title: Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer.
abstract: OBJECTIVE
  The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer.
  
  
  METHODS
  A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival.
  
  
  RESULTS
  The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001).
  
  
  CONCLUSIONS
  The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.
authors.affiliation: Department of Obstetrics and Gynecology and Section of Basic Reproductive Sciences, University of Colorado Denver and Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Lubna
authors.initials: L
authors.lastname: Qamar
authors.forename: Erin
authors.initials: E
authors.lastname: Deitsch
authors.forename: Aaron N
authors.initials: AN
authors.lastname: Patrick
authors.forename: Miriam D
authors.initials: MD
authors.lastname: Post
authors.forename: Monique A
authors.initials: MA
authors.lastname: Spillman
authors.forename: Ritsuko
authors.initials: R
authors.lastname: Iwanaga
authors.forename: Andrew
authors.initials: A
authors.lastname: Thorburn
authors.forename: Heide L
authors.initials: HL
authors.lastname: Ford
authors.forename: Kian
authors.initials: K
authors.lastname: Behbakht
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D005260:Female; D018398:Homeodomain Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016513:Mice, SCID; D010051:Ovarian Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011379:Prognosis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014183:Transplantation, Heterologous
chemical_list: D000912:Antibodies, Neoplasm; D018398:Homeodomain Proteins; D012333:RNA, Messenger; C099236:SIX1 protein, human