Host genetic variants associated with COVID-19 reconsidered in a Slovak cohort.

We present the results of an association study involving hospitalized coronavirus disease 2019 (COVID-19) patients with a clinical background during the 3rd pandemic wave of COVID-19 in Slovakia. Seventeen single nucleotide variants (SNVs) in the eleven most relevant genes, according to the COVID-19 Host Genetics Initiative, were investigated. Our study confirms the validity of the influence of LZTFL1 and 2'-5'-oligoadenylate synthetase (OAS)1/OAS3 genetic variants on the severity of COVID-19. For two LZTFL1 SNVs in complete linkage disequilibrium, rs17713054 and rs73064425, the odds ratios of baseline allelic associations and logistic regressions (LR) adjusted for age and sex ranged in the four tested designs from 2.04 to 2.41 and from 2.05 to 3.98, respectively. The OAS1/OAS3 haplotype 'gttg' carrying a functional allele G of splice-acceptor variant rs10774671 manifested its protective function in the Delta pandemic wave. Significant baseline allelic associations of two DPP9 variants in all tested designs and two IFNAR2 variants in the Omicron pandemic wave were not confirmed by adjusted LR. Nevertheless, adjusted LR showed significant associations of NOTCH4 rs3131294 and TYK2 rs2304256 variants with severity of COVID-19. Hospitalized patients' reported comorbidities were not correlated with genetic variants, except for obesity, smoking (IFNAR2), and hypertension (NOTCH4). The results of our study suggest that host genetic variations have an impact on the severity and duration of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the differences in allelic associations between pandemic waves, they support the hypothesis that every new SARS-CoV-2 variant may modify the host immune response by reconfiguring involved pathways.

Differential gene expression of immunity and inflammation genes in colorectal cancer using targeted RNA sequencing.

Introduction: Colorectal cancer (CRC) is a heterogeneous disease caused by molecular changes, as driver mutations, gene methylations, etc., and influenced by tumor microenvironment (TME) pervaded with immune cells with both pro- and anti-tumor effects. The studying of interactions between the immune system (IS) and the TME is important for developing effective immunotherapeutic strategies for CRC. In our study, we focused on the analysis of expression profiles of inflammatory and immune-relevant genes to identify aberrant signaling pathways included in carcinogenesis, metastatic potential of tumors, and association of Kirsten rat sarcoma virus (KRAS) gene mutation. Methods: A total of 91 patients were enrolled in the study. Using NGS, differential gene expression analysis of 11 tumor samples and 11 matching non-tumor controls was carried out by applying a targeted RNA panel for inflammation and immunity genes containing 475 target genes. The obtained data were evaluated by the CLC Genomics Workbench and R library. The significantly differentially expressed genes (DEGs) were analyzed in Reactome GSA software, and some selected DEGs were used for real-time PCR validation. Results: After prioritization, the most significant differences in gene expression were shown by the genes TNFRSF4, IRF7, IL6R, NR3CI, EIF2AK2, MIF, CCL5, TNFSF10, CCL20, CXCL11, RIPK2, and BLNK. Validation analyses on 91 samples showed a correlation between RNA-seq data and qPCR for TNFSF10, RIPK2, and BLNK gene expression. The top differently regulated signaling pathways between the studied groups (cancer vs. control, metastatic vs. primary CRC and KRAS positive and negative CRC) belong to immune system, signal transduction, disease, gene expression, DNA repair, and programmed cell death. Conclusion: Analyzed data suggest the changes at more levels of CRC carcinogenesis, including surface receptors of epithelial or immune cells, its signal transduction pathways, programmed cell death modifications, alterations in DNA repair machinery, and cell cycle control leading to uncontrolled proliferation. This study indicates only basic molecular pathways that enabled the formation of metastatic cancer stem cells and may contribute to clarifying the function of the IS in the TME of CRC. A precise identification of signaling pathways responsible for CRC may help in the selection of personalized pharmacological treatment.

