Cancers Of Epithelial Origin Research Articles

Preclinical Evaluation of 111In-Labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-Positive Cancer Models.

Epidermal growth factor receptor I (EGFR) is overexpressed in most cancers of epithelial origin. Antibody drug conjugates (ADCs) with PEGylated-maytansine (PEG-DM1) show promise in vitro and in vivo. However, in vivo biodistribution data for ADCs with PEG-DM1 have not been reported. Development of methods to understand the real-time in vivo behavior of these ADCs is needed to move these compounds to the clinic. Methods: Here we have used noninvasive small-animal SPECT/CT imaging and ex vivo biodistribution to understand the in vivo behavior of PEG6-DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG6-DM1. We generated immunoconjugates with low (nimotuzumab-PEG6-DM1-Low) and high (nimotuzumab-PEG6-DM1-High) drug-to-antibody ratios. The drug-to-antibody of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High was 3.5 and 7.3, respectively. Quality control was performed using ultraviolet spectrophotometry, size-exclusion high-performance liquid chromatography, bioanalyzer, biolayer interferometry, and flow cytometry in EGFR-positive DLD-1 cells. These immunoconjugates were conjugated with DOTA and radiolabeled with 111In. The in vitro binding and internalization rates of 111In-nimotuzumab, 111In-nimotuzumab-PEG6-DM1-Low, and 111In-nimotuzumab-PEG6-DM1-High were characterized. Furthermore, the pharmacokinetics, biodistribution, and imaging characteristics were evaluated in normal and DLD-1 tumor-bearing mice. Results: Flow cytometry and biolayer interferometry showed a trend toward decreasing EGFR affinity with increasing number of PEG6-DM1 on the antibody. Despite the lower overall cellular binding of the PEG6-DM1 radioimmunoconjugates, internalization was higher for PEG6-DM1 ADCs than for the non-PEGylated ADC in the following order: 111In-nimotuzumab-PEG6-DM1-High > 111In-nimotuzumab-PEG6-DM1-Low > 111In-nimotuzumab. Nuclear uptake of 111In-nimotuzumab-PEG6-DM1-High was 4.4-fold higher than 111In-nimotuzumab. Pharmacokinetics and biodistribution showed that 111In-nimotuzumab-PEG6-DM1-High had the slowest blood and whole-body clearance rate. Uptake in DLD-1 tumors of 111In-nimotuzumab was similar to 111In-nimotuzumab-PEG6-DM1-Low but was significantly higher than for 111In-nimotuzumab-PEG6-DM1-High. Tumor-to-background ratios for 111In-nimotuzumab and 111In-nimotuzumab-PEG6-DM1-Low were higher than for 111In-nimotuzumab-PEG6-DM1-High. Conclusion: The results show that conjugation of multiple PEG6-DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG6-DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of 111In-nimotuzumab-PEG6-DM1-High, with a slow overall whole-body clearance rate. These data provide insights for evaluating the pharmacokinetics and normal -tissue toxicity and in determining dosing rate of PEGylated ADCs.

Conditional ablation of p130Cas/BCAR1 adaptor protein impairs epidermal homeostasis by altering cell adhesion and differentiation

Backgroundp130 Crk-associated substrate (p130CAS; also known as BCAR1) is a scaffold protein that modulates many essential cellular processes such as cell adhesion, proliferation, survival, cell migration, and intracellular signaling. p130Cas has been shown to be highly expressed in a variety of human cancers of epithelial origin. However, few data are available regarding the role of p130Cas during normal epithelial development and homeostasis.MethodsTo this end, we have generated a genetically modified mouse in which p130Cas protein was specifically ablated in the epidermal tissue.ResultsBy using this murine model, we show that p130Cas loss results in increased cell proliferation and reduction of cell adhesion to extracellular matrix. In addition, epidermal deletion of p130Cas protein leads to premature expression of “late” epidermal differentiation markers, altered membrane E-cadherin/catenin proteins localization and aberrant tyrosine phosphorylation of E-cadherin/catenin complexes. Interestingly, these alterations in adhesive properties in absence of p130Cas correlate with abnormalities in progenitor cells balance resulting in the amplification of a more committed cell population.ConclusionAltogether, these results provide evidence that p130Cas is an important regulator of epidermal cell fate and homeostasis.

