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Tumor dissemination is one of the most-investigated steps of tumor progression, which in recent decades led to the rapid development of liquid biopsy aiming to analyze circulating tumor cells (CTCs), extracellular vesicles (EVs), and circulating nucleic acids in order to precisely diagnose and monitor cancer patients. Flow cytometry was considered as a method to detect CTCs; however, due to the lack of verification of the investigated cells’ identity, this method failed to reach clinical utility. Meanwhile, imaging flow cytometry combining the sensitivity and high throughput of flow cytometry and image-based detailed analysis through a high-resolution microscope might open a new avenue in CTC technologies and provide an open-platform system alternative to CellSearch®, which is still the only gold standard in this field. Hereby, we shortly review the studies on the usage of flow cytometry in CTC identification and present our own representative images of CTCs envisioned by imaging flow cytometry providing rationale that this novel technology might be a good tool for studying tumor dissemination, and, if combined with a high CTC yield enrichment method, could upgrade CTC-based diagnostics.

There is a great need to develop sensitive and specific methods for quantitative analysis of caspase-3 activities in cell apoptosis. Herein, we report a new method for sensitive detection of caspase-3 enzyme activities and inhibitor screening based on dual maleimide (DuMal) labeling quantitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Evaluation of caspase-3 activities is performed using MS analysis of the enzymatic product of the peptide probe, which fuses a caspase-3 cleavable peptide segment (DEVD) and a quantifiable "ID tag" (a peptide segment of FRGLRGFKC labeled by maleimide). The DuMal labeling technique features non-isotopic tagging, rapid reactions, and reproducible quantitation. We have achieved quantitative analysis of the enzyme activities with a limit of detection (LOD) and limit of quantitation (LOQ) of caspase-3 down to 0.11 nM and 0.29 nM respectively and a proof-of-concept demonstration of its inhibitor screening. Our method has further been tested for caspase-3 activities in a Parkinson's disease cellular model, suggesting a useful tool for protease activity research.

Breast cancer metastasis is the leading cause of cancer deaths in women and is difficult to combat due to the long periods in which disseminated cells retain a potential to be re-activated and start the relapse. Assessing the number and molecular profile of circulating tumor cells (CTCs) in breast cancer patients, especially in early breast cancer, should help in identifying the possibility of relapse in time for therapeutic intervention to prevent or delay recurrence. While metastatic breast cancer is considered incurable, molecular analysis of CTCs still have a potential to define particular susceptibilities of the cells representing the current tumor burden, which may differ considerably from the cells of the primary tumor, and offer more tailored therapy to the patients. In this review we inspect the routes to metastasis and how they can be linked to specific features of CTCs, how CTC analysis may be used in therapy, and what is the current status of the research and efforts to include CTC analysis in clinical practice.

Background: Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment (TME). Estrogen receptor alpha 36 (ERα36), the alternatively spliced variant of ERα, is described as an unfavorable factor when expressed in cancer cells. ERα can be expressed also in CAFs; however, the role of ERα36 in CAFs is unknown. Methods: Four CAF cultures were isolated from chemotherapy-naïve BC patients and characterized for ERα36 expression and the NanoString gene expression panel using isolated RNA. Conditioned media from CAF cultures were used to assess the influence of CAFs on triple-negative breast cancer (TNBC) cells using a matrigel 3D culture assay. Results: We found that ERα36high CAFs significantly induced the branching of TNBC cells in vitro (p < 0.001). They also produced a set of pro-tumorigenic cytokines compared to ERα36low CAFs, among which hepatocyte growth factor (HGF) was the main inducer of TNBC cell invasive phenotype in vitro (p < 0.001). Tumor stroma rich in ERα36high CAFs was correlated with high Ki67 expression (p = 0.041) and tumor-associated macrophages markers (CD68 and CD163, p = 0.041 for both). HGF was found to be an unfavorable prognostic factor in TCGA database analysis (p = 0.03 for DFS and p = 0.04 for OS). Conclusions: Breast cancer-associated fibroblasts represent distinct subtypes based on ERα36 expression. We propose that ERα36high CAFs could account for an unfavorable prognosis for TNBC patients.

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)–enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients.