Expression of OCT4 isoforms is reduced in primary colorectal cancer.

Introduction: Colorectal cancer (CRC) is one of the most common types of cancer worldwide. The carcinogenesis of CRC is indeed complex, and there are many different mechanisms and pathways that contribute to the development of malignancy and the progression from primary to metastatic tumors. The OCT4A, encoded by the POU5F1 gene, is a transcription factor responsible for the phenotype of stem cells, maintaining pluripotency and regulation of differentiation. The POU5F1 gene is made up of five exons that can create numerous isoforms through alternative promoter or alternative splicing. In addition to OCT4A, other isoforms called OCT4B are also translated into protein; however, their role in cells has been unclear. The aim of our work was to investigate the expression patterns of OCT4 isoforms in primary and metastatic CRC, providing us with useful information about their role in the development and progression of CRC. Methods: Surgical specimens from a total of 78 patients were collected and isolated from primary tumors (n = 47) and metastases (n = 31). The relative gene expression of OCT4 isoforms was investigated using the RT-qPCR method together with the TaqMan probes for particular OCT4 isoforms. Results: Our results suggest significantly downregulated expression of the OCT4A and OCT4Bs isoforms in both primary (p = 0.0002 and p < 0.0001, respectively) and metastatic tumors (p = 0.0006 and p = 0.00051, respectively) when compared with the control samples. We also observed a correlation between reduced expression of all OCT4 isoforms and both primary and left-sided tumors (p = 0.001 and p = 0.030, respectively). On the other hand, the expression of all OCT4 isoforms was significantly upregulated in metastases compared with primary tumors (p < 0.0001). Discussion: Unlike previous reports, we found out that the expression of OCT4A, OCT4Bs, and all OCT4 isoforms was significantly reduced in primary tumors and metastases compared with control samples. On the other hand, we supposed that the expression rate of all OCT4 isoforms may be related to the cancer type and side, as well as to liver metastases. However, further studies are required to investigate the detailed expression patterns and significance of individual OCT4 isoforms in carcinogenesis.

Hypermethylated GRIA4, a potential biomarker for an early non-invasive detection of metastasis of clinically known colorectal cancer.

Introduction: Colorectal cancer (CRC) can develop through several dysregulated molecular pathways, including the serrated pathway, characterized by CpG island methylator (CIMP) phenotype. Although the tumor tissue is a commonly tested material, sample types such as stool or plasma, bring a new, non-invasive approach. Several cancer-related methylated genes have been identified in CRC patients, including gene GRIA4, showing promising diagnostic potential. The aim of our study was to develop a sensitive droplet digital PCR (ddPCR) assay to examine GRIA4 hypermethylation status in CRC patients and evaluate its diagnostic potential in tissue and liquid biopsy samples. Methods: In total, 23 patients participated in this study, 7 patients with primary CRC and 16 patients with liver metastasis of clinically known CRC. We obtained tumor and non-tumor tissues (N=17), blood samples pre- and post-surgery (N=22), and blood of five volunteers without a personal cancer history. We have developed and optimized a ddPCR assay for GRIA4 hypermethylation detection, from tissue and plasma samples. Results: We detected significantly increased GRIA4 methylation in tumor tissues compared to their adjacent non-tumor tissue, p<0.0001. Receiver operating characteristic (ROC) analysis defined cutoff values to separate primary tumors and metastases from non-tumor colon/rectum, specifically 36.85% for primary tumors and 34.81% for metastases. All primary tumors were above this threshold. When comparing the methylation levels of metastatic vs. non-tumor tissue, a smaller increase was observed in liver metastasis versus colon tissue (3.6× gain; p=0.001), then in liver metastasis versus adjacent liver tissue (17.4× gain; p<0.0001). On average, GRIA4 hypermethylation in primary tumor plasma was 2.8-fold higher (p=0.39), and in metastatic plasma, 16.4-fold higher (p=0.0011) compared to healthy individuals. Hypermethylation in metastatic plasma was on average 5.9 times higher (p=0.051) than in primary tumor plasma. After tumor removal surgery, average hypermethylation decrease in plasma was 1.6× for primary (p=0.037) and 4.5× for metastatic patients (p=0.023). Discussion: Based on our data, it can be inferred that GRIA4 serves as a tissue specific biomarker for the colon/rectum tissue, thus is suitable for cancer classification. This biomarker showed the potential to be an attractive target for early non-invasive detection of metastases of clinically known CRC, although additional analysis has to be performed.