PAR1 signaling on tumor cells limits tumor growth by maintaining a mesenchymal phenotype in pancreatic cancer

Protease activated receptor-1 (PAR1) expression is associated with disease progression and overall survival in a variety of cancers. However, the importance of tumor cell PAR1 in pancreatic ductal adenocarcinomas (PDAC) remains unexplored. Utilizing orthotopic models with wild type and PAR1-targeted PDAC cells, we show that tumor cell PAR1 negatively affects PDAC growth, yet promotes metastasis. Mechanistically, we show that tumor cell-specific PAR1 expression correlates with mesenchymal signatures in PDAC and that PAR1 is linked to the maintenance of a partial mesenchymal cell state. Indeed, loss of PAR1 expression results in well-differentiated pancreatic tumors in vivo, with enhanced epithelial characteristics both in vitro and in vivo. Taken together, we have identified a novel growth inhibitory role of PAR1 in PDAC, which is linked to the induction, and maintenance of a mesenchymal-like phenotype. The recognition that PAR1 actively limits pancreatic cancer cell growth suggest that the contributions of PAR1 to tumor growth differ between cancers of epithelial origin and that its targeting should be applied with care.

Resident Memory-Like Tumor-Infiltrating Lymphocytes (TILRM): Latest Players in the Immuno-Oncology Repertoire.

Resident memory T cells (TRM) are a recently identified subset of long-lived memory T cells that are characterized in terms of their unique surface phenotype combined with a non-recirculating pattern of localization to non-lymphoid, peripheral tissues. TRM have quickly become a key area of focus in understanding immune responses to microbial infection in so-called “barrier” tissues, and appear to be particularly critical for protection against repeat exposure at the same site. More recently, tumor-infiltrating T cells with canonical TRM features are being identified in human cancers, in particular cancers of epithelial origin, and their presence is broadly found to be associated with favorable long-term prognosis. Moreover, recent studies have shown that these “resident memory-like” tumor-infiltrating lymphocytes (referred to herein as TILRM) are uniquely activated in melanoma patients undergoing PD-1 directed checkpoint blockade therapy. Accordingly, there is much interest at present regarding the biology of these cells and their precise role in anti-cancer immunity. Herein, we review the current state of the literature regarding TILRM with a specific emphasis on their specificity, origins, and relationship to conventional pathogen-specific TRM and speculate upon the way(s) in which they might contribute to improved prognosis for cancer patients. We discuss the growing body of evidence that suggests TILRM may represent a population of bona-fide tumor-reactive T cells and the attractive possibility of leveraging this cell population for future immunotherapy.

Increased p63 Expression in Canine Perianal Gland Tumours.

Introductionp63 is a homologous molecule of p53 and was recently identified as playing important roles in several key cellular processes, including epithelial development and proliferation. Since then, several studies have demonstrated altered p63 expression in various cancers of epithelial origin. Canine perianal gland tumour is one of the most common skin neoplasms in dogs; however, the molecular characteristics of this tumour remain poorly understood. The objective of the present study was to analyse and compare the expression of p63 in canine perianal gland adenomas and carcinomas.Material and MethodsHaematoxylin and eosin-stained slides were examined and immunohistochemistry was conducted for a total of 65 samples. Immunohistochemical data for p63 expressions were compared between groups using the Mann-Whitney U test.ResultsThe p63 expression level was increased in perianal gland carcinomas compared to that in the adenoma samples (P < 0.0001). The percentage of cells expressing p63 was higher in perianal gland carcinomas than in adenomas, although the intensity of immunostaining did not differ significantly between the two groups.Conclusionp63 is a candidate factor contributing to the malignant transformation and progression of canine perianal gland tumours.

1196 Forced expression of the reprogramming factors leads to attenuation of melanoma malignancy through MET-like transdifferentiation and suppression of MAPK and AKT pathways

