title: In depth evaluation of the prognostic and predictive utility of PTEN immunohistochemistry in colorectal carcinomas: performance of three antibodies with emphasis on intracellular and intratumoral heterogeneity.
abstract: BACKGROUND
  Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) loss of function is frequently detected in advanced colorectal cancer. Its detection is thought to have prognostic significance and it is being considered to predict responsiveness to anti-EGFR therapy. Unfortunately, while immunohistochemical assessment of PTEN expression is widespread, it lacks standardization and the results are hardly comparable across the available publications.
  
  
  METHODS
  Retrospectively collected, formalin-fixed and paraffin-embedded colorectal tumor tissue samples from 55 patients were combined into tissue microarray (TMA) blocks. We used three different PTEN antibodies to determine the frequency, intensity and intracellular pattern of PTEN immunohistochemical labeling: Neomarkers, Dako and CellSignaling. We evaluated the aforementioned parameters in selected regions of colorectal cancers and in their lymph node metastases by using three scoring methods that take into consideration both staining frequency and intensity (H1-H3-score). We also evaluated intracellular localization.
  
  
  RESULTS
  The Dako and CellSignaling antibodies stained predominantly cytoplasms, while the Neomarkers antibody specifically stained cell nuclei. PTEN H-scores were significantly lower in all tumor areas as compared to the normal colonic mucosa based on staining with the DAKO and CellSignaling antibodies. Intratumoral regional differences or differences between matching tumors and metastases were not detected with any of the antibodies. Neither Dako, neither CellSignaling, nor the Neomarkers antibodies revealed a significant correlation between PTEN expression and pT, Dukes/MAC and clinical stage. KRAS status, histological grade correlated with PTEN H-scores based on staining with the Neomarkers antibody. PTEN H-scores did not correlate with MMR status. PTEN H-scores did not show any correlation with relapse-free survival based on staining with either antibody.
  
  
  CONCLUSIONS
  While PTEN expression decreased in colorectal cancer according to two antibodies, neither of the three applied PTEN antibodies could justify significant correlation with clinicopathological data, nor had prognostic value. Thus, we might conclude that immunohistochemical PTEN investigation remains a challenge requiring more standardized evaluation on larger number of cases to clarify its utility as a prognostic and predictive tool in CRC. The standardization of immunohistochemical method is key in the evaluation process, which is further discussed.
authors.affiliation: Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary.
authors.forename: Emese Irma
authors.initials: EI
authors.lastname: Ágoston
authors.affiliation: Department of Pathology and Experimental Cancer Research, Semmelweis University, 26 Üllői út, Budapest, 1085, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Micsik
authors.affiliation: Department of Pathology, Semmelweis University, 93 Üllői út, Budapest, 1091, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Ács
authors.affiliation: Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Fekete
authors.affiliation: Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary.
authors.forename: Oszkár
authors.initials: O
authors.lastname: Hahn
authors.affiliation: Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Baranyai
authors.affiliation: Department of Surgery and Oncological Surgery, Uzsoki Teaching Hospital, 196 Róna utca, Budapest, 1145, Hungary.
authors.forename: Kristóf
authors.initials: K
authors.lastname: Dede
authors.affiliation: Department of Oncology, Szent István Hospital, 1 Nagyvárad tér, Budapest, 1097, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Bodoky
authors.affiliation: Department of Surgery and Oncological Surgery, Uzsoki Teaching Hospital, 196 Róna utca, Budapest, 1145, Hungary.
authors.forename: Attila
authors.initials: A
authors.lastname: Bursics
authors.affiliation: Department of Pathology, Semmelweis University, 93 Üllői út, Budapest, 1091, Hungary.
authors.forename: Janina
authors.initials: J
authors.lastname: Kulka
authors.affiliation: Department of Pathology and Experimental Cancer Research, Semmelweis University, 26 Üllői út, Budapest, 1085, Hungary.
authors.forename: Tibor
authors.initials: T
authors.lastname: Krenács
authors.affiliation: MTA-TTK Lendület Cancer Biomarker Research Group, Magyar tudósok körútja 2, Budapest, 1117, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Győrffy
authors.affiliation: Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary. hl@seb1.sote.hu.
authors.forename: László
authors.initials: L
authors.lastname: Harsányi
authors.affiliation: Department of Pathology, Semmelweis University, 93 Üllői út, Budapest, 1091, Hungary. cac@korb2.sote.hu.|MTA-TTK Lendület Cancer Biomarker Research Group, Magyar tudósok körútja 2, Budapest, 1117, Hungary. cac@korb2.sote.hu.
authors.forename: A Marcell
authors.initials: AM
authors.lastname: Szász
mesh_terms: D000368:Aged; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor / Q000378:metabolism*; D003106:Colon / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D011237:Predictive Value of Tests; D011379:Prognosis; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012189:Retrospective Studies; D046888:Tissue Array Analysis
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Colorectal cancer; Immunohistochemistry; Intratumoral heterogeneity; PTEN; Predictive marker; Prognostic marker; Protein expression