title: Identification and Validation of Differential Phosphorylation Sites of the Nuclear FOXL2 Protein as Potential Novel Biomarkers for Adult-Type Granulosa Cell Tumors.
abstract: Granulosa cell tumor (GCT) is a rare form of ovarian cancer classified as a sex cord-stromal tumor. The c.402C→G missense mutation in the FOXL2 gene that changes cysteine 134 to tryptophan (C134W) is found in more than 97% of adult-type GCTs, and the C134W FOXL2 mutant is hyperphosphorylated. We identified three differential phosphorylation sites, at serine 33 (S33), tyrosine 186 (Y186), and serine 238 (S238), of the C134W mutant by tandem mass spectrometry. Among these sites, antibodies were raised against the pS33 and pY186 epitopes using specific peptides, and they were tested by immunostaining tissue microarrays of archival adult-type GCT specimens, other tumors, and normal tissues. The pS33 antibody showed greater sensitivity and specificity for the detection of adult-type GCTs than that of the other phospho and nonphospho antibodies. The specificity of the pS33 antibody to the pS33 epitope was further confirmed by enriching the pS33 peptide by affinity chromatography using the immobilized antibody, followed by mass spectrometric and western blot analyses from whole cell lysates of the adult-type GCT cell line, KGN. pS33 FOXL2 immunostaining levels were significantly higher in adult-type GCTs than those in other tumors and tissues. The receiver operating characteristic curve analysis of pS33 FOXL2 showed high sensitivity (1.0) and specificity (0.76) to adult-type GCTs with a cutoff score of >30% positive cells, and the area under the curve was 0.96. This suggests the potential of pS33 FOXL2 to serve as a new biomarker for the diagnosis of adult-type GCT.
authors: †Department of Obstetrics and Gynecology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Korea.
authors: Dae-Shik
authors: DS
authors: Suh
authors: Hoon Kyu
authors: HK
authors: Oh
authors: Jae-Hong
authors: JH
authors: Kim
authors: Seeun
authors: S
authors: Park
authors: Eunkyoung
authors: E
authors: Shin
authors: Kangseok
authors: K
authors: Lee
authors: Yong-Hak
authors: YH
authors: Kim
authors: Jeehyeon
authors: J
authors: Bae
mesh_terms: D000328:Adult; D000906:Antibodies / Q000276:immunology; D015415:Biomarkers / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006106:Granulosa Cell Tumor / Q000378:metabolism*; D006801:Humans; D008297:Male; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D000906:Antibodies; D015415:Biomarkers; C421604:FOXL2 protein, human; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins
keywords: Diagnosis; FOXL2; granulosa cell tumor; phosphorylation; serine 33