Francesca Merchionne

Docetaxel versus paclitaxel for antiangiogenesis.

Cytoskeleton-toxic chemotherapeuticals, such as vinblastine and paclitaxel, display antiangiogenic activity. This study was designed to compare paclitaxel to its analog docetaxel and assess their doses still antiangiogenic in vitro and in vivo. Human endothelial cell functions involved in angiogenesis, namely proliferation, chemotaxis, morphogenesis, and secretion of matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA) were studied in vitro upon exposure to docetaxel and paclitaxel, whereas their effect on angiogenesis was studied in vivo by using the chick embryo chorioallantoic membrane (CAM) model. Proliferation of mouse embryo fibroblasts and human Kaposis sarcoma, breast and endometrial carcinoma, and lymphoid tumor cells was also studied. In vitro, 0.5, 0.75, and 1 nM docetaxel and 2, 3, and 4 nM paclitaxel, i.e., non-cytotoxic doses, impacted all endothelial cell functions, but not protease secretion, in a dose-dependent fashion, whereas they did not affect the proliferation of other cells, except those of Kaposis sarcoma. No apoptosis was induced by 0.5 nM docetaxel and 2 nM paclitaxel, and moderate apoptosis was induced by 1 nM docetaxel and 4 nM paclitaxel. The antiangiogenic effect rapidly disappeared on drug suspension and was accompanied ultrastructurally by thin lesions of cytoskeleton in the form of slight and equally reversible depolymerization and accumulation of microfilaments. Massive endothelial cell apoptosis with evident cytotoxicity and irreversibility were associated with 2 nM docetaxel and 5 nM paclitaxel, although these higher doses were ineffective on other cells except Kaposis sarcoma cells. In vivo, 1, 2, and 3 nM docetaxel and 4, 8, and 12 nM paclitaxel displayed a dose-dependent antiangiogenic activity. We suggest that very low docetaxel and paclitaxel doses selectively cause organic and functional damage of endothelial cells and that docetaxel is four times stronger. Their antiangiogenic activity could be applied to treat Kaposis sarcoma and cancers.

αvβ3 integrin engagement modulates cell adhesion, proliferation, and protease secretion in human lymphoid tumor cells

Abstract Objective The mechanisms used by human lymphoproliferative diseases to invade locally and metastasize are thought to be similar to those developed by solid tumors, including cell proliferation and secretion of extracellular matrix-degrading enzymes following adhesion to extracellular matrix proteins. Hence, the ability of Namalwa (Burkitts lymphoma), U266 (multiple myeloma), and CEM (T-cell lymphoblastic leukemia) cells to interact with the extracellular matrix components vitronectin and fibronectin was determined. Fresh bone marrow plasma cells from patients with multiple myeloma also were studied. Materials and Methods Engagement of α v β 3 integrin, formation and protein composition of focal adhesion contacts on the cell surface, phosphorylation of several signal transduction proteins in the contacts, cell proliferation, and enzyme secretion were studied following adhesion to vitronectin and fibronectin. Results All three lines adhered to immobilized vitronectin and fibronectin. Adhesion was fully prevented by neutralizing monoclonal anti-α v β 3 integrin antibody. Integrin engagement caused the formation of phosphorylated pp60 src /focal adhesion kinase complexes and the aggregation of focal adhesion plaques containing the β 3 integrin subunit, the cytoskeletal proteins vinculin, cortactin, and paxillin, the tyrosine kinases focal adhesion kinase and pp60 src , the adapter protein Grb-2, and the mitogen-activated protein kinase ERK-2. Free and immobilized vitronectin and fibronectin stimulated the proliferation of cells under serum-free conditions and the production and release of urokinase-type plasminogen activator, and increased the release of the activated forms of matrix metalloproteinase-2 and matrix metalloproteinase-9 in an α v β 3 integrin-dependent manner. Similar results were obtained in myeloma plasma cells. Conclusions The demonstrated ability of lymphoid tumor cells to interact with the extracellular matrix components vitronectin and fibronectin via α v β 3 integrin can be interpreted as evidence of a novel mechanism for their invasion and spreading. This interaction allows them to adhere to the substratum and enhances their proliferation and protease secretion.

Vascular endothelial growth factor and its receptors in multiple myeloma

Multiple myeloma (MM) progresses from an avascular to a vascular phase (active MM) accompanied by a significant increase in microvessel density in the bone marrow. This article summarizes the literature concerning the specific role played by vascular endothelial growth factor (VEGF) in this process. Recent applications of antiangiogenic agents that interfere with VEGF signaling and block MM progression are also described.

