Soluble PD-L1 as a diagnostic and prognostic biomarker in resectable gastric cancer patients.

BACKGROUND: In this study, we compared programmed death-ligand 1 (PD-L1) expression in primary tissue samples and its soluble form (sPD-L1) concentration in matched preoperative plasma samples from gastric cancer patients to understand the relationship between tissue and plasma PD-L1 expression and to determine its diagnostic and prognostic value. METHODS: PD-L1 expression in tissue was assessed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), and sPD-L1 concentration in plasma was quantified by ELISA. The levels of the CD274 gene, which encodes for PD-L1 protein, were examined as part of bulk tissue RNA-sequencing analyses. Additionally, we evaluated the association between sPD-L1 levels and various laboratory parameters, disease characteristics, and patient outcomes. RESULTS: GC patients had significantly higher levels of sPD-L1 in their plasma (71.69 pg/mL) compared to healthy controls (35.34 pg/mL) (p < 0.0001). Moreover, sPD-L1 levels were significantly correlated with tissue PD-L1 protein, CD274 mRNA expression, larger tumor size, advanced tumor stage, and lymph node metastasis. Elevated sPD-L1 levels (> 103.5 ng/mL) were associated with poor overall survival (HR = 2.16, 95%CI 1.15-4.08, p = 0.017). Furthermore, intratumoral neutrophil and dendritic cell levels were directly correlated with plasma sPD-L1 concentration in the GC patients. CONCLUSIONS: sPD-L1 was readily measurable in GC patients, and its level was associated with GC tissue PD-L1 expression, greater inflammatory cell infiltration, disease progression, and survival. Thus, sPD-L1 may be a useful minimally invasive diagnostic and prognostic biomarker in GC patients.

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.

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.

Collagen Family as Promising Biomarkers and Therapeutic Targets in Cancer.

Despite advances in cancer detection and therapy, it has been estimated that the incidence of cancers will increase, while the mortality rate will continue to remain high, a fact explained by the large number of patients diagnosed in advanced stages when therapy is often useless. Therefore, it is necessary to invest knowledge and resources in the development of new non-invasive biomarkers for the early detection of cancer and new therapeutic targets for better health management. In this review, we provided an overview on the collagen family as promising biomarkers and on how they may be exploited as therapeutic targets in cancer. The collagen family tridimensional structure, organization, and functions are very complex, being in a tight relationship with the extracellular matrix, tumor, and immune microenvironment. Moreover, accumulating evidence underlines the role of collagens in promoting tumor growth and creating a permissive tumor microenvironment for metastatic dissemination. Knowledge of the molecular basis of these interactions may help in cancer diagnosis and prognosis, in overcoming chemoresistance, and in providing new targets for cancer therapies.

Increased Dkk-1 plasma levels may discriminate disease subtypes in myeloproliferative neoplasms.

Alterations in the bone marrow niche induced by abnormal production of cytokines and other soluble factors have been associated with disease progression in classical BCR-ABL1 negative myeloproliferative neoplasms (MPN). Variations in circulating proteins might reflect local disease processes and plasma proteome profiling could serve to identify possible diagnostic and prognostic biomarkers. We employed a human cytokine array to screen for 105 distinct analytes in pooled plasma samples obtained from untreated young MPN patients (<35 years) with different clinical phenotypes and driver mutations, as well as from healthy individuals. Among molecules that exhibited significantly increased levels in MPN patients versus controls, the top of the list was represented by Dickkopf-related protein 1 (Dkk-1), which also showed the highest potential for discrimination between MPN subtypes. In the next step, a quantitative ELISA was used to measure plasma Dkk-1 levels in 30 young-onset MPN-10 essential thrombocythemia (ET), 10 polycythemia vera (PV), 10 pre-fibrotic primary myelofibrosis (pre-PMF)-and 10 controls. The results suggested that plasma Dkk-1 levels could differentiate ET from pre-PMF, in JAK2 V617F-positive as well as in CALR-positive patients, and also ET from PV in JAK2 V617F-positive patients.

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.

A link between the driver mutations and dysregulated apoptosis in BCR-ABL1 negative myeloproliferative neoplasms.

