grade and glioma morphology remain two cornerstones of treatment guidelines and ongoing clinical trials [3, 4]. Glioma grading is based on the definition of few morphological features, including mitotic count, observation of increased cellularity with nuclear atypia, necrosis and microvascular proliferation. The diagnostic reproducibility of morphologic criteria for LGG is still influeced by a considerable inter-observer variability among pathologists [5, 6]. One of the major determinants of such variability concerns the proliferative activity, as WHO grading criteria does not define strict mitotic figure cut-offs to distinguish grade II from grade III tumors. Mitotic count is based on the observation of the number of mitoses per 10 high power fields (mitoses/10HPF) on standard Hematoxylin and Eosin (H&E) stained slides. The accuracy of this method can be influenced by both technical artifacts and experience of the pathologist performing the count. To overcome these inconveniences, phospho-histone-H3 (PHH3) and MIB1/Ki-67 have been proposed as alternative assays of cellular proliferation. PHH3 is a mitosis-specific antibody, which highlights the cell nucleus during the late-G2 and all mitotic stages, but negligibly at any other time (including apoptosis) [7, 8]. Conversely, Ki-67 is the most reliable marker of cell proliferation, which stains cells in all phases of cell cycle except G0. In LGG, Colman et al.  compared the prognostic significance of standard H&E-based mitotic count (number of mitoses per 10 HPF), PHH3- based mitotic index (number of mitoses per 1000 cells), and Ki-67 proliferation index (number of positive nuclei per 1000 cells), identifying prognostic cut-off for each method (≤3 versus > 3/10 HPF for H&E, ≤ 4 versus > 4/ 1000 cells for PHH3 and ≤ 9 versus > 9 for Ki-67) and proposing PHH3 as the most useful and clinical relevant proliferation marker. The same cut-off for PHH3 was recently  adopted by the same group to demonstrate a PHH3 prognostic role only in specific subsets defined by the presence or absence of IDH mutation.
Based on the univariate analysis results, three grades were designed for prediction of the biological behavior of PUC-Ta. The univariate analysis results for PUC-T1 revealed that only tumor stage influenced the biological behavior. Thus, once the tumor had invaded the lamina propria/submucosa, the histological parameters had an insignificant impact on the clinical outcome. Therefore, our new grading system was designed focusing on the prediction of PUC-Ta tumors. To design a new grading system with more objective and reproducible, yet sim- pler parameters, we chose mitotic level, mitotic count, capillary proliferation, and nuclear pleomorphism as important histological parameters, based on the univari- ate analysis. All four of these parameters not only had a statistically significant influence on both recurrence and progression of PUC-Ta, but were also quantifiable. Add- itionally, divergent histology was also selected as one of the parameters in our grading system; even though it showed an insignificant p -value in both recurrence and progression of PUC-Ta, it was statistically significant in terms of progression in PUC-T1 tumors.
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were defined as ANO1 positive. ANO1s were more frequently detected in unresectable patients. Tumor size, mitotic count and risk level were associated with ANO1 detection in resectable GIST patients. The presence of ANO1 significantly correlated with poor disease-free survival (15.3 versus 19.6 months, p = 0.038). Most patients turned ANO1-negative after surgery and inversely, all 21 patients with recurrence turned ANO1-positive with high ANO1 expression levels. Moreover, in the neoadjuvant setting, decline of ANO1 expression level correlated with the response of imatinib.
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In conclusion, to our knowledge, this is the first study on the RacGAP1 expression in GISTs as well as its association with clinicopathological prognostic factors, Ki-67 proliferation index and risk stratification. RacGAP1 positivity was significantly associated with tumor location, high mitotic count, high-degree cellularity and high Ki-67 proliferation index in univariate anal- ysis. In addition, multivariate analysis support- ed the relation of higher degree of cellularity and RacGAP1 overexpression in patients with GISTs. While RacGAP1 scores were significantly higher in high risk than in low risk groups, only one-third and half of patients with RacGAP1 overexpression were classified into the high- risk group based on AFIP and modified NIH cri- teria, respectively and approximately 10% were categorized into very-low risk group. However, great majority of patients with high Ki-67 prolif- eration index were categorized in the high risk group along with multivariate analysis confirm- ing the higher Ki-67 proliferation index and higher mitotic count as the determinants of high-risk category in AFIP and modified NIH risk stratification, respectively. Accordingly, albeit identified in a lower percentage of overall cases in our cohort when compared to RacGAP1 expression, high Ki-67 index seems to be supe- rior to RacGAP1 expression in terms of specific- ity for high risk status in GISTs. Hence, given the great heterogeneity and unpredictability of behavior of GISTs, the utility of RacGAP1 expression as a potential predictive marker in GISTs should be further explored and validated in larger cohorts of risk stratified patients with long-term follow-up data.
