Gut diversity and the resistome as biomarkers of febrile neutropenia outcome in paediatric oncology patients undergoing hematopoietic stem cell transplantation.

The gut microbiota of paediatric oncology patients undergoing a conditioning regimen before hematopoietic stem cell transplantation is recently considered to play role in febrile neutropenia. Disruption of commensal microbiota and evolution of opportune pathogens community carrying a plethora of antibiotic-resistance genes play crucial role. However, the impact, predictive role and association of patient´s gut resistome in the course of the therapy is still to be elucidated. We analysed gut microbiota composition and resistome of 18 paediatric oncology patients undergoing hematopoietic stem cell transplantation, including 12 patients developing febrile neutropenia, hospitalized at The Bone Marrow Transplantation Unit of the National Institute of Children´s disease in Slovak Republic and healthy individuals (n = 14). Gut microbiome of stool samples obtained in 3 time points, before hematopoietic stem cell transplantation (n = 16), one week after hematopoietic stem cell transplantation (n = 16) and four weeks after hematopoietic stem cell transplantation (n = 14) was investigated using shotgun metagenome sequencing and bioinformatical analysis. We identified significant decrease in alpha-diversity and nine antibiotic-resistance genes msr(C), dfrG, erm(T), VanHAX, erm(B), aac(6)-aph(2), aph(3)-III, ant(6)-Ia and aac(6)-Ii, one week after hematopoietic stem cell transplantation associated with febrile neutropenia. Multidrug-resistant opportune pathogens of ESKAPE, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli found in the gut carried the significant subset of patient's resistome. Over 50% of patients treated with trimethoprim/sulfamethoxazole, piperacillin/tazobactam and amikacin carried antibiotic-resistance genes to applied treatment. The alpha diversity and the resistome of gut microbiota one week after hematopoietic stem cell transplantation is relevant predictor of febrile neutropenia outcome after hematopoietic stem cell transplantation. Furthermore, the interindividual diversity of multi-drug resistant opportunistic pathogens with variable portfolios of antibiotic-resistance genes indicates necessity of preventive, personalized approach.

High Diversity but Monodominance of Multidrug-Resistant Bacteria in Immunocompromised Pediatric Patients with Acute Lymphoblastic Leukemia Developing GVHD Are Not Associated with Changes in Gut Mycobiome.

Graft-versus-host disease (GvHD) is a severe complication after hematopoietic stem cell transplantation (HSCT). Our study focused on identifying multidrug-resistant (MDR) gut bacteria associated with GvHD-prone guts and association with gut microbiota (GM) diversity, bacteriome, and mycobiome composition in post-HSCT patients. We examined 11 pediatric patients with acute lymphoblastic leukemia (ALL), including six with GvHD, within three time points: seven days pre-HSCT, seven days post-, and 28 days post-HSCT. The gut microbiome and its resistome were investigated using metagenomic sequencing, taxonomically classified with Kraken2, and statistically evaluated for significance using appropriate tests. We observed an increase in the abundance of MDR bacteria, mainly Enterococcus faecium strains carrying msr(C), erm(T), aac(6')-li, dfrG, and ant(6)-la genes, in GvHD patients one week post-HSCT. Conversely, non-GvHD patients had more MDR beneficial bacteria pre-HSCT, promoting immunosurveillance, with resistance genes increasing one-month post-HSCT. MDR beneficial bacteria included the anti-inflammatory Bacteroides fragilis, Ruminococcus gnavus, and Turicibacter, while most MDR bacteria represented the dominant species of GM. Changes in the gut mycobiome were not associated with MDR bacterial monodominance or GvHD. Significant α-diversity decline (Shannon index) one week and one month post-HSCT in GvHD patients (p < 0.05) was accompanied by increased Pseudomonadota and decreased Bacteroidota post-HSCT. Our findings suggest that MDR commensal gut bacteria may preserve diversity and enhance immunosurveillance, potentially preventing GvHD in pediatric ALL patients undergoing HSCT. This observation has therapeutic implications.