The current understanding of BCR-ABL1 negative myeloproliferative neoplasms pathogenesis is centred on the phenotypic driver mutations in JAK2, MPL, or CALR genes, and the constitutive activation of JAK-STAT pathway. Nonetheless, there is still a need to better characterize the cellular processes that are triggered by these genetic alterations, such as apoptosis that might play a role in the pathological expansion of the myeloid lineages and, especially, in the morphological anomalies of the bone marrow megakaryocytes. In this article we will explore the connection between the driver mutations in MPN and the abnormal apoptosis that might be translated in new therapeutic strategies.

Epigenetics in gastric carcinogenesis: TET genes as important players.

Epigenetic processes including aberrant promoter methylation of tumor suppressor gene play a key role in gastric carcinogenesis. TET proteins are involved in DNA demethylation; many cancers, haematological or solid, present loss-of-function mutations and aberrant expression/regulation of TET. In gastric cancer there are few studies reporting a decreased expression of TET and associations between these proteins and signaling pathways involved in carcinogenesis. Identifying connections between aberrant expression of TET, disruption of the balance between DNA methylation and demethylation and their association with gastric carcinogenesis might be useful for the development of novel therapeutic approaches.

Thrombopoietin receptor down-modulation by JAK2 V617F: restoration of receptor levels by inhibitors of pathologic JAK2 signaling and of proteasomes.

The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.

Molecular variants of human papilloma virus 16 E2, E4, E5, E6 and E7 genes associated with cervical neoplasia in Romanian patients.

The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p < 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis.

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.

Schizophrenia polygenic risk scores, clinical variables and genetic pathways as predictors of phenotypic traits of bipolar I disorder.

AIM: We investigated the predictive value of polygenic risk scores (PRS) derived from the schizophrenia GWAS (Trubetskoy et al., 2022) (SCZ3) for phenotypic traits of bipolar disorder type-I (BP-I) in 1878 BP-I cases and 2751 controls from Romania and UK. METHODS: We used PRSice-v2.3.3 and PRS-CS for computing SCZ3-PRS for testing the predictive power of SCZ3-PRS alone and in combination with clinical variables for several BP-I subphenotypes and for pathway analysis. Non-linear predictive models were also used. RESULTS: SCZ3-PRS significantly predicted psychosis, incongruent and congruent psychosis, general age-of-onset (AO) of BP-I, AO-depression, AO-Mania, rapid cycling in univariate regressions. A negative correlation between the number of depressive episodes and psychosis, mainly incongruent and an inverse relationship between increased SCZ3-SNP loading and BP-I-rapid cycling were observed. In random forest models comparing the predictive power of SCZ3-PRS alone and in combination with nine clinical variables, the best predictions were provided by combinations of SCZ3-PRS-CS and clinical variables closely followed by models containing only clinical variables. SCZ3-PRS performed worst. Twenty-two significant pathways underlying psychosis were identified. LIMITATIONS: The combined RO-UK sample had a certain degree of heterogeneity of the BP-I severity: only the RO sample and partially the UK sample included hospitalized BP-I cases. The hospitalization is an indicator of illness severity. Not all UK subjects had complete subphenotype information. CONCLUSION: Our study shows that the SCZ3-PRS have a modest clinical value for predicting phenotypic traits of BP-I. For clinical use their best performance is in combination with clinical variables.

Combination treatment for myeloproliferative neoplasms using JAK and pan-class I PI3K inhibitors.

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.

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.

Targets in MPNs and potential therapeutics.

Philadelphia-negative classical Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hemopathies that emerge in the hematopoietic stem cell (HSC) compartment. MPN driver mutations are restricted to specific exons (14 and 12) of Janus kinase 2 (JAK2), thrombopoietin receptor (MPL/TPOR) and calreticulin (CALR) genes, are involved directly in clonal myeloproliferation and generate the MPN phenotype. As a result, an increased number of fully functional erythrocytes, platelets and leukocytes is observed in the peripheral blood. Nevertheless, the complexity and heterogeneity of MPN clinical phenotypes cannot be solely explained by the type of driver mutation. Other factors, such as additional somatic mutations affecting epigenetic regulators or spliceosomes components, mutant allele burdens and modifiers of signaling by driver mutants, clonal architecture and the order of mutation acquisition, signaling events that occur downstream of a driver mutation, the presence of specific germ-line variants, the interaction of the neoplastic clone with bone marrow microenvironment and chronic inflammation, all can modulate the disease phenotype, influence the MPN clinical course and therefore, might be useful therapeutic targets.

