Disentangling the Cellular Composition of FLCN-Mutated Tumors in Birt-Hogg-Dubé Syndrome by Spatial Transcriptomics

Birt-Hogg-Dubé (BHD) syndrome is a hereditary cancer predisposition syndrome caused by pathogenic variants in the folliculin (FLCN) gene and is associated with an increased risk of multifocal renal tumors. FLCN-mutated tumors (FMTs) often exhibit morphologic heterogeneity with mixed morphologic features resembling renal oncocytoma and chromophobe renal cell carcinoma, yet the molecular basis underlying the heterogeneous morphologic features and the morphologic-genomic correlations remain poorly defined. In our prior work, we identified mutually exclusive expressions of L1 cell adhesion molecule (L1CAM) and forkhead box I1 (FOXI1) labeling the 2 morphologically distinct cellular populations in BHD-associated FMTs, leading to the hypothesis that these 2 tumor compartments may have distinct molecular features and may reflect different nephron epithelial differentiation states. In this follow-up study, we tested this hypothesis using L1CAM and FOXI1 as morphology-guided markers for spatial transcriptomic profiling of the distinct tumor compartments in FMTs with the NanoString GeoMX Digital Spatial Profiler. Six FMTs from 3 patients with BHD and 3 normal kidney tissues were analyzed. L1CAM+ and FOXI1+ areas of illumination were collected from tumor areas with various tumor compositions, including L1CAM+ dominant, FOXI1+ dominant, and mixed tumor areas. Spatial transcriptomic analysis identified distinct gene expression signatures in L1CAM+ and FOXI1+ FMT compartments independent of the local tumor compositions. FOXI1+ tumor cells showed robust enrichment for intercalated cell-associated gene signatures. In contrast, L1CAM+ tumor cells exhibited a heterogeneous transcriptional profile, with partial overlap across a spectrum of renal tubular epithelial cell types rather than a definitive principal cell-like identity. Despite these compartment-specific differences, both compartments share expression of a panel of tumor signature genes, including the glycoprotein nmb (GPNMB) gene, and a core of cancer-related biological functions and signaling pathways. Together, these findings refined the prior dichotomous model of BHD-associated renal tumors and support a model in which L1CAM+ and FOXI1+ tumor compartments represent divergent evolutionary or differentiation states with a common FLCN-mutant neoplastic transcriptional program. This spatial transcriptomic profiling provides molecular evidence for the morphologic heterogeneity of FMTs and insights into the tumor biology of BHD-associated FMTs.