Evaluation of specific lymphocyte subsets is important in understanding the microenvironment in cancer and holds promise as a prognostic parameter in invasive breast cancer. To address this, we used digital image analysis to integrate cell abundance, distance metrics, neighbourhood relationships and sample heterogeneity into comprehensive assessment of immune infiltrates. Lymphocyte and macrophage subpopulations were
The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 cases with breast cancer, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.Robust assessment of kinase phosphorylation in tumor biopsy samples is necessary to understand how mutations translate into aberrant oncogenic signaling, and it has been successfully used as pharmacodynamic marker to monitor the effect of therapeutic inhibition of cancer-related pathways.
Erythropoietin (Epo) plays a dual role as an erythropoiesis-stimulating hormone and a locally produced cytoprotectant in various vertebrate tissues. Splice variants and engineered derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis suggest that alternative receptors, different from the 'classical' homodimeric receptor involved in haematopoiesis, mediate neuroprotective Epo functions. Previous studies on grasshoppers demonstrated neuroprotective and neuroregenerative effects of Epo that involved similar transduction pathways as in mammals. To advance the characterization of yet unidentified neuroprotective Epo receptors, we studied the neuroprotective potency of the human non-erythropoietic Epo splice variant EV-3 in primary cultured locust brain neurons. We demonstrate that EV-3, like Epo, protects locust neurons from hypoxia-induced apoptotic death through activation of the Janus kinase/signal transducer and activator of transcription transduction pathway. Using the fluorescent dye FM1-43 to quantify endocytotic activity we show that both Epo and EV-3 increase the number of fluorescently labelled endocytotic vesicles. This reveals that binding of Epo to its neuroprotective receptor induces endocytosis, as it has been described for the mammalian homodimeric Epo-receptor expressed by erythroid progenitors. Reduction in Epo-stimulated endocytotic activity following pre-exposure to EV-3 indicated that both Epo and its splice variant bind to the same receptor on locust neurons. The shared neuroprotective potency of Epo and EV-3 in insect and mammalian neurons, in the absence of erythropoietic effects of EV-3 in mammals, suggests a greater similarity of the unidentified nervous Epo receptors (or receptor complexes) across phyla than between mammalian haematopoietic and neuroprotective receptors. Insects may serve as suitable models to evaluate the specific protective mechanisms mediated by Epo and its variants in non-erythropoietic mammalian tissues.
Generation of multiple mRNAs by alternative splicing is well known in the group of cytokines and has recently been reported for the human erythropoietin (EPO) gene. Here, we focus on the alternatively spliced EPO transcript characterized by deletion of exon 3 (hEPOΔ3). We show co-regulation of EPO and hEPOΔ3 in human diseased tissue. The expression of hEPOΔ3 in various human samples was low under normal conditions, and distinctly increased in pathological states. Concomitant up-regulation of hEPOΔ3 and EPO in response to hypoxic conditions was also observed in HepG2 cell cultures. Using LC-ESI-MS/MS, we provide first evidence for the existence of hEPOΔ3 derived protein EV-3 in human serum from healthy donors. Contrary to EPO, recombinant EV-3 did not promote early erythroid progenitors in cultures of human CD34+ haematopoietic stem cells. Repeated intraperitoneal administration of EV-3 in mice did not affect the haematocrit. Similar to EPO, EV-3 acted anti-apoptotic in rat hippocampal neurons exposed to oxygen-glucose deprivation. Employing the touch-screen paradigm of long-term visual discrimination learning, we obtained first in vivo evidence of beneficial effects of EV-3 on cognition. This is the first report on the presence of a naturally occurring EPO protein isoform in human serum sharing non-erythropoietic functions with EPO.
A multitude of cancer-associated genomic aberrations converge into activation of a limited repertoire of oncogenic ‘core’ pathways. Assessment of the activation status of such key signaling cascades is necessary to characterize pathway dependency of tumors, to predict response to specific pathway inhibitors, and to track drug effects in pharmacodynamic studies. We established a duplex immunohistochemistry (IHC) tool for the combined assessment of two key pathways involved in breast cancer biology, PI3K/AKT and MAPK. Validation of the duplex IHC assay was performed by tracking activation kinetics of phospho-AKT and phospho-MAPK in Heregulin (HRG-≥1) stimulated cultured MDA-MB453 cells by Western Blotting and immunostaining of immediately paraformaldehyde-fixed cells. In addition, one-sided inhibition of PI3K/AKT and MAPK signaling cascades was performed using the MEK1-inhibitor PD98059 and the PI3K inhibitor LY294002. Xenograft tumors were used to define the acceptable cold ischemia time for complex tissues. Consistently, we found a narrow time window of <10 minutes processing time (cold ischemia), in which AKT and MAPK phosphorylation could be accurately detected, where the pMAPK assay was more susceptible to time-dependent decrease in signal than the pAKT assay. In line with this, breast cancer samples obtained from immediately fixed fine needle punch biopsies (n=19) consistently showed strong staining for pMAPK and pAKT, while signals clearly decreased in the corresponding routinely processed surgical resection specimens. Knowing the appropriate processing conditions for multiplexed assessment, we studied pathway activation patterns in a series of invasive breast cancer (n=37) of commercially acquired samples, for which the provider (Indivumed, Hamburg, Germany) guarantees a cold ischemia time <10 minutes and highly standardized, tightly controlled embedding process. 1/37 cases clearly showed predominant AKT activation, 2/37 cases had predominant MAPK activation, and 8/37 cases exhibited concomitant activation of both pathways throughout the tumor, indicating constitutive pathway activity. The remaining 26 cases showed a striking heterogeneity of pathway activation patterns, some with marked pMAPK signals at the interface of tumor to stromal/inflammatory cells, suggesting pathway activation in response to microenvironment interaction. Consistently, activated macrophages showed a strong pMAPK signal. The predominantly pAKT activated case was negative for PTEN by IHC and qRT-PCR. The pMAPK activated cases were ER/PR positive and HER2 negative. In conclusion, duplex IHC for concomitant detection of pAKT and pMAPK can be robustly applied to immediately processed breast cancer fine needle biopsies, whereas the use in surgical resection specimens is likely to be inaccurate unless the material has been processed under tightly controlled conditions. The observed staining patterns suggest distinctive activation patterns pointing to genomic aberrations causing constitutive pathway activation. As tumor heterogeneity and inflammatory cells are likely to influence any pathway analysis on homogenized human breast cancer tissue, we recommend to consider morphological assessment to localize pathway activation to relevant cell populations. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-04.
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