Prevention of metastasis is the final aim to conquer cancers including melanoma. It has been demonstrated that some of the malignant nature of tumor cells link to acquired mesenchymal properties, namely epithelial-mesenchymal transition (EMT), not only in cancers of epithelial origin, but also in melanoma. Thus, suppression of the mesenchymal natures in melanoma could be therapeutically beneficial. We have previously demonstrated that forced expression of the reprogramming factors, OCT4, SOX2, KLF4, c-MYC, and LIN28, which have been used for generation of induced-pluripotent stem cells (iPS), partially reversed human squamous cell carcinoma (SCC) cells from mesenchymal to epithelial properties (MET, or reversed EMT). In addition, attenuation of malignancy in SCC was obtained in vitro and in vivo. From these findings, we next addressed whether this method would be applied for melanoma. Forced expression of the reprogramming factors in both human and mouse melanoma led to drastic change in the cellular morphology from spindle to polygonal shape. The melanocyte signature genes, such as MITF and TYR, were strikingly down-regulated in the reprogramming factors-induced cells (RICs.) Similar to SCC-RICs, EMT genes including ZEB, SNAI2 and TWIST1 were decreased in melanoma-RICs. We also found that, although MEK/ERK, and AKT were constitutively phosphorylated in the parental melanoma cells, the RICs showed obviously decreased phosphorylation of these kinases. The in vitro invasion activity of melanoma-RICs was significantly suppressed as compared with the parent counterparts. Furthermore, the number of lung metastatic foci was obviously reduced in mice injected with RICs compared to those with the parental melanoma. Collectively, melanoma-RICs showed attenuated malignancy through EMT-like transdifferentiation and suppression of RAF-MEK-ERK and RAF-AKT signaling pathways.

C/EBPɑ is crucial determinant of epithelial homeostasis by preventing epithelial‐to‐mesenchymal transition

Extracellular signals such as transforming growth factor beta (TGF‐β) have been shown to influence both tumor initiation and metastasis. TGF‐β has been demonstrated to induce epithelial‐to‐mesenchymal transition (EMT) in cancers of epithelial origin by promoting molecular and phenotypical changes resulting in pro‐metastatic characteristics. Using global RNA‐sequencing analysis, we identified CCAAT/enhancer binding protein alpha (C/EBPα) as one of most TGF‐β‐mediated downregulated transcription factors in human mammary epithelial cells. Here, we show that upon TGF‐β pathway activation, SMAD3 binding to the CEBPA locus is enriched while SMAD3‐knockdown cells fail to repress CEBPA expression. Constitutive C/EBPα expression impaired TGF‐β‐driven EMT by inhibiting the expression of known EMT factors including N‐cadherin, MMP‐2 or ZEB1, and by maintaining E‐cadherin expression. Conversely, depletion of C/EBPα expression alone was sufficient to induce mesenchymal‐like morphology and molecular features. Moreover, cells that had undergone TGF‐β‐induced EMT reverted to an epithelial‐like state upon C/EBPα re‐expression. TGF‐β‐mediated disruption of epithelial spheroids architecture could also be rescued by the introduction of C/EBPα. By using an established mouse model of breast cancer EMT‐driven metastasis, we show that mice injected with tumor organoids constitutively expressing C/EBPα display a dramatic reduction of metastatic lesions in their lungs compared to controls. Taken together, these results show that C/EBPα is required for maintaining epithelial homeostasis by repressing the expression of key mesenchymal markers, thus preventing EMT‐mediated tumorigenesis. These data suggest that C/EBPα is an epithelial “gatekeeper” whose expression is required to prevent unwarranted mesenchymal transition, thereby supporting an important role for EMT in mediating breast cancer metastasis.Support or Funding InformationThis work was supported by a grant from the Dutch Cancer Foundation (KWF).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An interplay between MicroRNA and SOX4 in the regulation of epithelial–Mesenchymal transition and cancer progression

MicroRNAs (miRNAs) are abundant class of small, noncoding RNAs which are emerging as a potential therapeutic target for various cancers. Sex-determining region Y-related high-mobility group box 4 (SOX4) is an important member of SOX family and a crucial master regulator of epithelial–mesenchymal transition (EMT) that has been implicated in tumor growth and progression. In cancers of epithelial origin and in some others, growing evidence has revealed a close association between miRNAs and SOX4. Most miRNAs are reported to modulate SOX4 by directly binding to SOX4 3'-untranslated region, thereby regulating cancer cell proliferation, invasion, and EMT. In this review, we highlight the interaction of miRNAs with SOX4 in various cancers and discuss the possibility of combined miRNA-SOX4-based therapeutic approach that may serve as a targeted therapy for several cancers.

