The impact of obesity, age, and gender on plasmatic levels of selected glycoprotein biomarkers and miRNA-499 in OSA patients.

BACKGROUND: The current obstructive sleep apnea (OSA) diagnostic uses polysomnography or limited polygraphy and requires specialized personnel and technical equipment. Glycoprotein biomarkers and microRNAs are being explored as a possible new method for screening. We aimed to evaluate whether certain biomarkers and microRNA, previously identified as related to OSA, could be influenced by factors such as gender, age, and obesity level in patients with OSA. METHODS: In this retrospective analytical study, patients with moderate to severe OSA (n = 130) were compared with the control group. Serum levels of selected biomarkers and microRNA were taken from both groups. The group of OSA patients was then stratified by gender, obesity level, and age to see the possible influence of those variables on biomarker levels. RESULTS: Levels of all studied biomarkers - C-reactive protein (CRP), high-sensitivity troponin I (hsTnI), pentraxin-3 (PTX-3), and microRNA-499 were significantly higher in patients with OSA compared to the control group. In the OSA group only hsTnI showed a statistically significant relationship with gender. Levels of CRP and hsTnI showed a significant dependence on the level of obesity. Dependency on age was proven for hsTnI. CRP, PTX-3, and microRNA-499 did not have any statistically significant relationship with age. CONCLUSION: We found that serum levels of pentraxin-3 and microRNA-499 in patients with moderate to severe obstructive sleep apnoea are independent of gender, obesity, and age. CRP was affected by the level of obesity and hsTnI was influenced by all 3 variables. We consider these findings important for further research of OSA biomarkers.

Pluripotency Stemness and Cancer: More Questions than Answers.

Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.

A novel nonsense mutation in the ß-subunit of the epithelial sodium channel causing Liddle syndrome.

PURPOSE: Liddle syndrome is a hereditary form of arterial hypertension caused by mutations in the genes coding of the epithelial sodium channel - SCNN1A, SCNN1B and SCNN1G. It is characterised by early onset of hypertension and variable biochemical features such as hypokalaemia and low plasma concentrations of renin and aldosterone. Phenotypic variability is large and, therefore, LS is probably underdiagnosed. Our objective was to examine a family suspected from Liddle syndrome including genetic testing and evaluate clinical and biochemical features of affected family members. MATERIALS AND METHODS: Thirteen probands from the Czech family, related by blood, underwent physical examination, laboratory tests, and genetic testing. Alleles of SCNN1B and SCNN1G genes were examined by PCR amplification and Sanger sequencing of amplicons. RESULTS: We identified a novel mutation in the ß-subunit of an epithelial sodium channel coded by the SCNN1B gene, causing the nonsense mutation in the protein sequence p.Tyr604*. This mutation was detected in 7 members of the family. The mutation carriers differed in the severity of hypertension and hypokalaemia which appeared only after diuretics in most of them; low aldosterone level (< 0.12 nmol/l) was, however, present in all. CONCLUSIONS: This finding expands the spectrum of known mutations causing Liddle syndrome. Hypoaldosteronemia was 100% sensitive sign in the mutation carriers. Low levels are observed especially in the Caucasian population reaching 96% sensitivity. Assessment of plasma aldosterone concentration is helpful for differential diagnosis of arterial hypertension. CONDENSED ABSTRACT: Liddle syndrome is a hereditary form of arterial hypertension caused by mutations in the genes encoding the epithelial sodium channel's α-, ß- and γ-subunit. It is usually manifested by early onset of hypertension accompanied by low potassium and aldosterone levels. We performed a physical examination, laboratory tests and genetic screening in 13 members of a Czech family. We found a new mutation of the SCNN1B gene which encodes the ß-subunit of the epithelial sodium channel. We describe the variability of each family member phenotype and point out the relevance of using aldosterone levels as a high sensitivity marker of Liddle syndrome in Caucasians.

Prodrug suicide gene therapy for cancer targeted intracellular by mesenchymal stem cell exosomes.

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.

One-step nucleic acid amplification vs ultrastaging in the detection of sentinel lymph node metastasis in endometrial cancer patients.

