213 research outputs found
Effect of growth factors on the expression of 6-phosphofructo-2- kinase/fructose-2,6-bisfosfatase in Rat-1 fibroblasts
The activation of glycolytic flux is a biochemical characteristic of growing cells. Several reports have demonstrated the role of fructose 2,6-bisphosphate in this process. In this paper we show that the levels of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6PF2K/Fru-2,6-P2ase) mRNA are modulated in response to serum and growth factors and this effect is due to regulation of its transcription rate. The modulation of the expression of this enzyme by growth factors differs according their mitogenic effect; both lysophosphatidic acid and epidermal growth factor, when added alone, increased the mRNA levels, but endothelin had no effect. Furthermore, cAMP, which acts as an antimitogenic signal in Rat-1 fibroblasts, produced a decrease in 6PF2K/Fru-2, 6-P2ase mRNA and inhibited the effects of lysophosphatidic acid and epidermal growth factor on 6PF2K/Fru-2,6-P2ase expression. PD 098059, a specific inhibitor of the activation of the mitogen-activated protein kinase, was able to prevent the effect of EGF on 6PF2K/Fru-2, 6-P2ase gene expression. These results imply that activation of mitogen-activated protein kinase is required for the stimulation of the transcription of 6PF2K/Fru-2,6-P2ase by EGF
Lestaurtinib Inhibition of the JAK/STAT Signaling Pathway in Hodgkin Lymphoma Inhibits Proliferation and Induces Apoptosis
Standard cytotoxic chemotherapy for Hodgkin Lymphoma (HL) has changed little in 30 years; the treatment for patients with relapsed or refractory disease remains challenging and novel agents are under development. JAK/STAT constitutive activation plays an important role in the pathogenesis of HL. Lestaurtinib is an orally bioavailable multikinase inhibitor that has recently been shown to inhibit JAK2 in myeloproliferative disorders. The potential role of Lestaurtinib in HL therapy is unknown. We have analyzed the effect of Lestaurtinib treatment in five HL cell lines from refractory patients, L-428, L-1236, L-540, HDML-2 and HD-MY-Z. At 48 h, a dose-dependent cell growth inhibition (23%–66% at 300 nM) and apoptotic increment (10%–64% at 300 nM) were observed. Moreover, Lestaurtinib inhibited JAK2, STAT5 and STAT3 phosphorylation and reduced the mRNA expression of its downstream antiapoptotic target Bcl-xL. In addition, we have analyzed the effect of Lestaurtinib treatment in lymph nodes from four classic HL patients. We observed a decrease in cell viability at 24 hours of treatment in three patients (mean decrease of 27% at 300 nM). Our findings provide, for the first time, a molecular rationale for testing JAK2 inhibitors, specifically Lestaurtinib, in HL patients
Comparative Analysis of TCR-gamma Gene Rearrangements by Genescan and Polyacrylamide Gel-electrophoresis in Cutaneous T-cell Lymphoma
Demonstrating T-cell clonality has become an important approach supporting a diagnosis of malignant T-cell neoplasms. A comparative study between Genescan analysis, polyacrylamide gel and agarose gel electrophoresis in visualizing X-cell receptor gamma gene rearrangement was performed on 25 biopsy specimens from 18 patients with different forms of cutaneous T-cell lymphomas. Clonality was detected in 17 biopsy specimens when PCR products were evaluated by Genescan analysis. Seventeen showed discrete bands when visualized in polyacrylamide gel and 14 cases were clonal when visualized with agarose gel. In five cases, a clonal population was seen in the gels, but not with Genescan. On sequencing the PCR products we demonstrated nonclonality of these five samples. Our results confirm that PCR-Genescan is a useful, reliable and specific screening method for detecting dominant clones in patients with T-cell lymphoma
Study of Regulatory T-Cells in Patients with Gastric Malt Lymphoma: Influence on Treatment Response and Outcome
Purpose
FOXP3+ regulatory T cells (Treg) play an essential role in modulating host responses to tumors and infections. The role of these cells in the pathogenesis of MALT lymphomas remains unknown. The aims of the study were to quantify the number of infiltrating FOXP3+ and CD3+ cells in patients with gastric MALT lymphoma at diagnosis and to study kinetics of these cells and CD20+ tumor cells after treatment and during long-term follow-up.