New therapies in multiple myeloma

The melphalan-prednisone regimen has been considered as standard therapy for patients with multiple myeloma (MM) for many years. Recently, high-dose chemotherapy with stem-cell support has extended progression-free survival and increased overall survival, and it is now considered conventional therapy in younger patients. However, most patients relapse and the salvage treatment is not very effective. New active drugs, including immunomodulatory agents, thalidomide (Thal) and lenalidomide, and the proteasome inhibitor bortezomib, have shown promising anti-myeloma activity. These novel treatments are aimed at overcoming resistance of tumour cells to conventional chemotherapy, acting both directly on myeloma cells and indirectly by blocking the interactions of myeloma cells with their local microenvironment and suppressing growth and survival signals induced by autocrine and paracrine loops in the bone marrow. Thal has been widely studied, mostly in combination regimens in patients with relapsed MM and, more recently, in front-line therapy, showing efficacy in terms of response rate and event-free survival. Bortezomib has been found to possess remarkable activity, especially in combination with other chemotherapeutic agents, in relapsed/refractory and newly diagnosed MM, as well as in patients presenting adverse prognostic factors. Lenalidomide, in combination with dexamethasone, is showing high overall response rates in relapsed and refractory MM and promising results also in first-line therapy. In this paper, the results of the most significant trials with Thal, bortezomib and lenalidomide are reported. Several ongoing clinical studies will hopefully allow the identification of the most active combinations capable of improving survival in patients with MM.

Target Therapies in Pancreatic Carcinoma

Pancreatic ductal adenocarcinoma (PDAC) occurs in the majority of cases with early locoregional spread and distant metastases at diagnosis, leading to dismal prognosis and limited treatment options. Traditional cytotoxic chemotherapy provides only modest benefit to patients with PDAC. Identification of different molecular pathways, overexpressed in pancreatic cancer cells, has provided the opportunity to develop targeted therapies (monoclonal antibodies and small-molecule inhibitors) and peculiar new class of taxanes with a crucial therapeutic role in this cancer setting. A phase III trial has shown that erlotinib in combination with gemcitabine was clinically irrelevant and skin toxicity can be a positive prognostic factor. Moreover, the combination of cetuximab or erlotinib with radiotherapy in advanced pancreatic cancer has shown to be synergistic and a reversal of radio-resistance has been suggested by inhibition of VEGF/EGFR pathway. To overcome EGFR-inhibition therapy resistance several alternative pathways targets are under investigation (IGF- 1R, MMPs, Hedgehog proteins, m-TOR, MEK, COX-2) and provide the rationale for clinical use in phase II/III studies. Also nab-paclitaxel, a new taxanes class, uses high pancreatic albumin-binding protein SPARC levels to act in cancer cells with a less toxic and more effective dose with respect to classic taxanes. Understanding of molecular pathogenesis of pancreatic adenocarcinoma continues to expand. However, many promising data in preclinic and phase I/II trials did not yield promise in phase III trials, suggesting that identification of predictive biomarkers for these new agents is mandatory. The knowledge of biologic and molecular aspects of pancreatic cancer can be the basis for future therapeutic developments.

Proteolytic Activity of Human Lymphoid Tumor Cells.Correlation with Tumor Progression

Matrix metalloproteinase (MMP) expression and production are associated with advanced-stage tumor and contribute to tumor progression, invasion and metastases. The current study was designed to determine the expression and production of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by human lymphoid tumor cells. Changes in expression and production were also investigated during tumor progression of multiple myeloma and mycosis fungoides. In situ hybridization analysis revealed that lymphoblastic leukemia B cells (SB cell line), multiple myeloma (MM) cells (U266 cell line) and lymphoblastic leukemia T cells (CEM and Jurkat cell lines) express constitutively the mRNA for MMP-2 and/or MMP-9. We demonstrated by gelatin-zymography of cell culture medium that both enzymes were secreted in their cleaved (activated) form. In situ hybridization of bone marrow plasma cells and gelatin- zymography of the medium showed that patients with active MM (diagnosis, relapse, leukemic progression) express higher levels of MMP-2 mRNA and protein than patients with non-active MM (complete/objective response, plateau) and with monoclonal gammopathies of undetermined significance (MGUS). MMP-9 expression and secretion was similar in all patient groups. In patients with mycosis fungoides (MF), the expression of MMP-2 and MMP-9 mRNAs was significantly upregulated with advancing stage, in terms of lesions both positive for one of two mRNAs and with the greatest intensity of expression. Besides MF cells, the MMP-2 and/or MMP-9 mRNAs were expressed by some stromal cell populations (microvascular endothelial cells, fibroblasts, macrophages), suggesting that these cells cooperate in the process of tumor invasion. Our studies identify MMPs as an important class of proteinases involved in the extracellular matrix (ECM) degradation by human lymphoid tumors, and suggest that MMPs inhibitors may lead to important new treatment for their control.

