstein M, Ralser DJ, Holzel M, Ritter M, Ellinger J, et al. Hypermethylated SHOX2 in circulating cell-free DNA post renal cell carcinoma surgery as TNM-independent biomarker for recurrence risk. Am J Transl Res. 2024;16(1):304–13. Additional Declarations No competing interests reported. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6688444","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475982688,"identity":"10d55086-9c9c-4dff-9e6c-936dc3589bfc","order_by":0,"name":"Xu Zhen","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Zhen","suffix":""},{"id":475982689,"identity":"f3ee9568-bf91-4377-a448-887e3bb23f0e","order_by":1,"name":"Qiang Ma","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Ma","suffix":""},{"id":475982691,"identity":"313113c7-ae61-41ce-ace7-38cc1a94bfd2","order_by":2,"name":"Wenjin Yang²","email":"","orcid":"","institution":"Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Wenjin","middleName":"","lastName":"Yang²","suffix":""},{"id":475982692,"identity":"7bcde840-2ee4-4e74-827e-721236e16a57","order_by":3,"name":"Hongjun Yin","email":"","orcid":"","institution":"Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Hongjun","middleName":"","lastName":"Yin","suffix":""},{"id":475982696,"identity":"89586b91-4406-456c-bf9e-b522d6a4470c","order_by":4,"name":"Rui Si","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Si","suffix":""},{"id":475982698,"identity":"4ac2ce79-331b-4a03-be15-903c38882e9d","order_by":5,"name":"Lina Dai","email":"","orcid":"","institution":"Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Dai","suffix":""},{"id":475982699,"identity":"aa07ef96-409f-4137-9e1e-5ff86f82794a","order_by":6,"name":"Hongqian Guo","email":"","orcid":"","institution":"Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Hongqian","middleName":"","lastName":"Guo","suffix":""},{"id":475982700,"identity":"f381ec87-5406-40b0-be6c-0d9708dc93eb","order_by":7,"name":"Changwei Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACxmYGNgiLmfkAgwSIcYB4LWwJxGkBAqgWBh4DCE1IC3M787MHP3fUJvaz83x7YNnGIMd3I4HxcwFeh7GZG/aeOZ44s5l3u4FkG4Ox5I0EZukZeLXwsEnwth3L3XCYd5sEUEvihhsJbMw8BLRI/gVq2X+Y5xlISz1RWqR522pyNzADrQNqSTAgrIXNTFq27UD9jMNsZhIS5yQMZ5552CyNT4th/+Fnkm/b6oz5gQxpiTIbeb7jyQc/49XSAKYOg0lmCXBkMjbg0cDAIA+h6iCu/IBX7SgYBaNgFIxUAAAJ7UWuMUvKQQAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Changwei","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2025-05-17 17:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6688444/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6688444/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85647748,"identity":"ef3ec972-c21d-49db-82c7-1eda6e0a0ec1","added_by":"auto","created_at":"2025-06-30 08:50:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":473798,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/9299563a699f75e48d5f1a58.jpg"},{"id":85647749,"identity":"e2a0a173-b40d-4965-aba2-8299e3b80781","added_by":"auto","created_at":"2025-06-30 08:50:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":352631,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/e9387a6a5dc6ac2cd6bcebc3.jpg"},{"id":85649528,"identity":"3a6544c1-1a62-4864-a188-24d187686c76","added_by":"auto","created_at":"2025-06-30 08:58:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":260698,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/0193b90551be67d23cdb5a44.jpg"},{"id":85647752,"identity":"9bb2cc6a-cb62-4287-9eae-4b42a580266b","added_by":"auto","created_at":"2025-06-30 08:50:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353117,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/0ee4f9abcafad0b63bf1ff6c.jpg"},{"id":85649529,"identity":"8cb22b04-c2f2-48bf-848f-99361a864b0e","added_by":"auto","created_at":"2025-06-30 08:58:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":480189,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/5eb7a0f3fd14bbc433f249b4.jpg"},{"id":85649924,"identity":"87177e8c-9bb6-4250-ace7-3c08e8df763a","added_by":"auto","created_at":"2025-06-30 09:06:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":610731,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/50f0efb209a2d7059b06b132.jpg"},{"id":85647755,"identity":"94e0c631-2a3d-478d-a4ec-94330aec06e4","added_by":"auto","created_at":"2025-06-30 08:50:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":283173,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"F7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/43841509e98ed9beb6acfbbd.jpg"},{"id":96913365,"identity":"54a8028f-352e-4923-ae39-f5dfd5cb668e","added_by":"auto","created_at":"2025-11-27 13:59:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3434985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6688444/v1/a20f50fa-bc63-4a7a-8c3c-e5d78901b46e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSHOX2-Driven Lymphatic Metastasis in pT1 Non-Clear Cell Renal Cell Carcinoma: A Biomarker-Guided