Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients.Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR ¼ 2.914 (confidence interval 0.9286-9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stemlike biology have undergone a widespread epithelial-mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed.Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer.
There has been an exponential growth in the application of AI in health and in pathology. This is resulting in the innovation of deep learning technologies that are specifically aimed at cellular imaging and practical applications that could transform diagnostic pathology. This paper reviews the different approaches to deep learning in pathology, the public grand challenges that have driven this innovation and a range of emerging applications in pathology. The translation of AI into clinical practice will require applications to be embedded seamlessly within digital pathology workflows, driving an integrated approach to diagnostics and providing pathologists with new tools that accelerate workflow and improve diagnostic consistency and reduce errors. The clearance of digital pathology for primary diagnosis in the US by some manufacturers provides the platform on which to deliver practical AI. AI and computational pathology will continue to mature as researchers, clinicians, industry, regulatory organizations and patient advocacy groups work together to innovate and deliver new technologies to health care providers: technologies which are better, faster, cheaper, more precise, and safe.
Stromal-derived intratumoural heterogeneity (ITH) has been shown to undermine molecular stratification of patients into appropriate prognostic/predictive subgroups. Here, using several clinically relevant colorectal cancer (CRC) gene expression signatures, we assessed the susceptibility of these signatures to the confounding effects of ITH using gene expression microarray data obtained from multiple tumour regions of a cohort of 24 patients, including central tumour, the tumour invasive front and lymph node metastasis. Sample clustering alongside correlative assessment revealed variation in the ability of each signature to cluster samples according to patient-of-origin rather than region-of-origin within the multi-region dataset. Signatures focused on cancer-cell intrinsic gene expression were found to produce more clinically useful, patient-centred classifiers, as exemplified by the CRC intrinsic signature (CRIS), which robustly clustered samples by patient-of-origin rather than region-of-origin. These findings highlight the potential of cancer-cell intrinsic signatures to reliably stratify CRC patients by minimising the confounding effects of stromal-derived ITH.
Cytoskeletal remodeling events are tightly regulated by signal transduction systems that impinge on adhesion components and modulators of cellular architecture. We have previously shown that the Ste20-like kinase (SLK) can induce apoptosis through the induction of actin disassembly and cellular retraction (Sabourin, L. A., Tamai, K., Seale, P., Wagner, J., and Rudnicki, M. A. (2000) Mol. Cell. Biol. 20, 684 -696). Using immunofluorescence studies, we report that SLK is redistributed with adhesion components at large podosome-like adhesion sites in fibronectin-stimulated fibroblasts. However, in vitro kinase assays demonstrate that its activity is not modulated following fibronectin stimulation. Double immunofluorescence studies in exponentially growing or spreading fibroblasts show that SLK is associated with the microtubule network and can be coprecipitated with ␣-tubulin. Furthermore, the stimulation of adhesion site formation by microtubule-disrupting agents induces the relocalization of SLK with unpolymerized ␣-tubulin to large vinculin-containing adhesion complexes. Using microinjection studies, we show that ectopic expression of activated SLK induces the disassembly of actin stress fibers, a process that can be inhibited by dominant negative Rac1. Significantly, endogenous SLK can be colocalized with Rac1 in spreading cells on FN. Finally, the overexpression of SLK by adenoviral infection inhibits cell spreading on fibronectin. These results suggest that SLK is part of a microtubuleassociated complex that is targeted to adhesion sites and implicated in the regulation of cytoskeletal dynamics.
We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G 2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G 2 upstream of H1 kinase activation.Cell cycle progression is monitored through kinase-mediated signal transduction and the binding of various cyclin proteins to their respective cyclin-dependent kinases (Cdks (2, 3)). The activity of a cyclin/Cdk complex is regulated by cycles of expression and destruction of the cyclin subunit (reviewed in Ref. 4). G 1 progression is regulated, in part, by cyclins D and E and their respective cyclin-dependent kinases in a complex pathway that results in the retinoblastoma protein phosphorylation, and consequently, the production of cyclin A, leading to S phase entry (reviewed in Ref. 5). Cyclin B synthesis initiates at the end of S phase (6, 7) and forms a complex with p34 cdc2/cdk1 . This complex has been termed MPF 3 (maturation promoting factor or mitosis promoting factor) and is required for mitotic entry (reviewed in Ref. 8). During interphase, cytosolic MPF is kept inactive by inhibitory phosphorylation of cdc2 on Thr-14 and Tyr-15 by Myt1 and Wee1, respectively (9 -11). Activation of this complex is triggered by the Cdc25C phosphatase through cdc2 dephosphorylation of . Following dephosphorylation of these residues, MPF is believed to phosphorylate and further activate Cdc25C, resulting in full activation of MPF through an autocatalytic feedback loop (15,16). This results in the translocation of MPF from the cytoplasm to the nucleus at the beginning of mitosis (17), where it phosphorylates histone H1 (18) and induces changes in the microtubule network (19) and actin filaments (20).In Xenopus, polo-like kinase (Plx1) has been shown to phosphorylate and activate Cdc25 (21), and polo-like kinase kinase (xPlkk1) has been shown to be a direct activator of Plx1 (22). However, this may be an o...
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