BACKGROUND AND OBJECTIVES: Utilisation of the one-step nucleic acid amplification (OSNA) molecular biology method for the detection of the metastatic involvement of sentinel lymph nodes (SLNs) in endometrial cancer (EC) patients. A comparison with histopathological ultrastaging and a description of the clinical consequences. METHODS: Surgically treated EC patients underwent detection of SLNs. Nodes greater than 5 mm were cut into sections 2-mm thick parallel to the short axis of the node. Odd sections were examined according to the OSNA method, while even ones according to an appropriate ultrastaging protocol. Nodes less than or equal to 5 mm were cut into halves along the longitudinal axis with one half examined according to the OSNA method and the other half by ultrastaging. RESULTS: Fifty-eight patients were included and 135 SLNs were acquired. Both ultrastaging and OSNA agreed on 116 results. According to the OSNA method, 20.69% more patients were classified into International Federation of Gynecology and Obstetrics (FIGO) stage III. When comparing the results of the OSNA method to the conclusions of ultrastaging as a reference method, sensitivity of 90.9%, specificity of 85.5% and concordance of 85.9% were attained. CONCLUSIONS: The results of the OSNA method showed a higher frequency of detection of micrometastases and included 20.69% more patients into FIGO stage III.

Urothelial Cancer Stem Cell Heterogeneity.

Urothelial carcinoma is a tumor type featuring pronounced intertumoral heterogeneity and a high mutational and epigenetic load. The two major histopathological urothelial carcinoma types - the non-muscle-invasive and muscle-invasive urothelial carcinoma - markedly differ in terms of their respective typical mutational profiles and also by their probable cells of origin, that is, a urothelial basal cell for muscle-invasive carcinomas and a urothelial intermediate cell for at least a large part of non-muscle-invasive carcinomas. Both non-muscle-invasive and muscle-invasive urothelial carcinomas can be further classified into discrete intrinsic subtypes based on their typical transcriptomic profiles. Urothelial carcinogenesis shows a number of parallels to a urothelial regenerative response. Both of these processes seem to be dominated by specific stem cell populations. In the last years, the nature and location of urothelial stem cell(s) have been subject to many controversies, which now seem to be settled down, favoring the existence of a largely single urothelial stem cell type located among basal cells. Basal cell markers have also been amply used to identify urothelial carcinoma stem cells, especially in muscle-invasive disease, but they proved useful even in some non-muscle-invasive tumors. Analyses on molecular nature of urothelial carcinoma stem cells performed till now point to their great heterogeneity, both during the tumor development and upon intertumoral comparison, sexual dimorphism providing a special example of the latter. Moreover, urothelial cancer stem cells are endowed with intrinsic plasticity, whereby they can modulate their stemness in relation to other tumor-related traits, especially motility and invasiveness. Such transitional modulations suggest underlying epigenetic mechanisms and, even within this context, inter- and intratumoral heterogeneity becomes apparent. Multiple molecular aspects of urothelial cancer stem cell biology markedly influence therapeutic response, implying their knowledge as a prerequisite to improved therapies of this disease. At the same time, the notion of urothelial cancer stem cell heterogeneity implies that this therapeutic benefit would be most probably and most efficiently achieved within the context of individualized antitumor therapy.

Sarcoma Stem Cell Heterogeneity.