The Potential of Liquid Biopsy in Detection of Endometrial Cancer Biomarkers: A Pilot Study.

Endometrial cancer belongs to the most common gynecologic cancer types globally, with increasing incidence. There are numerous ways of classifying different cases. The most recent decade has brought advances in molecular classification, which show more accurate prognostic factors and the possibility of personalised adjuvant treatment. In addition, diagnostic approaches lag behind these advances, with methods causing patients discomfort while lacking the reproducibility of tissue sampling for biopsy. Minimally invasive liquid biopsies could therefore represent an alternative screening and diagnostic approach in patients with endometrial cancer. The method could potentially detect molecular changes in this cancer type and identify patients at early stages. In this pilot study, we tested such a detection method based on circulating tumour DNA isolated from the peripheral blood plasma of 21 Slovak endometrial cancer patients. We successfully detected oncomutations in the circulating DNA of every single patient, although the prognostic value of the detected mutations failed to offer certainty. Furthermore, we detected changes associated with clonal hematopoiesis, including DNMT3A mutations, which were present in the majority of circulating tumour DNA samples.

HLA-G 5'URR regulatory polymorphisms are associated with the risk of developing gliomas.

BACKGROUND: Human leukocyte antigen G (HLA-G) belongs to non-classical MHC class I molecules that is involved in the suppression of immune response. As HLA-G plays important role in the maintenance of fetal tolerance, its overexpression has been associated with tumor progression. For the regulation of HLA-G levels, genetic variants within the 5' upstream regulatory region (5'URR) are of crucial importance. Our study aimed to analyze the association between 16 HLA-G 5'URR variants, sHLA-G level and clinical variables in glioma patients. METHODS: We investigated 59 patients with gliomas (mean age 54.70 ± 15.10 years) and 131 healthy controls (mean age 41.45 ± 9.75 years). Patient's blood was obtained on the day of surgical treatment. The HLA-G 5'URR polymorphisms were typed by direct sequencing and the plasma level of sHLA-G assessed by ELISA. RESULTS: Haploblock within HLA-G 5'URR consisting of -762T, -716G, -689G, -666T, -633A, followed by -486C and -201A alleles were significantly more frequent in patients with gliomas than in the controls (p < 0.05). No correlation of HLA-G 5'URR variants with sHLA-G plasma level was found. Analysis of HLA-G 5'URR variants with main clinical variables in patients with grade IV gliomas revealed that haploblock carriers of -762CT, -716TG, -689AG, -666GT, -633GA, -486AC, -477GC, -201GA followed by -369AC carriers tend to have lower age at onset as compared to other genotype carriers (p = 0.04). CONCLUSION: Our results suggest genetic association of HLA-G 5'URR variants with risk of developing gliomas and possible contribution of HLA-G to disease pathology.

Maternal Copy Number Imbalances in Non-Invasive Prenatal Testing: Do They Matter?

Non-invasive prenatal testing (NIPT) has become a routine practice in screening for common aneuploidies of chromosomes 21, 18, and 13 and gonosomes X and Y in fetuses worldwide since 2015 and has even expanded to include smaller subchromosomal events. In fact, the fetal fraction represents only a small proportion of cell-free DNA on a predominant background of maternal DNA. Unlike fetal findings that have to be confirmed using invasive testing, it has been well documented that NIPT provides information on maternal mosaicism, occult malignancies, and hidden health conditions due to copy number variations (CNVs) with diagnostic resolution. Although large duplications or deletions associated with certain medical conditions or syndromes are usually well recognized and easy to interpret, very little is known about small, relatively common copy number variations on the order of a few hundred kilobases and their potential impact on human health. We analyzed data from 6422 NIPT patient samples with a CNV detection resolution of 200 kb for the maternal genome and identified 942 distinct CNVs; 328 occurred repeatedly. We defined them as multiple occurring variants (MOVs). We scrutinized the most common ones, compared them with frequencies in the gnomAD SVs v2.1, dbVar, and DGV population databases, and analyzed them with an emphasis on genomic content and potential association with specific phenotypes.