Abstract B115: MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts

Abstract Triple-negative breast cancer (TNBC) is a highly metastatic disease with very poor long-term prognosis. Because of the lack of well-defined molecular targets, traditional chemotherapy is currently the only therapeutic regimen for TNBC. Within this agent class, eribulin has been approved for metastatic breast cancer and the prespecified subgroup analysis in phase 3 clinical trial (study301) revealed that eribulin demonstrated the greatest therapeutic benefit on overall survival of TNBC patients among all other breast cancer subtypes. Folate receptor alpha (FRA) is a GPI-linked membrane glycoprotein that has limited expression in normal tissue, but is highly overexpressed on a large number of cancers of epithelial origin, including non-small cell lung cancer (NSCLC), ovarian cancer, and TNBC. These features make FRA an attractive target for the treatment of TNBC using targeted therapeutic approaches. Farletuzumab, a humanized monoclonal antibody targeting FRA, has been investigated in multiple clinical trials enrolling patients overexpressing FRA. MORAb-202, a novel antibody-drug conjugate (ADC) consisting of farletuzumab conjugated to eribulin, targets FRA overexpressed by a variety of tumor types including TNBC. Upon binding and internalization, it is hypothesized that the liberated eribulin elicits both a direct cytotoxic effect on FRA-positive tumor cells and a bystander effect on FRA-negative tumor cells and, importantly, tumor-associated stromal cells within the tumor microenvironment. To address this hypothesis, we have evaluated the in vivo efficacy of MORAb-202 against a FRA-positive TNBC patient-derived xenograft (PDX) model. Single administration of 5 mg/kg MORAb-202 mediated tumor regression statistically compared to vehicle control (P&amp;lt;0.0001). Additionally, one animal treated with MORAb-202 showed complete and durable tumor regression. In order to examine the effects of MORAb-202 treatment on the tumor microenvironment, we harvested tumors both prior to treatment and five days post-treatment. Immunofluorescent staining was used to assess the target-specific engagement of MORAb-202 to FRA-positive regions of the tumor. Strong membrane staining of MORAb-202 was observed, while no staining was seen in the adjacent stromal regions. MORAb-202 also had demonstrable effects on the tumor microenvironment via reduction of the number of FRA-negative cancer-associated fibroblasts (CAF) present. To further evaluate whether these effects on CAF are part of the mode of action (MOA) of MORAb-202, we tested the antitumor efficacy and effects on the tumor microenvironment in a surrogate FRA-positive in vivo model. Tumors were isolated on days 3, 5, 7, and 9 post-treatment. The reduction in CAF, visualized by alpha-smooth muscle actin staining, was directly proportional to overall tumor reduction. In both studies, single-dose MORAb-202 showed dramatically greater efficacy than molar equivalent dosing of eribulin alone. Histologic analyses revealed unique modes of action of MORAb-202 over antibody or payload alone. These results suggest a potential benefit of MORAb-202 in the treatment of TNBC. Citation Format: Katherine A. Rybinski, Christopher Maddage, Xin Cheng, Earl Albone, Luigi Grasso, Keiji Furuuchi, Toshimitsu Uenaka. MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B115.

EpCAM expression in squamous cell carcinoma of the uterine cervix detected by monoclonal antibody to the membrane-proximal part of EpCAM

BackgroundEpithelial cell adhesion molecule (EpCAM) is a promising biomarker for squamous cell carcinoma (SCC) of the uterine cervix, because it is over-expressed in various cancers of epithelial origin. However, EpCAM expression reported in previous immunohistochemistry (IHC) studies was inconsistent. We hypothesize that the membrane-distal part of EpCAM may be lost during tissue preparation, leaving only the membrane-proximal part of EpCAM available for antibody binding and IHC staining.MethodsTwo new anti-EpCAM MAbs to the membrane-proximal part (WC-2) and the membrane-distal part (WC-1) of EpCAM were generated and characterized. WC-2 was selected for its ability to detect EpCAM in cervical tissues by IHC. One hundred thirty-five archival paraffin-embedded tissues previously diagnosed as cervical SCC (n=44), high-grade (HSIL) (n=43), or low-grade (LSIL) (n=48) squamous intraepithelial lesions were examined. IHC score was collected, recorded, and analyzed for distribution, intensity, and percentage of cancer cells stained for EpCAM.ResultsEpCAM expression was consistently detected on cervical tissues by WC-2, but not by WC-1. EpCAM was expressed with high IHC score in the majority of cervical SCC (37/44), but not in normal epithelial area adjacent to SCC. EpCAM was also highly expressed on precancerous lesion of the cervix, particularly in HSIL. More importantly, EpCAM expression could be used to distinguish between HSIL and LSIL, according to staining distribution. HSIL tissues displayed EpCAM expression in two-thirds to full thickness of the epithelium, while in LSIL the staining was limited to the lower one-third of the thickness. The IHC score of EpCAM expression was strongly correlated with cervical cancer and grades of precancerous lesions (r=0.875, p<0.001).ConclusionOnly the anti-EpCAM MAb to the membrane-proximal part is able to detect EpCAM on paraffin-embedded cervical cancer tissues. A strong positive correlation between EpCAM expression level and the grades of SILs provides the possibility that EpCAM can be used to predict prognosis and severity in these patients.