The role of circulating tumor cells (CTCs), tumor microenvironment (TME), and the immune system in the formation of metastasis is evident, yet the details of their interactions remain unknown. This study aimed at exploring the immunotranscriptome of primary tumors associated with the status of CTCs in breast cancer (BCa) patients. The expression of 730 immune-related genes in formalin-fixed paraffin-embedded samples was analyzed using the multigenomic NanoString technology and correlated with the presence and the phenotype of CTCs. Upregulation of 37 genes and downregulation of 1 gene were observed in patients characterized by a mesenchymal phenotype of CTCs when compared to patients with epithelial CTCs. The upregulated genes were involved in NF-kappa B signaling and in the production of type I interferons. The clinical significance of the differentially expressed genes was evaluated using The Cancer Genome Atlas (TCGA) data of a breast invasive carcinoma (BRCA) cohort. Five of the upregulated genes&mdash, PSMD7, C2, IFNAR1, CD84, and CYLD&mdash, were independent prognostic factors in terms of overall and disease-free survival. To conclude, our data identify a group of genes that are upregulated in BCa patients with mesenchymal CTCs and reveal their prognostic potential, thus indicating that they merit further investigation.

The amplification of estrogen receptor alpha (ER&alpha, ) encoded by the ESR1 gene has been described as having a prognostic role in breast cancer patients. However, increased dosage of the ESR1 gene (tested by real-time PCR) is also observed in ER-negative breast cancers, which might suggest the expression of alternative isoforms of ER&alpha, (other than classical ER&alpha, of 66 kDa). In the current work, we have investigated the ESR1 gene dosage in 402 primary breast cancer patients as well as the expression of ER&alpha, isoforms&mdash, ER&alpha, 66 and ER&alpha, 36&mdash, on mRNA and protein levels. The obtained results were correlated with clinicopathological data of the patients. Results showed that increased ESR1 gene dosage is not related to ESR1 gene amplification measured by fluorescent in situ hybridization (FISH), but it correlates with the decreased expression of ER&alpha, 66 isoform (p = 0.01). Interestingly, the short ER isoform ER&alpha, 36 was expressed in samples with increased ESR1 gene dosage, suggesting that genomic aberration might influence the expression of that particular isoform. Similarly to ESR1 increased gene dosage, high ER&alpha, 36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ER&alpha, 66 level in breast tumors.

// Dominika Piasecka 1, 4 , Kamila Kitowska 1 , Dominika Czaplinska 2 , Kamil Mieczkowski 1 , Magdalena Mieszkowska 1 , Lukasz Turczyk 1 , Andrzej C. Skladanowski 1 , Anna J. Zaczek 2 , Wojciech Biernat 3 , Radzislaw Kordek 4 , Hanna M. Romanska 4 , Rafal Sadej 1 1 Department of Molecular Enzymology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Poland 2 Department of Cell Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Poland 3 Department of Pathomorphology, Medical University of Gdansk, Poland 4 Department of Pathology, Medical University of Lodz, Poland Correspondence to: Rafal Sadej, email: rsadej@gumed.edu.pl Hanna M. Romanska, email: hanna.romanska@gmail.com Keywords: progesterone receptor, FGFR2, breast cancer Received: April 12, 2016 Accepted: November 07, 2016 Published: November 12, 2016 ABSTRACT We have recently demonstrated that, fibroblast growth factor 2 (FGFR2), signalling via ribosomal S6 kinase 2 (RSK2), promotes progression of breast cancer (BCa). Loss of progesterone receptor (PR), whose activity in BCa cells can be stimulated by growth factor receptors (GFRs), is associated with poor patient outcome. Here we showed that FGF7/FGFR2 triggered phosphorylation of PR at Ser294, PR ubiquitination and subsequent receptor`s degradation via the 26S proteasome pathway in BCa cells. We further demonstrated that RSK2 mediated FGF7/FGFR2-induced PR downregulation. In addition, a strong synergistic effect of FGF7 and progesterone (Pg), reflected in the enhanced anchorage-independent growth and cell migration, was observed. Analysis of clinical material demonstrated that expression of PR inversely correlated with activated RSK (RSK-P) ( p = 0.016). Patients with RSK-P(+)/PR(–) tumours had 3.629-fold higher risk of recurrence ( p = 0.002), when compared with the rest of the cohort. Moreover, RSK-P(+)/PR(–) phenotype was shown as an independent prognostic factor ( p = 0.006). These results indicate that the FGF7/FGFR2-RSK2 axis promotes PR turnover and activity, which may sensitize BCa cells to stromal stimuli and contribute to the progression toward steroid hormone negative BCa.