Vitamin D and circulating tumor cells in primary breast cancer.

Background: Circulating tumor cells (CTCs) contribute to the metastatic cascade and represent an independent survival predictor in breast cancer (BC) patients. Vitamin D has pleiotropic effects, and its low concentrations are associated with breast cancer and metastasis. The aim of this study was to assess plasma vitamin D in primary BC patients in relation to CTCs. Methods: This study included 91 non-metastatic BC patients (stage I-III) and 24 healthy donors. Blood samples for the analyses were drawn at the time of surgery. CTCs were assessed using a quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-to-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, and ZEB1). Total 25-OH vitamin D was measured in plasma using ELISA. Plasma cytokines and angiogenic factors were measured by enzyme-linked immunoassay. Results: CTCs were detected in 30 (33%) patients. Patients with detectable CTCs in peripheral blood had significantly lower vitamin D concentrations in comparison to patients without detectable CTCs ((mean ± SD) 8.50 ± 3.89 µg/L for CTC-positive vs 9.69 ± 3.49 µg/L for CTC-negative patients, p = 0.03). The mean ( ± SD) vitamin D plasma level was 9.3 ± 3.65 µg/L for breast cancer patients compared to 18.6 ± 6.8 for healthy donors (p < 0.000001). There was no association between plasma vitamin D and other patient/tumor characteristics. Plasma vitamin D levels are inversely correlated with plasma TGF-ß1, TGF-ß2, IL ß, IL-5, and eotaxin (all p < 0.05). Patients with vitamin D above the median had a better overall survival (hazard ratio (HR) = 0.36, 95% CI 0.16-0.80, p = 0.017), and combined analysis showed the best survival for CTC-negative patients with vitamin D levels above the median as compared to patients with opposite characteristics (HR = 0.18, 95% CI 0.05-0.63, p = 0.004). Conclusions: Low vitamin D could be a consequence and hence a biomarker of a more invasive disease. Alternatively, vitamin D could be associated with survival because of its role in tumor dissemination. Whether its supplementation affects the metastatic cascade should be tested in animal experiments and interventional studies.

Uncovering Microbial Composition in Human Breast Cancer Primary Tumour Tissue Using Transcriptomic RNA-seq.

Recent research studies are showing breast tissues as a place where various species of microorganisms can thrive and cannot be considered sterile, as previously thought. We analysed the microbial composition of primary tumour tissue and normal breast tissue and found differences between them and between multiple breast cancer phenotypes. We sequenced the transcriptome of breast tumours and normal tissues (from cancer-free women) of 23 individuals from Slovakia and used bioinformatics tools to uncover differences in the microbial composition of tissues. To analyse our RNA-seq data (rRNA depleted), we used and tested Kraken2 and Metaphlan3 tools. Kraken2 has shown higher reliability for our data. Additionally, we analysed 91 samples obtained from SRA database, originated in China and submitted by Sichuan University. In breast tissue, the most enriched group were Proteobacteria, then Firmicutes and Actinobacteria for both datasets, in Slovak samples also Bacteroides, while in Chinese samples Cyanobacteria were more frequent. We have observed changes in the microbiome between cancerous and healthy tissues and also different phenotypes of diseases, based on the presence of circulating tumour cells and few other markers.

Prognostic role of matrix metalloproteinase 9 in early breast cancer.

