Abbreviations ALCL Anaplastic Large Cell Lymphoma ALDO A Aldolase A ALK+ Anaplastic Lymphoma Kinase positive AML12 Alpha Mouse Liver 12 CD30 Cluster of Differentiation 30 CM Conditioned Media CS ...Citrate Synthase ECAR Extracellular Acidification Rate EFS Event Free Survival GFP Green Fluorescent Protein GLUT1 Glucose Transporter 1 HD Healthy Donors MEF-1 Mouse Embryonic Fibroblasts-1 miR-122-5p microRNA-122-5p miRNA microRNA NC Negative Control NPM Nucleophosmin PCA Principal Component Analysis PKM2 Pyruvate Kinase M2 RT-qPCR Real-Time quantitative-PCR sRNA-seq Small RNA-sequencing analysis S-EVs Small Extracellular Vesicles Dear Editor, Anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL) is an aggressive peripheral T-cell lymphoma representing 10%-15% of pediatric lymphoid neoplasms. ...in a panel of ALCL cell lines, miR-122-5p was not expressed, while alpha mouse liver 12 (AML12) cells and S-EVs showed high miR-122-5p levels (Supplementary Figure S1D). Interestingly, we observed significantly higher transaminase values at diagnosis in ALCL patients with miR-122-5p levels above the median value (Supplementary Figure S1F). ...we hypothesized that the increased circulating levels of miR-122-5p might result from disease-related liver function impairment in patients with advanced ALCL. Principal Component Analysis; PKM2: Pyruvate Kinase M2; RNA-seq: RNA-sequencing analysis; RT-qPCR: Real Time-quantitative PCR; S-EVs: Small Extracellular Vesicles; SEM:
Mantle Cell Lymphoma (MCL) is still an incurable B-cell malignancy characterized by poor prognosis and frequent relapses. B Cell Receptor (BCR) signaling inhibitors, in particular of the kinases BTK ...and PI3Kγ/δ, have demonstrated clinically meaningful anti-proliferative effects in B cell tumors. However, refractoriness to these drugs may develop, portending a dismal prognosis. Protein kinase CK1α is an emerging pro-growth enzyme in B cell malignancies. In multiple myeloma, this kinase sustains β-catenin and AKT-dependent survival and is involved in the activation of NF-κB in B cells. In this study, we analyzed the role of CK1α on MCL cell survival and proliferation, on the regulation of BCR-related BTK, NF-κB, PI3K/AKT signaling cascades and the effects of CK1α chemical inhibition or gene silencing in association with the BTK inhibitor Ibrutinib or the PI3Kγ/δ inhibitor Duvelisib. CK1α was found highly expressed in MCL cells as compared to normal B cells. The inactivation/loss of CK1α caused MCL cell apoptosis and proliferation arrest. CK1α sustained BCR signaling, in particular the NF-κB, AKT and BTK pathways by modulating the phosphorylation of Ser 652 on CARD11, Ser 536 p65 on NF-κB, Ser 473 on AKT, Tyr 223 on BTK, as well as the protein levels. We also provided evidence that CK1α-mediated regulation of CARD11 and BTK likely implicates a physical interaction. The combination of CK1α inhibition with Ibrutinib or Duvelisib synergistically increased cytotoxicity, leading to a further decrease of the activation of BCR signaling pathways. Therefore, CK1α sustains MCL growth through the regulation of BCR-linked survival signaling cascades and protects from Ibrutinib/Duvelisib-induced apoptosis. Thus, CK1α could be considered as a rational molecular target for the treatment of MCL, in association with novel agents.
Inflammatory bowel diseases (IBDs; both ulcerative colitis UC and Crohn’s colitis CC) are well-established predisposing pathological conditions for colorectal cancer (CRC) development. In IBDs, both ...the endoscopy and the histology assessment of CRC precursors (i.e., dysplasia, also defined as intraepithelial neoplasia) are associated with low interobserver consistency, and no reliable dysplasia-specific biomarker is available. The programmed cell death 4 (PDCD4) tumor suppressor gene is involved in sporadic colorectal oncogenesis, but scanty information is available on its involvement in IBD-associated colorectal oncogenesis. One hundred twenty tissue samples representative of active and inactive IBD and of flat dysplasia were obtained from 30 cases of UC and 30 of CC who undergone colectomy. Twenty additional biopsy samples obtained from patients with irritable bowel syndrome acted as normal controls. PDCD4 expression was assessed by immunohistochemistry; the expression of miR-21 (a major PDCD4 regulator) was investigated by quantitative real-time PCR and in situ hybridization in different series of a hundred samples. Tissue specimens from both controls and inactive IBD consistently featured strong PDCD4 nuclear immunostain; conversely, lower PDCD4 nuclear expression was featured by both active IBD and IBD-associated dysplastic lesions. Significant PDCD4 down-regulation distinguished IBD-associated dysplasia (
p
< 0.001) versus active IBD. In both active IBD and dysplasia, PDCD4 down-regulation was significantly associated with miR-21 up-regulation. PDCD4 nuclear down-regulation (which parallels miR-21 up-regulation) is involved in the molecular pathway of IBD-associated carcinogenesis. PDCD4 nuclear expression may be usefully applied as ancillary maker in the histological assessment of IBD-associated dysplastic lesions.