We have previously demonstrated that fibroblast growth factor receptor 2 (FGFR2) activates ribosomal s6 kinase 2 (RSK2) in mammary epithelial cells and that this pathway promotes in vitro cell growth and migration. Potential clinical significance of FGFR2 and RSK2 association has never been investigated. Herein, we have undertaken an evaluation of a possible relationship between FGFR2/RSK2 interdependence and disease outcome in breast cancer (BCa) patients. The clinical analysis was complemented by an in vitro investigation of an involvement of RSK2 in the regulation of FGFR2 function. Primary tumour samples from 152 stage I–III BCa patients were examined for FGFR2 and RSK2 gene and protein expression. FGFR2 showed a positive correlation with RSK2 at both protein (p = 0.003) and messenger RNA (mRNA) (p = 0.001) levels. Lack of both FGFR2 and activated RSK (RSK-P) significantly correlated with better disease-free survival (DFS) (p = 0.01). Patients with tumours displaying immunoreactivity for either or both FGFR2 and RSK-P had 4.89-fold higher risk of recurrence when compared to the FGFR2/RSK-P-negative subgroup. FGFR2-RSK2 interactions were verified by co-immunoprecipitation and internalization assays in HB2 mammary epithelial cell line (characterized by high endogenous FGFR2 and RSK2 expression). In vitro analyses revealed that FGFR2 and RSK2 formed an indirect complex and that activated RSK exerted a significant impact on fibroblast growth factor 2 (FGF2)-triggered internalization of FGFR2. Our results suggest that the FGFR2-RSK2 signalling pathway is involved in pathophysiology of BCa and evaluation of FGFR2/RSK-P expression may be useful in disease prognostication. Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-5266-9) contains supplementary material, which is available to authorized users.

Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with different EMT status in order to establish their clinical significance in early breast cancer patients. We have obtained CTCs-enriched blood fraction from 83 breast cancer patients in which we have tested the expression of epithelial, mesenchymal and general breast cancer CTCs markers (MGB1/HER2/CK19/CDH1/CDH2/VIM/PLS3), cancer stem cell markers (CD44, NANOG, ALDH1, OCT-4, CD133) and cluster formation gene (plakoglobin). We have shown that in the CTCs-positive patients, epithelial, epithelial-mesenchymal and mesenchymal CTCs markers were detected at a similar rate (in 28%, 24% and 24%, respectively). Mesenchymal CTCs were characterized by the most aggressive phenotype (significantly higher expression of CXCR4, uPAR, CD44, NANOG, p &lt, 0.05 for all), presence of lymph node metastases (p = 0.043), larger tumour size (p = 0.023) and 7.33 higher risk of death in the multivariate analysis (95% CI 1.06&ndash, 50.41, p = 0.04). Epithelial-mesenchymal subtype, believed to correspond to highly plastic and aggressive state, did not show significant impact on survival. Gene expression profile of samples with epithelial-mesenchymal CTCs group resembled pure epithelial or pure mesenchymal phenotypes, possibly underlining degree of EMT activation in particular patient&rsquo, s sample. Molecular profiling of CTCs EMT phenotype provides more detailed and clinically informative results, proving the role of EMT in malignant cancer progression in early breast cancer.

Introduction. Triple-negative breast cancer (TNBC), representing over 15% of all breast cancers, has a poorer prognosis than other subtypes. There is no effective targeted treatment available for the TNBC sufferers. Ribosomal S6 kinases (RSKs) have been previously proposed as drug targets for TNBC based on observations that 85% of these tumors express activated RSKs. Materials and methods. Herein we examined an involvement of RSK1 (p90 ribosomal S6 kinase 1) in a regulation of TNBC growth and metastatic spread in an animal model, which closely imitates human disease. Mice were inoculated into mammary fat pad with 4T1 cells or their RSK1-depleted variant. We examined tumor growth and formation of pulmonary metastasis. Boyden chamber, wound healing and soft agarose assays were performed to evaluate cells invasion, migration and anchorage-independent growth. Results. We found that RSK1 promoted tumor growth and metastasis in vivo . After 35 days all animals inoculated with control cells developed tumors while in the group injected with RSK1-negative cells, there were 75% tumor-bearing mice. Average tumor mass was estimated as 1.16 g and 0.37 g for RSK1-positive vs. -negative samples, respectively (p < 0.0001). Quantification of the macroscopic pulmonary metastases indicated that mice with RSK1-negative tumors developed approximately 85% less metastatic foci on the lung surface (p < 0.001). This has been supported by in vitro data presenting that RSK1 promoted anchorage-independent cell growth and migration. Moreover, RSK1 knock-down corresponded with decreased expression of cell cycle regulating proteins, i.e. cyclin D3, CDK6 and CDK4. Conclusions. We provide evidence that RSK1 supports tumor growth and metastatic spread in vivo as well as in vitro migration and survival in non-adherent conditions. Further studies of RSK1 involvement in TNBC progression may substantiate our findings, laying the foundations for development of anti-RSK1-based therapeutic strategies in the management of patients with TNBC.