MMP9 is involved in extracellular matrix degradation during various physiological and pathological conditions, including tumorigenesis. The present study aimed to assess the prognostic role of intratumoral MMP9 and to determine its association with circulating tumor cells (CTCs) in patients with early breast cancer. A total of 318 patients with primary breast cancer (PBC) were enrolled into the present study. Specimens were subjected to immunohistochemistry analysis, using the MMP9 monoclonal antibody. MMP9 expression was scored using a weighted histoscore (WH). The results demonstrated that the mean WH ± SEM for MMP9 expression was significantly higher in breast tumor cells compared with tumor associated stromas (132.0±5.2 vs. 50.8±3.7; P<0.00001). Furthermore, a positive association was observed between MMP9 expression, the hormone positive status and proliferation index of analysed breast cancer tumour cells. Notably, the prognostic role of MMP9 was not observed in tumor cells [hazard ratio (HR) =0.96; 95% confidence interval (CI), 0.58-1.59; P=0.864] or tumor associated stroma (HR=1.29; 95% CI, 0.60-2.78; P=0.547). Subgroup analysis demonstrated that patients that were HR negative or triple negative, with low MMP9 expression in tumor cells and stroma had a significantly improved disease-free survival than patients with high MMP9 expression. Taken together, the results of the present study demonstrated that high MMP9 expression in PBC was associated with favorable tumor characteristics. However, the prognostic value of MMP9 was limited to only the HR negative and CTC epithelial-to-mesenchymal transition positive subgroups. Thus, analyzing MMP9 tumor expression may help identify patients with increased risk of disease recurrence in these subgroups.

miR-205-5p Downregulation and ZEB1 Upregulation Characterize the Disseminated Tumor Cells in Patients with Invasive Ductal Breast Cancer.

BACKGROUND: Dissemination of breast cancer (BC) cells through the hematogenous or lymphogenous vessels leads to metastatic disease in one-third of BC patients. Therefore, we investigated the new prognostic features for invasion and metastasis. METHODS: We evaluated the expression of miRNAs and epithelial-to-mesenchymal transition (EMT) genes in relation to CDH1/E-cadherin changes in samples from 31 patients with invasive ductal BC including tumor centrum (TU-C), tumor invasive front (TU-IF), lymph node metastasis (LNM), and CD45-depleted blood (CD45-DB). Expression of miRNA and mRNA was quantified by RT-PCR arrays and associations with clinico-pathological characteristics were statistically evaluated by univariate and multivariate analysis. RESULTS: We did not verify CDH1 regulating associations previously described in cell lines. However, we did detect extremely high ZEB1 expression in LNMs from patients with distant metastasis, but without regulation by miR-205-5p. Considering the ZEB1 functions, this overexpression indicates enhancement of metastatic potential of lymphogenously disseminated BC cells. In CD45-DB samples, downregulated miR-205-5p was found in those expressing epithelial and/or mesenchymal markers (CTC+) that could contribute to insusceptibility and survival of hematogenously disseminated BC cells mediated by increased expression of several targets including ZEB1. CONCLUSIONS: miR-205-5p and potentially ZEB1 gene are promising candidates for markers of metastatic potential in ductal BC.

Increased Stromal Infiltrating Lymphocytes Are Associated with the Risk of Disease Progression in Mesenchymal Circulating Tumor Cell-Positive Primary Breast Cancer Patients.

Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30-10.37, p < 0.001 for CD3high; HR = 6.17, 95% CI 2.75-13.80, p < 0.001 for CD8high; HR = 6.93, 95% CI 2.86-16.81, p < 0.001 for CD45ROhigh). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048-11.427, p < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

Plasma Nucleosomes in Primary Breast Cancer.

When cells die, nucleosomes composed of DNA and histone proteins enter the extracellular space and end eventually in the circulation. In plasma, they might serve as a nonspecific marker of cell death, potentially useful for noninvasive monitoring of tumor dynamics. The aim of this study was to analyze circulating nucleosomes in relation to patient/tumor characteristics and prognosis in primary breast cancer. This study included 92 patients with breast cancer treated with surgery for whom plasma isolated was available in the biobank. Plasma nucleosomes were detected in samples taken in the morning on the day of surgery using Cell Death Detection ELISA kit with anti-histone and anti-DNA antibodies. Circulating nucleosomes were positively associated with the systemic inflammatory index (SII), but not with other patient/tumor characteristics. Patients with high SII in comparison to low SII had higher circulating nucleosomes (by 59%, p = 0.02). Nucleosomes correlated with plasma plasminogen activator inhibitor-1, IL-15, IL-16, IL-18, and hepatocyte growth factor. Patients with lower nucleosomes had significantly better disease-free survival (HR = 0.46, p = 0.05). In a multivariate analysis, nucleosomes, hormone receptor status, HER2 status, lymph node involvement, and tumor grade were independent predictors of disease-free survival. Our data suggest that plasma nucleosomes in primary breast cancer are associated with systemic inflammation and might have a prognostic value. The underlying mechanisms require further studies.

