SAT-512 - A Virtual Experiment-Driven Translational Framework Supports Independent Clonal Origins and a Putative CXCL12/CXCR4 Paracrine Axis in Ipsilateral Pheochromocytoma and Aldosterone-Producing Adenoma

Background: Ipsilateral coexistence of pheochromocytoma (PHEO) and aldosterone-producing adenoma (APA) is exceptionally rare. Mechanistic investigation of potential cross-lesion interplay is often limited by scarce viable tissue for conventional functional assays.

Methods: A 62-year-old man with refractory hypertension underwent unilateral adrenalectomy after dual-tracer PET-CT (68Ga-DOTATATE and 68Ga-Pentixafor) identified two biologically distinct ipsilateral adrenal nodules. PHEO tissue underwent somatic targeted sequencing. CYP11B2-positive APA regions were isolated by laser-capture microdissection for whole-exome sequencing with Sanger confirmation of the key driver. Peripheral blood sequencing excluded phenotype-related pathogenic/likely pathogenic germline variants. To address wet-lab constraints, we applied a three-layer virtual experiment pipeline integrating residue-level structural modeling, pathway/network analysis, and public transcriptomic mining (TCGA-PCPG, GSE64957).

Results: Pathology confirmed collision lesions (2.9-cm PHEO, 1.0-cm APA). Genomics identified non-overlapping lesion-specific alterations: PHEO harbored VHL c.475A>G (p.K159E, VAF 11.83%), whereas APA harbored canonical KCNJ5 p.G151R (VAF 30.96%, Sanger-confirmed), with no VHL alteration in APA, supporting independent clonal origins. Structural analysis of VHL p.K159E showed reduced local contacts (20 to 12), decreased predicted VBC affinity (mCSM-PPI2 ΔΔG_affinity = -1.26 kcal/mol), and destabilization (DynaMut2 ΔΔG_stability = -0.77 kcal/mol), consistent with weakened peripheral interface microenvironment. KCNJ5 p.G151R modeling supported pore/selectivity microenvironment perturbation rather than formation of a new direct salt bridge. At pathway level, TCGA-PCPG showed a positive EPAS1-CXCL12 association (Pearson R=0.25, p=0.00071). In GSE64957, CXCR4 expression was enriched in aldosterone-lineage samples (ZG/APA > ZF; APA ≈ ZG), indicating receptor availability compatible with paracrine input.

Conclusions: This virtual experiment-driven framework supports genetically independent ipsilateral PHEO and APA and yields a testable cross-lesion model in which pseudohypoxia-linked PHEO signaling may contribute to a CXCL12-enriched, pro-survival vascular niche permissive for CXCR4-positive aldosterone-lineage cells. This strategy is scalable for mechanism generation in rare endocrine collision tumors with limited tissue.

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