Risk Stratification and Therapeutic Strategy\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) accounted for 434,419 new cases and 155,702 deaths globally in 2022[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Histologically, RCC is classified into clear cell RCC (ccRCC) and non-clear cell RCC (nccRCC). As the predominant subtype (70\u0026ndash;75% of RCC), ccRCC has been extensively studied through both basic research and large-scale randomized controlled/prospective phase III trials, yielding significant advances in molecular mechanisms and clinical management[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast, nccRCC\u0026mdash;encompassing papillary, chromophobe, and other rare subtypes with marked heterogeneity\u0026mdash;constitutes 25\u0026ndash;30% of RCC cases[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, no standardized first-line therapy exists for metastatic nccRCC, and current treatment protocols predominantly extrapolate from ccRCC guidelines due to the absence of successful phase III trials[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent imaging advancements have increased the detection of small renal masses (SRMs; ≦4 cm) in nccRCC, accounting for 48\u0026ndash;66% of renal tumors[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].However, despite favorable prognosis in pT1-stage RCC, lifelong surveillance remains necessary due to reported late recurrences[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], including rare cases of metastatic SRMs[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdjuvant therapy demonstrates efficacy for postoperative patients with microscopic residual disease (MRD) undetectable by imaging[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], The ASCO Annual Meeting has emphasized MRD detection in guiding RCC management. While circulating tumor DNA (ctDNA) analysis shows promise for identifying high-risk patients, its clinical utility in RCC is limited by economic constraints and low detection rates[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoth SRMs and MRD populations exhibit elevated recurrence risks, yet nccRCC surgical strategies largely mirror ccRCC protocols due to insufficient evidence. This study compares clinicopathological features between pT1 nccRCC and ccRCC to elucidate prognostic disparities, preliminarily explore molecular mechanisms, and propose optimized perioperative strategies for early nccRCC with high lymph node metastasis (LNM) risk. Our objectives are to: (1) address the lack of prognostic biomarkers in nccRCC; (2) develop novel therapeutic approaches to mitigate metastasis risk; and (3) improve survival outcomes for high-risk nccRCC populations.\u003c/p\u003e\n\u003ch3\u003eStudy Design and Ethics\u003c/h3\u003e\n\u003cp\u003eThis single-center retrospective cohort study received approval from Nanjing Gulou Hospital Ethics Committee. Inclusion criteria: pathologically confirmed pT1 RCC (AJCC 8th edition) treated with partial nephrectomy, complete clinical data, and endpoint events defined as metastasis, recurrence, or cancer-specific death. Patients receiving neoadjuvant therapy were excluded. The final cohort comprised 528 RCC cases (96 nccRCC; 432 ccRCC), all treated via robotic-assisted surgery.\u003c/p\u003e \u003cp\u003eFrom TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we obtained STAR-counts data and clinical information for nccRCC subtypes (KICH and KIRP). After data cleaning (excluding incomplete records), 355 cases were stratified into LN+ (n\u0026thinsp;=\u0026thinsp;20) 、LN- (n\u0026thinsp;=\u0026thinsp;89) and LNx(n\u0026thinsp;=\u0026thinsp;246) groups.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eSurvival analysis (Kaplan-Meier method), multivariate Cox regression, and Spearman correlation were performed using SPSS 26.0 and R 4.2.2. TCGA differential gene expression analysis employed DESeq2 (threshold: |log2FC|\u0026gt;1, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Gene set variation analysis (GSVA) utilized the ssGSEA algorithm (method='ssgsea'). Statistical significance was set at \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eTwenty paraffin-embedded specimens from robotic partial nephrectomy patients (2016\u0026ndash;2024) with pT1 nccRCC and LND (10 LN+, 10 LN-) were analyzed. SHOX2 staining used Proteintech polyclonal antibody (Cat No.16366-1-AP; optimized dilution 1:1500). Standardized protocols included:\u003c/p\u003e \u003cp\u003eDeparaffinization : Xylene (2\u0026times;20 min) \u0026rarr; ethanol series (100%\u0026rarr;60%);Antigen retrieval : TE buffer (pH 9.0) microwaved 10 min;Primary antibody incubation: 1.5 h at RT; Signal detection: HRP-conjugated secondary antibody (30 min) \u0026rarr; DAB (2\u0026ndash;5 min); Counterstaining: Hematoxylin (3 min) \u0026rarr; ethanol dehydration \u0026rarr; xylene clearing.