Epithelial-to-Mesenchymal and Mesenchymal-to-Epithelial Transition in Mesenchymal Tumors: A Paradox in Sarcomas?

The epithelial-to-mesenchymal transition (EMT) is a reversible process comprised of various subprograms via which epithelial cells reduce their intercellular adhesions and proliferative capacity while gaining a mesenchymal phenotype with increased migratory and invasive properties. This process has been well described in several carcinomas, which are cancers of epithelial origin, and is crucial to metastatic tumor cell dissemination and drug resistance. In contrast, the precise role of EMT-related processes in tumors originating from mesenchymal tissues, such as bone and soft-tissues sarcomas, is still largely unclear. In fact, although the existence of the EMT in sarcomas appears paradoxical because these cancers are, by definition, mesenchymal ab initio, accumulating evidence suggests that many sarcomas can undergo EMT-related processes, which may be associated with aggressive clinical behavior. These processes may be especially operative in certain sarcoma subtypes, such as carcinosarcomas displaying a biphenotypic morphology with characteristics of both mesenchymal and epithelial tumors. In this review, we discuss findings regarding the potential existence of EMT-related processes in sarcomas and propose that sarcomas can reside in a metastable state, enabling them to become either more mesenchymal or epithelial under specific conditions, which likely has important clinical implications. Cancer Res; 77(17); 4556-61. ©2017 AACR.

Racial disparities in the utilization of preventive health services among older women with early-stage endometrial cancer enrolled in Medicare.

To assess differences in the receipt of preventive health services by race/ethnicity among older women with endometrial cancer enrolled in Medicare, we conducted a retrospective population‐based cohort study of women diagnosed with endometrial cancer from 2001 to 2011 in the Surveillance Epidemiology and End Results (SEER)‐Medicare database. Women with stage I or II endometrial cancer of epithelial origin were included. The exposure was race/ethnicity (Non‐Hispanic [NH] White, NH Black, Hispanic, and NH Asian/Pacific Islander [PI]). The services examined were receipt of influenza vaccination and screening tests for diabetes mellitus, hyperlipidemia, and breast cancer. We used multivariate logistic regression to estimate odds ratios with 95% confidence intervals (CI) adjusted for age, region, and year of diagnosis. A total of 13,054 women were included. In the 2 years after diagnosis, receipt of any influenza vaccine ranged from 45% among NH Black women to 67% among NH White women; receipt of a mammogram ranged from 65% among NH Black women to 74% among NH White women. Relative to NH White women, NH Black women had a lower likelihood of receiving both influenza vaccination (adjusted odds ratio [aOR] 0.40, 95% CI 0.33–0.44) and screening mammography (aOR 0.64, 95% CI 0.52–0.79). Hispanic women also were less likely to receive influenza vaccination than NH White women (aOR 0.61, 95% CI 0.51–0.72). There were no significant differences across racial groups for diabetes or cholesterol screening services. Among older women with early‐stage endometrial cancer, racial disparities exist in the utilization of some preventive services.