Small-scale variants and large deletions in BRCA1/2 genes in Slovak high-grade serous ovarian cancer.

The role of PARP inhibitors is to prevent the polymerase from repairing the single-strand break that occurred due to tumor growth and thus induce cell apoptosis when the homologous recombination deficiency (HRD) system is disabled. The eliminated system can be monitored especially in patients with serous ovarian epithelial tumors. Current studies still show the highest progression-free survival (PFS) in the examined groups with BRCA mutant status, even though they are also effective in the case of a disrupted HRD system, apart from BRCA genes. The study cohort consists of women diagnosed with high-grade serous ovarian cancer (HGSOC), after at least two lines of chemotherapy and after relapse of the disease, as determined by ESMO standards and guidelines. The commercially available tool SOPHIA DDM™ (SophiaGenetics, Switzerland) was used to classify the variants after sequencing. The most common variants (pathogenic or likely pathogenic) were in BRCA1 c.1067 A>G (rs1799950) and c.5266dupC (rs80357906) and in BRCA2 c.9976 A>T (rs11571833). Large deletions were detected in one and three cases in the BRCA1 and BRCA2 genes, respectively. A mutation in the BRCA1/2 genes was confirmed in 50% of the examined patients. In the study, we focused on the identification of mutated BRCA genes by a commercially available Sophia DDM™ system to identify a pathogenic or probable pathogenic variant in a cohort of patients with HGSOC in the Slovak population, which could result in better management and stratification of the individual.

DNA Polymorphisms in Pregnant Women with Sticky Platelet Syndrome.

Sticky platelet syndrome (SPS) is a thrombophilia caused by the increased aggregability of platelets in response to the addition of low concentrations of epinephrine (EPI) and/or adenosine diphosphate (ADP). Some of the single nucleotide polymorphisms (SNP), alleles and haplotypes of platelet glycoprotein receptors were proved to have a role in the etiology of thrombotic episodes When comparing SPS and the control group, in VEGFA rs3025039, the p value for both CC vs. TT and CT vs. TT analyses was <0.001. Interestingly, no minor TT genotype was present in the SPS group, suggesting the thrombotic pathogenesis of recurrent spontaneous abortions (RSA) in these patients. Moreover, we found a significant difference in the presence of AT containing a risky A allele and TT genotype of ALPP rs13026692 (p = 0.034) in SPS patients when compared with the controls. Additionally, we detected a decreased frequency of the GG (CC) genotype of FOXP3 rs3761548 in patients with SPS and RSA when compared with the control group (p value for the CC (GG) vs. AA (TT) 0.021). This might indicate an evolutionary protective mechanism of the A (T) allele in the SPS group against thrombotic complications in pregnancy. These results can be used for antithrombotic management in such pregnant patients.

The Common Haplotype GATGACA in Surfactant-Protein B Gene Is Protective for Respiratory Distress Syndrome in Preterm Neonates.

