Abstract.Pioneering clinical studies in de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab have suggested that HER2 gene-amplification can take place also in a basal-like molecular background to generate basal/HER2 + tumors intrinsically resistant to trastuzumab. Here, we first investigated the unique histogenesis of the basal/HER2 + phenotype in breast carcinomas. The presence of basal CK5/CK6 cytokeratin expression in HER2 + tumors revealed a significant overlap in the histological features of HER2 + /CK5/6 + and basal-like breast carcinomas. Basal/ HER2 + tumors were typically poorly differentiated, highgrade invasive ductal carcinomas with large geographic necrosis, pushing margins of invasion, syncytial arrangement of tumor cells, ribbon-or festoon-like architecture, squamous metaplasia, stromal lymphocytic infiltrates, high mitotic index and strong p53 positivity. Secondly, we performed low-scale proteomic approaches in JIMT-1 cells, a unique model of HER2-gene amplified trastuzumab-resistant breast carcinoma with a basal-like phenotype, to develop biomarker signatures that may differentiate trastuzumab-responsive from non-responsive tumors. When applying antibody-based array technology to the extracellular milieu of trastuzumabrefractory JIMT-1 and trastuzumab-sensitive SKBR3 cell cultures, JIMT-1 cells were found to secrete higher amounts of several growth factors including amphiregulin, EGF, IGFBP-6, PDGF-AA, neurotrophins, TGFß and VEGF. Semiquantitative signaling node multi-target sandwich ELISAs revealed that JIMT-1 cells drastically overactivate RelA, the prosurvival subunit of NF-κB as compared to trastuzumabsensitive luminal/HER2 + SKBR3 cells. When simultaneously assessing the activation status of 42 receptor tyrosine kinases (RTK) using a human phospho-RTK array, JIMT-1 cells were found to constitutively display hyperactivation of the insulin-like growth factor-I receptor (IGF-1R). High-content immunofluorescence imaging revealed that activated IGF-1R mainly localized at focal adhesion-like structures in JIMT-1 cells. In vitro wound healing assays suggested that this functional reorganization of the JIMT-1 cytoskeletal reorganization may account for an exacerbated trastuzumab-refractory 'migratogenic' phenotype. Forthcoming studies should validate the notion that identification of basal-like immunophenotypes and/or basal-like molecular signatures within HER2 + breast carcinomas may provide rapid means to define subgroups of breast cancer patients likely to display resistance to trastuzumab ab initio.
Abstract. 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-mTOR Ser2481 ) during the G 1 /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-mTOR Ser2481 . Parallel experiments examining site-specific phosphorylation (i.e., Serine 10 and Serine 28) of the G 2 /M marker Histone H3 (PP-H3) revealed that PP-H3 Ser10/Ser28 staining efficiently detected mitotic cells from prophase until the beginning of anaphase, but not during late anaphase, telophase and cytokinesis. PP-mTOR Ser2481 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-mTOR Ser2481 immunostaining for improving the efficiency of mitotic counting using. Anti-PPmTOR Ser2481 -labeled mitotic figures (MFs) were easily seen and permitted a quick identification of mitotic hotspots in formalinfixed cancer tissues, even at low magnification. Importantly, average mitotic counts were significantly higher when using PP-mTOR Ser2481 staining than with the hematoxylin and eosin (H&E) protocol in breast cancer core biopsies. Mitotic count based on PP-mTOR Ser2481 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-mTOR Ser2481 as a novel high-contrast immunohistochemical mitosis marker in larger populations of human breast carcinomas.
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