\u003c/p\u003e \u003cp\u003eTwo pathologists independently scored specimens using semi-quantitative criteria : Intensity Score (IS): 0 (none), 1 (weak), 2 (moderate), 3 (strong);Proportion Score (PS): 0 (0%), 1 (1\u0026ndash;25%), 2 (26\u0026ndash;50%), 3 (51\u0026ndash;75%), 4 (76\u0026ndash;100%). Total Score (TS): IS\u0026thinsp;+\u0026thinsp;PS (range 0\u0026ndash;7). Inter-rater reliability met acceptable standards (\u003cem\u003eKappa\u0026thinsp;\u0026gt;\u0026thinsp;0.6\u003c/em\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eClinical Characteristics\u003c/h2\u003e \u003cp\u003eBoth groups exhibited negative surgical margins without renal vein/perirenal fat invasion. Propensity score matching was deemed unnecessary (SMD\u0026thinsp;\u0026lt;\u0026thinsp;0.2; Fig.\u0026nbsp;1A).\u003c/p\u003e \u003cp\u003eAmong 528 participants, 96 (18.2%) had non-clear cell renal carcinoma, while 432 (81.8%) had clear cell renal carcinoma. The non-clear cell group had a lower mean age (52.58\u0026thinsp;\u0026plusmn;\u0026thinsp;14.42 years vs 55.01\u0026thinsp;\u0026plusmn;\u0026thinsp;13.23 years; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.111\u003c/em\u003e) and similar BMI (24.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82 vs 24.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.821\u003c/em\u003e). Tumor size showed no difference between groups (3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44 cm vs 3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 cm; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.169\u003c/em\u003e). Gender distribution was comparable (34.38% male vs 38.19% male; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.484\u003c/em\u003e). The non-clear cell group had higher rates of metastasis or death (6.25% vs 0.69%; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), lymph node metastasis (5.21% vs 0.46%; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.001\u003c/em\u003e), and high-grade Fuhrman classification (31.25% vs 21.99%; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.054\u003c/em\u003e). No differences were observed in renal capsule penetration (20.83% vs 24.36%; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.462\u003c/em\u003e)(Fig.\u0026nbsp;1B).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurvival Analysis (dup: abstract ?)\u003c/h3\u003e\n\u003cp\u003eThe nccRCC cohort demonstrated significantly lower 5-year DFS (93.2% vs 99.2%; log-rank \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), indicating intrinsic biological aggressiveness (Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLymph Node Metastasis Prognostication\u003c/h2\u003e \u003cp\u003eUnivariate Cox regression identified LNM as the strongest predictor of poor outcomes in nccRCC (HR\u0026thinsp;=\u0026thinsp;38.63, 95% CI 6.92-215.73, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). This association persisted in multivariate analysis adjusting for Fuhrman grade and renal capsule involvement (adjusted HR\u0026thinsp;=\u0026thinsp;36.44, 95% CI 4.67-284.47, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDifferential Gene Expression\u003c/h3\u003e\n\u003cp\u003eVolcano plot analysis revealed SHOX2 as the most significantly upregulated gene in LN\u0026thinsp;+\u0026thinsp;versus LN- groups (log2FC\u0026thinsp;=\u0026thinsp;6.01, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e; Fig.\u0026nbsp;2B).\u003c/p\u003e\n\u003ch3\u003eSurvival Analysis\u003c/h3\u003e\n\u003cp\u003eUsing TCGA database, 178 nccRCC patients with high SHOX2 expression and 177 patients with low SHOX2 expression were stratified. Patients in the high-expression group exhibited significantly shorter survival time compared to the low-expression group (HR\u0026thinsp;=\u0026thinsp;2.896, 95% CI 1.329\u0026ndash;6.31, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). Samples were dichotomized into high- and low-expression groups based on median gene expression. Survival differences between groups were assessed using the log-rank test in Kaplan-Meier analysis. The Kaplan-Meier curves, analyzed by log-rank test and univariate Cox regression, demonstrated statistically significant prognostic relevance of SHOX2 (log-rank p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with higher expression correlating with poorer outcomes. When sorted by ascending SHOX2 expression (Fig.\u0026nbsp;4A), the intermediate scatterplot revealed a left-to-right trend of increasing mortality and decreasing survival time. The heatmap (Fig.\u0026nbsp;4B) illustrates gene expression patterns, confirming SHOX2 as a risk factor. Time-dependent ROC analysis showed AUC values of 0.809 (95% CI 0.7-0.918) at 1 year, 0.707 (95% CI 0.605\u0026ndash;0.809) at 3 years, and 0.658 (95% CI 0.544\u0026ndash;0.773) at 5 years (Fig.\u0026nbsp;4C).