Whilst the role of eukaryotic translation initiation factors (eIFs) has already been investigated in several human cancers, their role in endometrial cancer (EC) is relatively unknown. In the present retrospective study, 279 patients with EC (1180 samples) were included (mean age: 63.0 years, mean follow-up: 6.1 years). Samples were analysed for expression of 7 eIFs subunits (eIF2&alpha, eIF3c, eIF3h, eIF4e, eIF4g, eIF5, eIF6) through immunohistochemistry and western blotting. Fifteen samples of healthy endometrium served as controls. Density and intensity were assessed and mean combined scores (CS) calculated for each patient. Upon immunohistochemistry, median eIF5 CS were significantly higher in EC as compared with non-neoplastic tissue (NNT, p &lt, 0.001), whilst median eIF6 CS were significantly lower in EC (p &lt, 0.001). Moreover, eIF5 (p = 0.002), eIF6 (p = 0.032) and eIF4g CS (p = 0.014) were significantly different when comparing NNT with EC grading types. Median eIF4g CS was higher in type II EC (p = 0.034). Upon western blot analysis, eIF4g (p &lt, 0.001), peIF2&alpha, (p &lt, 0.001) and eIF3h (p &lt, 0.05) were significantly overexpressed in EC, while expression of eIF3c was significantly reduced in EC as compared with NNT (p &lt, 0.001). The remaining eIFs were non-significant. Besides tumour stage (p &lt, 0.001) and patient&rsquo, s age (p &lt, 0.001), high eIF4g CS-levels were independently associated with poor prognosis (HR: 1.604, 95%CI: 1.037&ndash, 2.483, p = 0.034). The other eIFs had no prognostic significance. Notably, the independent prognostic significance of eIF4g was lost when adding tumour type. Considering the difficulties in differentiating EC type I and II, eIF4g may serve as a novel prognostic marker indicating patient outcome.

The aim of this study was to analyze the occurrence of TOP2A gene amplification and chromosome 17 polysomy in patients with early breast cancer and to correlate the status of these alterations with the prognostic significance expressed as patients' clinical features and survival. Such concurrent analyses of TOP2A gene status and chromosome 17 polysomy have not been performed before. Study group included 149 consecutive stage I-III patients administered standard multimodality treatment. TOP2A abnormalities were examined by standard fluorescence in situ hybridization (FISH) and developed by our group quantitative real-time PCR (qPCR). TOP2A amplification and deletion assessed by FISH were found in 23% and 7% of the tumours, respectively, and by qPCR in 31% and 11% of the tumours, respectively. Chromosome 17 polysomy was detected in 40% of the cases. TOP2A amplification (by qPCR) correlated with shorter disease-free survival (p = 0.03) and overall survival (p = 0.047), and the prognostic value of TOP2A was confirmed in the multivariate analysis (HR = 3.22, 95% CI 1.09-9.56, p = 0.03). TOP2A gene amplification, but not chromosome 17 polysomy, carries negative prognostic information in early breast cancer. Given the aforementioned results, qPCR might serve as a prognostic tool in determining the patient's prognosis.

The occurrence of either regional or distant metastases is an indicator of poor prognosis for cancer patients. The mechanism of their formation has not yet been fully uncovered, which limits the possibility of developing new therapeutic strategies. Nevertheless, the discovery of circulating tumor cells (CTCs), which are responsible for tumor dissemination, and cancer stem cells (CSCs), required for tumor growth maintenance, shed light on the metastatic cascade. It seems that CTCs and CSCs are not necessarily separate populations of cancer cells, as CTCs generated in the process of epithelial-mesenchymal transition (EMT) can bear features characteristic of CSCs. This article describes the mechanisms of CTC and CSC formation and characterizes their molecular hallmarks. Moreover, we present different types of EMT occurring in physiological and pathological conditions, and we demonstrate its crucial role in providing CTCs with a CSC phenotype. The article delineates molecular changes acquired by cancer cells undergoing EMT that facilitate metastasis formation. Deeper understanding of those processes is of fundamental importance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation.

