Breast cancer circulating tumor cells with mesenchymal features-an unreachable target?

Circulating tumor cells (CTCs) mediate dissemination of solid tumors and can be an early sign of disease progression. Moreover, they show a great potential in terms of non-invasive, longitudinal monitoring of cancer patients. CTCs have been extensively studied in breast cancer (BC) and were shown to present a significant phenotypic plasticity connected with initiation of epithelial-mesenchymal transition (EMT). Apart from conferring malignant properties, EMT affects CTCs recovery rate, making a significant portion of CTCs from patients' samples undetected. Wider application of methods and markers designed to isolate and identify mesenchymal CTCs is required to expand our knowledge about the clinical impact of mesenchymal CTCs. Therefore, here we provide a comprehensive review of clinical significance of mesenchymal CTCs in BC together with statistical analysis of previously published data, in which we assessed the suitability of a number of methods/markers used for isolation of CTCs with different EMT phenotypes, both in in vitro spike-in tests with BC cell lines, as well as clinical samples. Results of spiked-in cell lines indicate that, in general, methods not based on epithelial enrichment only, capture mesenchymal CTCs much more efficiently that CellSearch® (golden standard in CTCs detection), but at the same time are not much inferior to Cell Search®, though large variation in recovery rates of added cells among the methods is observed. In clinical samples, where additional CTCs detection markers are needed, positive epithelial-based CTCs enrichment was the most efficient in isolating CTCs with mesenchymal features from non-metastatic BC patients. From the marker side, PI3K and VIM were contributing the most to detection of CTCs with mesenchymal features (in comparison to SNAIL) in non-metastatic and metastatic BC patients, respectively. However, additional data are needed for more robust identification of markers for efficient detection of CTCs with mesenchymal features.

Skin Microbiome in Prurigo Nodularis.

Prurigo nodularis (PN) is a chronic condition characterized by the presence of nodular lesions accompanied by intense pruritus. The disease has been linked to several infectious factors, but data on the direct presence of microorganisms in the lesions of PN are scarce. The aim of this study was to evaluate the diversity and composition of the bacterial microbiome in PN lesions by targeting the region V3-V4 of 16S rRNA. Skin swabs were obtained from active nodules in 24 patients with PN, inflammatory patches of 14 patients with atopic dermatitis (AD) and corresponding skin areas of 9 healthy volunteers (HV). After DNA extraction, the V3-V4 region of the bacterial 16S rRNA gene was amplified. Sequencing was performed using the Illumina platform on the MiSeq instrument. Operational taxonomic units (OTU) were identified. The identification of taxa was carried out using the Silva v.138 database. There was no statistically significant difference in the alpha-diversity (intra-sample diversity) between the PN, AD and HV groups. The beta-diversity (inter-sample diversity) showed statistically significant differences between the three groups on a global level and in paired analyses. Staphylococcus was significantly more abundant in samples from PN and AD patients than in controls. The difference was maintained across all taxonomic levels. The PN microbiome is highly similar to that of AD. It remains unclear whether the disturbed composition of the microbiome and the domination of Staphylococcus in PN lesions may be the trigger factor of pruritus and lead to the development of cutaneous changes or is a secondary phenomenon. Our preliminary results support the theory that the composition of the skin microbiome in PN is altered and justify further research on the role of the microbiome in this debilitating condition.

Higher platelet counts correlate to tumour progression and can be induced by intratumoural stroma in non-metastatic breast carcinomas.

BACKGROUND: Platelets support tumour progression. However, their prognostic significance and relation to circulating tumour cells (CTCs) in operable breast cancer (BrCa) are still scarcely known and, thus, merit further investigation. METHODS: Preoperative platelet counts (PCs) were compared with clinical data, CTCs, 65 serum cytokines and 770 immune-related transcripts obtained using the NanoString technology. RESULTS: High normal PC (hPC; defined by the 75th centile cut-off) correlated with an increased number of lymph node metastases and mesenchymal CTCs in the 70 operable BrCa patients. Patients with hPC and CTC presence revealed the shortest overall survival compared to those with no CTC/any PC or even CTC/normal PC. Adverse prognostic impact of hPC was observed only in the luminal subtype, when 247 BrCa patients were analysed. hPC correlated with high content of intratumoural stroma, specifically its phenotype related to CD8+ T and resting mast cells, and an increased concentration of cytokines related to platelet activation or even production in bone marrow (i.e. APRIL, ENA78/CXCL5, HGF, IL16, IL17a, MDC/CCL22, MCP3, MMP1 and SCF). CONCLUSIONS: Preoperative platelets evaluated alone and in combination with CTCs have prognostic potential in non-metastatic BrCa and define patients at the highest risk of disease progression, putatively benefiting from anti-platelet therapy.