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePathway Correlation\u003c/h2\u003e \u003cp\u003essGSEA revealed positive correlations between SHOX2 and Extracellular matrix (ECM) (r\u0026thinsp;=\u0026thinsp;0.401, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e)/Epithelial-mesenchymal transition (EMT) pathways (r\u0026thinsp;=\u0026thinsp;0.381, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; Fig.\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIHC Validation\u003c/h2\u003e \u003cp\u003eROC analysis demonstrated SHOX2's predictive value for LNM (AUC\u0026thinsp;=\u0026thinsp;0.77, 95% CI 0.54\u0026ndash;0.99). At optimal cutoff (TS\u0026thinsp;≧\u0026thinsp;3.5; Youden index: sensitivity 70.0%, specificity 90.0%), SHOX2 positivity was observed in 7/10 LN\u0026thinsp;+\u0026thinsp;versus 1/10 LN- cases ( \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e; Fig.\u0026nbsp;6B-C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eKey Findings\u003c/h2\u003e \u003cp\u003eThis study first reports a 4.1% incidence of LNM in Chinese patients with pT1 nccRCC, significantly higher than in ccRCC counterparts (0.4%; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). This observation aligns with prior studies demonstrating elevated pathological LNM rates in nccRCC versus ccRCC[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], potentially attributable to the predilection of nccRCC for lymphatic dissemination [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although early-stage pT1 nccRCC generally exhibits favorable postoperative outcomes, the presence of metastatic risk persists despite low pathological stage. Both univariate and multivariate Cox proportional hazards analyses identified LNM as an independent risk factor for adverse prognosis in nccRCC.\u003c/p\u003e \u003cp\u003eRecent advancements in systemic therapies\u0026mdash;including molecular targeted agents and immune checkpoint inhibitors (ICIs)\u0026mdash;have significantly improved survival outcomes in ccRCC through combination regimens [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e],However, the management of nccRCC remains extrapolated from ccRCC protocols due to insufficient randomized controlled trial data. nccRCC exhibits distinct immune evasion mechanisms: VEGF-targeted inhibition upregulates PD-L1 expression on lymphocytes, while dual pathway blockade enhances lymphocyte infiltration and activity via lymphotoxin beta receptor (LTBR) signaling[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].The 2022 EAU guidelines recommend cabozantinib over sunitinib for papillary RCC based on prolonged progression-free survival [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].Although ICI-targeted combinations demonstrate superior survival in ccRCC [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], their efficacy in nccRCC requires validation through dedicated trials.\u003c/p\u003e \u003cp\u003eLymph node dissection (LND) remains crucial for high-risk RCC management[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with studies demonstrating survival benefits in locally advanced high-risk RCC (median overall survival 43.10 vs 40.52 months, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nevertheless, LND carries significant risks: Suzuki et al. reported a 9.7% incidence of chylous leakage associated with LND[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. 99mTc-sulfur colloid lymphoscintigraphy provides high diagnostic accuracy for nodal staging (sensitivity 89%, specificity 93% [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]), utilizing SPECT imaging to track radiotracer transport through lymphatic endothelial cells. However, its clinical application requires judicious consideration of radiation exposure and cost-effectiveness. The dual challenges of selecting appropriate candidates for lymphoscintigraphy and determining optimal LND timing underscore the need for improved risk stratification tools.\"\u003c/p\u003e \u003cp\u003eWhile early surgical intervention achieves favorable outcomes in most pT1 nccRCC cases, a subset with high-risk features demonstrates significantly greater LNM propensity compared to ccRCC (4.1% vs 0.4%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The presence of MRD in these patients may drive subclinical progression, as current therapeutic strategies fail to adequately address high-risk nodal disease.