Pilot study of gene mutations associated with Lynch syndrome in Slovak patients with breast cancer.

BACKGROUND: Lynch syndrome (LS) is an autosomal dominant inherited disorder which causes an increased risk of cancer, especially colorectal and endometrial carcinomas. Recent studies have shown an association between LS and breast cancer as well. The aim of our study is to highlight the possible presence of mutations in genes associated with LS in patients with breast cancer and the need to include the examination of Lynch-associated genes in patients with a family history of breast cancer as well as in patients with recurrent breast cancer, as well as with the occurrence of other Lynch-associated cancer. MATERIALS AND METHODS: We analyzed tumor tissue samples from 78 patients with primary breast cancer. Our samples were tested with a gene panel associated with the risk of developing breast cancer, while in our study we focused primarily on the occurrence of mutations in mismatch-repair genes. DNA isolated from tumor tissue was sequenced using next generation sequencing (NGS) and analyzed using the Ingenuity Variant Analysis tool. To confirm the germline mutation, we examined the patient's blood sample using NGS sequencing. RESULTS: As a result of our analysis, we managed to identify a mutation in the PMS2 gene in one patient's breast tumor tissue. The presence of this mutation indicates that the resulting cancer may be a consequence of LS. As for pathogenicity, this was probably a pathogenic variant, as we detected deletions in the exon region, which led to frameshift mutation. Moreover, we also identified single-nucleotide pathogenic variants in the TP53 and PIK3CA genes. To definitively establish the diagnosis of LS in the patient, we examined a blood sample, where we also identified a mutation of the PMS2 gene. CONCLUSION: LS is underdiagnosed in many Lynch-associated cancers. However, in the case of a familial occurrence of breast cancer and other Lynch-associated genes, it is important to think about a possible diagnosis of LS and, if the patient meets the diagnostic criteria, to carry out a genetic examination of Lynch-associated genes.

CXCR4-SDF-1 interaction potentially mediates trafficking of circulating tumor cells in primary breast cancer.

BACKGROUND: Cytokines are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play key role in tumor dissemination and are an independent survival predictor in breast cancer patients. The aim of this study was to assess correlation between CTCs and plasma cytokines in primary breast cancer (PBC) patients. METHODS: This study included 147 chemotherapy naïve PBC patients. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoetic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (Twist1, Snail1, Slug, Zeb1) and epithelial (Ck19) gene transcripts by qRT-PCR. The concentrations of 51 plasma cytokines were measured using multiplex bead arrays. RESULTS: CTCs were detected in 25.2% patients. CTCs exhibiting only epithelial markers (CTC_EP) and only EMT markers (CTC_EMT) were present evenly in 11.6% patients, while CTCs co-expressing both markers were detected in 2.0% patients. Patients with presence of CTC_EP in peripheral blood had significantly elevated levels of plasma IFN-α2, IL-3, MCP-3, ß-NGF, SCF, SCGF-ß, TNF-ß and SDF-1 compared to patients without CTC_EP. CTC_EP exhibited overexpression of SDF-1 receptor and CXCR4, but not other corresponding cytokine receptor, and in multivariate analysis SDF-1 was independently associated with CTC_EP. There was an inverse correlation between CTC_EMT and plasma cytokines CTACK, ß-NGF and TRAIL, while presence of either subtype of CTCs was associated with increased level of TGF-ß2. CONCLUSION: Using cytokine profiling, we identified cytokines associated with CTCs subpopulations in peripheral blood of PBC. Our data suggest that CXCR4-SDF-1 axis is involved in mobilization and trafficking of epithelial CTCs.

Relationship between circulating tumor cells and epithelial to mesenchymal transition in early breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. Epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue. METHODS: This study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. Expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score. RESULTS: CTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT. CONCLUSIONS: In this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features.

Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer.

Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.

Predictors of inferior outcome in community acquired bacterial meningitis.

The aim of this study was to assess mortality and sequellae within cases from Nationwide survey of community acquired meningitis and identify risk factors for inferior outcome. Risk factors such as underlying disease (diabetes mellitus, cancer, trauma, neonatal age, splenectomy, alcoholism, sepsis, other infections), etiology, clinical symptoms and outcome (death, improvement and cured after modifications of ATB therapy, cured without change of therapy, cured with neurologic sequellae) were recorded and analysed with univariate analysis (chi2 or t test for trends, CDC Atlanta 2004). Analysing risk factors for inferior outcome (death or cured with neurologic sequellae), we compared patients who died or survived with neurologic sequellae to all patients with community acquired bacterial meningitis. Univariate analysis showed that trauma (p<0.05), alcohol abuse (p<0.05), diabetes, S. aureus (p<0.05) and gram-negative etiology (A. baumannii, Ps. aeruginosa or Enterobacteriaceae) (36% vs. 11,9%, p<0.05) were predicting inferior outcome. Analysing risk factors for treatment failure (death or failed but cured after change of antibiotic treatment) prior sepsis (34.1% vs. 13.9%, p<0.01) and gram-negative etiology (25% vs. 11.9%, p<0.02) were statistically significant predictors of treatment failure. Neisseria meningitis had less failures (p<0.05). Concerning infection associated mortality again diabetes mellitus (p<0.05), alcoholism (p<0.05) staphylococcal and gram-negative etiology (p<0.05) were significant predictors of death. N. meningitis had surprisingly less treatment failures (appropriate and rapid initial therapy). Neurologic sequellae were more common in patients with alcohol abuse (p<0.05), craniocerbral trauma (p<0.05) and less common in meningitis with pneumococcal etiology (p<0.05).