Chimeric virus-like particles presenting tumour-associated MUC1 epitope result in high titers of specific IgG antibodies in the presence of squalene oil-in-water adjuvant: towards safe cancer immunotherapy.

BACKGROUND: Immunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP-based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy. RESULTS: In this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen. CONCLUSIONS: The obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens.

Alpha-smooth muscle actin-positive cancer-associated fibroblasts secreting osteopontin promote growth of luminal breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) have been shown to support tumor development in a variety of cancers. Different markers were applied to classify CAFs in order to elucidate their impact on tumor progression. However, the exact mechanism by which CAFs enhance cancer development and metastasis is yet unknown. METHODS: Alpha-smooth muscle actin (α-SMA) was examined immunohistochemically in intratumoral CAFs of nonmetastatic breast cancers and correlated with clinicopathological data. Four CAF cell lines were isolated from patients with luminal breast cancer (lumBC) and classified according to the presence of α-SMA protein. Conditioned medium (CM) from CAF cultures was used to assess the influence of CAFs on lumBC cell lines: MCF7 and T47D cells using Matrigel 3D culture assay. To identify potential factors accounting for promotion of tumor growth by α-SMAhigh CAFs, nCounter PanCancer Immune Profiling Panel (NanoString) was used. RESULTS: In luminal breast cancer, presence of intratumoral CAFs expressing high level of α-SMA (13% of lumBC group) correlated with poor prognosis (p = 0.019). In in vitro conditions, conditioned medium obtained from primary cultures of α-SMA-positive CAFs isolated from luminal tumors was observed to enhance growth of lumBC cell line colonies in 3D Matrigel, in contrast to CM derived from α-SMA-negative CAFs. Multigene expression analysis indicated that osteopontin (OPN) was overexpressed in α-SMA-positive CAFs in both clinical samples and in vitro models. OPN expression was associated with higher percentage of Ki67-positive cells in clinical material (p = 0.012), while OPN blocking in α-SMA-positive CAF-derived CM attenuated growth of lumBC cell line colonies in 3D Matrigel. CONCLUSIONS: Our findings demonstrate that α-SMA-positive CAFs might enhance tumor growth via secretion of OPN.

EGFR as a stable marker of prostate cancer dissemination to bones.

BACKGROUND: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men. Although 5-year survival in patients with localised disease reaches nearly 100%, metastatic disease still remains incurable. Therefore, there is a need for markers indicating metastatic dissemination. METHODS: EGFR overexpression (EGFRover) was tracked in 1039 primary tumours, circulating tumour cells from 39 d'Amico high-risk patients and metastatic samples from 21 castration-resistant PCa cases. EGFR status was compared to clinical parameters and multiple molecular factors were assessed using immunohistochemistry and gene ontology analysis. The functional aspect of EGFR was evaluated by plating PC-3 cells on soft and rigid matrices. RESULTS: EGFRover was found in 14% of primary tumours, where it was associated with shorter metastasis-free survival and was an independent indicator of worse overall survival. EGFRover correlated with a pro-migratory and pro-metastatic phenotype of tumour cells as well as rich collagen fibre content. All circulating tumour cells (detected in 13% of cases) were positive for EGFR, independent of their EMT-related phenotype. EGFRover was more prevalent in castration-resistant bone metastases (29% of patients) and supported growth of human PCa cells on rigid matrices mimicking bone stiffness. CONCLUSIONS: EGFRover is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.

Circulating Tumor Cells in Early and Advanced Breast Cancer; Biology and Prognostic Value.

