Gisele W. B. Colleoni

Structural profiles of TP53 gene mutations predict clinical outcome in diffuse large B-cell lymphoma: An international collaborative study

The purpose of this study is to correlate the presence of TP53 gene mutations with the clinical outcome of a cohort of patients with diffuse large B-cell lymphoma (DLBCL) assembled from 12 medical centers. TP53 mutations were identified in 102 of 477 patients, and the overall survival (OS) of patients with TP53 mutations was significantly worse than those with wild-type TP53 (P < .001). However, subsets of TP53 mutations were found to have different effects on OS. Mutations in the TP53 DNA-binding domains were the strongest predictors of poor OS (P < .001). Mutations in the Loop-Sheet-Helix and Loop-L3 were associated with significantly decreased OS (P = .002), but OS was not significantly affected by mutations in Loop-L2. A subset of missense mutations (His158, His175, Ser245, Gln248, His273, Arg280, and Arg282) in the DNA-binding domains had the worst prognosis. Multivariate analysis confirmed that the International Prognostic Index and mutations in the DNA-binding domains were independent predictors of OS. TP53 mutations also stratified patients with germinal center B cell-like DLBCL, but not nongerminal center B cell-like DLBCL, into molecularly distinct subsets with different survivals. This study shows the prognostic importance of mutations in the TP53 DNA-binding domains in patients with DLBCL.

TGFβR2 aberrant methylation is a potential prognostic marker and therapeutic target in multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in MM. The aberrant methylation is one of the most frequent epigenetic alterations in human genome. This study evaluated the aberrant methylation status of 20 genes in 51 MM samples by quantitative methylation‐specific PCR (QMSP) and compared the methylation profile with clinicopathological characteristics of the patients. The QMSP analyses showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%) and TGFβR2 (39.2%) are frequently hypermethylated in MM while aberrant methylation of RARβ (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%) and THBS1 (4.1%) are rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. Hypermethylation of ESR1 was correlated positively with isotype IgA, while aberrant methylation of THBS1 correlated negatively with isotype IgG. Furthermore, hypermethylation of DCC and TGFβR2 were correlated with poor survival. The multivariate analysis showed ISS and TGFβR2 hypermethylation strongly correlated with poor outcome. This study represents the first quantitative evaluation of promoter methylation in MM and our data provide evidence that TGFβR2 hypermethylation, besides ISS, may be useful as prognostic indicator in this disease.

Prognostic significance of vascular endothelial growth factor immunoexpression in the context of adverse standard prognostic factors in multiple myeloma

Abstract:  Objectives: Vascular endothelial growth factor (VEGF) acts in several steps of multiple myeloma (MM) pathogenesis and it is an important mediator of tumor angiogenesis. The aim of this study was to examine the prognostic significance of VEGF immunoexpression in the context of standard prognostic factors present in a cohort of advanced MM patients. Methods: Fifty untreated MM patients were enrolled from May 2000 to December 2002. Bone marrow sections were subjected to morphologic assessment and immunohistochemical studies with antibodies against CD34 and VEGF. Angiogenesis was measured by microvessel density (MVD) and stratified into high (MVD ≥ 20) and low angiogenesis status (MVD < 20). VEGF immunoreactivity was examined on the basis of intensity and percentage of positive plasma cells (PC). Results: Ninety‐four percent of patients presented advanced disease at diagnosis. Median PC marrow infiltration was 80%. Twelve percent of patients presented plasmablastic morphology. Low angiogenesis was present in 27% of patients, while high angiogenesis was present in 73%. Twenty‐nine percent of patients had VEGF < 10% and 71% had VEGF ≥ 10%. Weak‐intensity VEGF was observed in 34% of cases, while 37% had moderate/strong VEGF intensity. Although VEGF had prognostic impact on overall survival (OS) and event‐free survival (EFS) in univariate analysis, multivariate analysis identified only plasmablastic morphology and elevated serum lactate dehydrogenase (LDH) level as independent prognostic factors to predict OS (P = 0.04 and P = 0.02, respectively). With regard to EFS, although VEGF showed statistical trend to influence survival (P = 0.08), the parameters of independent prognostic value were also plasmablastic morphology (P = 0.01) and elevated LDH level (P = 0.01). Conclusion: Our findings underline the frequent expression of VEGF in advanced‐stage MM and the greater prognostic information of simple and readily available factors, namely plasmablastic morphology and elevated LDH. Moreover, despite the absence of prognostic importance in multivariate analysis, VEGF and its receptors remain promising therapeutic targets in MM.

SAGE analysis highlights the importance of p53csv, ddx5, mapkapk2 and ranbp2 to multiple myeloma tumorigenesis

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2 and RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74%, 96%, and 21% of the MM samples, respectively. Analysis of differential expression using SAGE could identify genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

Confirmation of the utility of the International Staging System and identification of a unique pattern of disease in Brazilian patients with multiple myeloma

Multiple myeloma (MM) is one of the most frequent hematologic malignancies, and its incidence varies worldwide. Except for occasional case series or correlative biological studies, little is known about the incidence and clinical features of MM in Latin America. In Brazil, national estimates for the

Primary Breast Lymphoma: An Uncommon but Curable Disease

Primary malignant breast lymphoma (PBL) is a rare disease with an incidence of 0.04-0.5% of all malignant breast neoplasms. The majority of cases are B-cell lymphomas and the most common histologic type is diffuse large B-cell lymphoma (DLCL). In this study, we report our experience with three cases of PBL. The treatment was the same currently indicated for early stage aggressive NHL, i.e. anthracycline based chemotherapy followed by the involved field radiation therapy. Unfortunately, two patients underwent mastectomy to carry out correct diagnosis. The three patients are alive without any evidence of relapse after 24, 67 and 135 months of follow-up. Considering that aggressive NHL is very sensitive to chemotherapy, mastectomy should be avoided to preserve the quality of life of these patients, once surgery does not change the good prognosis of PBL.