\u003c/p\u003e \u003cp\u003eOur findings position SHOX2 as a central mediator of lymphatic metastasis in nccRCC, extending its known pan-cancer oncogenic roles (e.g., WASF3 activation in breast cancer[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; STAT3 signaling in gastric cancer[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; Hepatocellular carcinoma: Associates with early recurrence[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; Lung cancer: Validated as a prognostic biomarker in NSCLC[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]) to RCC pathogenesis. Specifically, the strong correlation between SHOX2 and EMT/ECM pathways (r\u0026thinsp;=\u0026thinsp;0.381/0.401, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) suggests conserved mechanisms across malignancies. Our TCGA analysis of nccRCC (n\u0026thinsp;=\u0026thinsp;355) identified SHOX2 as the most differentially expressed gene between LN\u0026thinsp;+\u0026thinsp;and LN- groups (log2FC\u0026thinsp;=\u0026thinsp;6.01, FDR\u0026thinsp;=\u0026thinsp;0.003). Elevated SHOX2 expression correlated with reduced survival (HR\u0026thinsp;=\u0026thinsp;2.896, 95% CI 1.329\u0026ndash;6.31, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), suggesting its prognostic utility in nccRCC.\u003c/p\u003e \u003cp\u003eIn bladder cancer models, SHOX2 overexpression induces EMT, enhancing migratory capacity[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].Although direct evidence in RCC remains limited, these findings suggest SHOX2 promotes lymphatic metastasis through conserved mechanisms involving ECM degradation and EMT activation.\u003c/p\u003e \u003cp\u003eMethylation of SHOX2 (mSHOX2) occurs at cytosine-phosphate-guanine (CpG) islands within the promoter region. A single-center prospective study demonstrated that mSHOX2 serves as a molecular biomarker for predicting poor prognosis in RCC patients at high risk of metastasis. Multivariable Cox analysis confirmed mSHOX2 as an independent risk factor for recurrence, with preoperative mSHOX2 levels significantly elevated in RCC patients with unfavorable outcomes. Furthermore, mSHOX2 in ctDNA showed significant correlation with lymph node metastasis in RCC[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].Hypomethylation of mSHOX2 may contribute to upregulated SHOX2 mRNA expression in tumor tissues[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].The U.S. Food and Drug Administration has approved an mSHOX2 detection kit for lung cancer diagnosis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], suggesting its potential utility in perioperative therapeutic decision-making for high-risk nccRCC patients. These findings collectively support mSHOX2 as a biomarker for identifying nccRCC patients at high risk of lymph node metastasis.\u003c/p\u003e \u003cp\u003eIHC analysis of 20 pT1 nccRCC specimens from patients undergoing lymph node dissection revealed significantly higher SHOX2 positivity in the LN\u0026thinsp;+\u0026thinsp;group compared to the LN- group (70% vs. 10%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). This indicates SHOX2's potential as a prognostic marker for screening MRD in high-risk nccRCC patients, enabling early personalized interventions to prevent adverse outcomes.\u003c/p\u003e \u003cp\u003eBased on existing evidence, we propose the following clinical pathway :Preoperative screening: Detect mSHOX2 levels to identify high-risk populations for lymph node metastasis. For patients with elevated mSHOX2 levels or suspicious lymph nodes on contrast-enhanced CT/MRI, perform 99mTc-sulfur colloid lymphoscintigraphy to confirm nodal status;\u003c/p\u003e \u003cp\u003eIntraoperative management: Implement LND for confirmed high-risk cases, balancing therapeutic benefits against surgical risks;\u003c/p\u003e \u003cp\u003ePostoperative surveillance: Quantify SHOX2 expression in resected specimens. For patients with SHOX2-positive IHC scores (TS\u0026thinsp;≧\u0026thinsp;3.5), initiate contrast-enhanced abdominal CT every 6 months and consider early targeted-immunotherapy combinations.The recently reported IUNU-RC-102 trial (ASCO-GU 2025) further supports the feasibility of adjuvant toripalimab-axitinib in high-risk nccRCC, demonstrating preliminary efficacy in reducing recurrence rates. Our SHOX2-driven stratification strategy could synergize with such regimens to optimize patient selection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical Translational Significance\u003c/h2\u003e \u003cp\u003eIn accordance with the EAU guidelines, we have developed a perioperative optimization strategy (Fig.\u0026nbsp;7) comprising sequential phases:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePreoperative assessment\u003c/h2\u003e \u003cp\u003epreoperative mSHOX2 testing to identify high-risk populations for lymph node metastasis, followed by 99mTc-sulfur colloid lymphoscintigraphy in mSHOX2-elevated cases to confirm nodal status; Intraoperative decision-making:meticulous lymph node evaluation guides selective LND in high-risk patients, balanced against overtreatment risks;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePostoperative surveillance\u003c/h2\u003e \u003cp\u003ePostoperative management integrates IHC quantification of tumor SHOX2 expression with periodic mSHOX2 monitoring, prompting six-monthly contrast-enhanced abdominal CT for early