Optimal protocol for PTEN immunostaining; role of analytical and preanalytical variables in PTEN staining in normal and neoplastic endometrial, breast, and prostatic tissues.
In some tumors, phosphatase and tensin homolog (PTEN) inactivation may have prognostic importance and predictive value for targeted therapies. Immunohistochemistry (IHC) may be an effective method to demonstrate PTEN loss. It was claimed that PTEN IHC showed poor reproducibility, lack of standardization, and variable effects of preanalytical factors. In this study, we developed an optimal protocol for PTEN IHC, with clone 6H2.1, by checking the relevance of analytical variables in normal tissue and tumors of endometrium, breast, and prostate. Pattern and intensity of cellular staining and background nonspecific staining were quantified and subjected to statistical analysis by linear mixed models. The proposed protocol showed a statistically best performance (P < .05) and included a high target retrieval solution, 1:100 primary antibody dilution (2.925 mg/L), FLEX diluent, and EnVisionFLEX+ detection method, with a sensitivity and specificity of 72.33% and 78.57%, respectively. Staining specificity was confirmed in cell lines and animal models. Endometrial carcinomas with PTEN genetic abnormalities showed statistically lower staining than tumors without alterations (mean histoscores, 34.66 and 119.28, respectively; P = .01). Controlled preanalytical factors (delayed fixation and overfixation) did not show any statistically significant effect on staining with optimal protocol (P > .001). However, there was a trend of significance for decreased staining and fixation under high temperature. Moreover, staining was better in endometrial aspirates than in matched hysterectomy specimens, subjected to less controlled preanalytical variables (mean histoscores, 80 and 40, respectively; P = .002). A scoring system combining intensity of staining and percentage of positive cells was statistically associated with PTEN alterations (P = .01).
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Oscar
O
Maiques
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Maria
M
Santacana
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Joan
J
Valls
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Judit
J
Pallares
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Cristina
C
Mirantes
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Sónia
S
Gatius
Department of Obstetric and Gynecology, University Hospitals Gasthuisberg, Leuven 3000, Belgium.
Diego Andrés
DA
García Dios
Department of Obstetric and Gynecology, University Hospitals Gasthuisberg, Leuven 3000, Belgium.
Frederic
F
Amant
Research and Development Dako, DK-2600 Glostrup, Denmark.
Hans Christian
HC
Pedersen
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain.
Xavier
X
Dolcet
Department of Pathology and Molecular Genetics/Oncologic Pathology group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida 25198, Spain. Electronic address: fjmatiasguiu.lleida.ics@gencat.cat.
Xavier
X
Matias-Guiu
D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D001940:Breast / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D016889:Endometrial Neoplasms / Q000378:metabolism / Q000473:pathology; D004717:Endometrium / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051059:PTEN Phosphohydrolase / Q000032:analysis* / Q000378:metabolism; D011467:Prostate / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results
D014408:Biomarkers, Tumor; D051059:PTEN Phosphohydrolase
Immunohistochemistry; PTEN; Preanalytical variables; Protocol