Background: Respiratory distress syndrome (RDS), a disorder of primary surfactant deficiency resulting in pulmonary insufficiency, remains a significant problem for preterm neonates. Associations between genetic variants of surfactant proteins and RDS have been reported, but haplotypes of the surfactant protein B gene (SFTPB) have not been studied. The aim of the study was to prove the hypothesis that certain haplotypes of SFTPB may be protective or risk factors for RDS. Methods: The study was performed with 149 preterm infants, born <34 weeks of gestation, with 86 infants with mild RDS or without RDS (control group) and 63 infants with severe RDS (patient group). RDS was considered severe if multiple doses of exogenous surfactant and/or mechanical ventilation within the first 72 h of life were needed. The venous blood sample was used for the analysis of gene polymorphisms associated with RDS, genotyping, and haplotype estimation. Multivariate logistic regression analysis and the odds ratio were calculated to detect the contribution of the studied variables to the development of RDS. Results: A new association of the common single nucleotide polymorphism (SNP) rs2304566 with RDS in premature infants was detected. Analysis of rs2304566 polymorphisms using a logistic regression model showed that there are two significant predictors inversely related to the occurrence of RDS (Apgar score of 5 min, CT and TT genotype in rs2304566 polymorphism). Gestational age, birth weight, and sex have border significance. Moreover, in the patient group, the frequency of the GATGACA haplotype in the SFTPB gene was lower (p = 0.037), and the GATGGCA haplotype was higher (p = 0.059) in comparison with the control group. Conclusion: The common haplotype GATGACA of the SFTPB gene can be protective against RDS in preterm infants. The trend of a higher frequency of GATGGCA in the SFTPB gene in infants with severe RDS suggests that this haplotype may be a risk factor for RDS susceptibility.

Cancer stem cell marker expression and methylation status in patients with colorectal cancer.

The number of individuals diagnosed with colorectal cancer (CRC) has been on an alarming upward trajectory over the past decade. In some countries, this cancer represents one of the most frequently diagnosed types of neoplasia. Therefore, it is an important demand to study the pathology underlying this disease to gain insights into the mechanism of resistance to treatment. Resistance of tumors to chemotherapy and tumor aggressiveness have been associated with a minor population of neoplastic cells, which are considered to be responsible for tumor recurrence. These types of neoplastic cells are known as cancer stem cells, which have been previously reported to serve an important role in pathogenesis of this malignant disease. Slovakia has one of the highest incidence rates of CRC worldwide. In the present study, the aim was to classify the abundance of selected stem cell markers (CD133, CD166 and Lgr5) in CRC tumors using flow cytometry. In addition, the methylation status of selected genomic regions of CRC biomarkers (ADAMTS16, MGMT, PROM1 (CD133), LGR5 and ALCAM) was investigated by pyrosequencing in a cohort of patients from Martin University Hospital, Martin, Slovakia. Samples from both primary tumors and metastatic tumors were tested. Analysis of DNA methylation in the genomic regions of indicated five CRC biomarkers was also performed, which revealed the highest levels of methylation in the A disintegrin and metalloproteinase with thrombospondin motifs 16 and O6-methyguanine-DNA methyl transferase genes, whereas the lowest levels of methylation were found in genes expressing prominin-1, leucine-rich repeat-containing G-protein-coupled receptor 5 and activated leukocyte cell adhesion molecule. Furthermore, tumor tissues from metastases showed significantly higher levels of CD133+ cells compared with that in primary tumors. Higher levels of CD133+ cells correlated with TNM stage and the invasiveness of CRC into the lymphatic system. Although relatively small number of samples was processed, CD133 marker was consider to be important marker in pathology of CRC.

Alzheimer's Disease-Associated SNP rs708727 in SLC41A1 May Increase Risk for Parkinson's Disease: Report from Enlarged Slovak Study.

SLC41A1 (A1) SNPs rs11240569 and rs823156 are associated with altered risk for Parkinson's disease (PD), predominantly in Asian populations, and rs708727 has been linked to Alzheimer's disease (AD). In this study, we have examined a potential association of the three aforementioned SNPs and of rs9438393, rs56152218, and rs61822602 (all three lying in the A1 promoter region) with PD in the Slovak population. Out of the six tested SNPs, we have identified only rs708727 as being associated with an increased risk for PD onset in Slovaks. The minor allele (A) in rs708727 is associated with PD in dominant and completely over-dominant genetic models (ORD = 1.36 (1.05-1.77), p = 0.02, and ORCOD = 1.34 (1.04-1.72), p = 0.02). Furthermore, the genotypic triplet GG(rs708727) + AG(rs823156) + CC(rs61822602) might be clinically relevant despite showing a medium (h ≥ 0.5) size difference (h = 0.522) between the PD and the control populations. RandomForest modeling has identified the power of the tested SNPs for discriminating between PD-patients and the controls to be essentially zero. The identified association of rs708727 with PD in the Slovak population leads us to hypothesize that this A1 polymorphism, which is involved in the epigenetic regulation of the expression of the AD-linked gene PM20D1, is also involved in the pathoetiology of PD (or universally in neurodegeneration) through the same or similar mechanism as in AD.

