The prognostic abilities of breast cancer gene expression signatures are due mostly to the detection of proliferation activity. One of the strongest, yet simple and well-reproducible proliferation-associated prognostic factors is the mitotic activity index (MAI). However: a) counting mitotic figures is regarded by many histopathologists as cumbersome and time-consuming, and b) most available immunohistochemical markers are much weaker predictors than the MAI. We have investigated the spatio-temporal sub-cellular distribution of the Serine 2481-autophosphorylated form of mTOR (PP-mTORSer2481) during the G1/S-to-M-phase transition both in cultured cancer cells and in cancer tissue specimens. Using a high-resolution, automated confocal high-content imaging system, we observed that mitotic cells notably accumulated a distinct pattern of nuclear and cytoplasmic immunolabelings of PP-mTORSer2481. Parallel experiments examining site-specific phosphorylation (i.e., Serine 10 and Serine 28) of the G2/M marker Histone H3 (PP-H3) revealed that PP-H3Ser10/Ser28 staining efficiently detected mitotic cells from prophase until the beginning of anaphase, but not during late anaphase, telophase and cytokinesis. PP-mTORSer2481 staining associated near and between separating chromosomes not only during early mitotic stages but also to the midzone and to midbody at ana/telophase through cytokinesis. We then evaluated the usefulness of PP-mTORSer2481 immunostaining for improving the efficiency of mitotic counting using. Anti-PP-mTORSer2481-labeled mitotic figures (MFs) were easily seen and permitted a quick identification of mitotic hotspots in formalin-fixed cancer tissues, even at low magnification. Importantly, average mitotic counts were significantly higher when using PP-mTORSer2481 staining than with the hematoxylin and eosin (H&E) protocol in breast cancer core biopsies. Mitotic count based on PP-mTORSer2481 immunostaining increased tumor grade by one grade in 2 of 9 breast carcinomas. These findings warrant forthcoming studies to confirm both the accuracy and the prognostic value of PP-mTORSer2481 as a novel high-contrast immunohistochemical mitosis marker in larger populations of human breast carcinomas.

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