Liver fibrosis is a key pathological precondition for hepatocellular carcinoma in which the severity is confidently correlated with liver cancer. Liver fibrosis, characterized by gradual cell loss ...and excessive extracellular matrix deposition, can be reverted if detected at the early stage. The gold standard for staging and diagnosis of liver fibrosis is undoubtedly biopsy. However, this technique needs careful sample preparation and expert analysis. In the present work, an ex vivo, minimally destructive, label-free characterization of liver biopsies is presented. Through a custom-made experimental setup, liver biopsies of bile-duct-ligated and sham-operated mice were measured at 8, 15, and 21 days after the procedure. Changes in impedance were observed with the progression of fibrosis, and through data fitting, tissue biopsies were approximated to an equivalent RC circuit model. The model was validated by means of 3D hepatic cell culture measurement, in which the capacitive part of impedance was proportionally associated with cell number and the resistive one was proportionally associated with the extracellular matrix. While the sham-operated samples presented a decrease in resistance with time, the bile-duct-ligated ones exhibited an increase in this parameter with the evolution of fibrosis. Moreover, since the largest difference in resistance between healthy and fibrotic tissue, of around 2 kΩ, was found at 8 days, this method presents great potential for the study of fibrotic tissue at early stages. Our data point out the great potential of exploiting the proposed needle setup in clinical applications.
Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) ...mouse model of the β regulatory subunit of CK2. CK2β
mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2β
animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2β
mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins.
assays highlight that B cells lacking CK2β have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2β
mice suggesting the importance of the β subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.
Ependymomas are rare primary central nervous system tumors. They can form anywhere along the neuraxis, but in adults, these tumors predominantly occur in the spine and less frequently intracranially. ...Ependymal tumors represent a heterogenous group of gliomas, and the WHO 2016 classification is based essentially on a grading system, with ependymomas classified as grade I, II (classic), or III (anaplastic). In adults, surgery is the primary initial treatment, while radiotherapy is employed as an adjuvant treatment in some cases of grade II and in all cases of anaplastic ependymoma; chemotherapy is reserved for recurrent cases. In recent years, important and interesting advances in the molecular characterization of ependymomas have been made, allowing for the identification of nine molecular subgroups of ependymal tumors and moving toward subgroup-specific patients with improved risk stratification for treatment-decisions and future prospective trials. New targeted agents or immunotherapies for ependymoma patients are being explored for recurrent disease. This review summarizes recent molecular advances in the diagnosis and treatment of intracranial ependymomas including surgery, radiation therapy and systemic therapies.