Circulating miRNA expression over the course of colorectal cancer treatment.

Colorectal cancer (CRC) is the third-most common cancer type in males and the second-most common cancer type in females, and has the second-highest overall mortality rate worldwide. Approximately 50% of patients in stage I-III develop metastases, mostly localized to the liver. All physiological conditions occurring in the organism are also reflected in the levels of circulating microRNAs (miRNAs/miRs) in patients. miRNAs are a class of small, non-coding, single-stranded RNAs consisting of 18-25 nucleotides, which have important roles in various cellular processes. The aim of the present study was to evaluate a panel of seven circulating miRNAs (miR-106a-5p, miR-210-5p, miR-155-5p, miR-21-5p, miR-103a-3p, miR-191-5p and miR-16-5p) as biomarkers for monitoring patients undergoing adjuvant treatment of CRC. Total RNA was extracted from the plasma of patients with CRC prior to surgery, in the early post-operative period (n=60) and 3 months after surgery (n=14). The levels of the selected circulating miRNAs were measured with the miRCURY LNA miRNA PCR system and fold changes were calculated using the standard ∆∆Cq method. DIANA-miRPath analysis was used to evaluate the role of significantly deregulated miRNAs. The results indicated significant upregulation of miR-155-5p, miR-21-5p and miR-191-5p, and downregulation of miR-16-5p directly after the surgery. In paired follow-up samples, the most significant upregulation was detected for miR-106a-5p and miR-16-5p, and the most significant downregulation was for miR-21-5p. Pathway analysis outlined the role of the differentially expressed miRNAs in cancer development, but the same pathways are also involved in wound healing and regeneration of intestinal epithelium. It may be suggested that these processes should also be considered in studies investigating sensitive and easily detectable circulating biomarkers for recurrence in patients.

Congenital Afibrinogenemia and Hypofibrinogenemia: Laboratory and Genetic Testing in Rare Bleeding Disorders with Life-Threatening Clinical Manifestations and Challenging Management.

Congenital fibrinogen disorders are rare pathologies of the hemostasis, comprising quantitative (afibrinogenemia, hypofibrinogenemia) and qualitative (dysfibrinogenemia and hypodysfibrinogenemia) disorders. The clinical phenotype is highly heterogeneous, being associated with bleeding, thrombosis, or absence of symptoms. Afibrinogenemia and hypofibrinogenemia are the consequence of mutations in the homozygous, heterozygous, or compound heterozygous state in one of three genes encoding the fibrinogen chains, which can affect the synthesis, assembly, intracellular processing, stability, or secretion of fibrinogen. In addition to standard coagulation tests depending on the formation of fibrin, diagnostics also includes global coagulation assays, which are effective in monitoring the management of replacement therapy. Genetic testing is a key point for confirming the clinical diagnosis. The identification of the precise genetic mutations of congenital fibrinogen disorders is of value to permit early testing of other at risk persons and better understand the correlation between clinical phenotype and genotype. Management of patients with afibrinogenemia is particularly challenging since there are no data from evidence-based medicine studies. Fibrinogen concentrate is used to treat bleeding, whereas for the treatment of thrombotic complications, administered low-molecular-weight heparin is most often. This review deals with updated information about afibrinogenemia and hypofibrinogenemia, contributing to the early diagnosis and effective treatment of these disorders.

