Integrated approach to the study of mantle cell lymphoma

Il linfoma a cellule del mantello (MCL) è un linfoma non Hodgkin B caratterizzato dalla proliferazione di linfociti “maturi” che esprimono CD5, con uno spettro morfologico e fenotipico più ampio di quanto inizialmente descritto quando fu riconosciuto come entità. Sebbene alcuni pazienti abbiano un decorso clinico indolente, in molti il comportamento è aggressivo con scarsa risposta ai convenzionali regimi chemioterapici. La caratteristica genetica tipica è la traslocazione t(11;14)(q13;q32) che comporta l’iper-espressione della Ciclina D1, considerato l’evento oncogenico iniziale. Il MCL può inoltre avere alterazioni cromosomiche e molecolari secondarie che alterano ad esempio il funzionamento del controllo del ciclo cellulare, della senescenza cellulare, della risposta al danno del DNA e molte altre. La conoscenza di questi meccanismi e del loro ruolo nella crescita tumorale è fondamentale per individuare modelli prognostici dell’evoluzione clinica della malattia, nonché per lo sviluppo di nuove terapie che abbiamo come bersaglio mirato proprio le vie alterate nei MCL. Il nostro progetto si è svolto in due fasi, una di raccolta dati da analisi del profilo proteico e fosfoproteico delle linee cellulari di MCL e una seconda di validazione su tessuto umano fissato in formalina e di sviluppo di modelli murini.

Mantle cell lymphoma (MCL) is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes expressing CD5 that may show a spectrum of morphological and phenotypic features broader than initially described. Although some patients may follow an indolent clinical evolution, in most of them the tumour has an aggressive behaviour with poor response to conventional chemotherapy. The genetic hallmark is the t(11;14)(q13;q32) translocation leading to the overexpression of cyclin D1, which is considered the initial oncogenic event. In addition to this translocation, MCL may carry a high number of secondary chromosomal and molecular alterations that target regulatory elements of the cell cycle machinery and senescence (BMI1/INK4/ARF/CDK4/RB1), DNA damage response pathways (ATM/CHK2/p53), and cell survival signals. The knowledge of these mechanisms and their influence on the behaviour of the tumour are facilitating the development of prognostic models with a more precise prediction of the clinical evolution of the patients. This information coupled with the availability of a new generation of innovative drugs targeting basic molecular process of the tumour cells, should facilitate the design of new therapeutic protocols able to overcome the resistance of this aggressive lymphoma to conventional treatments and improve the life expectancy of the patients. The goal of our project has been to integrate information from high-throughput techniques (proteomic approach), find good candidate “drivers” of MCL pathogenesis and validate them by independent techniques, especially by testing their functional importance in model systems, hopefully findings novel biomarkers that could be used in diagnostic practice. PhosphoScan analysis identified 421 unique peptides, corresponding to 341 proteins. Among these, several activated protein kinases were found. Interestingly, several identified proteins mapped to cytogenetic loci that have been reported to be altered in MCL by previous literature. Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes. For validation on tissues we characterised 40 MCL cases according to most recent criteria in order to recognize biological and prognostic variants; several proteins recognized by proteomics studies were also studied in tissues by means of immunohistochemistry. The final part of the project was focused on the attempt to establish experimental models by tumor xenografts. NOD/SCID mice were first injected by several ways with primary MCL cell in an effort to establish a model for further experimental work, but they developed GVHD and died. Then we’ve found that injecting MCL cell lines into Rag -/- γ -/- mice we were able to grow big tumoral masses.