Sarcomas represent an extensive group of divergent malignant diseases, with the only common characteristic of being derived from mesenchymal cells. As such, sarcomas are by definition very heterogeneous, and this heterogeneity does not manifest only upon intertumoral comparison on a bulk tumor level but can be extended to intratumoral level. Whereas part of this intratumoral heterogeneity could be understood in terms of clonal genetic evolution, an essential part includes a hierarchical relationship between sarcoma cells, governed by both genetic and epigenetic influences, signals that sarcoma cells are exposed to, and intrinsic developmental programs derived from sarcoma cells of origin. The notion of this functional hierarchy operating within each tumor implies the existence of sarcoma stem cells, which may originate from mesenchymal stem cells, and indeed, mesenchymal stem cells have been used to establish several crucial experimental sarcoma models and to trace down their respective stem cell populations. Mesenchymal stem cells themselves are heterogeneous, and, moreover, there are alternative possibilities for sarcoma cells of origin, like neural crest-derived stem cells, or mesenchymal committed precursor cells, or - in rhabdomyosarcoma - muscle satellite cells. These various origins result in substantial heterogeneity in possible sarcoma initiation. Genetic and epigenetic changes associated with sarcomagenesis profoundly impact the biology of sarcoma stem cells. For pediatric sarcomas featuring discrete reciprocal translocations and largely stable karyotypes, the translocation-activated oncogenes could be crucial factors that confer stemness, principally by modifying transcriptome and interfering with normal epigenetic regulation; the most extensively studied examples of this process are myxoid/round cell liposarcoma, Ewing sarcoma, and synovial sarcoma. For adult sarcomas, which have typically complex and unstable karyotypes, stemness might be defined more operationally, as a reflection of actual assembly of genetically and epigenetically conditioned stemness factors, with dedifferentiated liposarcoma providing a most thoroughly studied example. Alternatively, stemness can be imposed by tumor microenvironment, as extensively documented in osteosarcoma. In spite of this heterogeneity in both sarcoma initiation and underlying stemness biology, some of the molecular mechanisms of stemness might be remarkably similar in diverse sarcoma types, like abrogation of classical tumor suppressors pRb and p53, activation of Sox-2, or inhibition of canonical Wnt/ß-catenin signaling. Moreover, even some stem cell markers initially characterized for their stem cell enrichment capacity in various carcinomas or leukemias seem to function quite similarly in various sarcomas. Understanding the biology of sarcoma stem cells could significantly improve sarcoma patient clinical care, leading to both better patient stratification and, hopefully, development of more effective therapeutic options.

Serum Vitamin D Status, Vitamin D Receptor Polymorphism, and Glucose Homeostasis in Healthy Subjects.

Low vitamin D status has been frequently associated with impaired glucose metabolism. We examined associations between 25-hydroxyvitamin D (25-OH-D) and several parameters of glucose homeostasis in virtually healthy subjects, and explored possible interaction with vitamin D receptor (VDR) polymorphism. Nondiabetic subjects without chronic medication or any known significant manifest disease were selected from large general-population based population survey. Insulin sensitivity and ß cell secretion were calculated by homeostasis model assessment (HOMA) and soluble isoform of receptor for advanced glycation end-products (sRAGE) using commercial ELISA. Subjects were also genotyped for rs2228570 polymorphism of VDR. After adjustment for potential confounders, we observed a significant relationship between 25-OH-D and fasting glycemia (ß coefficient=-5.904; p=0.002) or insulin sensitivity (ß=0.042; p=0.001), but not with ß cell secretion or sRAGE. We found also an interaction with VDR polymorphism. Subjects with low 25-OH-D and AA genotype had significantly lower insulin sensitivity than those with GG genotype plus highest 25-OH-D concentrations (107.3% vs. 183.9%, p=0.021). In conclusion, low vitamin D status was in virtually healthy subjects associated with decreased insulin sensitivity, namely in those with GG genotype of rs2228570 VDR polymorphism.

A more sensitive detection of micrometastases of NSCLC in lymph nodes using the one-step nucleic acid amplification (OSNA) method.

BACKGROUND: Detection of tumor cells in lymph nodes (LNs) removed during the treatment of pulmonary tumor by radical surgery is limited by the possibilities of standard histopathological methods. The goal of this study was to obtain more accurate pTNM status by a more sensitive detection of micrometastases in LNs. METHODS: A total of 885 LNs, an average of 13.8 LNs per patient, were removed during 64 surgeries. LNs from the same zone were pooled together as a group, five groups of LNs were examined in each patient. A total of 320 groups of LNs were examined. One-step nucleic acid amplification (OSNA) method was compared to standard histopathological examination with haematoxylin-eosin (H&E) staining and CK19 immunohistochemistry, specifically by an ultimate analysis of all intraoperatively removed LNs. RESULTS: Identical results for H&E and OSNA examinations were recorded in 286 groups of LNs (89.4%). In total, positive examinations were recorded in 27 groups of LNs (8.4%) using the OSNA method, which were H&E negative. In seven groups of LNs (2.2%), the H&E examination was positive, while OSNA method produced negative results. CONCLUSIONS: The OSNA examination led to a higher pTNM stage classification in 14 (21.9%) patients. The clinical significance remains the subject of follow-up research.

ABC gene expression profiles have clinical importance and possibly form a new hallmark of cancer.