Abstract 3787: EpCAM- and EpCAM+ circulating tumor cells in metastatic prostate and breast cancer patients: a multicenter study

Abstract Introduction: The presence of circulating tumor cells (CTC) enumerated by the CellSearch system in blood of patients with cancers of epithelial origin is strongly associated with a poor prognosis of these patients. CTC are enriched by targeting the EpCAM antigen, raising the question which EpCAM subtypes are present as well in patients. In the EU-FP7 CTCTrap program, we investigated the presence of EpCAM- CTC in blood samples after depletion of EpCAM+ CTC by CellSearch. Studies were performed and validated at the participating laboratories by distribution and analysis of blood samples spiked with cancer cells and by testing blood from 73 metastatic prostate and 22 metastatic breast cancer patients. Methods: Blood samples were processed according to the standard operating procedures and tools developed in the program (https://www.utwente.nl/tnw/mcbp/protocolsandtools/). First, CellSearch was performed for EpCAM+ CTC, followed by filtration and fluorescent labeling of the blood discarded by CellSearch for EpCAM- CTC. To validate the procedures across the 6 participating clinical sites, 3x 3 tubes with aliquots of healthy donor blood, spiked with either PC3, MDA-MB-231 or no cells, were prepared at one site and shipped to all other sites for simultaneous processing. Metastatic prostate and breast cancer patients were processed with the same procedures. Results: 27% of PC3 cells were recovered by CellSearch and 21% by filtration, leaving 52% unaccounted for. For MDA, 26% were recovered by CellSearch and 18% by filtration, leaving 56% accounted for. Differences in recovery between sites were not significant. In patients both EpCAM+ and EpCAM- CTC were detected (see table). Conclusion: In a multicenter study EpCAM+ and EpCAM- CTC were present in blood of metastatic prostate and breast cancer patients. Spiking experiments showed that the developed methods can be further improved to increase the CTC yield. Molecular characterization of the CTC subtypes and relation with clinical outcome is ongoing. % metastatic cancer patients with EpCAM+ and EpCAM- CTC% prostate cancer patients (n=73)% breast cancer patients (n=22)CellSearch CTC EpCAM+CellSearch CTC EpCAM+# CTC01-4&amp;gt;4Total01-4&amp;gt;4TotalMicrosieve CTC EpCAM-011819381859321-4671629991432&amp;gt;48619331414936Total252154412732 Citation Format: Sanne de Wit, Kiki C. Andree, Joost F. Swennenhuis, Elisabetta Rossi, Mariangela Manicone, Riccardo Vidotto, Rita Zamarchi, Marianna Alunni-Fabbroni, Elisabeth K. Trapp, Marie Tzschaschel, Brigitte Rack, Rita Lampignano, Hans Neubauer, Tanja Fehm, Marianne Oulhen, Emeline Colomba, Françoise Farace, Mateus Crespo, Penelope Flohr, Gemma Fowler, Mariane Sousa Fontes, Johann S. de Bono, Leon WMM Terstappen. EpCAM- and EpCAM+ circulating tumor cells in metastatic prostate and breast cancer patients: a multicenter study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2017-3787

Cytokeratin AE1/AE3 mimicry in glioblastoma

INTRODUCTION : The diagnosis and treatment of intracranial tumors requires a multidisciplinary approach. A key moment in this process is the pathological verification of the tumor type. This process, although aided by immunohistochemistry (IHC), can often be difficult and misleading. MATERIALS AND METHODS : Ten histologically confirmed cases of glioblastoma multiforme (GBM) were reviewed for their IHC reaction with the anti-glial fibrillary acidic protein (GFAP) glial marker and the CK AE1/AE3 antibody cocktail, whose main use in neuropathology is to either prove or rule out metastatic cancer of epithelial origin, the primary location of which may not be known or even suspected. RESULTS : All ten pathologically verified cases of GBM were diagnostically positive for GFAP, with eight of them also revealing CK AE1/AE3 expression with variable intensity. Out of the CK AE1/AE3 positive cases, five (50% in total) gave a low to intermediate non-diagnostic positive reaction, while the other three cases (30% in total) gave a strong positive reaction with possible diagnostic value. Cells, across all GBM cases, that tested positive for CK AE1/AE3, regardless of the strength of the reaction, were also positive for GFAP on neighboring IHC serial slides. CONCLUSION : The presented results reveal CK AE1/AE3 expression in a great portion of GBM cases, which may be caused by three-dimensional mimicry between the CK AE1/AE3 and GFAP target molecules. This therefore necessitates the need for a careful interpretation of the results. CK AE1/AE3, however, remains a useful tool in neuropathology, regardless of the possibility of false positivity in GBM cells.