Comparison of postsurgical and community acquired bacterial meningitis--analysis of 372 cases within a nationwide survey.

The aim of this study was to assess if differences in etiology and risk factors among 372 cases of bacterial meningitis acquired after surgery (PM) or in community (CBM) have impact on outcome of infected patients. Among 372 cases of bacterial meningitis within last 17 years from 10 major Slovak hospitals, 171 were PM and 201 CBM. Etiology, risk factors such as underlying disease, cancer, diabetes alcoholism, surgery, VLBW, ENT infections, trauma, sepsis were recorded and mortality, survival with sequellae, therapy failure were compared in both groups. Significant differences in etiology and risk factors between both groups were reported. Those after neurosurgery had more frequently Coagulase negative staphylococci (p<0.001), Enterobacteriaceae (p=0.01) and Acinetobacter baumannii (p=0.0008) isolated from CSF and vice versa Streptococcus pneumoniae (p<0.001), Neisseria meningitis (p<0.001) and Haemophillus influenza (p=0.0009) were more commonly isolated from CSF in CBM. Neurosurgery (p<0.001), sepsis (p=0.006), VLBW neonates (p=0.00002) and cancer (p=0.0007) were more common in PM and alcohol abuse (p<0.001) as well as otitis/sinusitis (p<0.001) and Roma ethnic group (p=0.001) in CAM. Initial treatment success was significantly more frequently observed among CAM (p<0.001) but cure after modification was more common in PM (p=0.002). Therefore outcome in both groups was similar (14.6% vs. 12.4%, p=NS).

Meningococcal meningitis is still the commonest neuroinfection in the community in tropics: overview of 62 cases.

Within last 17 years we went through all charts of bacterial meningitis within our nationwide survey and among 372 cases we found 62 cases of MM, in 12 cases with meningococcal disease (with shock, petechial effusions or disseminated intravascular coagulation or digital gangrenes). MM was usually observed in young adults without any of investigated risk factors like neoplasia, ENT (ear, nose, throat) focuses, elderly age, sepsis, diabetes, alcoholism, trauma, neonatal VLBW etc. Trauma, diabetes mellitus, alcohol abuse and chronic sinusitis/otitis were significantly less frequently found as a risk factor for MM. Mortality was very low, only 4.8% and was lower than overall mortality in CBM (12.4%, NS). Also the proportion of neurologic sequellae (9.7%) and initial treatment failure (8.1%) were comparable or even lower. This positive outcome results are probably because all N. meningitis strains were susceptible to penicillin, chloramphenicol, cefotaxim, cotrimoxazol or ciprofloxacin. Other reason for low mortality was that most cases received oral antibiotic immediately, even before admission (50 of 62). 95.2% of cases survived, 90.3% without any transient neurological residual symptoms.

Prospective national survey of viridans streptococcal bacteraemia risk factors, antibacterial susceptibility and outcome of 120 episodes.

The aim of this study was to prospectively investigate 120 cases of viridans streptococcal bacteraemia (VSB) in 117 patients in major university hospitals in Slovakia in 2000-2002 (3 y) for antibacterial susceptibility, risk factors and outcome. From 127 episodes, 16 (13%) of VSB were caused by PEN-R strains and 13 (10%) by ERY-R strains. 32 cases had cancer as underlying disease (20 haematological), 41 had endocarditis and 35 were elderly (>65 y of age) patients. Concerning mortality, 29 of 127 patients died (24%). There were several risk factors associated with mortality. Solid tumour as underlying disease (p<0.02), stroke (p<0.002), concomitant lung infection (p<0.01), endoscopic procedure (p<0.036), intubation (p<0.0008), ventilatory support (p<0.002), and coma (p<0.009) were associated with more deaths. A comparison of 115 bacteraemias to 13 bacteraemias caused by erythromycin-resistant strains of Streptococcus viridans was performed. There were no significant differences in underlying disease, risk factors and mortality. Erythromycin resistance in bacteraemias caused by S. viridans did not have significant impact on outcome of the patients, nor did it show specific relation to analysed risk factors in our study. 14.5% of VSB were cause by PEN-resistant viridans streptococci. Risk factors for penicillin resistance were ventilatory support (p<0.01), intubation (p<0.001) and resistance to other antibiotics: 8 of 16 (50%) of PEN-R VSB were resistant also to erythromycin or cotrimoxazole or tetracycline compared with 9% of PEN-R VSB (p<0.005). Endoscopic procedures in the upper respiratory system were at risk for development of PEN-R VSB. There was also difference in outcome; 71% vs 22.5% (p<0.0002) of cases infected with PEN-R VSB died compared to PEN-S VSB. PEN-R is therefore clinically significant in VSB.