Adenosine triphosphate-binding cassette proteins constitute a large family of active transporters through extracellular and intracellular membranes. Increased drug efflux based on adenosine triphosphate-binding cassette protein activity is related to the development of cancer cell chemoresistance. Several articles have focused on adenosine triphosphate-binding cassette gene expression profiles (signatures), based on the expression of all 49 human adenosine triphosphate-binding cassette genes, in individual tumor types and reported connections to established clinicopathological features. The aim of this study was to test our theory about the existence of adenosine triphosphate-binding cassette gene expression profiles common to multiple types of tumors, which may modify tumor progression and provide clinically relevant information. Such general adenosine triphosphate-binding cassette profiles could constitute a new attribute of carcinogenesis. Our combined cohort consisted of tissues from 151 cancer patients-breast, colorectal, and pancreatic carcinomas. Standard protocols for RNA isolation and quantitative real-time polymerase chain reaction were followed. Gene expression data from individual tumor types as well as a merged tumor dataset were analyzed by bioinformatics tools. Several general adenosine triphosphate-binding cassette profiles, with differences in gene functions, were established and shown to have significant relations to clinicopathological features such as tumor size, histological grade, or clinical stage. Genes ABCC7, A3, A8, A12, and C8 prevailed among the most upregulated or downregulated ones. In conclusion, the results supported our theory about general adenosine triphosphate-binding cassette gene expression profiles and their importance for cancer on clinical as well as research levels. The presence of ABCC7 (official symbol CFTR) among the genes with key roles in the profiles supports the emerging evidence about its crucial role in various cancers. Graphical abstract.

Breast cancer epidemic in the early twenty-first century: evaluation of risk factors, cumulative questionnaires and recommendations for preventive measures.

Rapidly increasing incidence of breast cancer is a new social challenge resulting from a spectrum of internal and external risk factors which appear to be well accepted as an attribute of the early twenty-first century, being, however, new for female sub-populations compared to the past. These include altered socio-economical conditions such as occupational exposure, rotating shift work, specific environmental factors (increased pollution and environmental toxicity, altered dietary habits, quality and composition of meal) as well as consequently shifted and/or adapted physiologic factors such as lower age at menarche, late age of first full-term pregnancy, if any, shorter periods of breastfeeding and later menopause. Consolidated expert statements suggest that over 50 % of all breast cancer cases may be potentially prevented by risk reduction strategy such as regulation of modifiable risk factors. Currently available risk assessment models may estimate potential breast cancer predisposition, in general; however, they are not able to predict the disease manifestation individually. Further, current deficits in risk assessment and effective breast cancer prevention have been recently investigated and summarised as follows: gaps in risk estimation, preventive therapy, lifestyle prevention, understanding of the biology of breast cancer risk and implementation of known preventive measures. This paper overviews the most relevant risk factors, provides recommendations for improved risk assessment and proposes an extended questionnaire for effective preventive measures.

Clinical monitoring: infliximab biosimilar CT-P13 in the treatment of Crohn's disease and ulcerative colitis.

OBJECTIVE: The infliximab biosimilar CT-P13 (Remsima(®), Inflectra(®)) was approved in Europe for the treatment of inflammatory bowel disease (IBD) based on extrapolation of data from patients with rheumatic disease. Because there are limited published reports on clinical outcomes for IBD patients treated with CT-P13, we monitored responses to induction treatment with this biosimilar in patients with Crohn's disease (CD) or ulcerative colitis (UC) in centres across the Czech Republic. MATERIAL AND METHODS: Fifty-two patients with CD (n = 30) or UC (n = 22) were treated with 5 mg/kg CT-P13 for up to 14 weeks. Effectiveness of therapy was evaluated with the Crohn's Disease Activity Index (CDAI) or the Mayo Scoring System (MSS) in patients with CD or UC, respectively, before and after 14 weeks. Additional goals were to evaluate weight changes, serum C-reactive protein (CRP) levels, and complications/adverse events. RESULTS: In patients with CD, remission (CDAI <150) was achieved in 50.0% of cases, and partial response (≥70-point decrease in CDAI score from baseline) in the remaining 50.0%. In patients with UC, remission (total score on partial Mayo index ≤2 points) was achieved in 40.9% of cases, partial response (≥2-point decrease in partial Mayo score from baseline) in 54.5%, and no response in 4.5%. There were statistically significant improvements in CDAI, MSS and CRP serum levels after 14 weeks of therapy, and body weight increased. Four adverse events were identified (n = 1 each): lower-extremity phlebothrombosis, herpes labialis, pneumonia and allergic reaction. CONCLUSIONS: This prospective observational study provides evidence of the effectiveness of CT-P13 in IBD.