micrometastasis detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAdjuvant therapy\u003c/h2\u003e \u003cp\u003eEmerging evidence supporting targeted-immunotherapy combinations (e.g., toripalimab-axitinib) in high-risk cohorts warrants exploration of adjuvant regimens for SHOX2-overexpressing subgroups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInnovations and Limitations\u003c/h2\u003e \u003cp\u003eThis study provides the first Asian population-derived framework that synergizes molecular biomarkers (SHOX2/mSHOX2) with clinical stratification, addressing the historical overreliance on Eurocentric data in nccRCC management. By establishing actionable protocols for pT1 nccRCC\u0026mdash;a clinical scenario lacking consensus guidelines\u0026mdash;we bridge a critical knowledge gap. The retrospective single-center design inherently limits causal inference, necessitating validation through multicenter prospective cohorts to mitigate selection bias and enhance generalizability.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEarly surgical intervention for pT1 nccRCC is associated with a significantly elevated lymph node metastasis risk compared to ccRCC, mechanistically linked to SHOX2-mediated ECM remodeling and EMT. A precision medicine approach combining liquid biopsy (mSHOX2-guided lymphoscintigraphy), histopathological profiling (SHOX2 IHC scoring), and postoperative biomarker surveillance is proposed to optimize therapeutic decision-making. Validation through collaborative multicenter studies remains imperative to translate this biomarker-driven strategy into clinical practice.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003enccRCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-clear cell renal cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLNM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elymph node metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eccRCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClear cell renal cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHazards Ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConfidence Interval\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRenal cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSRMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSmall renal masses\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicroscopic residual disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ectDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCirculating tumor DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLNM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLymph node metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene set variation analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntensity Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProportion Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandardized Mean Difference\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially Expressed Genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003et\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003et-test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChi-square test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLN+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLymph node-positive\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLN\u0026minus;\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLymph node-negative\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICIs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmune checkpoint inhibitors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLTBR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elymphotoxin beta receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLND\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLymph node dissection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNonsmall-cell lung cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the General Program of National Natural Science Foundation of China (Grant No. 82172777) and Jiangsu Provincial Medical Key Discipline (Laboratory) Cultivation Unit (JSDW202221).\u003c/p\u003e\n\u003cp\u003eAuthor information\u003c/p\u003e\n\u003cp\u003eAuthor notes\u003c/p\u003e\n\u003cp\u003eXu Zhen contributed to this work.\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e¹Department of Urology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, 321 Zhongshan Road, Nanjing 210008, China\u003c/p\u003e\n\u003cp\u003eXu Zhen¹, Qiang Ma\u003csup\u003e1\u003c/sup\u003e, Changwei Ji\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e²Department of Urology, Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing 210008, China\u003c/p\u003e\n\u003cp\u003eWenjin Yang², Hongjun Yin\u003csup\u003e2\u003c/sup\u003e, Rui Si²,Lina Dai\u003csup\u003e2\u003c/sup\u003e,Hongqian Guo\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eStudy design: Z.X; Experimental operation: Z.X. and J.CW.; Data acquisition: Z.X.,D.LN and Y.WJ.; Data analysis:Z.X.,Y.HJ and S.R.; Research writing and revision of the manuscript: Z.X.,G.HQ.and M.Q.; Study supervision: J.CW.