Methods
FOXP3+, CD3+ and CD20+ cells were analyzed by immunohistochemistry and the number of cells was quantified using a micrometric ocular. Samples of 35 patients with gastric MALT lymphoma at diagnosis and after treatment were included. Diagnostic samples were compared to 19 cases of chronic gastritis and diffuse large B-cell lymphoma (DLBCL) of the stomach.
Results
The median number of FOXP3+ infiltrating cells was higher (27 cells/cm2) in gastric MALT patients than in DLBCL (10 cells; p = 0.162) but similar to chronic gastritis (20 cells; p = 0.605). No characteristic or specific distribution pattern of infiltrating FOXP3+ cells was found. Gastric MALT lymphoma patients responding to bacterial eradication therapy had higher number of FOXP3+ cells at study entry. Kinetics of both infiltrating FOXP3+ cells and tumor CD20+ cells were strongly dependent on the treatment administered.
Discussion
Gastric MALT lymphomas have a number of Treg cells more similar to chronic gastritis than to DLBCL. Patients with higher number of tumor infiltrating FOXP3+ cells at study entry seem to have better response to antibiotics. Kinetics of Treg and tumor cells are influenced by type of treatmen
Clinical Validation of the Vitro HPV Screening Assay for Its Use in Primary Cervical Cancer Screening
The advances in cervical cancer screening have mostly focused on high-risk human papillomavirus (HR HPV) detection as the primary screening tool in women over 30 years old. Although many new HPV assays have been commercialized during the last few years, only a few of them are validated according to international guidelines. Recently, new approaches for genotype-based risk-stratification and triage of HPV positive women have been developed. In this study, the Vitro HPV Screening assay targeting 14 oncogenic genotypes (HPV16, HPV18, and another 12 HR HPV genotypes) has demonstrated an appropriate clinical performance to be used as primary screening test. Additionally, the 12 HR HPV genotypes have been individually genotyped with a complementary assay performed by reverse dot blot hybridization of the PCR product from the HPV Screening assay. In conclusion, this n ew assay represents a unique integrated solution for cervical cancer screening with the extended HPV genotyping as a clinical tool for risk determination. Many scientific societies have issued guidelines to introduce population-based cervical cancer screening with HPV testing. The Vitro HPV Screening assay is a fully automatic multiplex real-time PCR test targeting the L1 GP5+/GP6+ region of HPV genome. The assay detects 14 high risk (HR) HPV genotypes, identifying individual HPV16 and HPV18 genotypes, and the HPV-positive samples for the other 12 HR HPV types are subsequently genotyped with the HPV Direct Flow Chip test. Following international guidelines, the aim of this study was to validate the clinical accuracy of the Vitro HPV Screening test on ThinPrep-collected samples for its use as primary cervical cancer screening, using as comparator the validated cobas (R) 4800 HPV test. The non-inferiority analysis showed that the clinical sensitivity and specificity of the Vitro HPV Screening assay for a diagnosis of cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) were not inferior to those of cobas (R) 4800 HPV (p = 0.0049 and p < 0.001 respectively). The assay has demonstrated a high intra- and inter-laboratory reproducibility, also among the individual genotypes. The Vitro HPV Screening assay is valid for cervical cancer screening and it provides genotyping information on HPV-positive samples without further sample processing in a fully automated workflow
Cell-Free DNA for Genomic Analysis in Primary Mediastinal Large B-Cell Lymphoma
