Zdenek Kolar

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM⁺ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.

Triple negative breast cancers (TNBC) are a morphologically and genetically heterogeneous group of breast cancers with uncertain prediction of biological behavior and response to therapy. Epithelial to mesenchymal transition (EMT) is a dynamic process characterized by loss of typical epithelial phenotype and acquisition of mesenchymal characteristics. Aberrant activation of EMT can aggravate the prognosis of patients with cancer, however, the mechanisms of EMT and role of microRNAs (miRNAs) in EMT activation is still unclear. The aim of our study was to analyze miRNA expression within areas of TNBCs with cellular morphology that may be related to the EMT process and discuss possible associations. Out of all 3953 re-examined breast cancers, 460 breast cancers were diagnosed as TNBC (11.64%). With regard to complete tumor morphology preservation, the tissue samples obtained from core—cut biopsies and influenced by previous neoadjuvant therapy were excluded. We assembled a set of selected 25 cases to determine miRNA expression levels in relation to present focal spindle cell and apocrine cell morphology within individual TNBCs. We used descriptive (histological typing and morphology), morphometric, molecular (microdissection of tumor and non-tumor morphologies, RNA isolation and purification, microchip analysis) and bioinformatic analysis (including pathway analysis). The results were verified by quantitative real-time PCR (RT-qPCR) on an extended set of 70 TNBCs. The majority of TNBCs were represented by high—grade invasive carcinomas of no special type (NST) with medullary features characterized by well-circumscribed tumors with central necrosis or fibrosis and frequent tendency to spindle-cell and/or apocrine cell transformation. Apocrine and spindle cell transformation showed a specific miRNA expression profile in comparison to other tumor parts, in situ carcinoma or non-tumor structures, particularly down-regulated expression of hsa-miRNA-143-3p and hsa-miRNA-205-5p and up-regulated expression of hsa-miR-22-3p, hsa-miRNA-185-5p, and hsa-miR-4443. Apocrine cell tumor morphology further revealed decreased expression of hsa-miR-145-5p and increased expression of additional 14 miRNAs (e.g. hsa-miR-182-5p, hsa-miR-3135b and hsa-miR-4417). Pathway analysis for target genes of these miRNAs revealed several shared biological processes (i.e. Wnt signaling, ErbB signaling, MAPK signaling, endocytosis and axon guidance), which may in part contribute to the EMT and tumor progression. We provide the first miRNA expression profiling of specific tissue morphologies in TNBC. Our results demonstrate a specific miRNA expression profile of apocrine and spindle cell morphology which can exhibit a certain similarity with the EMT process and may also be relevant for prognosis and therapy resistance of TNBC.

To date, no comprehensive prognostic or predictive marker profiling analysis has been performed in association with the age of patients with breast cancer. In the present study, 632 breast cancer tissue samples were analyzed for expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), B-cell lymphoma (Bcl)-2 protein, HER2 gene amplification, proliferation [as evaluated by proliferating cell nuclear antigen (PCNA) and Ki-67 index], tumor grade, histological type and molecular subtype. The data revealed correlations with the age of patients. A statistically significant positive correlation was identified between patient age and expression of ER (P<0.0001). There was no significant association between patient age and PR, HER2 protein expression, HER2 gene amplification or PCNA. A significant negative correlation between age and Ki-67 expression (P<0.0001) as well as grade of tumor (P=0.007) was identified. The spectrum of molecular subtypes differed according to age (P=0.0003). The highest incidence of aggressive triple-negative and HER2-positive breast cancer was present in patients aged between 20 and 39 years. Luminal A subtype was the most frequent cancer subtype in patients from age 40 onwards, where proliferation activity declined with age and expression of hormone receptors increased along with Bcl-2 expression. Aggressive forms of breast cancer were more common in younger patients. Prognostic and predictive markers have a complex age-specific distribution. The findings of less aggressive luminal A and B subtypes in older patients, and the positive correlation with ER, PR and Bcl-2 expression reveal the potential efficacy of Bcl-2 as a marker of hormone responsiveness in these patients.

The pathogenesis of prostate cancer (CaP) involves alterations in a gene structure of the androgen receptor (AR). The single nucleotide polymorphism AR-E211 G > A localized in exon 1 of the AR gene (G1733A) was detected using direct polymerase chain reaction and restriction digestion (PCR-RFLP) method on blood and tissue samples without prior DNA isolation. We used blood samples of patients with a diagnosis of benign prostatic hyperplasia (BPH) or CaP. From monitored group of CaP patients were selected specimen in formalin-fixed paraffin-embedded tissue blocks with morphology of BPH and CaP. The main objective of our study was to develop a method based the direct PCR-RFLP analysis from blood and tissue without prior DNA isolation for faster genotyping analysis of a large number of samples. We found no statistically significant differences in allelic % of the AR-E211 G > A polymorphism between BPH and CaP patients (p ≤ 0.8462). Genotyping of the AR-E211 G > A variant in blood was not identical with tumor tissue genotyping analysis. Significant agreement between blood and tissue AR-E211 G > A polymorphism only in non-tumor tissue focus was confirmed. Although we analyzed a limited number of the tissue samples, we suppose that a presence of the minor allele A may be associated with cancer transformation-induced changes of the modified AR gene.

Primary primitive neuroectodermal tumors (PNETs) are extremely rare in the lung and especially in adult women. We describe a case of PNET of the lung with aggressive behavior in 31-year-old woman. Diagnosis was based on histopathological and immunohistochemical studies, and confirmed by molecular genetic analysis of chromosome rearrangements in the EWSR1 gene region. Clinical follow-up, post-mortem findings, and differential diagnosis are also discussed.

Androgen receptor (AR) expression in prostate cancer (CaP) cells varies due to the multiple changes including epigenetic modifications such as DNA methylation and histone deacetylation. DNA methyltransferase and histone deacetylase inhibitors are promising for the treatment of CaP. The aim of our study was to analyze the 5-aza-2'-deoxycytidine (Aza‑dC) and sodium butyrate (NaB) effects on CaP cells with modified AR gene expression. The androgen-independent human prostate cancer cell lines PC3 (lacking a functional AR) and DU145 (strongly limited expression due to methylations in the AR gene) were used. PCR of bisulfite-modified DNA and RT-PCR with bisulfite-sequencing were used for AR gene analysis of DU145 and PC3 cells following their treatment with Aza-dC and/or NaB. Re-acetylated histones around the AR gene were detected by conventional PCR of immunoprecipitated DNA obtained from treated cells. In both cell lines without the AR expression, the combined treatment was followed with significant decrease of cell viability. The co-treatment of DU145 cells caused site-specific demethylation in the AR promoter region followed by gene re-expression and increased acetylation in histones H3 and H4. The co-treatment with Aza-dC and NaB was the most effective in demethylation and re-expression of the AR gene. In the AR gene promoter, the location and density of deme-thylated CpGs indicated the existence of distinct promoter hot spot that could be a target of AR gene inactivation therapy of CaP patients during androgen deprivation.

Background: Nestin is an intermediary filament protein, expressed in progenitor cells of neural and muscle origin and in activated endothelium. The expression of this protein in tumours can be associated with degree of differentiation, biologi...