Previous investigations have linked decreased nuclear expression of the cell cycle inhibitor p27 with poor outcome in prostate cancer. However, these reports are inconsistent regarding the magnitude of that association and its independence from other predictors. Moreover, cytoplasmic translocation of p27 has been proposed as a negative prognostic sign. Given the cost and accuracy limitations of manual scoring, particularly of tissue microarrays (TMAs), we determined if laser-based fluorescence microscopy could provide automated analysis of p27 in both nuclear and cytoplasmic locations, and thus clarify its significance as a prognostic biomarker.
We constructed TMAs covering 202 recurrent cases (rising PSA) and 202 matched controls without recurrence. Quadruplicate tumor samples encompassed 5 slides and 1,616 cancer histospots. Cases and controls matched on age, Gleason grade, stage and hospital. We immunolabeled epithelial cytoplasm with Alexa647®; p27 with Alexa488®; and nuclei with DAPI. Slides were scanned on an iCys® laser scanning cytometer. Nuclear crowding required a stereological approach - random arrays of circles (phantoms) were layered on images and the content of each phantom analyzed in scatterplots.
Both nuclear and cytoplasmic p27 were significantly lower in cases vs. controls (P=0.014, P=0.004, respectively). Regression models controlling for matching variables plus PSA showed strong linear trends for increased risk of recurrence with lower p27 in both nucleus and cytoplasm (highest vs lowest quartile, OR=0.35, P=0.006). Manual scoring identified an inverse association between p27 expression and tumor grade, but no independent association with recurrence.
In conclusion, we developed an automated method for subcellular scoring of p27 without the need to segment individual cells. Our method identified a strong relationship, independent of tumor grade, stage and PSA, between p27 expression – regardless of subcellular location - and prostate cancer recurrence. This relationship was not observed with manual scoring.