Association between endothelial NO synthase polymorphisms and arterial properties in the general population.

OBJECTIVE: Nitric oxide plays an important role in vascular biology. Several single nucleotide polymorphisms (SNP) in the endothelial nitric oxide gene (NOS3) have been previously associated with arterial hypertension. We investigated whether these SNPs might be associated with arterial phenotypes in the Czech general population. METHODS: We genotyped three NOS3 SNPs in 426 subjects not treated for arterial hypertension (mean age, 49.1 years; 55.9% women). Arterial properties were measured using applanation tonometry. In multivariate-adjusted analyses, we assessed the gene effects of rs3918226 (-665 C>T), rs1799983 (glu298asp G>T) and rs2070744 (786 T>C) on augmentation index (AIx), central augmentation pressure (AP) and aortic pulse wave velocity (PWV). RESULTS: Carriers of rs3918226 mutated T allele had marginally higher AIx (145.3 ± 2.5 vs. 140.2 ± 1.1%; P = 0.064) and significantly higher AP (12.7 ± 0.7 vs. 11.1 ± 0.3 mm Hg; P = 0.033). These associations were independent of potential confounding factors. Aortic PWV was not different in the two rs39182226 genotypes groups (P = 0.35). In single gene analyses, we did not observe any association between measured phenotypes and rs1799983 or rs2070744 (P ≥ 0.11). In haplotype analysis, we observed trend for higher PWV in haplotypes containing rs3918226 mutated T allele compared with other allelic combination (P ≤ 0.079). CONCLUSION: Mutated T allele of rs3918226 polymorphism in NOS3 gene was associated with parameters reflecting central arterial stiffness and wave reflection. We hypothesize that genetic modulation of intermediate arterial phenotypes might lead to higher blood pressure.

[Vitamin D in relation to head and neck cancer]. / Vitamin D ve vztahu ke karcinomu hlavy a krku.

Vitamin D was lately introduced as the important anti-cancer agent with therapeutic potential to be used in prevention and therapy of malignant tumors. Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer in the world. Prognosis of this cancer is in most patients poor. In attempts to improve therapy of this cancer, contemporary research is focused on extension of the etiological and pathobiological aspects of cancerogenesis of HNSCC. This fact provoked us to summarize present scientific knowledge about etiopathological, chemopreventive and therapeutic role of vitamin D in head and neck squamous cancer and outline the direction of further research in this interesting and from clinical point of view important field. Key words: head and neck squamous cell carcinoma - anti-tumor effect - vitamin D.

BRAF mutation, selected miRNAs and genes expression in primary papillary thyroid carcinomas and local lymph node metastases.

Mutations in cancer-related genes are now known to be accompanied by epigenetic events in carcinogenesis by modification of the regulatory pathways and expression of genes involved in the pathobiology. Such cancer-related mutations, miRNAs and gene expression may be promising molecular markers of the most common papillary thyroid carcinoma (PTC). However, there are limited data on their relationships. The aim of this study was to analyse the interactions between BRAF mutations, selected microRNAs (miR-21, miR-34a, miR-146b, and miR-9) and the expression of selected genes (LGALS3, NKX2-1, TACSTD2, TPO) involved in the pathogenesis of PTC. The study cohort included 60 primary papillary thyroid carcinomas (PTC) that were classified as classical (PTC/C; n=50) and invasive follicular variant (PTC/F; n=10), and 40 paired lymph node metastases (LNM). BRAF mutation status in primary and recurrent/persistent papillary thyroid carcinomas was determined. The mutation results were compared both between primary and metastatic cancer tissue, and between BRAF mutation status and selected genes and miRNA expression in primary PTC. Furthermore, miRNAs and gene expression were compared between primary PTCs and non-neoplastic tissue, and local lymph node metastatic tumor, respectively. All studied markers showed several significant mutual interactions and contexts. In conclusion, to the best our knowledge, this is the first integrated study of BRAF mutational status, the expression levels of mRNAs of selected genes and miRNAs in primary PTC, and paired LNM.

