Angelo Borsarelli Carvalho Brito

Associations Of VEGF and VEGFR2 Polymorphisms With Increased Risk and Aggressiveness Of Multiple Myeloma

Angiogenesis has been highlighted as a critical component in the progression of multiple myeloma (MM), and vascular endothelial growth factor (VEGF) as well as its type 2 receptor (VEGFR2) are thought to play a major role in the process. Single nucleotide polymorphisms (SNPs) have been described in VEGF and VEGFR2 genes, with quantitative or qualitative changes in encoded VEGF and VEGFR2. The roles of VEGF −2578C/A, −1154G/A, and −634G/C as well as VEGFR2 −604T/C and +1192G/A SNPs in the risk and manifestations of MM are still unknown; therefore, this study aimed to clarify this issue. DNA from 192 patients and 209 controls were analyzed by real-time polymerase chain reaction for identification of genotypes. The frequencies of VEGF −2578CC, VEGF −2578CC plus VEGF −634GG, and VEGF −2578CC plus VEGF −1154GG plus VEGF −634GG genotypes were higher in patients than in controls. Carriers of the respective genotypes had a 1.89-, a 5.52-, and a 4.91-fold increased risk for MM than others. VEGF −2578CC plus VEGFR2 +1192GG, VEGF −2578CC plus VEGF −634GG plus VEGFR2 +1192GG, and VEGF −1154GG plus VEGF −634GG plus VEGFR2 −604TT combined genotypes were more common in patients than in controls. Carriers of the respective genotypes had a 2.56-, a 10.97-, and a 14.10-fold increased risk for MM than others. An excess of VEGFR2 −604TT genotype was also seen in patients with stage II or III tumors when compared with those with stage I tumors. Our data suggest, for the first time, that inherited abnormalities in VEGF and VEGFR2 pathways influence the risk and aggressiveness of MM.

Dural lymphoma mimicking subdural haematoma on computerized tomography

A 69-year-old man was admitted for investigation of a 2-month history of left-sided weakness and headache with dizziness. There was no history of trauma. Initial cranial computerized tomography (CT) showed an extra-axial crescentshaped hyperdense lesion in the right frontoparietal region (top left). CT performed 1 month later showed no involution of the collection and persistence of its hyperdensity. Magnetic resonance imaging (MRI) revealed an extra-axial pachymeningeal lesion that was hypointense on T2 weighting (top right), with intense pachymeningeal and leptomeningeal enhancement on a T1-weighted image after contrast administration (bottom left); hypointensity on an apparent diffusion coefficient map (bottom right) confirmed restricted diffusion. These findings suggested meningeal lymphoma. Serum protein electrophoresis showed a monoclonal peak of immunoglobulin M (4 26 g/l) and bone marrow biopsy showed extensive interstitial infiltration by small lymphoplasmacytic cells. A diagnosis of lymphoplasmacytic lymphoma was made. The patient received six cycles of rituximab plus cyclophosphamide, vincristine and prednisone (R-CVP), and achieved regression of the meningeal lesions. The typical radiological picture of an acute subdural haematoma on CT (hyperdense extra-axial collection) in a patient without a history of trauma and the persistence of this abnormality on follow up CTs suggests an alternative diagnosis and the need for further radiological investigation with MRI.

Intracerebral Granulocytic Sarcoma in recurrence of Chronic Myeloid Leukemia

A 21-year-old male with a 2-year diagnosis of chronic myeloid leukemia in complete hematologic and cytogenetic responses was admitted to hospital with drowsiness, headache and seizures. Laboratory evaluation disclosed leucocitosis with 19.0% of peripheral blasts. Brain magnetic resonance imaging (MRI) (Figure 1) corroborated the diagnosis of granulocytic sarcoma (GS) in blast crisis. MRI performed 38 days after chemotherapy was indicative of tumor regression (Figure 2). GS is an extramedullary solid tumor composed of immature myeloid cells. It develops before, during or after the onset of myeloid leukemia. Intra-axial GS without involvement of skull or meninges is rare.