Detection of therapeutically relevant and concomitant rare somatic variants in colorectal cancer.

In colorectal cancer (CRC), clinically relevant biomarkers are known for genome-guided therapy that can be detected by both first and next generation methods. The aim of our work was to introduce a robust NGS assay that will be able to detect, in addition to standard predictive single nucleotide-based biomarkers, even rare and concomitant clinically relevant variants. Another aim was to identify truncating mutations in APC and pathogenic variants in TP53 to divide patients into potentially prognostic groups. A multigene panel with hotspots in 50 cancer-critical genes was used. Finally, 86 patients diagnosed with primary or metastatic colorectal cancer were enrolled. In total, there were identified 163 pathogenic variants, among them in the genes most recurrent mutated in CRC such as TP53 (49%), the RAS family genes KRAS and NRAS (47%), APC (43%), and PIK3CA (15%). In 30 samples, two driver mutations were present in one sample, 11 patients were without any mutations covered by this panel. In one patient, a novel variant in BRAF p.D594E was found, not previously seen in CRC, and was concomitant with KRAS p.G12A. In KRAS, a potentially sensitive mutation to anti-EGFR therapy p.A59T was found along with the PIK3CA missense variant p.E545K. It was possible to divide patients into groups based on the occurrence of truncating APC variant alone or concomitant with TP53 or KRAS. Our results demonstrate the potential of small multigene panels that can be used in diagnostics for the detection of rare therapeutically relevant variants. Moreover, the division of patients into groups based on the presence of APC and TP53 mutations enables this panel to be used in retrospective studies on the effectiveness of treatment with anti-EGFR inhibitors.

High-grade serous ovarian carcinoma and detection of inactivated BRCA genes from biopsy material of Slovak patients.

Ovarian cancer is the leading cause of mortality among all gynecological cancers in developed countries and its most common and most lethal type is the high-grade serous ovarian carcinoma (HGSC). At the molecular level, nearly half of all HGSCs exhibit ineffective homologous DNA recombination and disruption of DNA damage/repair pathway inactivation caused often by BRCA1 and BRCA2 gene mutation. Recently, the detection of BRCA1/2 mutations became important for personalized treatment of HGSC patients with the PARP-inhibitors in the defined clinical setting of relapse after positive adjuvant platinum-based chemotherapeutic response. Based on the selection of patients by regional oncologists, we attempted to verify the possibilities of BRCA1/2 mutation testing on archival formalin-fixed paraffin-embedded (FFPE) biopsy material from regional hospitals. In the study we used: a/ FFPE tumor resections of 97 patients sent to our laboratory, originally stored in archives of regional departments for a period of 1-3 years and retrieved on the principle to contain a maximum of non-necrotic tumor tissue, b/ next-generation sequencing (NGS) assay covering all known mutations in the BRCA1/2 genes on MiSeq (Illumina® platform), and c/ Sophia DDM® bioinformatics platform. After processing of FFPE samples, 5 cases were excluded due to the insufficient genomic DNA quantity. Bioinformatics results of NGS analyses of 92 patients' samples indicated 17.39% pathogenic mutations and 32.61% potentially pathogenic mutations in genes BRCA1/2. Overall, 50% pathogenic and potentially pathogenic mutations were detected in the patient's cohort. The relatively high incidence of BRCA1/2 mutations in our series may be influenced by various indicators including the selection of patients based on adjuvant therapy response as well as regional or population heterogeneity in their frequency. Based on the interdisciplinary cooperation, the use of archival biopsy material processed primarily and stored for a longer period in different laboratories without uniformly defined pre-analytical conditions allows identifying the HGSC patients who might better respond to the PARP-inhibition therapy.

Prevalence and significance of M541L single nucleotide polymorphism in the central European cohort of gastrointestinal stromal tumor patients.

