Raquel Malumbres

Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R-CHOP

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by variable clinical outcomes. Outcome prediction at the time of diagnosis is of paramount importance. Previously, we constructed a 6-gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies. However, the standard therapy has evolved into rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Herein, we evaluated the predictive power of a paraffin-based 6-gene model in R-CHOP-treated DLBCL patients. RNA was successfully extracted from 132 formalin-fixed paraffin-embedded (FFPE) specimens. Expression of the 6 genes comprising the model was measured and the mortality predictor score was calculated for each patient. The mortality predictor score divided patients into low-risk (below median) and high-risk (above median) subgroups with significantly different overall survival (OS; P = .002) and progression-free survival (PFS; P = .038). The model also predicted OS and PFS when the mortality predictor score was considered as a continuous variable (P = .002 and .010, respectively) and was independent of the IPI for prediction of OS (P = .008). These findings demonstrate that the prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment and that the model can be applied to routine FFPE tissue from initial diagnostic biopsies.

Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas

Interleukin-21 (IL-21), a member of the IL-2 cytokine family, has diverse regulatory effects on natural killer (NK), T, and B cells. In contrast to other cytokines that are usually immunostimulatory, IL-21 can induce apoptosis of murine B cells at specific activation-differentiation stages. This effect may be used for treatment of B-cell malignancies. Herein we report that diffuse large B-cell lymphoma (DLBCL) cell lines exhibit widespread expression of the IL-21 receptor (IL-21R) and that IL-21 stimulation leads to cell-cycle arrest and caspase-dependent apoptosis. IL-21 also induces apoptosis in de novo DLBCL primary tumors but does not affect viability of human healthy B cells. Furthermore, IL-21 promotes tumor regression and prolongs survival of mice harboring xenograft DLBCL tumors. The antilymphoma effects of this cytokine are dependent on a mechanism involving IL-21-activated signal transducer and activator of transcription 3 (STAT3) up-regulating expression of c-Myc. This up-regulation promotes a decrease in expression of antiapoptotic Bcl-2 and Bcl-X(L) proteins triggering cell death. Our results represent one of the first examples in which the STAT3-c-Myc signaling pathway, which can promote survival and oncogenesis, can induce apoptosis in neoplastic cells. Moreover, based on IL-21s potency in vitro and in animal models, our findings indicate that this cytokine should be examined in clinical studies of DLBCL.

Pharmacogenomic approach for the identification of novel determinants of acquired resistance to oxaliplatin in colorectal cancer

Oxaliplatin is a third-generation platinum agent used in colorectal cancer treatment. Oxaliplatin resistance acquisition is a complex process mainly based on alteration of genes and pathways involved in its mechanism of action. Therefore, our purpose was to perform a gene expression screening in an in vitro model to identify genes that could play a role in oxaliplatin resistance acquisition processes. Four colorectal cancer cell lines and their oxaliplatin-resistant derived sublines were compared. Microarray analysis was done using Human 19K Oligo Array Slides. RNA from cells were hybridized with a commercial RNA reference sample and labeled with both fluorochromes Cy3 and Cy5. Data were analyzed by hierarchical clustering method. Subsequently, quantitative real-time PCR (qRT-PCR) was used to corroborate microarray data, considering as positively validated those genes that showed significant differences in expression levels between groups and a correlation between microarray and qRT-PCR data. By microarray analysis, 32 candidate genes were identified. After validation process by qRT-PCR, the genes AKT1, CDK5, TRIP, GARP, RGS11, and UGCGL1 were positively validated. The 3 first genes proved to be involved in regulation of nuclear factor-κβ antiapoptotic transcription factor previously related to drug resistance, and the other 3 genes are novel finds. We have identified 6 genes related to oxaliplatin resistance acquisition. These findings are of paramount importance to understand these processes better and open new lines of study to elucidate the relevance of this pharmacogenomic approach into the clinic. [Mol Cancer Ther 2009;8(1):194–202]

Somatically mutated immunoglobulin IGHV@ genes without intraclonal heterogeneity indicate a postgerminal centre origin of primary intraocular diffuse large B-cell lymphomas

Primary intraocular lymphoma (PIOL) is a type of diffuse large B‐cell lymphoma (DLBCL) that is related to primary central nervous system lymphoma (PCNSL). Whether their pathogenesis is similar is presently unknown. Immunoglobulin heavy chain variable genes (IGHV@) somatic mutations were analysed in five patients with PIOL, one patient with concomitant PCNSL and one patient with systemic DLBCL involving the eye. Six in‐frame mutated clonal IGHV@ rearrangements were cloned from PIOL specimens. The sequences showed no evidence of antigen selection and revealed absence of intraclonal heterogeneity in four of five cases, suggesting that PIOL and PCNSL may differ in their ontogeny.