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cally significant (P=0.039; Figure 2). Here, the lower the Ki-67 expression, the longer the DFS. In addition, differ- ences in DFS among groups with different mitotic counts were statistically significant (P<0.01; Figure 3). It is noti- ced that the higher the mitotic count, more than 5/50 HPF, the poorer the DFS (P<0.01; Figure 3). Cox multivariate regression analysis demon- strated that the larger the tumor mitotic count, the high- er the risk of recurrence. This result suggested that mitotic count was an independent risk factor affecting progno- sis (P=0.003; Table 2). The life table method was used to calculate DFS among patients in the research at y1, y2 and y3, and values of 81.0%±6.1%, 59.2%±8.0%, and 47.3%±8.3% were obta- ined respectively.
Results: The patients included 52 men and 48 women. Their ages ranged from 27 to 82 years. Among the 85 patients who underwent curative resection, 44 (51.8%) developed disease recurrence (liver metastasis was the most common form of recurrence). The follow-up period ranged from 5 to 202 months (median: 33.2 months). The 1-, 3-, and 5-year DFS and OS rates were 85.2%, 53.8%, and 43.7%, and 91.5%, 66.6%, and 50.5%, respectively. Using multivariate analysis, it was found that high tumor cellularity, mitotic count >5/50 high-power field, and a Ki-67 index ⭌ 10% were three independent factors that were inversely associated with DFS. However, absence of tumor perforation, mitotic count < 5/50 high power field, and tumor with low cellularity were predictors of long-term favorable OS.
Altogether, there are many borderline malignant tumours within the spectrum of GISTs, ranging from indolent tumours to clearly malignant cancers. All GISTs should be considered as having some low malignant potential, and they should be described in terms of risk assessment, rather than using distinct benign and malignant categories.3 The only certain indication of malignancy is tumour spread beyond the organ of origin at the time of diagnosis.13 However, most primary GISTs larger than 5 cm in diameter and with a mitotic count higher than five per 50 HPF are likely to behave in a malignant way. Similarly, GISTs larger than 10 cm in diameter have a high risk of aggressive behaviour whatever the mitotic count, and GISTs of any size with a high mitotic count (more than ten per 50 HPF) are also deemed to be high-risk tumours.3
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exceptionally rare to non-existing [1,29]. Al- though the few published series pointed to a generally poor outcome in EGIST [29,30], the risk stratification of the “EGIST” has not been addressed separately. Notably, the same risk criteria as for their intramural and polypoid counterparts have been applied for EGIST. In a recent paper by Miettinen et al on a large series of omental GIST , 8% of the patients pre- sented with tumor rupture which is a higher per- centage compared to GIST in general. Most tu- mors in that study were large (>10 cm), but had a low mitotic count. Many of the patients with solitary tumors were long-term survivors (median, 129 months) indicating a generally indolent tumor behavior irrespective of the large tumor size. On the other hand, patients with multiple omental tumors had a poor median survival (8 months). Peritumoral fat invasion did not correlate with outcome in that study . A recent study on a small series of cases has shown a correlation between serosal penetra- tion and tumor progression . However, the methods used and the extent of the histopa- thological assessment of serosal penetration have not been described in details. In our ex- perience, the gross pattern in GIST, in particular the presence of no more than thin serosal cov- ering on the surface of extramural tumors repre- sents a significant predictor of peritoneal dis- ease recurrence, probably as a result of unde- tected microscopic serosal penetration (Agaimy et al, unpublished data). Thus it would be of prognostic relevance to carefully assess the presence or absence of normal tissue on the peritoneal aspect of tumors and to check for any evidence of serosal tears or old sealed focal rupture both grossly and histologically (Figure 1). The negative impact of tumor rupture and R1-resection on the disease-free survival has been confirmed in several studies . Notably, peritoneal dissemination represented the chief route of tumor spread in GIST in different series; disease recurrence involved the liver alone in 25%, the peritoneum alone in 33% and both in 28% in a large series published recently by Rut- kowsky et al .
