ALBERT

Description: Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. Objectives: We generated SAGE libraries from normal and neoplastic plasma cells with the aim of identifying genes differentially expressed in MM that could be useful as new potential diagnostic, prognostic markers or therapeutic targets. Material and methods: Normal plasma cells were obtained from palatine tonsils of 20 children who underwent tonsillectomy. MM SAGE library was obtained from bone marrow plasma cells of two IgGk newly diagnosed MM patients. Purified normal and neoplastic plasma cells were isolated after magnetic sorting of CD-138-positive cells. Results: We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 50 overexpressed genes in MM library. After comparison of unique tags expression levels, we selected 12 overexpressed (at least 10 times) genes in the MM library (ZFHX1B, XBP1, PIM2, LGALS1, RANBP2, P53CSV, DDX5, LSM5, JUND, MAPKAPK2, SP140 and NTN1) for further investigation. Expression of 10 out of 12 genes was validated by quantitative real-time PCR in purified plasma cells of 31 MM patients and three controls. XBP1, RANBP2, P53CSV, DDX5, MAPKAPK2 were overexpressed in at least 50% of MM cases. Comparative analyses of relative expression (2-D D CT) of the 10 genes in 31 MM cases and 3 controls showed statistically significant difference between cases and controls for all genes but PIM2 and ZHFX1B, confirming the SAGE data. Also, we could identify that RANBP2 and ZHFX1B have prognostic impact in MM OS (p = 0.040 and p = 0.0502, respectively). Multivariate analysis confirmed RANBP2 expression as a good independent prognostic marker. Conclusions: SAGE technique allowed the identification of genes differentially expressed in normal and neoplastic plasma cells. These genes may have important role in tumorigenesis or may be possible therapeutic targets for MM control, particularly P53CSV and MAPKAPK2, or prognostic impact, as RANBP2.

Description: Background: Cancer testis antigens have become the most extensively studied antigen group in the field of tumor immunology. Aims: This study aims to analyze global expression of 14 CT (cancer/testis) antigens in MM to identify possible prognostic markers and therapeutic targets. Patients and Methods: The expression of MAGEA1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA10, MAGEA12, BAGE1, MAGEC1/CT7, GAGE family, LAGE-1, PRAME, NY-ESO-1, SPA17 and SSX1 was studied by RT-PCR in: 15 normal tissues, one pool of 10 normal bone marrow samples, three normal tonsils and bone marrow aspirates from six normal donors, three monoclonal gammophaties of undetermined significance (MGUS), five solitary plasmacytomas, 39 MM samples (95% advanced stage) and MM cell line U266. CodeLink Human UniSet I Bioarrays 10,000 genes was used for arrays analyses. Results: SPA17 was positive in all normal tissues and was excluded for further analyses. MAGEC1/CT7 was positive in bone marrow aspirates from one MGUS and in one plasmacytoma. U266 cell line was positive for all CT antigens, except SSX1. The frequencies of CT antigens expression in MM patients were: MAGEC1/CT7 = 30/39 (77%); LAGE-1 = 19/39 (49%); MAGEA3/6 = 16/39 (41%); MAGEA2 = 14/39 (36%); GAGE family = 13/39 (33%); NY-ESO-1 = 13/39 (33%); BAGE-1 = 12/39 (28%); MAGEA1 = 10/39 (26%); PRAME = 9/39 (23%); SSX-1 = 10/39 (26%); MAGEA12 = 8/39 (20.5%); MAGEA4 and MAGEA10 = 0%. Cox’s regression model showed that GAGE family positivity and number of positive CT antigens 〉 6 were independent prognostic factors when all patients were analyzed. However, MAGEC1/CT7 expression was the only independent prognostic factor when non-transplanted patients where analyzed. Three samples predominantly positive (〉 6) and three samples predominantly negative (0 or 1) for the 13 analyzed CT antigens were submitted to microarrays analyses. 147 genes were overexpressed in predominantly positive CT antigens samples. Conclusions: Based on our findings, MAGEC1/CT7, MAGEA3/6 and LAGE-1 seem good candidates for immunotherapy, since together they are overexpressed in 85% of our MM cases. Besides, GAGE family expression, number of CT antigens 〉 6 and MAGEC1/CT7 seem to have impact on MM prognosis. Also, the results of arrays analyses corroborate the hypothesis that MM can be separate in two groups: predominantly positive and predominantly negative for CT antigens, meaning that these antigens may have important role for MM biology.

