Computer-based intensity measurement assists pathologists in scoring PTEN immunohistochemistry - Clinical associations in NSCLC patients of the ETOP Lungscape cohort

: INTRODUCTION PTEN loss is frequently observed in NSCLC and associated with both PI3K activation and tumoral immunosuppression. PTEN immunohistochemistry is a valuable read-out, but lacks standardized staining protocol and cut-off value. METHODS Following an external quality assessment (EQA) using SP218, 138G6 and 6H2.1 anti-PTEN antibodies, scored on webbook and tissue microarray, the ETOP cohort samples (n=2245 NSCLC patients, 8980 TMA cores) were stained with SP218. All cores were H-scored by pathologists and by computerized pixel-based intensity measurements calibrated by pathologists. RESULTS All 3 antibodies differentiated 6 PTEN+ versus 6 PTEN- cases on EQA. For 138G6 and SP218, high sensitivity and specificity was found for all H-score threshold values including prospectively defined 0, calculated 8 (pathologists) and calculated 5 (computer). High concordance among pathologists in setting computer-based intensities and between pathologists and computer in H-scoring was observed. Due to over-integration of the human eye, pixel-based computer H-scores were overall 54% lower. For all cut-off values, PTEN- was associated with smoking history, squamous cell histology and higher tumor stage (p<0.001). In adenocarcinomas, PTEN- was associated with poor survival. CONCLUSION Calibration of immunoreactivity intensities by pathologists, following computerized H-score measurements has the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multi-centre studies. ABSTRACT Introduction: Phosphatase and tensin homolog (PTEN) loss is frequently observed in NSCLC and associated with both phosphoinositide 3-kinase activation and tumoral immu-nosuppression. PTEN immunohistochemistry is a valuable readout, but lacks standardized staining protocol and cutoff value. Methods: After an external quality assessment using SP218, 138G6 and 6H2.1 anti-PTEN antibodies, scored on webbook and tissue microarray, the European Thoracic Oncology Platform cohort samples (n ¼ 2245 NSCLC patients, 8980 tissue microarray cores) were stained with SP218. All cores were H-scored by pathologists and by computerized pixel-based intensity measurements calibrated by pathologists. Results: All three antibodies differentiated six PTEN þ versus six PTEN- cases on external quality assessment. For 138G6 and SP218, high sensitivity and speci ﬁ city was found for all H-score threshold values including prospectively de ﬁ ned 0, calculated 8 (pathologists), and calculated 5 (computer). High concordance among pathologists in setting computer-based intensities and between pathologists and computer in H-scoring was observed. Because of over-integration of the human eye, pixel-based computer H-scores were overall 54% lower. For all cutoff values, PTEN- was associated with smoking history, squamous cell histology, and higher tumor stage ( p < 0.001). In adenocarcinomas, PTEN- was associated with poor survival. Conclusion: Calibration of immunoreactivity intensities by pathologists following computerized H-score measurements the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multicenter studies.