Prognostic Value of Tumor Tissue Up-regulated microRNAs in Clear Cell Renal Cell Carcinoma (ccRCC).

BACKGROUND/AIM: The management of patients with clear cell renal cell carcinoma (ccRCC) includes prognosis assessment based on TNM classification and biochemical markers. This approach stratifies patients with advanced ccRCC into groups of favorable, intermediate, and poor prognosis. The aim of the study was to improve prognosis estimation using microRNAs involved in the pathogenesis of ccRCC. PATIENTS AND METHODS: The study was based on a histologically-verified set of matched ccRCC FFPE tissue samples (normal renal tissue, primary tumor, metastasis, n=20+20+20). The expression of 2,549 microRNAs was analyzed using the SurePrint G3 Human miRNA microarray kit (Agilent Technologies). Prognostic value of significantly deregulated microRNAs was further evaluated on microRNA expression and clinical data of 475 patients obtained from TCGA Kidney Clear Cell Carcinoma (KIRC) database. RESULTS: There were 13 up-regulated and 6 down-regulated microRNAs in tumor tissues compared to control tissues. Among them, survival analysis revealed those with prognostic significance. Patients with high expression of miR-21, miR-27a, miR-34a, miR-106b, miR-210, and miR-342 showed significantly unfavorable outcome. The opposite was observed for miR-30e, patients with low expression had significantly shorter survival. CONCLUSION: The inclusion of these microRNAs in a prognostic panel holds the potential to enhance stratification scoring systems, on which the treatment of ccRCC patients is based.

Predictive Significance of Combined Plasmatic Detection of BRAF Mutations and S100B Tumor Marker in Early-Stage Malignant Melanoma.

BACKGROUND: Melanoma is the most aggressive skin cancer with ability to recur also after early-stage tumor surgery. The aim was to identify early-stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. METHODS: Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre-amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. RESULTS: The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild-type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). CONCLUSION: We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. SIGNIFICANCE: There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early-stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre- and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage.

The paradigm change from reactive medical services to 3PM in ischemic stroke: a holistic approach utilising tear fluid multi-omics, mitochondria as a vital biosensor and AI-based multi-professional data interpretation.

Worldwide stroke is the second leading cause of death and the third leading cause of death and disability combined. The estimated global economic burden by stroke is over US$891 billion per year. Within three decades (1990-2019), the incidence increased by 70%, deaths by 43%, prevalence by 102%, and DALYs by 143%. Of over 100 million people affected by stroke, about 76% are ischemic stroke (IS) patients recorded worldwide. Contextually, ischemic stroke moves into particular focus of multi-professional groups including researchers, healthcare industry, economists, and policy-makers. Risk factors of ischemic stroke demonstrate sufficient space for cost-effective prevention interventions in primary (suboptimal health) and secondary (clinically manifested collateral disorders contributing to stroke risks) care. These risks are interrelated. For example, sedentary lifestyle and toxic environment both cause mitochondrial stress, systemic low-grade inflammation and accelerated ageing; inflammageing is a low-grade inflammation associated with accelerated ageing and poor stroke outcomes. Stress overload, decreased mitochondrial bioenergetics and hypomagnesaemia are associated with systemic vasospasm and ischemic lesions in heart and brain of all age groups including teenagers. Imbalanced dietary patterns poor in folate but rich in red and processed meat, refined grains, and sugary beverages are associated with hyperhomocysteinaemia, systemic inflammation, small vessel disease, and increased IS risks. Ongoing 3PM research towards vulnerable groups in the population promoted by the European Association for Predictive, Preventive and Personalised Medicine (EPMA) demonstrates promising results for the holistic patient-friendly non-invasive approach utilising tear fluid-based health risk assessment, mitochondria as a vital biosensor and AI-based multi-professional data interpretation as reported here by the EPMA expert group. Collected data demonstrate that IS-relevant risks and corresponding molecular pathways are interrelated. For examples, there is an evident overlap between molecular patterns involved in IS and diabetic retinopathy as an early indicator of IS risk in diabetic patients. Just to exemplify some of them such as the 5-aminolevulinic acid/pathway, which are also characteristic for an altered mitophagy patterns, insomnia, stress regulation and modulation of microbiota-gut-brain crosstalk. Further, ceramides are considered mediators of oxidative stress and inflammation in cardiometabolic disease, negatively affecting mitochondrial respiratory chain function and fission/fusion activity, altered sleep-wake behaviour, vascular stiffness and remodelling. Xanthine/pathway regulation is involved in mitochondrial homeostasis and stress-driven anxiety-like behaviour as well as molecular mechanisms of arterial stiffness. In order to assess individual health risks, an application of machine learning (AI tool) is essential for an accurate data interpretation performed by the multiparametric analysis. Aspects presented in the paper include the needs of young populations and elderly, personalised risk assessment in primary and secondary care, cost-efficacy, application of innovative technologies and screening programmes, advanced education measures for professionals and general population-all are essential pillars for the paradigm change from reactive medical services to 3PM in the overall IS management promoted by the EPMA.

