Abnormal cell-clearance and accumulation of autophagic vesicles in lymphocytes from patients affected with Ataxia-Teleangiectasia.

Ataxia-Teleangiectasia (A-T) is a neurodegenerative disorder due to mutations in ATM gene. ATM in the nucleus ensures DNA repair, while its role in the cytosol is still poorly clarified. Abnormal autophagy has been documented in other neurodegenerative disorders, thus we evaluated whether alteration in this process may be involved in the pathogenesis of A-T by analyzing the autophagic vesicles and the genes implicated in the different stages of autophagy. Through transmission electron microscopy (TEM) and immunofluorescence analysis we observed an accumulation of APs associated with a LC3 puncta pattern, and a reduced number of ALs. We also documented an increased expression of genes involved in AP and lysosome biogenesis and function, and a decrease of Vps18 expression, involved in their vesicular trafficking and fusion. mTORC1-controlled proteins were hyperphosphorylated in A-T, in keeping with an increased mTOR inhibitory influence of autophagy. Betamethasone is able to promote the degradation of SQSTM1, a biomarker of autophagy. Collectively, our results indicate that in cells from A-T patients, the APs maturation is active, while the fusion between APs and lysosomes is inappropriate, thus implying abnormalities in the cell-clearance process. We also documented a positive effect of Betamethasone on molecules implicated in autophagosome degradation.

Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia.

Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease.

Human skin-derived keratinocytes and fibroblasts co-cultured on 3D poly ε-caprolactone scaffold support in vitro HSC differentiation into T-lineage committed cells.

In humans, the thymus is the primary lymphoid organ able to support the development of T cells through its three-dimensional (3D) organization of the thymic stromal cells. Since a remarkable number of similarities are shared between the thymic epithelial cells (TECs) and skin-derived keratinocytes and fibroblasts, in this study we used human keratinocytes seeded with fibroblasts on the 3D poly ε-caprolactone scaffold to evaluate their ability to replace TECs in supporting T-cell differentiation from human haematopoietic stem cells (HSCs). We observed that in the multicellular biocomposite, early thymocytes expressing CD7(+)CD1a(+), peculiar markers of an initial T-cell commitment, were de novo generated. Molecular studies of genes selectively expressed during T-cell development revealed that TAL1 was down-regulated and Spi-B was up-regulated in the cell suspension, consistently with a T-cell lineage commitment. Moreover, PTCRA and RAG2 expression was detected, indicative of a recombinant activity, required for the generation of a T-cell receptor repertoire. Our results indicate that in the multicellular biocomposite, containing skin-derived elements in the absence of thymic stroma, HSCs do start differentiating toward a T-cell lineage commitment. In conclusion, the construct described in this study exerts some properties of a lymphoid organoid, suitable for future clinical applications in cell-based therapies.

Role of the common γ chain in cell cycle progression of human malignant cell lines.

The γ-chain (γc) is a transducing element shared between several cytokine receptors whose alteration causes X-linked severe combined immunodeficiency. Recently, a direct involvement of γc in self-sufficient growth in a concentration-dependent manner was described, implying a direct relationship between the amount of the molecule and its role in cell cycle progression. In this study, we evaluate whether γc expression could interfere in cell cycle progression also in malignant hematopoietic cells. Here, we first report that in the absence of γc expression, lymphoblastoid B-cell lines (BCLs) die at a higher extent than control cells. This phenomenon is caspase-3 independent and is associated to a decreased expression of the antiapoptotic Bcl-2 family members. By contrast, increased expression of γc protein directly correlates with spontaneous cell growth in several malignant hematopoietic cell lines. We, also, find that the knockdown of γc protein through short interfering RNA is able to decrease the cell proliferation rate in these malignancies. Furthermore, an increased expression of all D-type cyclins is found in proliferating neoplastic cells. In addition, a direct correlation between the amount of γc and cyclins A2 and B1 expression is found. Hence, our data demonstrate that the amount of the γc is able to influence the transcription of genes involved in cell cycle progression, thus being directly involved in the regulatory control of cell proliferation of malignant hematopoietic cells.

Genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles.

Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre-existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down-regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N- and K-RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF-kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM.

Comparative analyses of DNA extraction methods for whole blood quantification of HCMV DNAemia in patients with hematological diseases: false negative cases in manual method.

Human cytomegalovirus (HCMV) DNA quantitation in whole blood (WB) by real-time or quantitative polymerase chain reaction (qPCR) is a highly sensitive and reproducible diagnostic procedure for monitoring HCMV DNAemia (DNAemia is the detection of DNA in samples of plasma, whole blood, isolated peripheral blood leukocytes or in buffy-coat specimens) in patients. We provided a comparative analysis of HCMV DNA extraction performance by two different techniques, one performed by an automated extractor and the other by a manual method. We observed that the automated extraction method allowed HCMV DNA detection in the presence of weak viremia while no differences are observed when the viral load is greater. Therefore, automated DNA extraction is a suitable and recommended protocol not only for early detection of HCMV infection but also for more accurate monitoring of HCMV DNAemia during post-therapy follow-up.

Role of IL-6/STAT3 Axis in Resistance to Cisplatin in Gastric Cancers.

Gastric cancer, the second most common cause of death worldwide, is characterized by poor prognosis and low responsiveness to chemotherapy. Indeed, multidrug resistance, based mainly on cellular and molecular factors, remains one of the most limiting factors of the current approach to gastric cancer (GC) therapy. We employed a comprehensive gene expression analysis through data mining of publicly available databases to assess the role of the signal transducer and activator of transcription 3 (STAT3) in gastric cancer drug efficiency. It has been proposed that gastric cancer cells are less sensitive to these drugs because they develop resistance to these agents through activating alternative signalling pathways responsible for overcoming pharmacological inhibition. Our study evaluated the hypothesis that activating STAT3 signalling in response to cisplatin reduces the reaction to the drug. Consistent with this hypothesis, inhibition of interleukin 6 (IL-6)/STAT3 in combination therapy with cisplatin prevented both STAT3 activation and more lethality than induction by a single agent. The data suggest that the IL-6/STAT3 axis block associated with cisplatin treatment may represent a strategy to overcome resistance.

Case report: Hematologic malignancies concomitant diagnosis of hairy cell leukemia and chronic lymphocytic leukemia: A rare association.

A case of concomitant hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) in a 50- year-old man was reported. Flow cytometry and droplet digital PCR (ddPCR) were used to detect the B-Raf proto-oncogene (BRAF) V600E mutation. The HCL population was the predominant component. The patient was first treated with cladribine and then with rituximab and achieved HCL partial remission. Importantly, the high sensitivity of our flow cytometric approach allowed the detection of a small population "P3," in addition to the typical HCL and CLL clones. The P3 clone changed over time, from an HCL-like to a CLL-like immunophenotype. This case is added to the few other cases of synchronous HCL and CLL already reported in the literature and underlines the importance of analyzing chronic lymphoproliferative disorders by highly sensitive diagnostic techniques, like the multicolor flow cytometry and ddPCR, to evaluate the possible association between HCL and CLL at diagnosis.

Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma.

INTRODUCTION: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established. METHODS: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR. RESULTS: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters. CONCLUSION: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study.

JAK2 V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR's technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.