Hand2 is a core transcription factor responsible for chromaffin cell differentiation. However, its potential utility in surgical pathology has not been studied. Thus, we aimed to investigate its expression in paragangliomas, other neuroendocrine neoplasms (NENs), and additional non-neuroendocrine tumors. We calibrated Hand2 immunohistochemistry on adrenal medulla cells and analyzed H-scores in 46 paragangliomas (PGs), 9 metastatic PGs, 21 cauda equina neuroendocrine tumors (CENETs), 48 neuroendocrine carcinomas (NECs), 8 olfactory neuroblastomas (ONBs), 110 well-differentiated NETs (WDNETs), 10 adrenal cortical carcinomas, 29 adrenal cortical adenomas, 8 melanomas, 41 different carcinomas, and 10 gastrointestinal stromal tumors (GISTs). Both tissue microarrays (TMAs) and whole sections (WSs) were studied. In 171 NENs, previously published data on Phox2B and GATA3 were correlated with Hand2. Hand2 was positive in 98.1% (54/55) PGs, but only rarely in WDNETs (9.6%, 10/104), CENETs (9.5%, 2/21), NECs (4.2%, 2/48), or ONBs (12.5%, 1/8). Any Hand2 positivity was 98.1% sensitive and 91.7% specific for the diagnosis of PG. The Hand2 H-score was significantly higher in primary PGs compared to Hand2-positive WDNETs (median 166.3 vs. 7.5; p < 0.0001). Metastatic PGs were positive in 88.9% (8/9). No Hand2 positivity was observed in any adrenal cortical neoplasm or other non-neuroendocrine tumors, with exception of 8/10 GISTs. Parasympathetic PGs showed a higher Hand2 H-score compared to sympathetic PGs (median H-scores 280 vs. 104, p < 0.0001). Hand2 positivity in NENs serves as a reliable marker of primary and metastatic PG, since other NENs only rarely exhibit limited Hand2 positivity.

1st Department of Pathology St Anne's University Hospital and Faculty of Medicine Masaryk University Pekarská 664 53 602 00 Brno stred Brno Czech Republic

4th Department of Internal Medicine Charles University Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové Sokolska 582 500 05 Hradec Kralove Czech Republic

Centre for Hypertension 3rd Department of Medicine Charles University 1st Faculty of Medicine and General University Hospital Prague Prague Czech Republic

Department of Internal Medicine 1st Faculty of Medicine Charles University and Military University Hospital Prague Czech Republic

Department of Neurosurgery and Neurooncology 1st Medical Faculty Charles University Military University Hospital Prague U Vojenske Nemocnice 1200 Praha 6 169 02 Prague Czech Republic

Department of Neurosurgery Charles University Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové Sokolska 582 500 05 Hradec Kralove Czech Republic

Department of Pathology Charles University 1st Faculty of Medicine and General University Hospital Prague Studnickova 2039 128 00 Nové Mesto Prague Czech Republic

Department of Pathology Military University Hospital Prague U Vojenske Nemocnice 1200 Praha 6 169 02 Prague Czech Republic

Department of Pathology The Regional Hospital Pardubice Pardubice Czech Republic

The Fingerland Department of Pathology Charles University Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové Sokolska 582 500 05 Hradec Kralove Czech Republic

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