VEGF gene polymorphisms and outcome of epithelial ovarian cancer patients

AIM Since VEGF polymorphisms were associated with variable protein production, we analyzed herein their roles in outcome of epithelial ovarian cancer (EOC) patients. METHODS Genotypes of 85 patients with primary EOC were identified in DNA by real-time PCR. Progression-free survival and overall survival were analyzed using Kaplan-Meier method, univariate Cox model and bootstrap resampling study. RESULTS At 60 months of follow-up, progression-free survival was shorter in patients with VEGF c.-2578 CC genotype compared with others (52.7 vs 82.2%; p = 0.04). Those patients had 2.15 more chance of presenting disease progression than others (p = 0.04); bootstrap study validated the result (p = 0.03). CONCLUSION Our data suggest that VEGF c.-2578C>A polymorphism acts as a prognostic factor in EOC.

Association of VEGFA-2578 C>A polymorphism with clinicopathological aspects and outcome in follicular lymphoma patients

Association of VEGFA -2578 C>A polymorphism with clinicopathological aspects and outcome in follicular lymphoma patients

WT1, p53 and p16 expression in the diagnosis of low- and high-grade serous ovarian carcinomas and their relation to prognosis

Objective To evaluate the diagnostic and prognostic value of the immunohistochemical expression of WT1, p53 and p16 in low- (LGSOCs) and high-grade serous ovarian carcinomas (HGSOCs). Results HGSOC had a significantly higher proportion of advanced stage disease, higher CA125 levels, higher proportion of post-surgery residual disease and higher recurrence or disease progression. WT1 was expressed in 71.4% of LGSOCs and in 57.1% of HGSOCs (p = 0.32). Focal and/or complete absence of p53 expression with negative p16 expression was found in 90.5% of LGSOCs, in contrast to the 88.1% of HGSOCs with diffuse or complete absence of p53 expression with positive p16 expression (<0.001). The IHC p53/p16 index and the morphological classification were closely matched (k = 0.68). In the univariate analysis, FIGO stage, post-surgery residual disease and histological grade were significantly associated with progression-free survival (PFS) and overall survival (OS). The IHC p53/p16 index was associated only with PFS. WT1 was not associated with PFS or OS. According to the multivariate analysis, advanced FIGO stage and presence of post-surgery residual disease remained independent prognostic factors for worst PFS, however these features had only a trend association with OS. Methods 21 LGSOC and 85 HGSOC stage I–IV cases were included. The morphological classification was assessed according to the World Health Organization (WHO) criteria. Immunohistochemistry (IHC) was performed in tissue microarray slides. IHC p53/p16 index was compared with the morphological classification. Conclusions The IHC p53/p16 index was a good marker for the differentiation of LGSOC and HGSOC, but the morphologic classification showed a better association with survival. FIGO stage and post-surgery residual disease remained the only independent prognostic factors for survival.

VEGF , VEGFR2 and GSTM1 polymorphisms in outcome of multiple myeloma patients treated with thalidomide-based regimens

VEGF , VEGFR2 and GSTM1 polymorphisms in outcome of multiple myeloma patients treated with thalidomide-based regimens

Polymorphisms in key regulator genes of the intrinsic apoptosis pathway in risk and clinical presentation of diffuse large B-cell lymphoma