Purpose: About 40% of non-small cell lung cancer (NSCLC) patients are candidates for systemic chemotherapy, despite the fact that at diagnosis most NSCLC are usually chemoresistant both in vivo and ex vivo. It is important to develop sufficient methods of prediction of the response to chemotherapy and to find molecular markers that may prognose this response. Therefore, a study on the relationship of p53 gene status to the ex vivo chemosensitivity of primary human NSCLC was performed. Methods: Three drug combinations (carboplatin/etoposide, cyclophosphamide/etoposide/epirubicin, and paclitaxel/carboplatin) were tested in a modified ATP cell viability assay. A group of 28 cases of primary human NSCLC was assessed. Results: Ex vivo chemosensitivity testing showed that tumors with p53 mutations were significantly more resistant to the cyclophosphamide/etoposide/epirubicin regimen than with normal p53 gene (P = 0.012). However, no correlation was observed for two other treatment regimens. Conclusion: Mutations in the p53 gene can lead to enhanced chemoresistance, confirming the hypothesis that the p53 gene may serve as a marker of tumor response to treatment in NSCLC. However, the data also illustrate that some additional factors might contribute to drug resistance of the examined tumors.

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.

Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are presumed to be key conditions for malignancy. Data concerning their role in endometrial cancer (EC) are scarce. We aimed to investigate the possible link between EMT and CSCs markers in EC samples.The study encompassed 156 primary tumour samples. Using RT-qPCR, we analyzed the expression of EMT-related genes, SNAIL and SLUG, and the CSCs marker CD133.SNAIL and SLUG correlated with each other (R=0.33; p=0.00003). All the studied genes were expressed in both normal and malignant endometrial tissue. Decreased SNAIL expression was found to correlate with post-menopausal status (p=0.002). Decreased SLUG expression was associated with shorter overall survival (p=0.01).SLUG expression could serve as a prognostic factor in EC. No correlation between the expression of EMT and CSCs markers was found, suggesting there to be no association between the EMT and CSC phenotype in endometrial cancer.

Breast cancers can metastasize via hematogenous and lymphatic routes, however in some patients only one type of metastases are detected, suggesting a certain proclivity in metastatic patterns. Since epithelial-mesenchymal transition (EMT) plays an important role in cancer dissemination it would be worthwhile to find if a specific profile of EMT gene expression exists that is related to either lymphatic or hematogenous dissemination. Our study aimed at evaluating gene expression profile of EMT-related markers in primary tumors (PT) and correlated them with the pattern of metastatic spread. From 99 early breast cancer patients peripheral blood samples (N = 99), matched PT (N = 47) and lymph node metastases (LNM; N = 22) were collected. Expression of TWIST1, SNAI1, SNAI2 and VIM was analyzed in those samples. Additionally expression of CK19, MGB1 and HER2 was measured in CTCs-enriched blood fractions (CTCs-EBF). Results were correlated with each other and with clinico-pathological data of the patients. Results show that the mesenchymal phenotype of CTCs-EBF correlated with poor clinico-pathological characteristics of the patients. Additionally, PT shared more similarities with LNM than with CTCs-EBF. Nevertheless, LNM showed increased expression of EMT-related markers than PT; and EMT itself in PT did not seem to be necessary for lymphatic dissemination.

Cohesins and cohesin-regulated genes are deregulated in numerous types of human cancer. However, data concerning their status and role in endometrial cancer are scarce. This study aimed to determine the clinical significance of double-strand-break repair protein rad21 homolog (RAD21) and runt-related transcription factor 1 (RUNX1) gene dosage and mRNA expression in endometrial cancer. RAD21 is a component of the cohesin complex, crucial for chromosome segregation and DNA repair. RUNX1 is the transcription factor implicated in RAD21 regulation. The study group included 144 endometrial cancer patients. RAD21 and RUNX1 expression profiles were measured by reverse-transcription quantitative PCR. RAD21 gene dosage was determined by quantitative PCR. RAD21 gene dosage was associated with RAD21 mRNA expression (ϱ=0.22; p=0.009). Furthermore, RAD21 expression strongly correlated with RUNX1 expression (ϱ=0.43; p

HER2 amplification and/or overexpression in breast cancer are adverse prognostic factors and can predict the response to trastuzumab therapy. As assessment of HER2 status in breast cancer is of great importance for clinical decision-making, it is crucial to have reliable methods to quantify HER2. In the present project, we have developed a modification of the direct-double-differential PCR method (dddPCR) for gene dosage quantification of HER2 in breast cancer samples. The study was also undertaken to establish the potential application of the newly developed dddPCR method for HER2 testing in a diagnostic laboratory. The dddPCR validity for determining HER2 amplification in comparison to IHC-based HercepTest for the detection of protein levels was performed. Modified dddPCR was proven to be highly reproducible and reliable. A statistically significant correlation between protein expression and gene dosage of HER2 was found in the examined material. The obtained results indicate that dddPCR provides a fast and reliable diagnostic tool for HER2 quantification and may be considered as a supplementary method to verify equivocal results obtained with IHC-based HercepTest. Notably, dddPCR can be performed in every basic molecular biology laboratory, and is inexpensive and time-saving.