Asymptomatic abdominal aortic aneurysms show histological signs of progression: a quantitative histochemical analysis.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a serious disease due to its covert nature, relatively high prevalence and fatal prognosis in the case of rupture. To obtain new insights into AAA pathogenesis, we examined the relationships between histopathology, multiplex in vitro immunoassay data, diameter and symptomatology. METHODS: In a prospective, non-randomised study, we evaluated samples from 6 normal infrarenal aortae and 65 AAA patients (65 walls, 55 thrombi). The AAA patients were either asymptomatic (n = 44), symptomatic (n = 7) or with ruptured AAA (n = 14). The AAA diameter was classified as small (<5 cm, n = 18), medium (5-7 cm, n = 26) and large (>7 cm, n = 21). We quantified the histopathology of the AAA wall and the adjacent thrombus. We assessed the expression of proteins in the same samples. RESULTS: Asymptomatic AAAs had walls with more abundant inflammatory infiltrates, lower amounts of PAI-1, a higher number of tPA-positive elements, a tendency towards decreased collagen content, whereas the adjacent thrombi had a greater concentration of VCAM-1 and MMP-2 when compared with symptomatic AAAs. Compared with the aneurysmatic aorta, the normal aorta contained less collagen and more elastin, actin, desmin and PAI-1-positive elements; in addition, it was more vascular. Medium-sized AAAs were the most actin and vimentin rich, and large AAAs were the most vascular. CONCLUSION: Our results show that asymptomatic AAA walls often have more potentially deleterious histopathological alterations than symptomatic AAA walls. This result indicates that a progression from an asymptomatic AAA to rupture can be expected and screening patients who are at risk of rupture could be beneficial.

Next Generation Sequencing Analysis and its Benefit for Targeted Therapy of Lung Adenocarcinoma.

BACKGROUND/AIM: Targeted therapy has become increasingly important in treating lung adenocarcinoma, the most common subtype of lung cancer. Next-generation sequencing (NGS) enables precise identification of specific genetic alterations in individual tumor tissues, thereby guiding targeted therapy selection. This study aimed to analyze mutations present in adenocarcinoma tissues using NGS, assess the benefit of targeted therapy and evaluate the progress in availability of targeted therapies over last five years. PATIENTS AND METHODS: The study included 237 lung adenocarcinoma patients treated between 2018-2020. The Archer FusionPlex CTL panel was used for NGS analysis. RESULTS: Gene variants covered by the panel were detected in 57% patients and fusion genes in 5.9% patients. At the time of the study, 34 patients (14.3% of patients) were identified with a targetable variant. Twenty-five patients with EGFR variants, 8 patients with EML4-ALK fusion and one patient with CD74-ROS1 fusion received targeted therapy. Prognosis of patients at advanced stages with EGFR variants treated by tyrosine kinase inhibitors and patients with EML4-ALK fusion treated by alectinib was significantly favorable compared to patients without any targetable variant treated by chemotherapy (p=0.0172, p=0.0096, respectively). Based on treatment guidelines applicable in May 2023, the number of patients who could profit from targeted therapy would be 64 (27.0% of patients), this is an increase by 88% in comparison to recommendations valid in 2018-2020. CONCLUSION: As lung adenocarcinoma patients significantly benefit from targeted therapy, the assessment of mutational profiles using NGS could become a crucial approach in the routine management of oncological patients.