Targeting MAGE-C1/CT7 Expression Increases Cell Sensitivity to the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cell Lines

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26–27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.

Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma.

OBJECTIVE This study aims to analyze the expression of cancer testis antigen 45 (CT45) in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in multiple myeloma (MM) patients. MATERIALS AND METHODS Expression of CT45 was studied in 20 normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain and fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon, and one pool of 10 normal bone marrow samples) and bone marrow aspirates from 3 monoclonal gammopathies of undetermined significance, 5 solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by reverse transcriptase polymerase chain reaction. RESULTS CT45 was positive in 3 of 20 (15%) normal tissues tested: lung, brain (both fetal and adult), and spinal cord. Among monoclonal gammopathies, CT45 was positive in 2 of 5 (40%) solitary plasmacytomas bone marrow aspirates, 10 of 61 (16%) MM bone marrow aspirates, and in the U266 MM cell line. CONCLUSIONS We did not find associations between bone marrow histology and CT45 expression. However, we demonstrated for the first time that positive expression of CT45 was associated with poor prognostic (International Staging System) and poor outcomes in MM patients, meaning that CT45-positive cases presented seven times more chance of worse evolution than the negative ones.

Bilateral Central Retinal Vein Occlusion Associated with Multiple Myeloma

Purpose: To report a case of simultaneous bilateral central retinal vein occlusion (CRVO) associated with multiple myeloma. Methods: A 65-year-old woman had sudden, painless loss of vision in both eyes for 20 days. Ophthalmologic examination revealed bilateral CRVO. Appropriate medical workup was conducted, and multiple myeloma was diagnosed as the underlying cause. Results: Clinical support and chemotherapy effectively controlled paraprotein production, leading to improvement of both systemic and ocular alterations. Conclusions: Many conditions have been noted to be associated with CRVO. Based on a Medline search, this is the first report of simultaneous bilateral CRVO as the first manifestation of multiple myeloma, illustrating the need for a primary care ophthalmologist to be involved in the basic assessment for associated underlying diseases in retinal disorders.

Can thalidomide be effective to treat plasma cell leptomeningeal infiltration

To the Editor: Dear Sir, In May 2000, a previously healthy, 52-yr-old woman was diagnosed with Salmon-Durie stage IIIA, IgG j multiple myeloma (MM). She was initially treated with six cycles of intravenous (i.v.) vincristine 0.4 mg days 1–4; i.v. doxorubicin 9 mg/m days + 198 1–4; P. O dexamethasone days 1–4, 9–12, 17–20 (VAD) every 4 weeks), the last interrupted because of Staphylococcus aureus endocarditis. At this time, during the infection treatment, she developed radicular symptoms associated with a paravertebral mass identified on computed tomography scan. The biopsy ruled out an abscess and showed a plasmacytoma. The patient underwent lumbar spinal irradiation (4000 cGy) followed by six cycles of i.v. cyclophosphamide 750 mg/m day 1: i.v. doxorubicin 50 mg/m day 1; vincristine 1, 4 mg/m day 1; P. O prednisone 60 mg/m days 1–5 (CHOP), achieving complete clinical response. In December 2000, few days before the harvest of peripheral stem-cell for autologous transplantation, she was confused and hemiparetic on physical examination. A cranial magnetic resonance imaging showed a left parietal mass. Cerebrospinal fluid (CSF) showed 250 cells/ mm, with 100% of plasma cells (Fig. 1). This finding was confirmed by flow cytometry analysis of CSF (CD45–, CD38+, j+). There were no other signs of systemic activity of the disease (no serum or urinary M protein, <5% plasma cells in the bone marrow). She was treated on oral dexamethasone (40 mg on days 1–4, 9–12, 17–20 with interval doses of 16 mg/d) and three doses of weekly intrathecal chemotherapy (methotrexate 12 mg and dexamethasone 2 mg). Despite a brief period of symptomatic relief, there was no CSF cleansing (CSF on 11/30/2001: 640 cells/mm, 100% plasma cell; CSF on 02/14/2002: 80 cells/ mm, 100% plasma cells) and the neurologic symptoms ended up progressing (see Table 1). The patient was then treated with thalidomide (Thal) 800 mg/d for 30 d with simultaneous cranial radiation, achieving total dose of 1000 cGy. Radiotherapy was interrupted because the patient’s focal deficits worsened and her mental status deteriorated, resulting in progression to death 3 months after detection of the central nervous system involvement. In this study, we describe a very unusual complication in MM with 53 cases previously related in the literature. Meningeal myelomatosis, defined as meningeal involvement by plasma cells in the CSF, although might be a presenting feature, has usually been described in the setting of pre-existing MM (1). It tends to occur in stage III disease and is associated with plasma cell leukemia in 20% of patients. Few patients were reported to relapse with meningeal compromising and limited disease outside the central nervous system (CNS) (2). We questioned the role of the paraspinal plasmacytoma as the seeding source of plasma cells into CSF (3). As the meningeal myelomatosis represents a dismal event, with 1– 2 months median survival despite aggressive local treatment, associated or not with systemic therapy (1), we tried to treat this patient with Thal and radiotherapy following intrathecal chemotherapy and high dose glucocorticoid. We were expecting a better outcome than that previously reported, assuming the usefulness of Thal in relapsed/ refractory MM patients (4), the high angiogenesis Fig. 1. Cytospin of CSF sample showing 100% of dysplasic plasmocytes, confirming the diagnosis of leptomenigeal infiltration in our patient, ·1000. Eur J Haematol 2003: 70: 198–199 Printed in UK. All rights reserved Copyright Blackwell Munksgaard 2003