Calcifying nested stromal-epithelial tumor (CNSET) is an extremely rare primary liver tumor of uncertain histogenesis that predominantly occurs in the pediatric age group and young adults. Knowledge regarding the molecular genetic profile of this entity remains limited, with only two molecular studies conducted to date, which identified pathogenic mutations in the CTNNB1 gene and TERT promoter mutations in all analyzable cases. A more aggressive biological potential than previously reported has been only recently unveiled as well. To further advance the understanding of pathogenic mechanisms of CNSET and to investigate the distinctiveness of this rare entity, we analyzed two cases using immunohistochemistry, next-generation sequencing (NGS), and methylation profiling. The latter was employed to compare the epigenetic landscape of CNSET with that of clinicopathologically similar entities, such as hepatoblastoma and solid pseudopapillary neoplasm (SPN) of the pancreas. Both CNSET cases occurred in women (aged 24 and 23 years) and measured 24 cm and 16 cm in diameter, respectively. Both cases showed similar histological features, being composed of organoid nests of bland spindled to epithelioid cells embedded in myofibroblastic stroma. Both cases were immunohistochemically positive for CD56, WT1, and CAM5.2 and negative for hepatocellular and neuroendocrine markers. Case 2 showed aberrant nuclear expression of β-catenin, while in case 1, there was cytoplasmic positivity only. Using Illumina TruSight Oncology 500 NGS panel, case 1 revealed a pathogenic mutation in the WT1 gene and a TERT promoter mutation, and case 2 had a CTNNB1 mutation. DNA methylation analysis showed that CNSET forms a distinct cluster, separate from other reference entities. Follow-up in case 2 revealed a disease-free status 21 months after partial hepatectomy. This study showed that the molecular landscape of CNSET of the liver is characterized by CTNNB1, TERT promoter, and WT1 gene mutations, with the latter representing a novel alteration. Similarly to CTNNB1, the WT1 gene plays a significant role in the Wnt signaling pathway. Given the metastatic potential and chemotherapy resistance of CNSET, understanding its molecular background is important for potential alternative targeted treatment. Methylation profiling confirms CNSET as a distinct entity, separate from hepatoblastoma and SPN of the pancreas.

Bioptical Laboratory Ltd Plzen Czech Republic

Cytopathos Ltd Bratislava Slovakia

Department of Pathology 1st Faculty of Medicine Charles University General University Hospital Prague Czech Republic

Department of Pathology Faculty of Medicine in Plzen Charles University Plzen Czech Republic

Department of Pathology Military University Hospital Prague Prague Czech Republic

Diagnostic Center of Pathology Unilabs Slovakia Ltd Bratislava Slovakia

Laboratorio Athena Cesena Italy

Medical Laboratory CSD Ltd Kiev Ukraine

The Fingerland Department of Pathology Charles University Faculty of Medicine in Hradec Králové and University Hospital Hradec Kralove Czech Republic

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