Ovarian cancer: current approaches to classification, diagnostics, staging and differential management of patients

Purpose of this study: systematic analysis of the data available in the modern scientific medical literature on contemporary aspects of the diagnosis, staging and differential treatment tactics of patients with epithelial ovarian tumors.&#x0D; Research method used in this article is the systematic analysis of the medical literature, both domestic and western.&#x0D; Results. This article attempts to summarize the experience of modern medicine and the world’s leading centers for the fight against ovarian cancer of epithelial origin and offer solutions relevant to everyday clinical practice. Problems of the modern classification of forms of the disease are described in detail; description of instrumental diagnostic methods also is given in thus article; problem of lack of effective proved themselves in clinical practice of screening methods also was disccused; and, thus, current understanding of the disease staging and differential tactics for treatment of patients are given.&#x0D; Conclusion. One of the most important directions of development of gynecological oncology is a decision the existing problems related to the diagnosis and treatment of ovarian cancer: namely, the development of effective screening programs, improving diagnosis and therapy methods, the development of measures of postoperative management of patients with ovarian cancer, including medical rehabilitation measures.

Abstract 063: Role of Ribosomal S6 Kinase 2 in Vascular Myogenic Tone and Blood Pressure Regulation

Smooth muscle (SM) contractile force is generated through phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase. However, emerging evidence also suggests that additional Ser/Thr kinases may contribute to the pathways that regulate SM contractility. Recently, we published that the 90 kDa ribosomal S6 kinase (RSK), extensively studied in cancers of epithelial origin, is a major new player in the regulation of SM contractility. RSKs are unusual kinases having two catalytic domains separated by a linker having multiple phosphorylation sites and are activated by a cascade of ERK phosphorylations and by PDK, which phosphorylates RSK at Ser227 resulting in transition to a fully active enzyme. The thromboxane agonist, U46619 leads to activation of ERK1/2 and PDK1 in SM. We find that 4 th order mesenteric arteries are significantly more dilated at a pressure of 80 mm Hg in Rsk2 -/- mice compared to wild-type (WT) (2.0±0.8 vs 12.8±4.5 %, p &lt;0.05, respectively). The contractile response to dose-dependent phenylephrine stimulation is significantly smaller in the absence of RSK2, p &lt;0.0001 two-way ANOVA, while EC50 for phenylephrine contractions did not differ. Using isolated arcades of mesenteric vessels, phosphorylation of myosin RLC20 and myosin phosphatase regulatory subunit and RSK Ser227 (PDK site) and Thr 577 (ERK site) increased with in 30secs following an increase in intraluminal pressure to 80 mm Hg to induce myogenic tone development. Mean baseline systolic blood pressure (SBP, mmHg) was significantly lower in Rsk2 -/- animals (WT=107.4±1.6 and RSK2 -/- =95.8±2.2, n=23, p&lt; 0.0002). In view of the lower SBP in the Rsk2 -/- animals, we examined the potential role for RSK signaling in the kidney, since RSK2 is known to phosphorylate NHE3. By immunohistochemistry, we show for the first time the expression of RSK2 in human kidney tubules. Lithium excretion, moles/kg/day, was increased in Rsk2-/- mice compared to WT (21.7±3.7 vs 48.6±7.9, respectively), indicative of defective renal Na + reabsorption. Taken together, RSK2 signaling in vascular SM and the kidney is a novel mechanism for the regulation of SM contractility and of BP. RSK2 may be a potential selective target in hypertension.

4.1N is involved in a flotillin-1/β-catenin/Wnt pathway and suppresses cell proliferation and migration in non-small cell lung cancer cell lines.

The membrane-cytoskeletal protein 4.1N has recently been proposed as a tumor suppressor in a number of cancers of epithelial origin, including non-small-cell lung cancer (NSCLC). However, the molecular mechanism associated with 4.1N tumor suppression remains has not been thoroughly characterized. In this study, 4.1N was shown to directly interactwith the lipid raft marker flotillin-1 through its FERM and U2 domains in several different NSCLC cell lines using immunoprecipitation, co-immunoprecipitation and pull-down assays. Moreover, 4.1N silencing/overexpression experiments in paired 95C/95D cells that are of homologous origin but varying endogenous 4.1N expression (high expression in 95C cells, low expression in 95D cells) indicated that 4.1N is involved in the suppression of cell proliferation and migration through a flotillin-1/β-catenin/Wnt pathway. Taken together, the findings of this study help to elucidate the novel tumor suppressor role of 4.1N in NSCLC.

