Hypermethylation of SHOX2 and RASSF1A in intraoperative pleural lavage fluid as a molecular signature of aggressive tumor biology and high histologic grade in stage I lung adenocarcinoma

BACKGROUNDS: DNA methylation profiling has emerged as a powerful adjunct for refining pathological accuracy. This study investigates the correlation between the SHOX2 and RASSF1A methylation status in intraoperative pleural lavage fluid (IPLF) and indices of tumor aggressiveness in stage I lung adenocarcinoma. METHODS: A total of 596 consecutive patients undergoing curative-intent resection for suspected stage I lung cancers were screened. 524 met criteria and formed the final cohort. Immediately after thoracoscopic access and before tumor manipulation, IPLF was collected. Genomic DNA was extracted and analyzed by quantitative methylation-specific PCR for SHOX2 and RASSF1A. Clinicopathologic variables, including demographics, imaging features, and histopathologic diagnoses were recorded. RESULTS: The results indicated that 97% of the IPLF samples achieved the minimal DNA concentration required for methylation assays. Optimal cut-off values were determined for SHOX2 and RASSF1A, revealing promoter hypermethylation in 14.3% and 7.0% of specimens, respectively. SHOX2/RASSF1A methylation status correlated significantly with multiple clinicopathologic variables, including sex, smoking history, radiological solid-dominant nodules, poor differentiation, tumor airway vascular permeation (TAVP), visceral pleural invasion (VPI), spread through air spaces (STAS), lymphovascular invasion (LVI), elevated Ki-67 labeling index, PD-L1 expression, and TP53 mutation. Multivariate logistic regression analysis revealed that SHOX2/RASSF1A methylation was independently associated with intermediate/high tumor grade (G2/3) (OR = 7.02, P < 0.05). CONCLUSION: Detection of SHOX2/RASSF1A hypermethylation in IPLF demonstrates significant association with aggressive biological behavior and higher tumor grade(G2/3) in stage I lung adenocarcinoma. This biomarker signature may enable refinement of intraoperative surgical strategies and inform postoperative therapeutic escalation.