BACKGROUND: Single nucleotide polymorphisms can create a genetic microenvironment in some tumors that affects the course of treatment, resistance, etc. Whether single nucleotide polymorphisms have an impact on gastrointestinal stromal tumor (GIST) development and disease progression is not yet accurately verified. KIT SNPM541L in exon 10 correlates with a worse prognosis of many cancers. The impact of KIT SNPM541L in GISTs is relatively unknown and, therefore, its analyses could have potential in patient therapy and could provide more detailed information on tumor character, clinical presentation, or tumor behavior in treatment. AIM: The aim of the study was the analysis of the biological and clinical significance of the KIT SNPM541L polymorphism in exon 10. MATERIALS AND METHODS: Paraffin sample tissues were obtained from the National GIST Register in Martin. Retrospective samples from 177 GIST patients were divided into several groups. Detection of SNPM541L was performed by Sanger sequencing. Statisitical analyses were performed to determine the prevalence of KIT SNPM541L in the Slovak GIST cohort, to search for correlation between c-KIT status and clinicopathological, molecular and biological data. RESULTS: Overall, 29 samples out of 177 showed KIT SNPM541L polymorphism. CONCLUSION: Our results do not support the association between KIT SNPM541L and increased risk of relapse in localized primary GISTs. Additionally, we found a positive correlation between KIT SNPM541L occurrence and earlier onset of relapse in PDGFRa and WT subgroup of GISTs.

Genomic profile and immune contexture in colorectal cancer-relevance for prognosis and immunotherapy.

Colorectal cancer (CRC) is one of the leading cancers in both genders. TNM staging system is still the most commonly used tumor classification and prognostic system. The disadvantage of TNM is that the prognostic information it provides is incomplete, and patients with the same histological tumor stages may differ significantly in the clinical outcome. Therefore, the identification of new prognostic parameters is crucial. The carcinogenic process that gives rise to an individual tumor is unique and tumor microenviroment should be taken into consideration. In CRC, T-cell infiltration is not homogenous, and recent studies are mostly focusing on memory T-cells and CD8 cells in predicting disease-free survival (DFS) and overall survival (OS). It seems that DFS and OS are not only dependent on microsatellite instable or stable status but mostly on the levels of expression of the immune signatures. Also, patients with high infiltration of cytotoxic and memory cells have significantly better outcome. This review consolidates current knowledge and recent research about importance of immune-cell-associated proteins, specific gene profiles of immune cells and immunotherapy in CRC. We also discussed cell-specific signatures in cancer treatment.

BRAF mutations in KIT/PDGFRA positive gastrointestinal stromal tumours (GISTs): Is their frequency underestimated?

BRAF V600E mutations in GISTs are considered to be one of the mutational events in KIT/PDGFRA negative or positive GISTs, respectively. BRAF mutated GISTs usually do not respond to imatinib treatment, even more GISTs with imatinib sensitive KIT mutation. However, they are almost phenotypically and morphologically identical with KIT/PDGFRA positive GISTs. In general, due to the small number of BRAF mutations in GIST and because of the rarity of concomitant BRAF/KIT or BRAF/PDGFRA mutations, their frequency may be depreciated. The aim of this study was BRAF mutation detection in KIT/PDGFRA positive GISTs and their verification by other molecular methods. We applied the sensitive droplet digital PCR on 35 randomly selected KIT/PDGFRA positive GISTs to detect V600E mutations. We have established two criteria for the evaluation of samples: false positive rate (FPR) based on the negative controls; Limit of Detection (LoD) based on the serial dilution of positive control from RKO cell line harboring heterozygous V600E mutation in constant wild-type DNA background. Results from ddPCR were verified by other molecular methods: allele-specific PCR, dideoxysequencing, competitive allele-specific TaqMan PCR (castPCR). FPR was determined as 5 (∼4.4) positive droplets, and LoD was assessed to 3.4293 copies/µL what is the method sensitivity of 0.0162 %. We identified eight KIT/PDGFRA positive patients with concomitant V600E mutation. The five of them were in coexistence with KIT mutation and three with PDGFRA mutation. We also included the liver metastasis, but data from primary tumour were not available. We achieved the very high sensitivity of the ddPCR method for detecting BRAF mutation in GISTs to have importance from the point of view of therapy.