Inflammation-Based Scores Increase the Prognostic Value of Circulating Tumor Cells in Primary Breast Cancer.

A correlation between circulating tumor cells (CTCs) and monocytes in metastatic breast cancer (BC), where CTCs and monocyte-to-lymphocyte ratio (MLR) were predictors of overall survival (OS), was recently shown. Herein, we aimed to assess the association between CTCs and the complete blood count (CBC)-derived inflammation-based scores in 284 primary BC patients. CTCs were determined in CD45-depleted peripheral blood mononuclear cells by real time-PCR. This method allowed us to detect a subset of CTCs with an epithelial-to-mesenchymal transition phenotype (CTC EMT), previously associated with inferior outcomes in primary BC. In the present study, CTC EMT positivity (hazard ratio (HR) = 2.4; 95% CI 1.20-4.66, p = 0.013) and elevated neutrophil-to-lymphocyte ratio (NLR) (HR = 2.20; 95% CI 1.07-4.55; p = 0.033) were associated with shorter progression-free survival (PFS) in primary BC patients. Multivariate analysis showed that CTC EMT-positive patients with NLR ≥ 3 had 8.6 times increased risk of disease recurrence (95% CI 2.35-31.48, p = 0.001) compared with CTC EMT-negative patients with NLR < 3. Similarly, disease recurrence was 13.14 times more likely in CTC EMT-positive patients with MLR ≥ 0.34 (95% CI 4.35-39.67, p < 0.001). Given its low methodological and financial demands, the CBC-derived inflammation-based score determination could, after broader validation, significantly improve the prognostication of BC patients.

Circulating tumor cells and breast cancer-specific mutations in primary breast cancer.

Circulating tumor cells (CTCs) play a pivotal role in tumor dissemination and progression, and are considered to be a critical part of the metastatic cascade. The aim of the present research article was to examine breast cancer-specific mutations in primary breast cancer (PBC) using targeted resequencing. A total of 78 patients with PBC were enrolled into this translational study. Reverse transcription-quantitative PCR assay for the expression of epithelial markers (CK19) or epithelial-to-mesenchymal transition (EMT)-related genes (TWIST1, SNAIL1, SLUG and ZEB1) was applied for identification of CTCs prior to surgery. Total DNA was isolated from fresh frozen primary tumors. Sequencing was performed by Agilent SureSelect target enrichment and Illumina paired-end sequencing on the MiSeq platform. The most commonly affected genes were TP53 (mutated in 21 tumors; 26.9%), followed by PIK3CA (mutated in 16 tumors; 20.5%) and BRCA1/2 (mutated in 7 tumors, BRCA1 n=2 and BRCA2 n=5; 9.0%). In our cohort, a significantly higher proportion of patients with epithelial CTCs harbored mutations in the BRCA1/2 genes in the tumor tissue. There were no mutations in specific genes associated with CTCs with the EMT phenotype. To the best of our knowledge, this study is the first to report a correlation between the presence of epithelial CTCs in the peripheral blood and mutations of the BRCA1/2 genes in primary tumor tissue.

