Increased mRNA expression for serotonin receptor 1B (HTR1B) is associated with thrombosis in BCR::ABL1-negative myeloproliferative neoplasms.

BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are clonal haematopoietic stem cell disorders characterized by specific driver mutations and an increased risk of both macrothrombosis and microthrombosis. Serotonin receptor type 1B (HTR1B) was found to be expressed by various solid tumours, and also primary bone marrow mononuclear cells from myelodysplastic neoplasm and acute myeloid leukaemia patients, representing a potential therapeutic target. In this study we assessed for the first time the expression levels of HTR1B mRNA in the peripheral blood mononuclear cells (PBMC) of 85 newly diagnosed MPN patients, consisting of 28 polycythemia vera, 25 essential thrombocythemia and 32 primary myelofibrosis cases. Levels of HTR1B expression between MPN subtypes and control group were not significantly different. However, at clinical data examination, it was observed that MPN patients with a recent history of major thrombosis and/or signs of impaired microcirculation exhibited significantly higher HTR1B expression levels compared to non-thrombotic MPNs and control group. Moreover, thrombotic MPN patients had significantly higher HTR1B expression than patients with recent thrombosis and absence of MPN diagnostic criteria. These findings suggest that increased levels of HTR1B expression in PBMC might be associated with thrombosis in MPN patients, but larger studies are needed for confirmation, including testing of the receptor protein expression level.

Leukemic conversion involving RAS mutations of type 1 CALR-mutated primary myelofibrosis in a patient treated for HCV cirrhosis: a case report.

Somatic frameshift mutations in exon 9 of calreticulin (CALR) gene are recognized as disease drivers in primary myelofibrosis (PMF), one of the three classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Type 1/type 1-like CALR mutations particularly confer a favorable prognostic and survival advantage in PMF patients. We report an unusual case of PMF incidentally diagnosed in a 68-year-old woman known with hepatitis C virus (HCV) cirrhosis who developed a progressive painful splenomegaly, without anomalies in blood cell counts. While harboring a type 1 CALR mutation, the patient underwent a leukemic transformation in less than 1 year from diagnosis, with a lethal outcome. Analysis of paired DNA samples from chronic and leukemic phases by a targeted next-generation sequencing (NGS) panel and single-nucleotide polymorphism (SNP) microarray revealed that the leukemic clone developed from the CALR-mutated clone through the acquisition of genetic events in the RAS signaling pathway: an increased variant allele frequency of the germline NRAS Y64D mutation present in the chronic phase (via an acquired uniparental disomy of chromosome 1) and gaining NRAS G12D in the blast phase. SNP microarray analysis showed five clinically significant copy number losses at regions 7q22.1, 8q11.1-q11.21, 10p12.1-p11.22, 11p14.1-p11.2, and Xp11.4, revealing a complex karyotype already in the chronic phase. We discuss how additional mutations, detected by NGS, as well as HCV infection and antiviral therapy, might have negatively impacted this type 1 CALR-mutated PMF. We suggest that larger studies are required to determine if more careful monitoring would be needed in MPN patients also carrying HCV and receiving anti-HCV treatment.

Collagen Family and Other Matrix Remodeling Proteins Identified by Bioinformatics Analysis as Hub Genes Involved in Gastric Cancer Progression and Prognosis.

Gastric cancer has remained in the top five cancers for over ten years, both in terms of incidence and mortality due to the shortage of biomarkers for disease follow-up and effective therapies. Aiming to fill this gap, we performed a bioinformatics assessment on our data and two additional GEO microarray profiles, followed by a deep analysis of the 40 differentially expressed genes identified. PPI network analysis and MCODE plug-in pointed out nine upregulated hub genes coding for proteins from the collagen family (COL12A1, COL5A2, and COL10A1) or involved in the assembly (BGN) or degradation of collagens (CTHRC1), and also associated with cell adhesion (THBS2 and SPP1) and extracellular matrix degradation (FAP, SULF1). Those genes were highly upregulated at the mRNA and protein level, the increase being correlated with pathological T stages. The high expression of BGN (p = 8 × 10-12), THBS2 (p = 1.2 × 10-6), CTHRC1 (p = 1.1 × 10-4), SULF1 (p = 3.8 × 10-4), COL5A1 (p = 1.3 × 10-4), COL10A1 (p = 5.7 × 10-4), COL12A1 (p = 2 × 10-3) correlated with poor overall survival and an immune infiltrate based especially on immunosuppressive M2 macrophages (p-value range 4.82 × 10-7-1.63 × 10-13). Our results emphasize that these genes could be candidate biomarkers for GC progression and prognosis and new therapeutic targets.

Kinetics and persistence of cellular and humoral immune responses to SARS-CoV-2 vaccine in healthcare workers with or without prior COVID-19.