\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCorrespondence to Changwei Ji.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed during the study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis retrospective study was approved by and the Ethics Committee of the Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School. All methods were carried out in accordance with relevant guidelines and regulation. Informed consent was obtained from of all subjects and/or their legal guardian(s).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotzer RJ, Powles T, Burotto M, Escudier B, Bourlon MT, Shah AY, Suarez C, Hamzaj A, Porta C, Hocking CM, et al. 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Clin Chem. 2019;65(4):559\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi N, Zeng Y, Tai M, Lin B, Zhu D, Luo Y, Ren X, Zhu X, Li L, Wu H, et al. Analysis of the Prognostic Value and Gene Expression Mechanism of SHOX2 in Lung Adenocarcinoma. Front Mol Biosci. 2021;8:688274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButtner T, Zarbl R, Krausewitz P, Strieth S, Kristiansen G, Eckstein M, Ralser DJ, Holzel M, Ritter M, Ellinger J, et al. Hypermethylated SHOX2 in circulating cell-free DNA post renal cell carcinoma surgery as TNM-independent biomarker for recurrence risk. Am J Transl Res. 2024;16(1):304\u0026ndash;13.\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":"Non-clear cell renal cell carcinoma, SHOX2, Lymph node metastasis, Biomarker validation, Precision medicine","lastPublishedDoi":"10.21203/rs.3.rs-6688444/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6688444/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNon-clear cell renal cell carcinoma (nccRCC), representing 25\u0026ndash;30% of RCC cases, exhibits higher lymph node metastasis (LNM) risk than clear cell RCC (ccRCC). Despite favorable outcomes in early-stage pT1 nccRCC, metastatic potential persists, necessitating improved risk stratification.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA single-center retrospective cohort of 528 pT1 RCC patients (96 nccRCC; 432 ccRCC) and TCGA data (n\u0026thinsp;=\u0026thinsp;355) were analyzed. Differential gene expression (DESeq2, log2FC\u0026thinsp;\u0026gt;\u0026thinsp;1, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), survival analyses (Kaplan-Meier/Cox regression), and immunohistochemistry (IHC) validated SHOX2 as a biomarker.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe LNM rate in pT1 nccRCC was significantly higher than in ccRCC (4.1% vs. 0.4%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). SHOX2 was markedly overexpressed in LNM\u0026thinsp;+\u0026thinsp;tumors (log2FC\u0026thinsp;=\u0026thinsp;6.01, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and independently predicted adverse prognosis (HR\u0026thinsp;=\u0026thinsp;2.896, 95% CI 1.329\u0026ndash;6.31, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). Functional enrichment linked SHOX2 to ECM remodeling (r\u0026thinsp;=\u0026thinsp;0.401, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and EMT activation (r\u0026thinsp;=\u0026thinsp;0.381, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). IHC validation demonstrated SHOX2\u0026rsquo;s predictive value for LNM (AUC\u0026thinsp;=\u0026thinsp;0.77, 95% CI 0.61\u0026ndash;0.93), with optimal cutoff\u0026thinsp;\u0026ge;\u0026thinsp;3.5 (70% sensitivity, 90% specificity). SHOX2 positivity was enriched in LNM\u0026thinsp;+\u0026thinsp;specimens (70% vs. 10%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eSHOX2 drives lymphatic dissemination in pT1 nccRCC via ECM-EMT crosstalk, serving as a robust biomarker. A tripartite strategy integrating preoperative mSHOX2 liquid biopsy, image-guided lymph node dissection, and postoperative targeted-immunotherapy (e.g., toripalimab-axitinib) is proposed to optimize outcomes. Multicenter validation is warranted for clinical translation.\u003c/p\u003e","manuscriptTitle":"SHOX2-Driven Lymphatic Metastasis in pT1 Non-Clear Cell Renal Cell Carcinoma: A Biomarker-Guided Risk Stratification and Therapeutic Strategy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 08:50:01","doi":"10.21203/rs.3.rs-6688444/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":"71f5dba4-3049-494b-a43a-c943d1ad1348","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-13T13:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-30 08:50:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6688444","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6688444","identity":"rs-6688444","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}