Fatal neonatal nephrocutaneous syndrome in 18 Roma children with EGFR deficiency.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with tyrosine-kinase signaling activity, involved in many cellular functions including cell growth and differentiation. Germ line loss-of-function mutations in EGFR lead to a severe neonatal skin disorder (Online Mendelian Inheritance in Man #131550). We report 18 premature Roma children from 16 families with birthweights ranging 440-1470 g and multisystem diseases due to the homozygous mutation c.1283G˃A (p.Gly428Asp) in EGFR. They presented with thin, translucent, fragile skin (14/15), skin desquamation (10/17), ichthyosis (9/17), recurrent skin infections and sepsis (9/12), nephromegaly (10/16) and congenital heart defects (7/17). Their prognosis was poor, and all died before the age of 6 months except one 13-year-old boy with a severe skin disorder, dentinogenesis imperfecta, Fanconi-like syndrome and secondary hyperaldosteronism. Management of ion and water imbalances and extremely demanding skin care may improve the unfavorable outcome of such patients.

Circulating tumor cells and drug history in primary breast cancer patients.

Aim: Different types of chronic medication may affect breast cancer prognosis. Circulating tumor cells (CTCs) play an important role in cancer metastasis formation. There is no evidence of how chronic medication affects CTCs and breast cancer prognosis. The aim of this study was to evaluate association between chronic medication and CTCs in patients with primary breast cancer. Methods: This study involved 414 patients with stage I-III primary breast cancer. Chronic drug history was collected from patients' medical records and included all drugs that were prescribed for patients over at least the last 6 months prior to CTCs evaluation. CTCs were detected using a quantitative real-time polymerase chain reaction (qRT-PCR)-based method at the time of breast surgery. Results: There was no association between CTCs, including their different subpopulations and chronic medication. Chronic medication using angiotensin-converting-enzyme inhibitors (ACEi), metformin, and insulin were associated with inferior disease-free survival (HR = 0.49, 95%CI 0.26-0.94, P = 0.007 for ACEi; HR = 0.27, 95%CI 0.08-0.91, P < 0.001 for metformin; and HR = 0.12, 95%CI 0.01-2.91, P < 0.001 for insulin) and this was most pronounced in patients with epithelial to mesenchymal transition (CTC_EMT) phenotype. In multivariate analysis, chronic administration of metformin and/or insulin was an independent predictor of inferior outcome. Conclusion: Our findings show that there was no association between chronically used medication and CTCs in primary breast cancer patients. However, administration of ACEi, metformin, and/or insulin could negatively affect prognosis of patients with CTC_EMT.

Circulating Tumor Cells With Epithelial-to-mesenchymal Transition Phenotypes Associated With Inferior Outcomes in Primary Breast Cancer.

BACKGROUND/AIM: Circulating tumor cells (CTCs) comprise a heterogeneous population of cancer cells with different clinical and biological value. The aim of this study was to evaluate the prognostic value of CTCs with an epithelial-mesenchymal transition (EMT) phenotype in primary breast cancer (PBC) patients. PATIENTS AND METHODS: This study included 427 primary breast cancer patients. RNA extracted from CD45-depleted peripheral blood mononuclear cell (PBMCs) was evaluated for the expression of EMT transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) by quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: In total, CTC EMT was detected in 77 (18.0%) patients. Patients without detectable CTC EMT in peripheral blood had significantly longer disease-free survival than patients with detectable CTC EMT. The prognostic value of CTC EMT was demonstrated in all subgroups of patients. CONCLUSION: CTCs with an EMT phenotype have a prognostic value in primary breast cancer.

A disintegrin and metalloprotease 23 hypermethylation predicts decreased disease-free survival in low-risk breast cancer patients.

A Disintegrin And Metalloprotease 23 (ADAM23), a member of the ADAM family, is involved in neuronal differentiation and cancer. ADAM23 is considered a possible tumor suppressor gene and is frequently downregulated in various types of malignancies. Its epigenetic silencing through promoter hypermethylation was observed in breast cancer (BC). In the present study, we evaluated the prognostic significance of ADAM23 promoter methylation for hematogenous spread and disease-free survival (DFS). Pyrosequencing was used to quantify ADAM23 methylation in tumors of 203 BC patients. Presence of circulating tumor cells (CTC) in their peripheral blood was detected by quantitative RT-PCR. Expression of epithelial (KRT19) or mesenchymal (epithelial-mesenchymal transition [EMT]-inducing transcription factors TWIST1, SNAI1, SLUG and ZEB1) mRNA transcripts was examined in CD45-depleted peripheral blood mononuclear cells. ADAM23 methylation was significantly lower in tumors of patients with the mesenchymal CTC (P = .006). It positively correlated with Ki-67 proliferation, especially in mesenchymal CTC-negative patients (P = .001). In low-risk patients, characterized by low Ki-67 and mesenchymal CTC absence, ADAM23 hypermethylation was an independent predictor of DFS (P = .006). Our results indicate that ADAM23 is likely involved in BC progression and dissemination of mesenchymal CTC. ADAM23 methylation has the potential to function as a novel prognostic marker and therapeutic target.