To the Editor Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for 30–40% of the newly diagnosed cases. Apoptosis is an important mechanism involved in lymphomagenesis, in which the pro-apoptotic B-cell lymphoma 2-associated X (Bax) and the anti-apoptotic Bcell lymphoma 2 (Bcl2) are the most active proteins [1]. It is already well established that the ability to induce apoptosis is variable in humans [2]. A single nucleotide polymorphism (SNP) located in the 5′-untranslated region of the BAX gene with a G→A substitution at 248 nucleotide position (rs4645878) has its allele ‘G’ associated with lower transcriptional activity when compared with ‘A’ allele [3]. Regarding BCL2 gene, a C→A substitution at 717 nucleotide position (rs2279115), located on the promoter region of the gene, has its AA genotype associated with increased Bcl2 expression in comparison with CC genotype [4]. Wang et al. [5] found excesses of the GA+AA genotype of BAX c. 248G>A and the AA genotype of BCL2 c.938C>A (rs2279115) SNPs in a mixed group of NHL patients compared to controls in China. The authors also found that the above mentioned genotypes were associated with dimension and stage of the tumour, respectively. Because DLBCL has a distinct behaviour in comparison with other types of NHL [6], and genetic variations in genes related to cellular homeostasis might have a crucial role in DLBCL development [7], the identification of BAX c. 248G>A and BCL2 c. 717C>A genotypes in patients with de novo DLBCL and controls was considered necessary to test whether SNPs influence the risk and clinical presentation of this aggressive NHL. The case group comprised 153 de novo DLBCL patients at diagnosis (median age: 56 years, range: 17–89 years, 70 males, 83 females, 140 Caucasians, 13 non-Caucasians), considering that 101 patients presented systemic symptoms seen at the Haematology and Haemotherapy Centre from December 2007 to June 2014. The diagnosis of DLBCL was established in accord with the World Health Organization criteria. R-IPI was calculated in each case as previously reported [8], and 16 patients were of very good R-IPI, 76 of good R-IPI, and 61 of poor R-IPI. The control group comprised 240 blood donors (median age: 49 years, range: 23–60 years, 124 males, 116 females,

Association of BAX G(-248)A and BCL2 C(-717)A polymorphisms with outcome in diffuse large B-cell lymphoma patients.

Belmonte, L., Parodi, C., Bar e, P., Malbran, A., Ruibal-Ares, B. & de Bracco, M.M. de E. (2007) Factors involved in the generation of memory CD8 + T cells in patients with X-linked lymphoproliferative disease (XLP). Clinical and Experimental Immunology, 147, 456–464. Belmonte, L., Parodi, C., Bast on, M., Coraglia, A., Felippo, M., Bar e, P., Malbran, A., Ruibal-Ares, B. & de Bracco, M.M. de E. (2009) Increased lymphocyte viability after non-stimulated peripheral blood mononuclear cell (PBMC) culture in patients with X-linked lymphoproliferative disease (XLP). Clinical Immunology, 133, 86–94. Cohen, J.I., Jaffe, E.S., Dale, J.K., Pittaluga, S., Heslop, H.E., Rooney, C.M., Gottschalk, S., Bollard, C.M., Rao, V.K., Narques, A., Burbele, P.D., Turk, S.P., Fulton, R., Wayne, A.S., Little, R.F., Cairo, M.S., El-Mallawany, N.K., Fowler, D., Sportes, C., Bishop, M.R., Wilson, W. & Strauss, S.E. (2015) Characterization and treatment of chronic active Epstein-Barr virus disease: a 28year experience in the United States. Blood, 117, 5835–5849. Coraglia, A., Belmonte, L., Parodi, C., Bast on, M., Bar e, P., Ruibal-Ares, B., Galassi, N. & de Bracco, M.M. de E. (2010) B-lymphocyte memory in X-linked lymphoproliferative disease (XLP). Current Immunology Reviews, 6, 323– 328. Czar, M.J., Kersh, E.N., Mijares, L.A., Lanier, G., Lewis, J., Yap, G., Chen, A., Sher, A., Duckett, C.S., Ahmed, R. & Schwartzberg, P.L. (2001) Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/ SAP. Proceedings of the National Academy of Sciences U.S.A., 98, 7449–7454. Malbran, A., Belmonte, L., Ruibal-Ares, B., Bar e, P., Massud, I., Parodi, C., Felippo, M., Hodinka, R., Haines, K., Nichols, K.E. & de Bracco, M.M. (2001) Loss of circulating CD27 + memory B cells and CCR4 + T cells occurring in association with elevated EBV loads in XLP patients surviving primary EBV infection. Blood, 103, 1625–1631. Milone, M.C., Tsai, D.E., Hodinka, R.L., Silverman, L.B., Malbran, A., Wasik, M.A. & Nichols, K.E. (2005) Treatment of primary Epstein-Barr virus infection in patients with X-linked lymphoproliferative disease using B-cell-directed therapy. Blood, 105, 994–996. Nichols, K.E., Harkin, D.P., Levitz, S., Krainer, M., Kolquist, K.A., Genovese, C., Bernard, A., Ferguson, M., Zuo, L., Snyder, E., Buckler, A.J., Wise, C., Ashley, J., Lovett, M., Valentine, M.B., Look, A.T., Gerald, W., Housman, D.E. & Haber, D.A. (1998) Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. Proceedings of the National Academy of Sciences U.S.A., 95, 13765–13770. Paquin Proulx, D., Aubin, E., Lemieux, R. & Bazin, R. (2010) Inhibition of B cell-mediated antigen presentation by intravenous immunoglobulins (IVIg). Clinical Immunology, 135, 422– 429. Qi, H., Cannons, J.L., Klauschen, F., Schwartzberg, P.L. & Germain, R.N. (2008) SAP-controlled TB interactions underlie germinal center formation. Nature, 455, 764–769.