Overexpression of cancer cell-derived immunoglobulin G correlates with poor prognosis in gastric cancer patients

Background: Various human epithelial origin cancers produce cytoplasmic immunoglobulin (Ig) G. Recent studies have elucidated that it was related with the tumorigenesis. This study was aimed to identify relationship between the cancer cell-derived IgG expression level and clinical parameters in gastric cancer (GC). Methods: Cancer cell-derived IgG expression was assessed using immunohistochemistry analysis in 231 primary GC tissues. The role of cancer cell-derived IgG on cell proliferation, migration and invasion were assessed. Results: Our results indicated that IgG was overexpressed in GC tissues (42.2%, 46/109) than in adjacent tissues (11.0%, 12/109, P vs. 37.2%, respectively). Upon univariate analysis, IgG positive group had significantly lower 5-year overall survival than the negative group (P=0.0361). Multivariate analysis showed IgG expression was an independent prognostic indicator for 5-year overall survival of patients with GC (P=0.018). Furthermore, knockdown of IgG by short interfering RNA (siRNA) resulted in reducing proliferation, migration and invasion of GC cell. Conclusions: Our results showed that cancer cell-derived IgG overexpression might be correlated with GC progression, and would be a novel biomarker to predict the prognosis of patients with GC.

Fibroblast activation and senescence in oral cancer

There is now compelling evidence that the tumour stroma plays an important role in the pathogenesis of cancers of epithelial origin. The pre-eminent cell type of the stroma is carcinoma-associated fibroblasts. These cells demonstrate remarkable heterogeneity with activation and senescence being common stress responses. In this review, we summarise the part that these cells play in cancer, particularly oral cancer, and present evidence to show that activation and senescence reflect a unified programme of fibroblast differentiation. We report advances concerning the senescent fibroblast metabolome, mechanisms of gene regulation in these cells and ways in which epithelial cell adhesion is dysregulated by the fibroblast secretome. We suggest that the identification of fibroblast stress responses may be a valuable diagnostic tool in the determination of tumour behaviour and patient outcome. Further, the fact that stromal fibroblasts are a genetically stable diploid cell population suggests that they may be ideal therapeutic targets and early work in this context is encouraging.

The T-box transcription factor 3 is a promising biomarker and a key regulator of the oncogenic phenotype of a diverse range of sarcoma subtypes.

Sarcomas represent a complex group of malignant neoplasms of mesenchymal origin and their heterogeneity poses a serious diagnostic and therapeutic challenge. There is therefore a need to elucidate the molecular mechanisms underpinning the pathogenesis of the more than 70 distinguishable sarcoma subtypes. The transcription factor TBX3, a critical developmental regulator, is overexpressed in several cancers of epithelial origin where it contributes to tumorigenesis by different molecular mechanisms. However, the status and role of TBX3 in sarcomas have not been reported. Here we show that a diverse subset of soft tissue and bone sarcoma cell lines and patient-derived sarcoma tissues express high levels of TBX3. We further explore the significance of this overexpression using a small interferring RNA approach and demonstrate that TBX3 promotes the migratory ability of chondrosarcoma, rhabdomyosarcoma and liposarcoma cells but inhibits fibrosarcoma cell migration. This suggested that TBX3 may play a key role in the development of different sarcoma subtypes by functioning as either an oncoprotein or as a brake to prevent tumour progression. To further explore this, TBX3 knockdown and overexpression cell culture models were established using chondrosarcoma and fibrosarcoma cells as representatives of each scenario, and the resulting cells were characterized with regard to key features of tumorigenesis. Results from in vitro and in vivo assays reveal that, while TBX3 promotes substrate-dependent and -independent cell proliferation, migration and tumour formation in chondrosarcoma cells, it discourages fibrosarcoma formation. Our findings provide novel evidence linking TBX3 to cancers of mesenchymal origin. Furthermore, we show that TBX3 may be a biomarker for the diagnosis of histologically dynamic sarcoma subtypes and that it impacts directly on their oncogenic phenotype. Indeed, we reveal that TBX3 may exhibit oncogene or tumour suppressor activity in sarcomas, which suggests that its role in cancer progression may rely on cellular context.