SARS-CoV-2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS-CoV-2 infection. No new infections were diagnosed during follow-up. At 6 months, post-vaccination or post-infection, despite a downward trend in the level of anti-S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live-virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow-up in persons vaccinated after prior infection. Anti-S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1-3) or low neutralizing activity (30%-40%) at 6 months, although with lower T-cell stimulation index (p = 0.046) and IFN-γ secretion (p = 0.0007) compared to those with preserved humoral responses.

Gastrointestinal cancer stem cells as targets for innovative immunotherapy.

The role of cancer stem cells in gastrointestinal cancer-associated death has been widely recognized. Gastrointestinal cancer stem cells (GCSCs) are considered to be responsible for tumor initiation, growth, resistance to cytotoxic therapies, recurrence and metastasis due to their unique properties. These properties make the current therapeutic trials against GCSCs ineffective. Moreover, recent studies have shown that targeting stem cell surface markers or stemness associated pathways might have an additional off-target effect on the immune system. Recent advances in oncology and precision medicine have opened alternative therapeutic strategies in the form of cancer immunotherapy. This approach differs from classical anti-cancer therapy through its mechanism of action involving the activation and use of a functional immune system against tumor cells, instead of aiming physically destruction of cancer cells through radio- or chemotherapy. New immunological approaches for GCSCs targeting involve the use of different immune cells and various immune mechanisms like targeting specific surface antigens, using innate immune cells like the natural killer and T cells, T-cell chimeric antigen receptor technology, dendritic cell vaccine, or immune checkpoint inhibitors. In this respect, better understandings of immune regulatory mechanisms that govern anti-tumor response bring new hope in obtaining long-term remission for cancer therapy.

Inflammation-Related Patterns in the Clinical Staging and Severity Assessment of Chronic Kidney Disease.

Chronic kidney disease (CKD) is an irreversible loss of kidney function, and it represents a major global public health burden due to both its prevalence and its continuously increasing incidence. Mineral bone disorders (MBDs) constitute a hallmark of CKD, and alongside cardiovascular complications, they underlie a poor prognosis for these patients. Thus, our study focused on novel CKD biomarker patterns and their impact on the clinical staging of the disease. As a first testing approach, the relative expression levels of 105 proteins were assessed by the Proteome Profiler Cytokine Array Kit for pooled CKD stage 2-4 serum samples to establish an overall view regarding the proteins involved in CKD pathogenesis. Among the molecules that displayed significant dysregulation in the CKD stages, we further explored the involvement of Dickkopf-related protein 1 (Dkk-1), a recognised inhibitor of the Wnt signalling pathway, and its crosstalk with 1,25OH2D3 (calcitriol) as new players in renal bone and vascular disease. The serum levels of these two molecules were quantified by an ELISA (76 samples), and the results reveal decreasing circulating levels of Dkk-1 and calcitriol in advanced CKD stages, with their circulating expression showing a downward trend as the CKD develops. In the next step, we analysed the inflammation and MBD biomarkers' expression in CKD (by xMAP array). Our results show that the molecules involved in orchestrating the inflammatory response, interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα), as well as the mineral biomarkers osteoprotegerin (OPG), osteocalcin (OC), osteopontin (OPN), and fibroblast growth factor 23 (FGF-23), correlate with Dkk-1 and calcitriol, raising the possibility of them being potential useful CKD biomarkers. These results reveal the impact of different biomarker patterns in CKD staging and severity, thus opening up novel approaches to be explored in CKD clinical management.

Long non-coding RNAs in brain tumours: Focus on recent epigenetic findings in glioma.

Glioma biology is a major focus in tumour research, primarily due to the aggressiveness and high mortality rate of its most aggressive form, glioblastoma. Progress in understanding the molecular mechanisms behind poor prognosis of glioblastoma, regardless of treatment approaches, has changed the classification of brain tumours after nearly 100 years of relying on anatomopathological criteria. Expanding knowledge in genetic, epigenetic and translational medicine is also beginning to contribute to further elucidating molecular dysregulation in glioma. Long non-coding RNAs (lncRNAs) and their main representatives, large intergenic non-coding RNAs (lincRNAs), have recently been under scrutiny in glioma research, revealing novel mechanisms of pathogenesis and reinforcing others. Among those confirmed was the reactivation of events significant for foetal brain development and neuronal commitment. Novel mechanisms of tumour suppression and activation of stem-like behaviour in tumour cells have also been examined. Interestingly, these processes involve lncRNAs that are present both during normal brain development and in brain malignancies and their reactivation might be explained by epigenetic mechanisms, which we discuss in detail in the present review. In addition, the review discusses the lncRNAs-induced changes, as well as epigenetic changes that are consequential for tumour formation, affecting, in turn, the expression of various types of lncRNAs.

New therapeutic options opened by the molecular classification of gastric cancer.