High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for genomic characterization in 20 PMBL patients by means of a custom NGS panel for gene mutations and low-pass whole-genome sequencing (WGS) for copy number analysis (CNA) in a real-life setting. Appropriate cfDNA to perform the analyses was obtained in 18/20 cases. The sensitivity of cfDNA to detect the mutations present in paired FFPE samples was 69% (95% CI: 60-78%). The mutational landscape found in cfDNA samples was highly consistent with that of the tissue, with the most frequently mutated genes being B2M (61%), SOCS1 (61%), GNA13 (44%), STAT6 (44%), NFKBIA (39%), ITPKB (33%), and NFKBIE (33%). Overall, we observed a 75% concordance to detect CNA gains/losses between DNA microarray and low-pass WGS. The sensitivity of low-pass WGS was remarkably higher for clonal CNA (18/20, 90%) compared to subclonal alterations identified by DNA microarray. No significant associations between cfDNA amount and tumor burden or outcome were found. cfDNA is an excellent alternative source for the accurate genetic characterization of PMBL cases
Gal-1 Expression Analysis in the GLIOCAT Multicenter Study: Role as a Prognostic Factor and an Immune-Suppressive Biomarker
Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The beta-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM
In silico validation of RNA-Seq results can identify gene fusions with oncogenic potential in glioblastoma
RNA-Sequencing (RNA-Seq) can identify gene fusions in tumors, but not all these fusions have functional consequences. Using multiple data bases, we have performed an in silico analysis of fusions detected by RNA-Seq in tumor samples from 139 newly diagnosed glioblastoma patients to identify in-frame fusions with predictable oncogenic potential. Among 61 samples with fusions, there were 103 different fusions, involving 167 different genes, including 20 known oncogenes or tumor suppressor genes (TSGs), 16 associated with cancer but not oncogenes or TSGs, and 32 not associated with cancer but previously shown to be involved in fusions in gliomas. After selecting in-frame fusions able to produce a protein product and running Oncofuse, we identified 30 fusions with predictable oncogenic potential and classified them into four non-overlapping categories: six previously described in cancer; six involving an oncogene or TSG; four predicted by Oncofuse to have oncogenic potential; and 14 other in-frame fusions. Only 24 patients harbored one or more of these 30 fusions, and only two fusions were present in more than one patient: FGFR3::TACC3 and EGFR::SEPTIN14. This in silico study provides a good starting point for the identification of gene fusions with functional consequences in the pathogenesis or treatment of glioblastoma
Multicenter Evaluation of the Fully Automated PCR-Based Idylla EGFR Mutation Assay on Forman-Fixed, Paraffin-Embedded Tissue of Human Lung Cancer
Before initiating treatment of advanced non-small-cell lung cancer with tyrosine kinase inhibitors (eg, erlotinib, gefitinib, osimertinib, and afatinib), which inhibit the catalytic activity of epidermal growth factor receptor (EGFR), clinical guidelines require determining the EGFR mutational status for activating (EGFR exons 18, 19, 20, or 21) and resistance (EGFR exon 20) mutations. The EGFR resistance mutation T790M should be monitored at cancer progression. The Idylla EGFR Mutation Assay, performed on the Idylla molecular diagnostics platform, is a fully automated (<2.5 hours turnaround time) sample-to-result molecular test to qualitatively detect 51 EGFR oncogene point mutations, deletions, or insertions. In a 15-center evaluation, Idylla results on 449 archived formalin-fixed, paraffin-embedded tissue sections, originating from non-small-cell lung cancer biopsies and resection specimens, were compared with data obtained earlier with routine reference methods, including next-generation sequencing, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays. When results were discordant, a third method of analysis was performed, when possible, to confirm test results. After confirmation testing and excluding invalids/errors and discordant results by design, a concordance of 97.6% was obtained between Idylla and routine test results. Even with <10 mm(2) of tissue area, a valid Idylla result was obtained in 98.9% of the cases. The Idylla EGFR Mutation Assay enables sensitive detection of most relevant EGFR mutations in concordance with current guidelines, with minimal molecular expertise or infrastructure
miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk
Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a−/− mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling
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