Introduction Circulating tumor cells (CTCs) were shown to confer poor prognosis to breast cancer (BC) patients, however their clinical utility may further improve upon characterization. It is believed that CTCs are not equally malignant as they carry different metastases seeding potential. The aim of this study was to analyze the expression of epithelial and breast cancer-specific transcripts, as well as invasion and metastasis genes in CTCs-enriched blood fraction of BC patients and correlate it with available clinicopathological data. Materials and methods Peripheral blood samples from of 27 BC patients (11 N(-) and 16 N(+)) were used for CTCs isolation by density gradient centrifugation followed by depletion of CD45-positive cells. Applying ΔΔCt method in real-time PCR with hydrolysis probes, expression of nine genes was analyzed (TWIST1, SNAIL, SLUG, cytokeratin 19 (CK19), vimentin, mammaglobin-1, HER2, CXCR4, uPAR). A sample was considered positive for a particular marker when its expression was higher than in the blood samples from healthy controls. Results In the analyzed group 26% of the samples expressed CK19, out of which 14% was positive for mammaglobin-1. Expression of TWIST1, SNAIL and SLUG was found in 77%, 92% and 11% of the samples, respectively and it was not restricted to CK19-positive samples. Expression of TWIST1 and SNAIL correlated with each other (p = 0.04), however only SNAIL was associated with CXCR4 (p = 0.003) and uPAR (p = 0.008) expression. There was no association between expression of any of the genes studied and clinicopathological characteristics of the patients. Conclusions CK19 can be detected in patients independently of T and N stage, which supports parallel model of cancer metastasis. EMT seems to be involved in CTCs dissemination. In particular, SNAIL seems to be the strongest factor conferring invasiveness and metastatic features, observed as increased expression of uPAR and CXCR4 in CTCs-enriched samples. Disclosure All authors have declared no conflicts of interest.

ErbB-1, -2, -3 and -4 proteins are growth factor receptors, encoded by the family of respective erbB protooncogenes. These receptor-encoding proto-oncogenes frequently undergo amplification, and less frequently, a deletion, in several human neoplasms. The role of the ErbB family in human endocrine neoplasms, including pheochromocytoma (PHEO), was not extensively tested and not previously established. The expression/overexpression of erbB oncogenes in pheochromocytoma tissue was determined only in a few cases, and to the best of our knowledge, their mutations (amplification or deletion) were not examined in any series of PHEO cases. We, therefore, used a double differential polymerase chain reaction (ddPCR) for determination of the amplification/deletion profiles of erbB-1, -2, -3 and -4 genes in formalin-fixed, paraffin embedded (FFPE) specimens of human PHEOs. We examined the average gene copy number (AGCN) of the genes in 36 samples of pheochromocytomas (2 extra-adrenal and 34 adrenal tumors). We found the mean AGCNs of the oncogenes equal 1.18 for erbB-1 [amplification was found in 11/35 cases (31%) and deletion in 6/35 cases (17%)], 2.00 for erbB-2 [amplification was found in 8/34 cases (24%), no deletion was found], 1.36 for erbB-3 [amplification was found in 4/36 cases (11%) and deletion in 1/36 cases (3%)], and 1.22 for erbB-4 [amplification was found in 5/30 cases (17%) and deletion in 1/30 cases (3%)]. A mutation(s) of any erbB oncogene was found in 25/36 (69%) samples tested. Some abnormalities of the erbB oncogenes showed interesting correlations with one another and with clinical features of the tumors. The frequent occurrence of amplifications and deletions of the erbB oncogenes in human pheochromocytoma implies the importance of the gene family in the development of these tumors.

e16539 Background: Endometrial cancer (EC) is the most frequent malignancy of the female genital tract, with approximately 88,068 cases registered each year in the European Union. Discoveries of re...

e16540 Background: Dysregulation of the ERBB signaling network occurs in many human cancers and is associated with more aggressive tumor behavior and poor outcome. The data about the status of the ...

Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.