Gastric cancer (GC) is one of the most lethal and aggressive cancers, being the third cause of cancer related death worldwide. Even with radical gastrectomy and the latest generation of molecular chemotherapeutics, the numbers of recurrence and mortality remains high. This is due to its biological heterogeneity based on the interaction between multiple factors, from genomic to environmental factors, diet or infections with various pathogens. Therefore, understanding the molecular characteristics at a genomic level is critical to develop new treatment strategies. Recent advances in GC molecular classification provide the unique opportunity to improve GC therapy by exploiting the biomarkers and developing novel targeted therapy specific to each subtype. This article highlights the molecular characteristics of each subtype of gastric cancer that could be considered in shaping a therapeutic decision, and also presents the completed and ongoing clinical trials addressed to those targets. The implementation of the novel molecular classification system will allow a preliminary patient selection for clinical trials, a mandatory issue if it is desired to test the efficacy of a certain inhibitor to the given target. This will represent a substantial advance as well as a powerful tool for targeted therapy. Nevertheless, translating the scientific results into new personalized treatment opportunities is needed in order to improve clinical care, the survival and quality of life of patients with GC.

Knockdown of KRT17 by siRNA induces antitumoral effects on gastric cancer cells.

BACKGROUND: Keratin 17 (KRT17) was shown to be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of various cancer types. Our previous studies identified KRT17 as a possible biomarker for gastric cancer by gene microarray, with an elevated expression that occurred early during tumorigenesis and increased during tumor progression. Based on these findings, we aimed to investigate KRT17 biological functions in gastric adenocarcinoma and its possible use as a rational molecular target for anticancer therapy. METHODS: We used RNA interference-mediated knockdown of KRT17 expression and analyzed the effects on cell proliferation, cell migration, and signal transduction in two gastric cell lines (AGS and NCI-N87) in vitro and on xenograft growth in vivo. RESULTS: The functional analysis of KRT17 knockdown cell lines showed a decreased cell proliferation (with 42.36% ± 3.2%) and migration ability (with 37.2% ± 6.2%) relative to scrambled siRNA control. The in vivo tumorigenicity on nude mice exhibited a significant decrease in tumor weight with 69.14% in xenografts obtained from AGS cells and 84.43% in xeno-NCI-N87 tumors. The analysis on KRT17 knockdown outcome on intracellular signaling identifies AKT/mTOR as the main affected pathway that sustains proliferation and survival, and also the AMPKα1/CREB pathway that was recently shown to induce organ protection and antiinflammatory response. CONCLUSIONS: Our results highlight KRT17 as a possible biomarker in gastric cancer promoting tumor growth, motility, and invasion, and suggest that KRT17 can be a valuable molecular target for development of anti-gastric cancer-specific therapies.

Therapies targeting cancer stem cells: Current trends and future challenges.

Traditional therapies against cancer, chemo- and radiotherapy, have multiple limitations that lead to treatment failure and cancer recurrence. These limitations are related to systemic and local toxicity, while treatment failure and cancer relapse are due to drug resistance and self-renewal, properties of a small population of tumor cells called cancer stem cells (CSCs). These cells are involved in cancer initiation, maintenance, metastasis and recurrence. Therefore, in order to develop efficient treatments that can induce a long-lasting clinical response preventing tumor relapse it is important to develop drugs that can specifically target and eliminate CSCs. Recent identification of surface markers and understanding of molecular feature associated with CSC phenotype helped with the design of effective treatments. In this review we discuss targeting surface biomarkers, signaling pathways that regulate CSCs self-renewal and differentiation, drug-efflux pumps involved in apoptosis resistance, microenvironmental signals that sustain CSCs growth, manipulation of miRNA expression, and induction of CSCs apoptosis and differentiation, with specific aim to hamper CSCs regeneration and cancer relapse. Some of these agents are under evaluation in preclinical and clinical studies, most of them for using in combination with traditional therapies. The combined therapy using conventional anticancer drugs with CSCs-targeting agents, may offer a promising strategy for management and eradication of different types of cancers.

MECHANISMS OF THROMBOSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS.

Thrombotic events are highly prevalent in systemic lupus erythematosus (SLE). Antiphospholipid antibodies play an essential role in promoting thrombosis by activating several intracellular signaling pathways (TLR4, p38MAPK, NFkB) in platelets, monocytes and endothelial cells. New therapeutic opportunities might be offered by addressing these molecular targets. Chronic inflammatory status, the degree of disease activity and accelerated atherosclerosis are also responsible for the thrombotic phenotype in patients with SLE. The aim of this review is to highlight thrombosis mechanisms and to look for possible connection between SLE, antiphospholipid antibodies and cancer, especially myeloproliferative neoplasms.

IL-6 and IL-11 as markers for tumor aggressiveness and prognosis in gastric adenocarcinoma patients without mutations in Gp130 subunits.