Summary Differences in human epithelial growth factor receptor 2 dysregulation in primary solid tumors and metastases may (at least partially) explain human epithelial growth factor receptor ...2–targeted therapeutic inconsistencies. Human epithelial growth factor receptor 2 status was tested in a series of 47 radically treated consecutive esophagogastric junction adenocarcinomas (male/female, 38/9; mean age, 67.9 years) in both primary cancers and paired synchronous nodal metastases. None of the patients received neoadjuvant therapy. For each case, 2 nonadjacent tissue samples from primary esophagogastric junction adenocarcinoma and 2 different metastatic nodes were considered (188 tissue samples in all). Human epithelial growth factor receptor 2 status was assessed by immunohistochemistry (PATHWAY-HER2/neu 4B5; Ventana Medical Systems, Milan, Italy) and dual chromogenic in situ hybridization (duoCISH; DAKO, Glostrup, Denmark). Immunohistochemistry staining scores were nil in 22 tumors (47%), 1 (21%) in 10, 2 (13%) in 6, and 3 (19%) in 9. Human epithelial growth factor receptor 2 gene amplification (25.5%) was associated with more differentiated phenotype (Fisher exact test, P = .039) and advanced tumor stage (Fisher exact test, P = .015). Significant agreement was observed between human epithelial growth factor receptor 2 protein expression (immunohistochemistry) and human epithelial growth factor receptor 2 gene's amplification (chromogenic in situ hybridization) ( κ = 0.84, P < .001). Both immunohistochemistry and chromogenic in situ hybridization documented an excellent intratumor agreement in human epithelial growth factor receptor 2 status ( κ = 0.75, P < .001; κ = 0.88, P < .001, respectively). Human epithelial growth factor receptor 2 status was comparable in primary versus metastatic nodal cancers by both immunohistochemistry and chromogenic in situ hybridization (Cohen Φ, both P < .001). In esophagogastric junction adenocarcinomas, human epithelial growth factor receptor 2 status (as assessed by immunohistochemistry and/or chromogenic in situ hybridization) is virtually unaffected by intratumor variability; it is consistent with findings in nodal metastases, and it reliably identifies patients with esophagogastric junction adenocarcinoma eligible for anti–human epithelial growth factor receptor 2 therapy.
Serine-threonine kinase CK2 is highly expressed and pivotal for survival and proliferation in multiple myeloma, chronic lymphocytic leukemia and mantle cell lymphoma. Here, we investigated the ...expression of α catalytic and β regulatory CK2 subunits by immunohistochemistry in 57 follicular (FL), 18 Burkitt (BL), 52 diffuse large B-cell (DLBCL) non-Hodgkin lymphomas (NHL) and in normal reactive follicles. In silico evaluation of available Gene Expression Profile (GEP) data sets from patients and Western blot (WB) analysis in NHL cell-lines were also performed. Moreover, the novel, clinical-grade, ATP-competitive CK2-inhibitor CX-4945 (Silmitasertib) was assayed on lymphoma cells. CK2 was detected in 98.4% of cases with a trend towards a stronger CK2α immunostain in BL compared to FL and DLBCL. No significant differences were observed between Germinal Center B (GCB) and non-GCB DLBCL types. GEP data and WB confirmed elevated CK2 mRNA and protein levels as well as active phosphorylation of specific targets in NHL cells. CX-4945 caused a dose-dependent growth-arresting effect on GCB, non-GCB DLBCL and BL cell-lines and it efficiently shut off phosphorylation of NF-κB RelA and CDC37 on CK2 target sites. Thus, CK2 is highly expressed and could represent a suitable therapeutic target in BL, FL and DLBCL NHL.
Summary Among patients with gastric cancer (GC) and gastroesophageal cancer (G-EC), HER2 amplification identifies those who may benefit from trastuzumab. HER2 status assessment, however, is ...influenced by preanalytic, analytic, and postanalytic variables. In a series of 5426 microarray cancer tissue cores obtained from 1040 GC/G-ECs (824 GC, 216 G-EC) and 720 synchronous nodal metastases, we evaluated both the performances of 2 different immunohistochemistry (IHC) protocols and the HER2 status intratumor variability. The prevalence of HER2 amplification and protein overexpression were assessed by chromogenic in situ hybridization and by 2 IHC protocols (CB11 and 4B5). HER2 was amplified in 114 (11%) of 1040 cases; in 6 (5.3%) of 114 cases, gene amplification only involved nodal metastasis. HER2 amplification prevailed in intestinal-type ( P = .001) and low-grade ( P < .001) tumors, showing no correlation with patients’ age/sex, tumor location, stage, and Ming histotype. Overall, 12.5% and 13.7% of cases IHC scored 2+/3+ using the CB11-IHC and the 4B5-IHC protocol, respectively. HER2 amplification was not associated with protein overexpression (score 0/1+) in 11.4% and 6.2% of cases using the CB11-IHC and the 4B5-IHC protocol, respectively. The 4B5-IHC protocol proved more sensitive than CB11-IHC (93.9% versus 88.6%) and just as specific (96.1% versus 96.9%). Tested by chromogenic in situ hybridization, intratumor HER2 status was “substantially” consistent in different tissue cores obtained from the same case ( κ = 0.78). Similar results were obtained for HER2 protein expression (CB11-IHC, κ = 0.78, and 4B5-IHC, κ = 0.83). Immunohistochemistry testing, however, fails in identifying about 10% of HER2 -amplified cancers, potentially excluding these patients from anti-HER2 therapy.
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