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are the most important prognostic indicators of GISTs. However, they do not always reliably pre- dict patient outcomes. The clinical behavior of GIST varies, and some small and mitotically inactive GISTs show aggressive behaviors . A reliable method to predict the prognosis of GIST is necessary for clinical management. Alteration of cell-cycle regulatory proteins has been implicated in the pathogenesis and tumor progression of various kinds of human cancers. Loss of p16 expression has been reported to be associated with progression to malignant disease . However, p16 overexpression was found in some tumors, and it was associated with the aggressiveness of disease subtypes [6-8]. Although there have been extensive stud- ies of p16 expression in GISTs, discrepancies still exist with respect to its prognostic value . Loss of p16 expression has been previ- ously reported as a negative prognostic factor in GISTs. Schneider-Stock et al.  did not find any correlation between p16 gene alteration and clinicopathologic variables, but p16 loss was associated with a poor prognosis and p16 expression was higher in the benign GISTs. Huang et al.  also demonstrated that com- plete loss of p16 expression preferentially affected intermediate- and high-risk groups, and they suggested that p16 deregulation might be involved in early tumorigenesis. Several other studies have confirmed this cor- relation and its implication for poor prognosis [21-23]. However, Haller et al.  demonstrat- ed that loss of chromosomal region 9p21 led to reduced mRNA and p16 expression in GISTs. Steigen et al.  also showed that patients with p16-expressing GISTs had a significantly worse OS than those without p16 expression. In addition, p16-expressing GISTs tended to have a larger size and a higher mitotic count (> 5/50 HPF) compared with those not expressing Table 2. Univariate and multivariate analyses of clinicopathologic factors affecting the survival of patients with gastrointestinal stromal tumors
completely inhibited using relatively high concentrations of the drugs. As described above, inhibition of PLK1 induced a metaphase arrest (Figure 2; Supplementary Video 3). Inhibition of AURKA causes defects in centrosome separation and spindle formation, resulting in mitotic arrest and apoptosis . Both live-cell imaging (Figure 4A) and flow cytometry (Figure 4B) supported that inhibition of AURKA induced mitotic arrest after metaphase plate formation (Supplementary Video 4). Co-inhibition of PLK1 and AURKA (using lower concentrations of the drugs) also delayed mitosis after the metaphase plate was formed (Figure 4D; Supplementary Video 5). In agreement with this, conventional immunostaining analysis indicated that the percentage of mitotic cells containing bipolar spindle remained unchanged after co-inhibition of PLK1 and AURKA (data not shown).
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spindle formation, chromosome segregation, and/or mitotic exit, by targeting PLK1, Aurora kinases, or the APC/C. All these drugs can trigger cell death, the preferred outcome in clinical use. However, they demonstrated limited clinical suc- cess in acute leukemias, due to the unfavorable balance be- tween dose-effectiveness and toxicity and the development of some of them has been stopped. The reported adverse effects could be prevented by reducing the inhibitor dose, thus indi- cating the need to develop effective combinatorial strategies, as observed in the promising combination of the Aurora A inhibitor MLN8237 with chemotherapy. The same success can be achieved by the identification of the right patients, which may benefit of a low inhibitor dose due to a high sen- sitivity. This approach, based on the concept of gene-to-drug synthetic lethality, has proven highly effective in cancer. Re- garding mitotic inhibitors in acute leukemias, preclinical data suggest a very good response of complex karyotype AML to PLK1 inhibition  and of cohesion-mutated AML to disruption of APC/C CDC20 function . Moreover, most mitotic inhibitors show a SAC-independent and TP53-inde- pendent activity, which represents a major breakthrough in the field, being TP53 abnormalities an absolute marker of poor prognosis. Mitotic inhibitors also favor numerical as well as structural chromosomal instability (CIN) and/or tetraploidy-mediated aneuploidy in surviving cells, which are indicative of cancer evolution, and frequently associate with chemoresistance . This downside can provide a therapeutic window for synthetic lethal approaches, in which exposure to one drug greatly sensitizes to treatment with a second agent (drug-to-drug synthetic lethality). This
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Abstract : Histopathological images are widely utilized by the pathologists to determine the severity of the cancer. Usually, the pathologists analyse histopathological images manually, which demands more time and effort. The quality of image interpretation can be improvised with the help of a computerized assisting system for image analysis. This idea enhances the accuracy of the system as the computerized system helps in making decisions, which the pathologist may recheck. Understanding the benefits, this article presents a histopathological image classification scheme meant for breast tissues in detecting the mitotic cells. The goal is achieved by pre- processing the image by performing top and bottom hat transformation followed by stain normalization. The nuclei are then located with the help of Localized Active Contour Model (LACM) combined with Lion Optimization (LO) algorithm. The shape, statistical and texture features are extorted from the areas of interest and the differentiation of mitotic cells from the non- mitotic ones is performed by Support Vector Machine (SVM) classifier. The work efficiency is tested with respect to the standard performance measures such as accuracy, sensitivity, specificity and time consumption.