Background: The biology and pathomechanism of bilateral breast cancers is not fully understood: the risk of developing second (metachronous) breast cancer is 4-6 times higher than that of first primary in general population, whereas for synchronous breast cancers the number of cases is about 100 times higher than could be expected by chance alone. We compared the morphological and immunohistochemical characteristics of primary synchronous (sBBC) vs. metachronous (mBBC) bilateral breast cancers), with special focus on intrinsic tumor phenotype. Methods: Tumor morphology and expression of 8 immunohistochemical markers were assessed in tissue microarrays containing primary breast tumor cores from 113 mBBC and 61 sBBC. Analyzed markers included hormone receptors (estrogen receptor, progesterone receptor), HER2, Ki67, cytokeratin 5/6, E-cadherin, vimentin and epidermal growth factor receptor. Surrogate intrinsic phenotypes were determined according to St Gallen 2011 criteria. Results: mBBChad higher incidence of ductal histology (p = 0.037), lower estrogen receptor expression (p = 0.047), and higher expression of cytokeratin 5/6 (p = 0.017) and of vimentin (p = 0.008); in multivariate analysis only vimentin retained the significance (p = 0.01). Ten (13%) and 11 (26%) of mBBCand sBBC, respectively, had luminal A phenotype, 39 (50%) and 15 (36%) were luminal B HER2-negative, 13 (17%) and 12 (28%) - luminal B HER2-positive, 3 (4%) and 2 (5%) - HER2-positive (not luminal), and 12 (16%) and 2 (5%) had triple negative phenotype (p = 0.07). Characteristics of mBBC and sBBCFeaturemBBC (%)sBBC (%)p valueDuctal histology98 (87)47 (77)0.037Grade 346 (41)17 (28)0.09Extensive intraductal component26 (23)18 (29)0.42Strong ER expression73 (67)48 (81)0.047Strong PR expression55 (49)38 (63)0.07HER2 positivity (2+, 3+)51 (46)29 (50)0.6Ki76 ≤14%24 (22)21 (35)0.06Surrogate intrinsic phenotype luminal A10 (13)11 (26) luminal B HER2-39 (50)15 (36) luminal B HER2+13 (17)12 (28)0.07triple negative12 (16)2 (5) HER2+ (non luminal)3 (4)2 (5) CK5/6 >10%28 (26)6 (11)0.017Vimentin >10%25 (23)4 (7)0.008E-cadherin (+)88 (83)49 (83)0.99EGFR (+)35 (32)11 (19)0.07 Conclusion: mBBC, compared to sBBC, are characterized by more aggressive phenotype, expressed by lower expression of estrogen receptor and stronger expression of cytokeratin /6 and vimentin; this does not, however, translate into substantial differences in the proportion of intrinsic tumor phenotypes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-30.

Introduction ERBB (EGFR, epidermal growth factor receptor) family consists of four tyrosine kinase receptors: ERBB1, ERBB2, ERBB3 and ERBB4, which form a complex network of interacting signal transduction pathways. Excessive activation of the ERBB network, often found in tumors, is associated with rapid growth, proliferation, cell survival and also poor patient prognosis. Data about the importance of ERBB network in endometrial cancer is scarce. The purpose of this study was to determine the gene dosages of all the ERBB genes in endometrial cancer in the context of mutual dependence as well as clinicopathological parameters. Materials and methods The study group included 144 premenopausal and postmenopausal endometrial cancer patients, staged I-IV. Fresh frozen specimens, derived from primary tumors, were used as material for molecular analysis. Relative gene dosages of ERBB1, ERBB2, ERBB3 and ERBB4 were assessed by SYBR Green-based quantitative PCR, using ΔΔCt method. Results Gene dosages of individual ERBB genes correlated with each other. Particularly strong correlation occurred in case of ERBB2 and ERBB3 (p = 0.002), ERBB2 and ERBB4 (p Summary Pattern of genetic aberrations of ERBB network varies depending on the stage of the disease. Deregulation of ERBB system is associated with more aggressive tumor characteristics and appears to be crucial in tumor progression. Disclosure All authors have declared no conflicts of interest.

Introduction Aldehyde dehydrogenase 1 (ALDH1) has been identified as a marker of breast cancer (BC) stem cells. High expression of ALDH1 was shown to correlate with poor outcome of BC patients. Breast cancer susceptibility gene 1 (BRCA1), a well-known tumor suppressor gene frequently mutated or lost in hereditary BC, has been recently demonstrated to influence stem cells fate in in vitro studies. Nevertheless, its role in sporadic tumors is still poorly investigated. This study was designed to examine the clinical relevance of BRCA1 and ALDH1 in a cohort of sporadic BC patients. Materials and methods The study group included 163 patients with sporadic BC in stage I-III. Protein expression was examined by immunohistochemistry (IHC) on tissue-microarrays applying anti-human mouse monoclonal antibodies against BRCA1 (MS110, Calbiochem) and ALDH1 (Clone 44, BD Bioscience). The intensity of staining, the percentage of stained cells and staining localization were documented and compared to molecular and clinico-pathological parameters. Results ALDH1 was strongly positive in 14.7% of carcinomas whereas BRCA1 protein was expressed in 46.6% and lost in 54.4% of the examined cases. Inverse correlation between expression levels of BRCA1 and ALDH1 was found (p = 0.007). High ALDH1 expression was associated with high histological tumor grade (p = 0.03). Interestingly, co-expression of BRCA1 and ALDH1 correlated with shorter disease free survival (p = 0.03). Disease recurrence occurred in 33% of BRCA1 + /ALDH1+ cases (12/4) in comparison to 8% of BRCA1-/ALDH1+ cases (23/2). No correlation was found between BRCA1/ALDH1 phenotype and molecular subtypes of BC. Conclusions It was found that in sporadic BC co-expression of BRCA1 and ALDH1 is associated with poorer prognosis compared to BRCA1-/ALDH1+ phenotype. However, further studies would be needed to confirm these results. Disclosure All authors have declared no conflicts of interest.

