A FISH-Based Three-Tier Classification of Chromosome 3 Alterations in Clear Cell Renal Cell Carcinoma: Diagnostic and Prognostic Implications Utility

Background: Clear cell renal cell carcinoma (CCRCC) is the predominant subtype of renal cell carcinoma and is characterized by frequent chromosome 3 alterations, including 3p deletion, monosomy, and aneuploidy. However, the absence of standardized fluorescence in situ hybridization (FISH) cut-off values has led to inconsistent reported frequencies and limited clinical integration of this accessible assay. This study aimed to establish clinically applicable cut-off values, propose a practical three-tier classification, and evaluate its diagnostic accuracy and clinicomolecular correlation with tumor aggressiveness. Methods: FISH using VHL (3p25.3) and CEP3 probes was performed on 1748 RCC cases (1655 CCRCC, 48 papillary RCC, 45 chromophobe RCC). Cut-off values were determined by combining ROC analysis with Youden’s index and the mean + 3SD method from normal renal tubular cells. A paired cohort of 97 CCRCC cases with targeted next-generation sequencing was stratified into three subgroups (3p intact, isolated 3p loss, broad chr3 change) for clinicomolecular comparison, including 3D principal component analysis. Results: Clinically applicable thresholds of 30% for 3p deletion and 20% for monosomy identified chromosome 3 alterations in 76.9% of CCRCC cases. The combination of both markers achieved superior diagnostic accuracy (AUC = 0.82). Aneuploidy was significantly associated with higher WHO/ISUP grade (p < 0.001) and older age (p = 0.006). The three-tier classification showed stepwise progression of aggressive features (older age, higher grade, larger tumor size) and increasing PBRM1 mutation frequency from the 3p intact to the broad chr3 change group. Conclusions: This study establishes standardized FISH cut-offs and a practical three-tier classification that captures a continuous spectrum of genomic instability and tumor aggressiveness in CCRCC. Routine 3p FISH provides a simple, cost-effective, and reproducible tool with substantial diagnostic and stratification value that complements more complex genomic profiling.