OSCC was until now, chiefly considered to be a disease affecting older individuals, with usage of tobacco being a major causative factor. However, there seems to be a change in the demographic trend, with OSCCs increasingly seen in younger individuals. This has led to increasing prevalence of ‘early-onset Squamous Cell Carcinoma (SCC) which may be arbitrarily defined as SCC occurring in individuals younger than 40 years of age . It is observed that there may be certain differences in the biological behaviour of tumours in younger adults. However, there is no known or proven explanation yet, attributable for these differences. In younger adults, OSCCs are sometimes seen to lack the typical association with tobacco and/or alcohol habit in addition to differences in the type and duration of habit. This raises the possibility of association of other etiological or risk factors such as viral infection and genetic susceptibility [1,2]. Differences in clinical behaviour are also observed in terms of recurrence, tendency for metastasis and survival rate. For the present analytical study, the null hypothesis was that there is no difference in clinicopathological characteristics of OSCC between individuals below and above 40 years of age. We compared two groups of individuals with OSCC categorized according to age as, below 40 and above 40 years. The objective was to assess differences in type of habit, histological features (grade of differentiation, mitotic index, and AgNOR count) and prognostic factors (lymph node metastasis and involvement of resected margins) between the two groups.
“Christ, Billy. This is money. I don’t give a damn about what floats your boat. For the most part it’s none of my business, but we got a good thing going here. We can win the pennant and you can go down in the record books as the best damn player in the last sixty years. Listen, Billy, you know what that could mean to this organization? I’m not here to judge you, son, but I am telling you this: I don’t care if you bat a goddamned thousand, we aren’t renewing your contract if you’re more interested in your goddam love life than you are in what’s best for this team, hell, not just for the team, but for all of baseball, and I wouldn’t count on any other franchise opening their arms for the game’s first openly gay player. I think you get my point.”
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The centromere is flanked on each side by constitutively condensed regions of chromatin known as pericentromeric heterochromatin (Sullivan, 2001). Rather than being nonfunctional blocks of silent condensed chromatin, these regions are in fact essential for centromere specification and contribute to sister chromatid cohesion and condensation during mitosis (Hendzel et al., 1997; Bernard et al., 2001). Pericentromeric regions are enriched with specific repressive epigenetic marks, including methylation of histone H3 at lysine 9 (H3K9me2 and H3K9me3) and histone H4 at lysine 20 (H4K20me3), and enrichment of heterochromatic factors including Heterochromatin Protein 1 (HP1, the homolog of yeast Swi6). Recruitment of HP1 to pericentromeric chromatin depends on the interaction of its chromodomain with the H3K9me3 modification, which is catalyzed by the histone methyltransferase enzyme Suv39h1 (Lachner et al., 2001). In turn, HP1 recruits the DNA methyltransferase 3b (DNMT3b), which methylates the cytosine (C) residues of cytosine-guanine (G) dinucleotides (CpG), leading to silencing and heterochromatinzation of pericentromeric DNA. These marks serve to maintain the condensed structure of the pericentric chromatin and maintain chromatid cohesion through recruitment of the cohesin protein complex (Nonaka et al., 2002; Amor et al., 2004). HP1 proteins are an integral component of condensed chromatin at pericentric regions, and loss of HP1 or Swi6 leads to loss of heterochromatin, centromeric instability and mitotic defects (Ekwall et al., 1996; Amor et al., 2004). Interestingly, HP1α has been shown to recruit ATRX to PCH in mESCs (Kourmouli et al., 2005), suggesting a specific function for ATRX at this genomic locus. Supporting this hypothesis, a growing number of chromatin remodeling enzymes have been found to maintain the structural integrity of PCH, with deficiencies leading to mitotic abnormalities (Lejeune et al., 2007; Bourgo et al., 2009).