Decreased methylation in the SNAI2 and ADAM23 genes associated with de-differentiation and haematogenous dissemination in breast cancers.

BACKGROUND: In breast cancer (BC), deregulation of DNA methylation leads to aberrant expressions and functions of key regulatory genes. In our study, we investigated the relationship between the methylation profiles of genes associated with cancer invasivity and clinico-pathological parameters. In detail, we studied differences in the methylation levels between BC patients with haematogenous and lymphogenous cancer dissemination. METHODS: We analysed samples of primary tumours (PTs), lymph node metastases (LNMs) and peripheral blood cells (PBCs) from 59 patients with sporadic disseminated BC. Evaluation of the DNA methylation levels of six genes related to invasivity, ADAM23, uPA, CXCL12, TWIST1, SNAI1 and SNAI2, was performed by pyrosequencing. RESULTS: Among the cancer-specific methylated genes, we found lower methylation levels of the SNAI2 gene in histologic grade 3 tumours (OR = 0.61; 95% CI, 0.39-0.97; P = 0.038) than in fully or moderately differentiated cancers. We also evaluated the methylation profiles in patients with different cancer cell dissemination statuses (positivity for circulating tumour cells (CTCs) and/or LNMs). We detected the significant association between reduced DNA methylation of ADAM23 in PTs and presence of CTCs in the peripheral blood of patients (OR = 0.45; 95% CI, 0.23-0.90; P = 0.023). CONCLUSION: The relationships between the decreased methylation levels of the SNAI2 and ADAM23 genes and cancer de-differentiation and haematogenous dissemination, respectively, indicate novel functions of those genes in the invasive processes. After experimental validation of the association between the lower values of SNAI2 and ADAM23 methylation and clinical features of aggressive BCs, these methylation profiles could improve the management of metastatic disease.

Down-regulation of traditional oncomiRs in plasma of breast cancer patients.

Deregulated expression of microRNAs has the oncogenic or tumor suppressor function in cancer. Since miRNAs in plasma are highly stable, their quantification could contribute to more precise cancer diagnosis, prognosis and therapy prediction. We have quantified expression of seven oncomiRs, namely miR-17/92 cluster (miR-17, miR-18a, miR-19a and miR-20a), miR-21, miR-27a and miR-155, in plasma of 137 breast cancer (BC) patients. We detected down-regulation of six miRNAs in patients with invasive BC compared to controls; however, only miR-20a and miR-27a down-regulations were statistically significant. Comparing miRNA expression between early and advanced stages of BC, we observed statistically significant decrease of miR-17 and miR-19a. We identified down-regulation of miR-17 and miR-20a in patients with clinical parameters of advanced BC (lymph node metastasis, tumor grade 3, circulating tumor cells, higher Ki-67-related proliferation, hormone receptor negativity and HER2 amplification), when compared to controls. Moreover, decreased level of miR-17 was found from low to high grade. Therefore, miR-17 could represent an indicator of advanced BC. Down-regulated miR-27a expression levels were observed in all clinical categories regardless of tumor progression. Hence, miR-27a could be used as a potential diagnostic marker for BC. Our data indicates that any changes in miRNA expression levels in BC patients in comparison to controls could be highly useful for cancer-associated pathology discrimination. Moreover, dynamics of miRNA expression changes could be used for BC progression monitoring.