Assessment of the Humoral Immune Response Following COVID-19 Vaccination in Healthcare Workers: A One Year Longitudinal Study.

The continuous variability of SARS-CoV-2 and the rapid waning of specific antibodies threatens the efficacy of COVID-19 vaccines. We aimed to evaluate antibody kinetics one year after SARS-CoV-2 vaccination with an mRNA vaccine in healthcare workers (HCW), with or without a booster. A marked decline in anti-Spike(S)/Receptor Binding Domain (RBD) antibody levels was registered during the first eight months post-vaccination, followed by a transitory increase after the booster. At three months post-booster an increased antibody level was maintained only in HCW vaccinated after a prior infection, who also developed a higher and long-lasting level of anti-S IgA antibodies. Still, IgG anti-nucleocapsid (NCP) fades five months post-SARS-CoV-2 infection. Despite the decline in antibodies one-year post-vaccination, 68.2% of HCW preserved the neutralization capacity against the ancestral variant, with a decrease of only 17.08% in the neutralizing capacity against the Omicron variant. Nevertheless, breakthrough infections were present in 6.65% of all participants, without any correlation with the previous level of anti-S/RBD IgG. Protection against the ancestral and Omicron variants is maintained at least three months after a booster in HCW, possibly reflecting a continuous antigenic stimulation in the professional setting.

Clinical Applications of Liquid Biopsy in Gastric Cancer.

Liquid biopsy represents an exciting new area in the field of cancer diagnosis and management, offering a less invasive and more convenient approach to obtain a time-point image of the tumor burden and its genomic profile. Samples collected from several body fluids, mostly blood, can be used to gain access to circulating tumor cells and DNA, non-coding RNAs, microRNAs, and exosomes, at any moment, offering a dynamic picture of the tumor. For patients with GC, the use of blood-based biopsies may be particularly beneficial since tissue biopsies are difficult to obtain and cause real distress to the patient. With advantages such as repeatability and minimal invasion, it is no wonder that the field of liquid biopsy has received tremendous attention. However, the abundance of studies, involving a wide range of assays with different principles, prevented for the moment the reproducibility of the results and therefore the translation into the clinic of liquid biopsy. In this review, we present the latest technical development and data on circulating biomarkers available through liquid biopsy in gastric cancer with an emphasis on their clinical utility in areas such as cancer screening, prognostic stratification, and therapeutic management.

A Case of Inherited t(4;10)(q26;q26.2) Chromosomal Translocation Elucidated by Multiple Chromosomal and Molecular Analyses. Case Report and Review of the Literature.

We present a complex chromosomal anomaly identified using cytogenetic and molecular methods. The child was diagnosed during the neonatal period with a multiple congenital anomalies syndrome characterized by: flattened occipital region; slight turricephaly; tall and broad forehead; hypertelorism; deep-set eyes; down slanting and short palpebral fissures; epicanthic folds; prominent nose with wide root and bulbous tip; microstomia; micro-retrognathia, large, short philtrum with prominent reliefs; low set, prominent ears; and congenital heart disease. The GTG banding karyotype showed a 46,XY,der(10)(10pter→10q26.2::4q26→4qter) chromosomal formula and his mother presented an apparently balanced reciprocal translocation: 46,XX,t(4;10)(q26;q26.2). The chromosomal anomalies of the child were confirmed by MLPA, and supplementary investigation discovered a quadruplication of the 4q35.2 region. The mother has a triplication of the same chromosomal fragment (4q35.2). Using array-CGH, we described the anomalies completely. Thus, the boy has a 71,057 kb triplication of the 4q26-q35.2 region, a 562 kb microdeletion in the 10q26.3 region, and a 795 kb quadruplication of the 4q35.2 region, while the mother presents a 795 kb triplication of the 4q35.2 region. Analyzing these data, we consider that the boy's phenotype is influenced only by the 4q partial trisomy. We compare our case with similar cases, and we review the literature data.

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.