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Classical anti-mitotic drugs have failed to translate their preclinical efficacy into clinical response in human trials. Their clinical failure has challenged the notion that tumor cells divide frequently at rates comparable to those of cancer cells in vitro and in xenograft models. Given the preponderance of interphase cells in clinical tumors, we asked whether targeting amplified centrosomes, which cancer cells carefully preserve in a tightly clustered conformation throughout interphase, presents a superior chemotherapeutic strategy that sabotages interphase- specific cellular activities such as migration. Herein we have utilized supercentrosomal N1E- 115 murine neuroblastoma cells as a test-bed to study interphase centrosome declustering induced by putative declustering agents such as Reduced-9-bromonoscapine (RedBr-Nos), Griseofulvin and PJ-34. We found tight “supercentrosomal” clusters in interphase and mitosis of ~80% of patients’ tumor cells with excess centrosomes. RedBr-Nos was the strongest declustering agent with a declustering index (DI) of 0.36, and completely dispersed interphase centrosome clusters in N1E-115 cells. Interphase centrosome declustering caused inhibition of neurite formation, impairment of cell polarization and Golgi organization, disrupted cellular protrusions and focal adhesion contacts - factors that are crucial pre-requisites for directional migration. Thus our data illustrate an interphase-specific potential anti-migratory role of
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knockdown , reduced progenitor division at the VS of Emx1-KO mice in vivo was observed: we found that the spatiotemporal regulation of the RGP cell cycle was affected in Emx1-KO mice (Figs. 6,7) and that an in- creased amount of RGPs failed to undergo INM. Inter- estingly however, the number of cells undergoing mitosis was not reduced: we found that RGPs still underwent mitosis, but at an ectopic position. This KO- phenotype is a novel finding and appears to be specific for BICD2 function in progenitor proliferation and dif- ferentiation . The changed morphology, chromatin con- densation and location of the nucleus and centrosome of sub-apical PH3+ progenitors in Emx1-KO mice hint at the presence of two distinct progenitor populations. One population of PH3+ progenitors retains a radial morph- ology but is impaired in mitotic progression, possibly be- cause the nucleus is stuck in the upper VZ and not able to reach the centrosome. In the second population of progenitors, in which the centrosome lies adjacent to the nucleus, mitotic progression is not impaired. This might be caused by a detachment from the VS, which is sup- ported by the more basal position of their nuclei and lack of radial morphology. Since we observe that centro- somes localized within apical processes of sub-apical mi- totic cells, it is possible that the centrosome migrates towards the nucleus when apical nuclear migration is impaired. This could cause a subsequent detachment from the VS, since centrioles at the VS are required for apical attachment by forming the basal body of the pri- mary cilium at the apical end feet [5, 22, 60].
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It consists of at least 11 core subunits and functions as an E3 ubiquitin ligase for targeting proteins via the 26S proteasome. APC/C promotes mitotic transitions through several key processes, which include the destruction of mitotic cyclins and inhibitors of chromosome separation as well as the regulation of DNA replication, centrosome dublication and mitotic spindle assembly (reviewed in (Pesin & Orr-Weaver, 2008)) APC/C activity is required for proper asymmetric localization of the adaptor protein Miranda and its associated cargo proteins Staufen, Prospero and Brat in Drosophila neuroblasts (Slack et al, 2007).
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Although protein phosphorylation plays a pivotal role in the regulation of cellular networks, many phosphorylation events remain undiscovered mainly due to technical limitations . The advent of mass spectrometry based proteomics along with developments in phosphopeptide enrichment methods has enabled large scale global phosphoproteomic studies [55; 82]. However, the number of phosphorylation sites identified on kinases is limited compared to other proteins due to their frequently low expression levels. In order to overcome this problem, small inhibitor based kinase enrichment strategies were developed resulting in the identification of more than 200 kinases from HeLa cell lysates [83; 84]. This method was also used recently to compare the phospho-kinomes during S-phase and M-phase of the cell cycle resulting in the identification of several hundreds of M-phase specific kinase phosphorylation sites . In the present study, we address the dynamics of the phospho- kinome during mitotic progression using large scale cell synchronization at three distinct mitotic stages, small inhibitor based kinase enrichment and SILAC based quantitative mass spectrometry. Thus we have determined the mitotic phosphorylation dynamics of more than 900 kinase phosphorylation sites, and identified distinctly regulated kinase interaction networks. Our results provide a valuable resource for the dynamics of the kinome during mitotic progression and give insight into the systems properties of kinase interaction networks.
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This work would not have been possible without the help and encouragement of many people. First, I would like to thank my advisor, Michael Lampson for giving me a chance to be part of his research group and to work in his lab. I thank him for being an excellent mentor, for knowing when to help me find the answers, and when to step back and let me figure them out on my own. Where my experience and knowledge were lacking, his were exhaustive; when I needed confidence in my work or myself, I could always count on his help and support. He taught me how to be a scientist, not just the skills and knowledge required to do research, but also how to be inquisitive and thorough. It has really been an honor and a pleasure working with him. I have also been extremely lucky to work closely with Richard Schultz. He has been an invaluable source in tackling the questions of my research, but also in planning my future career. I am extremely grateful for all that I was able to learn from him. I also thank my dissertation committee Mark Goulian, Tatyana Svitkina, Francis Luca and Greg Guild, for their
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