New symmetrical quinazoline derivatives selectively induce apoptosis in human cancer cells

In the search of new symmetrical derivatives with anticancer activity, we have looked for novel compounds able to induce a selective proapoptotic mechanism in cancer cells. The potential antitumoral activity of several quinazoline derivatives was evaluated in vitro examining their cytotoxic effects against human breast, colon and bladder cancer cell lines. The IC50 value of the compounds that showed cytotoxic activity was calculated. These compounds were tested for their ability to induce caspase-3 activation and nuclear chromatin degradation. Non-tumoral human cell lines were used to test the selectivity of the cytotoxic compounds against cancer cells. Several compounds showed no cytotoxicity in these cell lines. Finally, JRF12 (2,4-dibencilaminoquinazoline) was chosen as the best candidate and its mechanism of action was studied in more detail. A time dependent evaluation of apoptosis was performed in the three cancer cell lines, followed by an evaluation of the cell cycle regulation involvement that showed a decrease of cells in G1 phase and increase of cells in G2 phase before cell death. 2,4-dibencilaminoquinazoline treatment produces few changes in the expression of genes as evaluated by using oligonucleotide microarrays and Q-RT-PCR assays. In conclusion, 2,4-dibencilaminoquinazoline is a promising anticancer drug showing cytostatic and apoptotic effects mainly in a transcription independent manner.

MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia

The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the SET gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates SET expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of SET. These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of SET and MYC, RUNX1, and GATA2 in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to SET overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of SET expression in AML.

Expression of miRNAs in Lymphocytes: A Review

In this chapter, we provide a review on the functions of the most important miRNAs in lymphocytes. Most of them are involved in lymphopoiesis, immune response, and lymphoid malignancies, highlighting the importance of miRNAs in these cells.

Successful use of semi-nested PCR for the diagnosis of primary intraocular lymphoma

Primary intraocular lymphoma (PIOL) is a rare category of non-Hodgkin lymphoma, usually of diffuse large B-cell subtype, representing 51% of non-Hodgkin lymphomas and 51% of intraocular tumors. PIOL is considered to be a type of primary CNS lymphoma, although differences in disease pathogenesis have recently been suggested [1]. Monoclonal lymphocytic transformation is believed to occur either intraocularly, as a result of chronic intraocular antigenic stimulation, or extraocularly, whereby malignant cells are sequestered and protected from normal surveillance in the immuneprivileged intraocular environment. PIOL is characterised by invasion of a monoclonal population of lymphocytes into the sub-RPE space, retina, vitreous cavity and choroid. Patients may present with either isolated intraocular disease or with concurrent CNS involvement (5–25%). Ultimately, 60–85% of patients will develop concurrent CNS involvement, typically within 3 years of the diagnosis. PIOL is typically associated with a high mortality rate due to its progressive course and high rate of recurrence after treatment, with historical 5year survival rates of 10–29% after external beam radiation [2]. PIOL presents diagnostic and therapeutic challenges due to its highly variable clinical presentation and elusive histopathologic nature. Although recent advances, have improved diagnostic yields, a significant number of vitrectomies are non-diagnostic largely as a result of spontaneous cell lysis and poor sample yield. For this reason, more sensitive diagnostic testing is sorely needed. This case documents the use of a new semi-nested PCR technique to demonstrate monoclonality following failure of traditional histopathologic and molecular diagnostic methods in a patient with PIOL. A 71-year-old Hispanic female presented with 2 weeks of blurry vision and a visual acuity of 20/50 in the right eye and 20/60 in the left eye. There was no relevant ocular, family, or medical history, and review of systems was unremarkable. Exam revealed bilateral 2þ vitreous cellular infiltrate, and multifocal creamy yellow-white subretinal lesions, clinically consistent with PIOL [Figure 1(a)]. Diagnostic echography demonstrated focal and diffuse fundus thickening. The patient was referred to the Hematology and Oncology service, where a thorough systemic workup was negative for systemic or CNS involvement. The patient underwent par plana vitrectomy of the left eye for histopathological evaluation and confirmation of the suspected clinical diagnosis. The cytological evaluation and flow cytometry results were inconclusive, revealing a heterogenous T cell population, an immature atypical B cell population and a minor population of immature myeloid cells. Initial gene rearrangement studies based on the European BIOMED-2 collaborative study