Cross-Reactivity of TPIT Antibody Clone OTI2G1 in Chordoma: Structural Mechanisms and Diagnostic Implications.

Abstract

TPIT (TBX19 [T-box transcription factor 19]) immunohistochemistry is widely used to classify corticotroph tumors, yet unexpected nuclear staining has been observed in chordoma with certain commercial clones, raising concerns about diagnostic cross-reactivity. To investigate the molecular basis of cross-reactivity of the TPIT antibody clone OTI2G1 in chordoma and assess the potential diagnostic pitfall in tumors of the sellar region. We compared TBX19 and TBXT (Brachyury [T-box transcription factor T]) by T-box domain sequence alignment and structural modeling/superposition. Immunohistochemistry for TPIT (OTI2G1, CL6251) and Brachyury was performed on pituitary tissues, corticotroph tumors, and chordomas. Colocalization of OTI2G1 and Brachyury was quantified by multiplex immunofluorescence. TBX19 and Brachyury T-box domains showed marked structural similarity (Cα root mean square deviation, 0.071 Å), and the OTI2G1 immunogen (TBX19 amino acids [aa] 1-238) overlapped the Brachyury T-box. OTI2G1 showed diffuse nuclear staining in chordomas, whereas CL6251 showed negativity. Multiplex immunofluorescence demonstrated OTI2G1 signal in 77.6% of Brachyury-positive chordoma cells (95% CI, 77.5%-77.6%; P < .001). Diffuse nuclear "TPIT" staining with OTI2G1 in chordoma reflects cross-reactivity driven by conserved T-box structure rather than true TBX19 expression. For the differential diagnosis of sellar lesions, especially poorly differentiated chordoma versus pituitary neuroendocrine tumor, use of more specific clones (eg, CL6251) is recommended.