Pit 1

For all included participants, the archival slides of their surgical specimens were retrieved and the pathological diagnosis of PitNET was confirmed by an experienced neuropathologist. Their formalin-fixed, paraffin-embedded blocks of surgical specimens were retrieved and stained for the transcription factors SF-1, TPIT and Pit-1 using the Leica BOND III fully automated in-situ hybridization (ISH) staining system. The following commercially available antibodies and concentrations were used: TPIT (Sigma-Aldrich, 1:200), SF-1 (R&D, 1:250), and Pit-1 (Santa Cruz, 1:200). Re-staining for Ki-67 proliferation marker (Dako, 1:250), prolactin (Dako, 1:20), FSH (BioG, 1:20), GH (NeoMarker, 1:3000), ACTH (Dako, 1:40) and/or TSH (BioG, 1:20) were also performed if not done previously. The NF-PitNETs were classified into different subgroups according to both 2017 and 2022 WHO classifications.

Hematoxylin and eosin (HE) staining was performed for histological examination. Immunohistochemistry was performed using primary antibodies specific to anti-prion proteins: (mouse monoclonal 3F4 [1:400], mouse monoclonal 8G8 [specific for PrP at 95–110, 1:400]; Cayman, Ann Arbor, MI, USA, and rabbit monoclonal EP1802Y [1:1000]), mouse monoclonal anti-pituitary-specific positive transcription factor 1 (Pit-1; 1:100, Santa Cruz Biotechnology), rabbit polyclonal anti-T-Box Transcription Factor 19 (TBX19; 1:200, Atlas Antibodies, Bromma, Sweden), mouse monoclonal anti-steroidogenic factor 1 (SF-1; 1:100, Perseus Proteomics, Tokyo, Japan), and mouse monoclonal anti-chromogranin A (CgA; 1:100, Millipore). Double immunofluorescence staining was performed using a combination of mouse monoclonal Pit-1 and rabbit monoclonal EP1802Y antibodies or rabbit polyclonal TBX19 and mouse monoclonal 3F4 antibodies. Alexa 488-labeled anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa 546-labeled anti-mouse IgG (Invitrogen), Alexa 488-labeled anti-rabbit IgG (Invitrogen), and Alexa 546-labeled anti-rabbit IgG (Invitrogen) were used as secondary antibodies. The specimens were visualized using a Nikon A1R-A1 confocal microscope (Nikon, Tokyo, Japan).