title: A novel double staining strategy for improved detection of testicular carcinoma in situ cells in human semen samples.
abstract: Prompted by the recently reported expression of POU5F1 (OCT3/4) in epididymis, a panel of markers for carcinoma in situ (CIS) testis and testicular germ cell tumours (TGCT), including AP-2γ(TFAP2C), NANOG, OCT3/4, KIT, placental-like alkaline phosphatase (PLAP), M2A/PDPN and MAGE-A4 were examined by immunohistochemistry or in situ hybridisation in urogenital epithelia, which may interfere with detection of CIS cells in semen. In addition to OCT3/4, the expression of AP-2γ and NANOG or their variants was detected in urogenital epithelia, while other CIS markers, including PLAP/alkaline phosphatase were absent. A combination of immunocytological staining for AP-2γ or OCT3/4 and rapid cytochemical alkaline phosphatase reaction was subsequently developed. This approach was tested in 22 patients with TGCT. In 14 patients (63.6%), double stained cells were found and thus the method was proven suitable for the detection of CIS cells in semen. In conclusion, transcription factors related to pluripotency and undifferentiated state of cells, which most likely have several variants or modifications, are unexpectedly detected using currently available antibodies in urogenital epithelial cells which may be shed into semen. Combining the immunohistochemical nuclear markers with a rapid cytochemical alkaline phosphatase reaction for detection of CIS cells in ejaculates may provide a more reliable diagnostic method.
authors.affiliation: University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark. john.erik.nielsen@rh.regionh.dk
authors.forename: J E
authors.initials: JE
authors.lastname: Nielsen
authors.forename: D M
authors.initials: DM
authors.lastname: Kristensen
authors.forename: K
authors.initials: K
authors.lastname: Almstrup
authors.forename: A
authors.initials: A
authors.lastname: Jørgensen
authors.forename: I A
authors.initials: IA
authors.lastname: Olesen
authors.forename: G K
authors.initials: GK
authors.lastname: Jacobsen
authors.forename: T
authors.initials: T
authors.lastname: Horn
authors.forename: N E
authors.initials: NE
authors.lastname: Skakkebaek
authors.forename: H
authors.initials: H
authors.lastname: Leffers
authors.forename: E
authors.initials: E
authors.lastname: Rajpert-De Meyts
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D014408:Biomarkers, Tumor / Q000032:analysis*; D002278:Carcinoma in Situ / Q000175:diagnosis*; D018398:Homeodomain Proteins / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis; D008297:Male; D000071317:Nanog Homeobox Protein; D009373:Neoplasms, Germ Cell and Embryonal / Q000175:diagnosis*; D050814:Octamer Transcription Factor-3 / Q000032:analysis; D012661:Semen / Q000737:chemistry* / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*; D013736:Testicular Neoplasms / Q000175:diagnosis*; D013737:Testis / Q000201:enzymology; D050656:Transcription Factor AP-2 / Q000032:analysis*
chemical_list: D014408:Biomarkers, Tumor; D018398:Homeodomain Proteins; D007527:Isoenzymes; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C493679:TFAP2C protein, human; D050656:Transcription Factor AP-2; C055791:germ-cell AP isoenzyme; D000469:Alkaline Phosphatase