An Integrative Morphological and Genomic Analysis With a Refined FISH Threshold and Novel Kinase Fusions in a Large Asian Cohort of Spitzoid Neoplasms.

Differentiating atypical Spitz tumors (AST) from true Spitz melanomas (SM) and conventional melanomas with spitzoid features (MSF) remains a formidable diagnostic challenge. Because current molecular epidemiological data are overwhelmingly derived from Caucasian cohorts, the genomic landscape of Asian populations remains largely unexplored. To elucidate the molecular progression landscape and refine diagnostic criteria, we performed a comprehensive multimodal analysis-integrating histomorphology, immunohistochemistry (IHC), multi-probe fluorescence in situ hybridization (FISH), and targeted RNA/DNA-based next-generation sequencing (NGS)-on a cohort of 140 spitzoid neoplasms. This cohort, comprising 126 ASTs, 8 SMs, and 6 MSFs, represents the largest Asian cohort to date. Malignant phenotype strongly correlated with lesional asymmetry, deep atypical mitoses, a sheet-like growth pattern, diffuse PRAME positivity, and significant loss of p16 expression (64.3% in SM/MSF vs. 9.5% in ASTs; P<0.0001). Building upon established melanoma FISH criteria, we optimized a prognostic threshold of ≥2 FISH abnormalities specifically tailored for spitzoid neoplasms. We demonstrated that isolated single chromosomal aberrations (particularly MYB loss) are relatively stable events frequent in indolent ASTs, whereas our refined ≥2 threshold yielded 100% sensitivity and 91.7% specificity for predicting regional lymph node metastasis/local recurrence. Molecularly, NGS identified mutually exclusive initiating driver alterations (comprising kinase fusions and HRAS mutations) in 89.9% of true Spitz neoplasms, a remarkably high prevalence suggesting a distinct genetic background in Asian populations. We also characterized five entirely novel kinase fusions (ZNF24::ROS1, PCBP1::ROS1, NUMA1::RET, CBWD1::ALK, and TPR::NTRK1). Furthermore, NGS definitively segregated true Spitz neoplasms from morphologic mimics (MSF), which lacked fusions and were driven by canonical genomic alterations of the conventional melanoma pathway. Integrating these genomic landscapes validated a stepwise progression model. While isolated kinase fusions drove indolent ASTs, malignant SM invariably harbored concurrent pathogenic secondary alterations, demonstrating a profound reliance on CDKN2A/B, TP53, and CDK4 aberrations. Ultimately, we propose an integrated diagnostic algorithm combining morphologic evaluation, the refined FISH threshold, and comprehensive NGS profiling, providing a precise, evidence-based framework for pathway classification and clinical management of spitzoid neoplasms.