Genetic Variants in the FGB and FGG Genes Mapping in the Beta and Gamma Nodules of the Fibrinogen Molecule in Congenital Quantitative Fibrinogen Disorders Associated with a Thrombotic Phenotype.

Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bß, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bß, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.

Diagnostic Potential of MicroRNAs as Biomarkers in the Detection of Preeclampsia.

Preeclampsia is a multisystemic disorder that occurs in 5-8% of pregnant women and remains a leading cause of both maternal and fetal morbidity and mortality. The disease is characterized by the abnormal vascular response to placentation, but the exact pathophysiology and pathogenesis of preeclampsia remain unknown. Risk factors for preeclampsia include increased maternal age, obesity, multiple gestations, and a history of preeclampsia. Several studies have suggested that altered expression of some microRNAs (miRNAs) in placental tissue, and maternal circulation, may be associated with several types of pregnancy complications such as preeclampsia, preterm birth, and spontaneous abortion. It is assumed that these miRNAs play an important role in various cellular processes important for maintaining a healthy pregnancy, including promoting angiogenesis and the differentiation of trophoblast cells. In this review, we discuss the role of miRNAs as potential biomarkers of preeclampsia.

The Role of CADM1 and MAL Promoter Methylation in Inflammation and Cervical Intraepithelial Neoplasia.

Aims: This study investigated the presence of human papillomavirus (HPV) infection and the methylation status of the promoters of the cell adhesion molecule 1 (CADM1) gene and T lymphocyte maturation associated protein (MAL) gene in patients with cervicitis/inflammation and cervical intraepithelial neoplasia (CIN). Materials and Methods: Cervical specimens (n = 47) were collected from women with normal cervical cytology (n = 21) and those with cervical abnormalities (n = 26). The presence of HPV infection was confirmed by an HPV DNA test and an HPV mRNA test (Aptima HPV test). Methylation levels of the CADM1 and MAL promoters were evaluated by pyrosequencing. Results: Compared with the HPV DNA test, the Aptima HPV test improved specificity from 57% to 70% for the detection of inflammation and/or CIN type 1 (CIN1) or more advanced conditions (CIN1+). The methylation level of the CADM1 and MAL promoters was 1.5 times higher in inflammatory samples, compared with normal cervical cytology (p < 0.05). Conclusion: Selected 5'-C-phosphate-G-3' islands within the promoters of the CADM1 and MAL genes were differentially methylated in the inflammatory samples compared with the CIN samples. These results suggested that methylation likely occurred following tissue disruption, and the detection of persistent inflammation might be associated with a higher risk of lesion progression.

Comparison of clinical phenotype with genetic and laboratory results in 31 patients with congenital dysfibrinogenemia in northern Slovakia.

Congenital dysfibrinogenemia (CD) is a rare disorder of hemostasis. The majority of cases are caused by heterozygous missense mutations in one of the three fibrinogen genes. Patients with CD may experience bleeding and thrombosis, but many are asymptomatic. To better describe the clinical, laboratory, and genotypic picture of CD, we evaluated 31 patients from seven unrelated families using standard coagulation tests and genetic analysis. The clinical phenotype consisted of bleeding in 13/31 (42%) patients; other patients (18/31; 58%) were asymptomatic. Among patients with bleeding, symptoms were mostly in single anatomical sites, with variable intensity of bleeding. Compared to results from a previous large systematic survey, our results showed a similar mean bleeding score, but a higher incidence of bleeding episodes without thrombotic complications. In the present study, we identified three known pathogenic point mutations in the FGA (c.95G > A, c.104G > A) and FGB (c.586C > T) genes. The variants of CD identified in this cross-sectional study were either asymptomatic or had bleeding manifestations and showed similar laboratory features, irrespective of genotype. Results from genetic and clinical studies will continue to yield valuable information on the structure and function of the fibrinogen molecule.