Heterogeneity of Mesenchymal Markers Expression—Molecular Profiles of Cancer Cells Disseminated by Lymphatic and Hematogenous Routes in Breast 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

Altered circadian genes expression in breast cancer tissue according to the clinical characteristics.

Breast cancer has a multifactorial etiology. One of the supposed and novel mechanisms is an alteration of circadian gene expression. Circadian genes play a crucial role in many physiological processes. These processes, such as genomic stability, DNA repair mechanism and apoptosis, are frequently disrupted in breast tumors. To assess the significance of circadian gene expression in breast cancer, we carried out an analysis of CLOCK, BMAL1, NPAS2, PER1, PER2, PER3 and CRY1, CRY2, TIMELESS, CSNK1E expression by the use of the quantitative Real-Time PCR technique in tumor tissue and non-tumor adjacent normal tissue sampled from 107 women with a newly diagnosed disease. The obtained data were compared to the clinical and histopathological features. PER1, PER2, PER3, CRY2 were found to be significantly down-expressed, while CLOCK, TIMELESS were over-expressed in the studied tumor samples compared to the non-tumor samples. Only gene expression of CRY1 was significantly down-regulated with progression according to the TNM classification. We found significantly decreased expression of CRY2, PER1, PER2 genes in the ER/PR negative breast tumors compared to the ER/PR positive tumors. Additionally, expression of CRY2, NPAS2 genes had a decreased level in the poorly differentiated tumors in comparison with the well and moderately differentiated ones. Our results indicate that circadian gene expression is altered in breast cancer tissue, which confirms previous observations from various animal and in vitro studies

Expression of epithelial to mesenchymal transition-related markers in lymph node metastases as a surrogate for primary tumor metastatic potential in breast cancer

Abstract Background Breast cancers are phenotypically and genotypically heterogeneous tumors containing multiple cancer cell populations with various metastatic potential. Aggressive tumor cell subpopulations might more easily be captured in lymph nodes metastases (LNM) than in primary tumors (PT). We evaluated mRNA and protein levels of master EMT regulators: TWIST1, SNAIL and SLUG, protein levels of EMT-related markers: E-cadherin, vimentin, and expression of classical breast cancer receptors: HER2, ER and PgR in PT and corresponding LNM. The results were correlated with clinicopathological data and patients outcomes. Methods Formalin-fixed paraffin-embedded samples from PT and matched LNM from 42 stage II-III breast cancer patients were examined. Expression of TWIST1, SNAIL and SLUG was measured by reverse-transcription quantitative PCR. Protein expression was examined by immunohistochemistry on tissue microarrays. Kaplan-Meier curves for disease-free survival (DFS) and overall survival (OS) were compared using F-Cox test. Hazard ratios (HRs) with 95% confidence intervals (95% CI) were computed using Cox regression analysis. Results On average, mRNA expression of TWIST1, SNAIL and SLUG was significantly higher in LNM compared to PT (P TWIST1 and SNAIL in LNM was associated with shorter OS (P = 0.04 and P = 0.02, respectively) and DFS (P = 0.02 and P = 0.01, respectively), whereas their expression in PT had no prognostic impact. Negative-to-positive switch of SNAIL protein correlated with decreased OS and DFS (HR = 4.6; 1.1-18.7; P = 0.03 and HR = 3.8; 1.0-48.7; P = 0.05, respectively). Conclusions LNM are enriched in cells with more aggressive phenotype, marked by elevated levels of EMT regulators. High expression of TWIST1 and SNAIL in LNM, as well as negative-to-positive conversion of SNAIL confer worse prognosis, confirming the correlation of EMT with aggressive disease behavior. Thus, molecular profiling of LNM may be used as surrogate marker for aggressiveness and metastatic potential of PT.</p

Chemometric Evaluation of Urinary Steroid Hormone Levels as Potential Biomarkers of Neuroendocrine Tumors

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL−1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1–300 ng mL−1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed

Relative gene expression levels in CTC-enriched blood fractions from breast cancer patients.

<p>*Number of positive and negative samples for a particular gene, percentages of samples classified as marker-positive with the chosen cut-off levels as well as average expression with standard deviation (SD), minimal and maximal measured relative expression level.</p><p>**In the healthy control group only genes which expression was found in the samples are shown.</p

Relative expression level of <i>CXCR4</i> and <i>uPAR</i> in CTC-enriched blood fractions.

<p>CTC-enriched blood fractions positive for either <i>MGB1</i> or <i>HER2</i> were divided according to <i>VIM</i> and <i>CK19</i> expression status into three groups (<i>CK19−/VIM−</i>; <i>CK19+/VIM−</i>; <i>CK19−/VIM+</i>). Error bars depict standard error. * - statistically significant difference in <i>CXCR4</i> and <i>uPAR</i> relative expression level (<i>P</i><0.00001 for both) between <i>CK19−/VIM−</i> and <i>CK19−/VIM+</i>; ** - statistically significant difference in <i>CXCR4</i> (<i>P</i> = 0.00002) and <i>uPAR</i> (<i>P</i> = 0.00004) relative expression level between <i>CK19+/VIM</i>− and <i>CK19−/VIM+</i>.</p

Correlations between genes expression status in CTC-enriched blood fractions.

<p>Number of cases classified as positive (Pos.) or negative (Neg.) for a particular marker are shown. Statistically significant <i>P</i> values are given in bold.</p

Results of univariate and multivariate logistic regression analysis in relation to lymph node involvement.

<p>N- lymph node involvement absent, N+ lymph node involvement present. OR-overall risk; CI – confidence interval, NS-not statistically significant.</p

Correlation between CTC-enriched blood fractions gene expression status and clinicopathological parameters.

<p>Number of cases classified as positive (Pos.) or negative (Neg.) for a particular marker in each group are shown. Statistically significant <i>P</i> values are given in bold.</p><p>*CTC-enriched blood fraction phenotype – only <i>MBG1</i>+ and/or <i>HER2</i>+ fractions were considered</p><p>** <i>CK19−/VIM+</i> - mesenchymal phenotype; <i>CK19+/VIM−</i> - epithelial phenotype.</p