959PBAX AND BCL-2 POLYMORPHISMS MODULATING AGGRESSIVENESS AND PROGNOSIS IN DIFFUSE LARGE B-CELL LYMPHOMA

ABSTRACT Aim: Apoptosis, with participation of the pro-apoptotic BAX and the anti-apoptotic BCL-2 proteins, play a key role in outcome of patients with diffuse large B cell lymphoma (DLBCL). The ability to induce apoptosis is variable in humans, since several proteins enrolled in the process are encoded by polymorphic genes. The G wild allele of the BAX G(-248)A and the variant A allele of the BCL2 C(-717)A single nucleotide polymorphisms (SNPs) are related to lower transcriptional activity and higher BCL-2 protein expression, respectively, compared with the A variant and C wild alleles. Since the roles of these SNPs in clinical aspects and prognosis of DLBCL are still unknown, investigation of these were the aims of the present study. Methods: Our analysis included 154 consecutive DLBCL patients at diagnosis seen at the University Hospital from December 2007 to March 2014. Genomic DNA from peripheral blood was analyzed by polymerase chain reaction followed by enzymatic digestion for discrimination of distinct genotypes of each SNP. Multivariate analysis using the logistic regression model served to assess the associations between genotypes and clinical aspects. Overall survival (OS) was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analysed by the log-rank test. Results: The frequencies of BAX GG and BAX GG plus BCL-2 AA genotypes were higher in patients with stage IV tumors compared to those with tumors of I + II + III stages (95% vs. 77%, P = 0.01; 94% vs. 54%, P = 0.02), respectively. The median time of observation of patients was 22 months (range: 1-75). On univariate analysis, the presence of B symptoms (68% vs. 87%, P = 0.02), bone marrow involvement (53% vs. 79%, P = 0.009), high LDH levels (64% vs. 81%, P = 0.03), high risk disease (52% vs. 76%, P = 0.002), and stage IV (61% vs. 81% P = 0.01) were predictive of worse outcome at 24 months of follow up. Moreover, at the same time, patients with BCL2 CA + AA genotypes had worse outcome than others (68% vs. 88%, P = 0.04). Conclusions: Our data indicate, for the first time, that inherited abnormalities in intrinsic apoptosis pathway, related to the BAX G(-238)A and BCL2 C(-717)A SNPs, influence aggressiveness and outcome of DLBCL patients. Disclosure: All authors have declared no conflicts of interest.