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.

Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation.

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.

High-grade non-small cell lung carcinoma: a comparative analysis of the phenotypic profile in small biopsies with the corresponding postoperative material.

The most recent classification of the lung cancer expanded the diagnostic criteria of its histological subtypes and included its immunophenotypic profile. We performed the study to compare the reliability of selected markers in high-grade non-small cell lung carcinoma (NSCLC) in the oligobiopsies with the matched postoperative samples. We evaluated expression of p40, p63, TTF1, cytokeratin 5/6, cytokeratin 7, napsin A, desmoglein 3, desmocollin 3 and mucin secretion as detected by mucicarmine staining. The study cohort included 123 cases of poorly-differentiated NSCLC. The tissue oligobiopsy material was available in 38 cases. Tissue microarrays (TMAs) from all postoperative cases were constructed. Comparing the immunophenotype between postsurgical samples and oligobiopsies we found an almost perfect agreement for most of performed IHC reactions. The highest concordance of results was found for desmoglein 3, CK7, and p40, whereas the lowest - for desmocollin 3. Immunoprofile of the oligobiopsies corresponded well to that in the resection specimens. The most useful markers in poorly differentiated ADs are: TTF1 and napsin A, and for non-keratinizing SCCs: p40, p63, CK5/6 and desmoglein 3.

The Prognostic Significance of Eukaryotic Translation Initiation Factors (eIFs) in Endometrial Cancer.

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 (eIF2α, 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 < 0.001), whilst median eIF6 CS were significantly lower in EC (p < 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 < 0.001), peIF2α (p < 0.001) and eIF3h (p < 0.05) were significantly overexpressed in EC, while expression of eIF3c was significantly reduced in EC as compared with NNT (p < 0.001). The remaining eIFs were non-significant. Besides tumour stage (p < 0.001) and patient's age (p < 0.001), high eIF4g CS-levels were independently associated with poor prognosis (HR: 1.604, 95%CI: 1.037-2.483, 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.

Clinical and Biological Significance of ESR1 Gene Alteration and Estrogen Receptors Isoforms Expression in Breast Cancer Patients.

The amplification of estrogen receptor alpha (ERα) 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 ERα (other than classical ERα 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 ERα isoforms-ERα66 and ERα36-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 ERα66 isoform (p = 0.01). Interestingly, the short ER isoform ERα36 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 ERα36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ERα66 level in breast tumors.

Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains.

Resistance of Mycobacterium tuberculosis to rifampin (RMP), mediated by mutations in the rpoB gene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individual rpoB mutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout the rpoB gene among 115 Mycobacterium tuberculosis clinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discovered rpoB mutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results from in vitro mutagenesis experiments were combined with the assessment of the prevalence of rpoB mutations, and their reciprocal co-occurrences, across global M. tuberculosis populations. Twenty-two different types of mutations in the rpoB gene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50 and MIC90 values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenic M. tuberculosis H37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

Sensitive Detection of Single-Cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters.

As an important signaling molecule, hydrogen peroxide (H2O2) secreted externally by the cells influences cell migration, immunity generation, and cellular communications. Herein, we have developed a microfluidic approach with droplets in combination with Au nanoclusters for the sensitive detection of H2O2 secreted by a single cell. Isolated in the ultrasmall volume (4.2 nL) of a microdroplet, single-cell secreted H2O2 can initiate dramatic fluorescence changes of horseradish peroxidase-Au nanoclusters. We have demonstrated an ultrahigh sensitivity (200-400 attomole H2O2 directly measured from a single cell) with good specificity. It offers a useful research tool to study the cell-to-cell differences of H2O2 secretion at the single-cell level.

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria.

Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed.

The Landscape of Circulating Tumor Cell Research in the Context of Epithelial-Mesenchymal Transition.

The metastatic spread of cancer accounts for the vast majority of cancer-related deaths. It is mediated by tumor cells circulating in blood (called circulating tumor cells, CTCs), which escaped from their established niches. CTCs give a unique opportunity to look into the metastatic cascade and to study the molecular processes supporting the spread of tumor cells throughout the body. As current therapies are not sufficiently effective in treating metastatic disease, it is important to determine cellular and molecular features of cancer cells that "seed" new tumors in distant organs at early stages. In this review we focus on the role of the epithelial-mesenchymal transition program in providing a survival advantage to metastasizing tumor cells, especially CTCs, and put it in the context of clinical findings.