Identification of an Antigenic and Immunogenic Motif Expressed by Two 7-Mer Rituximab-Specific Cyclic Peptide Mimotopes : Implication for Peptide-Based Active Immunotherapy

Two 7-mer cyclic peptides—Rp15-C and Rp13-C—which bear the antigenic motif recognized by the anti-CD20 mAb rituximab, but have different motif-surrounding amino acids, show a comparable avidity for rituximab and inhibit the binding of rituximab to raft-associated CD20 and rituximab-induced membrane ceramide on human lymphoid Daudi cells. Their immunogenic profiles differed: Abs recognizing CD20 were induced in two and five of five BALB/c mice immunized with Rp15-C and Rp13-C, respectively. Analysis of immunogenic motif, performed by panning a 7-mer phage-display peptide library with purified anti-peptide IgGs, showed that the motif defined by anti-Rp15-C mostly included amino acids surrounding the rituximab-specific antigenic motif , whereas that defined by anti-Rp13-C was . These data indicate that their motif-surrounding amino acids can markedly influence the specificity of Abs, even when elicited with a short 7-mer peptide. Because these anti-peptide Abs are of IgG isotype, their specificity is likely to reflect how peptides are processed at the T cell level and suggest that, within a short peptide, the motifs defined by T cells during the initial phase and upon their stimulation may be different. Our findings may account for the failure of most forms of peptide-based immunotherapy in cancer and autoimmune diseases in which anti-mimotope Abs are expected to play a relevant therapeutic effect. They also suggest strategies to implement the specificity of peptide-induced Abs against the target Ag.

Challenges and Opportunities of MicroRNAs in Lymphomas

MicroRNAs (miRNAs) are small non-coding RNAs that control the expression of many target messenger RNAs (mRNAs) involved in normal cell functions (differentiation, proliferation and apoptosis). Consequently their aberrant expression and/or functions are related to pathogenesis of many human diseases including cancers. Haematopoiesis is a highly regulated process controlled by a complex network of molecular mechanisms that simultaneously regulate commitment, differentiation, proliferation, and apoptosis of hematopoietic stem cells (HSC). Alterations on this network could affect the normal haematopoiesis, leading to the development of haematological malignancies such as lymphomas. The incidence of lymphomas is rising and a significant proportion of patients are refractory to standard therapies. Accurate diagnosis, prognosis and therapy still require additional markers to be used for diagnostic and prognostic purpose and evaluation of clinical outcome. The dysregulated expression or function of miRNAs in various types of lymphomas has been associated with lymphoma pathogenesis. Indeed, many recent findings suggest that almost all lymphomas seem to have a distinct and specific miRNA profile and some miRNAs are related to therapy resistance or have a distinct kinetics during therapy. MiRNAs are easily detectable in fresh or paraffin-embedded diagnostic tissue and serum where they are highly stable and quantifiable within the diagnostic laboratory at each consultation. Accordingly they could be specific biomarkers for lymphoma diagnosis, as well as useful for evaluating prognosis or disease response to the therapy, especially for evaluation of early relapse detection and for greatly assisting clinical decisions making. Here we summarize the current knowledge on the role of miRNAs in normal and aberrant lymphopoiesis in order to highlight their clinical value as specific diagnosis and prognosis markers of lymphoid malignancies or for prediction of therapy response. Finally, we discuss their controversial therapeutic role and future applications in therapy by modulating miRNA.

Biological functions and therapeutic use of erythropoiesis‐stimulating agents: perplexities and perspectives

Randomized clinical studies, carried out in patients with haematological malignancies and with solid tumours, have consistently demonstrated that treatment with recombinant human erythropoietin (Epo) increases haemoglobin levels, reduces blood transfusion requirements, and improves the quality of life. In addition, identification of erythropoietin receptor (EpoR) expression on many types of non‐erythroid and cancer cells has spurred an interest in the extra‐haematological activities of Epo itself and other erythropoiesis‐stimulating agents (ESAs). Epo and its derivatives have emerged as major tissue‐protective cytokines in ischaemic and degenerative damage of cardiovascular, neurological and renal diseases, while their angiogenetic and immunomodulatory properties indicate that their therapeutic potential may extend well beyond erythropoiesis alone. Both preclinical and clinical data, however, have suggested that they may contribute to tumour progression and prejudice survival when administered to anaemic cancer patients, though the results are equivocal and the assumed mechanisms by which tumour growth could be promoted are not fully understood. While these findings offer new perspectives, they nonetheless demand caution in the employment of ESAs. Further, well‐designed experimental and clinical studies are warranted.

Bendamustine plus rituximab for relapsed or refractory diffuse large B cell lymphoma: A retrospective analysis

For patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) who are not eligible for intensive chemotherapy because of comorbidities, advanced age, or relapse after heavy salvage regimens, treatment options are very limited and prognosis is poor. We retrospectively analyzed 29 patients with relapsed/refractory DLBCL treated with combination bendamustine plus rituximab (BR) between July 2010 and January 2014 to evaluate overall response rate (ORR), progression-free survival (PFS), duration of response (DOR) and treatment safety. Twenty-eight patients were available for this analysis. ORR was 50% (14 patients), with 39.3% CR (11 patients), and 10.7% PR (3 patients). SD was reported in 2 patients (7.2%) and PD in 12 patients (42.8%). At the median follow up of 8 months (range 1-37.4 months), the median PFS was 8 months for all patients (95% CI 5.5-26.6). The median DOR was 24.7 months (95% CI 3.2-24.7). Grade 3/4 toxicity observed included hematologic events: lymphopenia (42.8%), neutropenia (32.1%), anemia (17.2%), and thrombocytopenia (14.2%). BR can be considered to have a role in the treatment of patients with relapsed/refractory DLBCL with limited therapeutic options, in that it can induce long-term remission in some patients with an acceptable toxicity profile.