Germline-Somatic Interplay Shapes Molecular Divergence in Peripheral Nerve Sheath Tumors

Peripheral nerve sheath tumors (PNSTs) arising in neurofibromatosis type 1 (NF1) show marked histological and clinical heterogeneity, ranging from benign to malignant PNSTs. Although germline NF1 variants underlie tumor predisposition, the mechanisms driving phenotypic and clinical heterogeneity remain incompletely defined. We performed integrated germline and somatic analyses of 208 samples (74 blood samples and 134 tumors) from a primary cohort of 74 patients with NF1. Using a custom NF-focused sequencing panel, we investigated NF1 variant types, co-occurring oncogenic alterations, and affected domains to assess genotype-phenotype associations. Germline analysis identified 55 pathogenic NF1 variants, including eight unreported variants, with recurrent NF1 microdeletions involving SUZ12 in high-grade and premalignant tumors. Among tumors from individuals with germline NF1 variants, biallelic NF1 inactivation occurred in 90/124 tumors (72.6%), most frequently through loss of heterozygosity. In addition, high-grade PNSTs frequently harbored co-alterations in CDKN2A, SUZ12, EED, or TP53. Analysis of the distribution of somatic NF1 variants across functional domains further revealed significant enrichment in the GRD in benign tumors, whereas variants involving the LRD, including the Sec14-PH subdomain, showed a trend toward enrichment in premalignant and malignant tumors. Notably, multi-lesion analysis of spatially and temporally distinct tumors from 30 individuals revealed pronounced intrapatient somatic heterogeneity: despite a shared germline NF1 background, individual tumors typically carried distinct NF1 second-hit events, consistent with lesion-specific molecular divergence rather than a single shared somatic driver across lesions. In contrast, microdissected histologically distinct components within individual tumors shared underlying NF1 alterations and showed sequential acquisition of additional alterations involving CDKN2A and EED, supporting stepwise molecular evolution during malignant progression. Together, these data reveal lesion-specific molecular divergence across distinct tumors and stepwise molecular evolution within individual tumors, underscoring the value of lesion-specific genomic evaluation in NF1-associated PNSTs.