BACKGROUND AND AIMS: A point mutation (gp130Y757F/Y759F) was identified as being responsible for aberrant activation of gp130 in mice and associated with gastric adenocarcinoma induction. As a result, we investigated the possible role of key point mutations in Tyr from IL6ST exon 17 that encode for the catalytic domain of gp130, and of its respective activators (IL-6 family member cytokines) in human gastric cancer initiation and development. METHOD: DNA, protein and plasma from 51 patients with gastric adenocarcinoma have been used in exploring gp130 status. We used sequencing analysis of IL6ST exon 17 in order to identify possible mutations that would lead to constitutive active forms of the receptor. The levels of gp130 activators (IL-6, IL-11, LIF) were analyzed by ELISA in plasma and mucosa of patients with gastric adenocarcinoma. RESULTS: Sequencing analysis did not identify mutations in gp130 key positions (Y759, Y767, Y814, Y905 and Y915). An increased IL-6 and IL-11 level in gastric mucosa was observed, correlated with staging, indicating these cytokines as gp130 activators in tumor epithelial cell. Those variations were consistent with increased IL-6 level in plasma. Furthermore, IL-6, but not IL-11 showed a significant correlation with patient's survival time, suggesting that tissue and plasma concentration of IL-6 might be a marker of tumor aggressiveness with prognostic value. CONCLUSIONS: According to our results, no mutations were detected in gp130 key positions in human gastric adenocarcinoma samples. However, gp130 activation may occur due to the increased level of IL-6 and IL-11 cytokines detected that can become valuable biomarkers.

Identification of potential biomarkers for early and advanced gastric adenocarcinoma detection.

BACKGROUND/AIMS: This study aimed to understand gradual biological variations during gastric tumorigenesis, and to identify the candidate genes that are involved in tumor progression and metastasis. METHODOLOGY: cDNA microarray data were obtained from 10 pair of cancerous and normal adjacent tissue from gastric adenocarcinoma patients. The samples were divided in primary and advanced gastric adenocarcinoma with lymph node metastasis. Validation of the microarray data was accomplished by quantitative RT-PCR on additional 41 samples. The significantly modified genes were grouped in clusters according to their functional annotation, and comparison was done regarding molecular mechanisms involved tumor progression. RESULTS: A total of 136 genes were up-regulated and 96 genes were down-regulated by at least fourfold in tumor tissue. The analysis of gene clusters revealed a complex remodelling of normal gastric epithelium morphology and function associated with the tumorigenesis and metastasis. A large number of proteases are being overexpressed, together with keratins, genes associated with morphogenesis and anti-apoptosis. Between the most significant down-regulated genes, there were genes involved in gastric motility and synthesis and genes related to metabolic and pro-apoptotic processes. We also report, the identification of seven genes, significant up-regulated, that seem to be associated with tumor progression: KRT17, COL10A2, KIAA1199, SPP1, IL11, S100A2, and MMP3. CONCLUSIONS: Our cDNA microarray study identified several genes that appeared to meet the criteria of a good biomarker, and may therefore be especially useful for the development of diagnostic tools, for the early detection, or for the prediction of tumor progression.

In vitro hepatic differentiation of human bone marrow mesenchymal stem cells under differential exposure to liver-specific factors.

Recent findings demonstrated that stem cells could be harvested from a patient and used to repair his or her own damaged liver. Additionally, stem cells may be manipulated in vitro to induce hepatic differentiation. The current study aims to determine the differentiation efficacy of various liver-specific factors (hepatocyte growth factor, Insulin-Transferrin-Selenium, dexamethasone, and nicotinamide) used for stem cell differentiation into hepatocyte-like cells. Human mesenchymal stem cells were exposed to different media containing these compounds added individually or in various combinations. Hepatic differentiation was assessed via quantitative reverse transcription-polymerase chain reaction and immunocytochemical staining for stemness or liver-specific genes and proteins, including albumin, cytokeratins 18 and 19, HepPar-1, alpha-fetoprotein, and nestin. In addition, functional tests for glycogen storage, urea production, glucose, and albumin synthesis were also performed. The expression profiles of albumin, alpha-fetoprotein, and cytokeratin 19 demonstrated that when hepatocyte growth factor, nicotinamide, or dexamethasone were added individually, incomplete hepatocyte differentiation was achieved; the obtained cell populations contained progenitors that expressed both hepatic (albumin) and biliary (cytokeratin 19) markers, as well as alpha-fetoprotein. Hepatocyte growth factor and nicotinamide were the factors with the most hepatogenic potential. When all factors were added to the culture, cells exhibited features that closely resembled human adult hepatocytes as determined by their gene expression patterns (albumin, HepPar-1, and alpha-fetoprotein, but not cytokeratin 19) and functional testing. These cells with hepatic function may become important tools for liver transplant procedures, liver development studies, and pharmacologic research.