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Zucchetto A.,Clinical and Experimental Onco Hematology Unit | Vaisitti T.,University of Turin | Benedetti D.,Clinical and Experimental Onco Hematology Unit | Tissino E.,Clinical and Experimental Onco Hematology Unit | And 12 more authors.
Leukemia | Year: 2012

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d CD38 and CD49d + CD38 - primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d + CD38 - CLL-derived cell line Mec-1. Results indicate that CD49d + CD38 - cells adhered more efficiently onto CD49d-specific substrates than CD49d CD38 cells (P0.001). Upon adhesion, CD49d + CD38 - cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d + CD38 - cells (P0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d + CD38 - cells were more resistant to serum-deprivation- induced (P0.001) and spontaneous (P0.03) apoptosis than the CD49d + CD38 -s counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells. © 2012 Macmillan Publishers Limited.

Macor P.,University of Trieste | Secco E.,University of Trieste | Mezzaroba N.,University of Trieste | Zorzet S.,University of Trieste | And 11 more authors.
Leukemia | Year: 2015

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications. © 2015 Macmillan Publishers Limited All rights reserved

CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.Leukemia advance online publication, 20 May 2016; doi:10.1038/leu.2016.88. © 2016 Macmillan Publishers Limited

CD49d is a negative prognosticator in chronic lymphocytic leukemia (CLL), expressed by ~40% of CLL cases and associated with aggressive, accelerated clinical courses. In this study, analyzing CD49d expression in a wide CLL cohort (n = 1200) belonging to different cytogenetic groups, we report that trisomy 12 CLL almost universally expressed CD49d and were characterized by the highest CD49d expression levels among all CD49d(+) CLL. Through bisulfite genomic sequencing, we demonstrated that, although CD49d(+)/trisomy 12 CLL almost completely lacked methylation of the CD49d gene, CD49d(-)/no trisomy 12 CLL were overall methylated, the methylation levels correlating inversely to CD49d expression (P = .0001). Consistently, CD49d expression was recovered in CD49d(-) hypermethylated CLL cells upon in vitro treatment with the hypomethylating agent 5-aza-2'-deoxycytidine. This may help explain the clinicobiological features of trisomy 12 CLL, including the high rates of cell proliferation and disease progression, lymph node involvement, and predisposition to Richter syndrome transformation.

Brenca M.,Centro Of Riferimento Oncologico | Rossi S.,Treviso Regional Hospital | Lorenzetto E.,Centro Of Riferimento Oncologico | Piccinin E.,Centro Of Riferimento Oncologico | And 6 more authors.
Molecular Cancer Therapeutics | Year: 2013

Epithelioid sarcoma is a rare soft tissue neoplasm that usually arises in the distal extremities of young adults. Epithelioid sarcoma presents a high rate of recurrences and metastases and frequently poses diagnostic dilemmas. We previously reported loss of tumor suppressor SMARCB1 protein expression and SMARCB1 gene deletion in the majority of epithelioid sarcoma cases. Unfortunately, no appropriate preclinical models of such genetic alteration in epithelioid sarcoma are available. In the present report, we identified lack of SMARCB1 protein due to a homozygous deletion of exon 1 and upstream regulatory region in epithelioid sarcoma cell line VAESBJ. Restoration of SMARCB1 expression significantly affected VAESBJ cell proliferation, anchorage-independent growth, and cell migration properties, thus supporting the causative role of SMARCB1 loss in epithelioid sarcoma pathogenesis. We investigated the translational relevance of this genetic background in epithelioid sarcoma and showed that SMARCB1 ectopic expression significantly augmented VAESBJ sensitivity to gamma irradiation and acted synergistically with flavopiridol treatment. In VAESBJ, both activated ERBB1/EGFR and HGFR/MET impinged on AKT and ERK phosphorylation. We showed a synergistic effect of combined inhibition of these 2 receptor tyrosine kinases using selective small-molecule inhibitors on cell proliferation. These observations provide definitive support to the role of SMARCB1 inactivation in the pathogenesis of epithelioid sarcoma and disclose novel clues to therapeutic approaches tailored to SMARCB1-negative epithelioid sarcoma. © 2013 American Association for Cancer Research.

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