Introduction Basal, mesenchymal and stem cell markers are recently the main focus of a scientific query in breast cancer. However, their clinical significance and relation to molecular subtypes different than basal-like still remain unrecognized. The objective of this study was to correlate CK 5/6 expression with epithelial-mesenchymal transition (EMT)-related and cancer stem cell markers, and to compare them to clinicopathological data and outcome of the patients. Materials and methods Fresh-frozen and FFPE tumor samples from 166 consecutive, stage I-III breast cancer patients were examined. Tumors were divided into 5 intrinsic subtypes based on the expression of ER, PgR, HER2, Ki-67 and/or tumor grade (according to St Gallen 2011 guidelines). Expression of TWIST1, SNAIL and SLUG was measured by RT-qPCR. Expression of CK 5/6, CD44, E-cadherin, vimentin, TWIST1, SNAIL and SLUG was analyzed by immunohistochemical staining of tissue microarrays. Results CK 5/6 staining correlated with vimentin (p Conclusions The expression of CK 5/6, vimentin and CD44 correlates with aggressive tumor characteristics and has the adverse prognostic impact in breast cancer, effect which is not restricted to basal-like or triple negative subtype. Poor prognosis of patients with CK 5/6-positive, vimentin-positive or CD44-positive tumors is not related to the activation of EMT process, which is believed to confer more aggressive phenotype. Further research is warranted to uncover the role of EMT process in different breast cancer subtypes. Disclosure All authors have declared no conflicts of interest.

Introduction C-myc, a protooncogene encoding a transcription factor, is commonly found amplified in breast carcinoma. C-myc amplification often leads to c-Myc overexpression and correlates with more aggressive course of the disease and poor prognosis. In hereby study c-Myc abnormal expression has been examined in the context of EMT (epithelial-mesenchymal transition) and CSCs (cancer stem cells) properties acquired by cells during carcinogenesis. Materials and methods Study group included 162 consecutive stage I-III breast cancer patients. EMT phenotype was defined as E-cadherin-negative and/or vimentin-positive. CSCs properties were determined based on ALDH1 (aldehyde dehydrogenase) staining. Expression of c-Myc, E-cadherin, vimentin and ALDH1 was analyzed by immunohistochemical staining of tissue microarrays (TMA-IHC) with the following antibodies: E-cadherin [Nch 38, Dako], vimentin [V9, Dako], ALDH1 [44/ALDH1, BD Biosciences], c-Myc [NCL-c-myc, Novocastra]. C-myc amplification was assessed by dddPCR (direct double differential). Based on the percentage of positively stained cells and the intensity of staining, samples were classified as positive or negative for a particular marker and compared to molecular and clinico-pathological data as well as patients' outcome. Results C-Myc expression occurred in 76% of cases (120/157). Median c-myc gene dosage was 0.84 and 0.64 in c-Myc positive and negative subgroup, respectively (p = 0.025). EMT was found in 23% (38/162) and ALDH1 positive result in 22% (36/161) of cases. C-Myc expression correlated with EMT phenotype (p = 0.009), with 65% of EMT-positive samples (24/37) occurring in c-Myc positive subgroup. 73% of ALDH1-positive cases (24/33) occurred in c-Myc positive subgroup (p = 0.012). No correlation between c-Myc status and patients' outcome was found. Conclusions C-Myc expression appears to be related to EMT phenotype and ALDH1 expression. As EMT renders tumor cells more invasive and metastatic and cells with increased ALDH1 activity possess stem/progenitor properties, the results are in compliance with the fact that c-Myc overexpression is characteristic for aggressive breast carcinoma. Disclosure All authors have declared no conflicts of interest.