Interactions between FGFR2 and RSK2-implications for breast cancer prognosis.

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.

Keratin 7 expression in lymph node metastases but not in the primary tumour correlates with distant metastases and poor prognosis in colon carcinoma.

Colorectal carcinoma (CRC) is one of the leading causes of cancer-related deaths worldwide. Alterations in keratin expression, including keratin 7 (K7), are frequent findings in multiple cancers, and they constitute a prognostic factor. The aim of our study was to evaluate the prognostic significance of K7 in the primary tumour and lymph node metastases in two separate cohorts of patients: the first one with lymph node involvement (LN+, 129 cases) and the second one free of LN metastases (LN-, 85 cases). Keratin 7 expression in CRC was analysed on tissue microarrays with immunohistochemistry and evaluated using the h-score. In the LN+ group K7 positivity was identified in 7/129 (5.4%) of primary tumours (PT) and lymph node metastases (LNM); concordance between them was 94% ( 0.396). Keratin 7 was expressed in 8/85 cases (9.4%) in the LN- group. K7 expression in LNM of the LN+ cohort correlated with shorter overall survival (OS) (p = 0.047) and presence of distant metastases at diagnosis (p = 0.005). Expression of K7 in the primary tumour in both cohorts did not correlate with survival. We conclude that the status of K7 expression in metastatic lymph nodes from CRC is a poor prognostic factor.

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration.

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.

PCR melting profile as a tool for outbreak studies of Salmonella enterica in chickens.

BACKGROUND: Salmonellosis is of great economic concern in all phases of the poultry industry, from production to marketing, leading to severe economic losses. Monitoring the source of the bacterial contamination has fundamental importance in the spreading of salmonellosis. RESULTS: We applied a ligation-mediated PCR method, PCR MP (PCR melting profile), to type S. enterica ssp. enterica ser. Enteritidis (56 strains) and 43 control strains classified to other serovars isolated from poultry. We demonstrated the PCR MP potential for salmonellosis spreading monitoring. Our rapid test presents higher discriminatory power (0.939 vs. 0.608) compared to current molecular subtyping tool such as pulsed-field gel electrophoresis (PFGE), which ineffectiveness underlies the high degree of clonality of S. Enteritidis. CONCLUSIONS: PCR MP was found to be a highly discriminating, sensitive and specific method that could be a valuable molecular tool, particularly for analyzing epidemiological links of limited number of S. enterica ser. Enteritidis strains.

CD99 correlates with low cyclin D1, high topoisomerase 2 status and triple negative molecular phenotype but is prognostically irrelevant in breast carcinoma.

CD99 is a protein initially described in the Ewing sarcoma family of tumors, but growing evidence has shown its expression in other tumors of mesenchymal, hematopoietic and even epithelial origin. Some articles report CD99 in metaplastic carcinoma of the breast, a subtype of breast carcinoma (BC) with pronounced epithelial to mesenchymal (EMT) phenotype. Our aim was to analyse the potential relationship between CD99 and selected EMT (vimentin, E-cadherin, Twist) and proliferation markers (Ki-67, c-myc, cyclin D1, topoisomerase 2), molecular subtypes of BC, as well as overall survival (OS) and progression-free survival (PFS). In a group of 122 cases CD99 membrane expression was seen in 14 (11.5%) cases: strong in 11 (9%) and moderate in 3 (2.5%). Expression of CD99 correlated with low cyclin D1 index, high level of topoisomerase 2 expression and lack of progesterone receptor (PR) but not with EMT characteristics. Additionally, strong expression of CD99 correlated with triple negative molecular BC phenotype. CD99 was prognostically irrelevant for OS and PFS. CD99 correlates with selected proliferative markers and low ER/PR receptor status but not with patients' outcome in BC. Further studies are required to explain precisely its role in molecular pathogenesis of BC.