Description: Introduction: Cancer testis (CT) antigens have become the most extensively studied antigen group in the field of tumor immunology. CT45 antigen expression was described in colon adenocarcinomas, germ cell tumors, Hodgkin’s lymphomas and, more recently, in multiple myeloma (MM). Aims: This study aims to analyze the expression of CT45 in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in MM. Patients and Methods: The expression of CT45 was studied in twenty normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain, fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon and one pool of ten normal bone marrow samples) and in bone marrow aspirates from three monoclonal gammopathies of undetermined significance (MGUS), five solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by RT-PCR. Results: CT45 was positive in three out of 20 (15%) normal tissues tested: lung, brain (both fetal and adult) and spinal cord. Among monoclonal gammopathies, CT45 was positive in two out of five (40%) solitary plasmacytomas’ bone marrow aspirates, 10 out of 61 (16%) MM bone marrow aspirates and in the U266 MM cell line. Six out of 10 (60%) CT45 positive MM cases were classified as International Staging System (ISS) 3 (p = 0.009). Six CT45-positive cases were classified as plasmacytic (PC) and four as polymorphic (PM). Median OS of the MM group was 21 months. Nine patients were submitted to autologous stem cell transplantation. All of the transplanted cases were CT45-negative. Univariate analysis showed that Durie-Salmon Staging System (Durie-Salmon IIIA: N = 35, median OS = 40 months; Durie-Salmon IIIB: N = 19, median OS = 12 months; log-rank p= 0.0139), b2microglobulin (b2microglobulin £ 5.5 mg/L: N = 27, median OS = 40 months; b2microglobulin 〉 5.5 mg/L: N = 24, median OS = 12 months, log-rank p= 0.0520, Breslow p = 0.0352, Tarone-Ware p = 0.0399), plasma cell morphology (PC: N = 38, median OS = not reached; PM: N = 11, median OS = 12 months; PB: N = 5, median OS = 1 month; log-rank p= 0.0037), transplantation proceedings (transplanted patients: N = 9, median OS = not reached; non-transplanted patients: N = 47, median OS = 14 months; p = 0.0064) and CT45 expression (CT45 expression negative: N = 46, median OS = 25 months; CT45 expression positive: N = 10, median OS = 3 months, log-rank p = 0.038 for all patients and CT45 expression negative: N = 37, median OS = 19 months; CT45 expression positive: N = 10, median OS = 3 months, p = 0.0245, only non-transplanted patients) had impact on OS. Cox Regression Model showed that only plasma cell morphology (p = 0.029, RR 5.288, CI 1.77704–15.7988), transplant proceedings (p = 0.0742, RR 0.1582, CI 0.0209–1.1976) and CT45 expression (p = 0.0016, RR 7.0403, CI2.0978–23.6278) were independent prognostic factors in MM patients survival. CT45-positive cases were associated with poor outcome and presented 7 times more chance of worse evolution then the negative ones. Conclusions: CT45 was expressed in only 16% of MM patients. However, we demonstrated for the first time that positive expression of CT45 was associated with high ISS scores and poor outcome in MM

Description: Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. Objectives: We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Material and Methods: Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. Results: 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, 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. Conclusions: Analysis of differential expression using SAGE could identify new genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

Description: Abstract 1908 Introduction: MAGE-C1/CT7 encodes for a cancer/testis antigen (CTA) frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. The expression of this CTA is restricted to malignant plasma cells and a positive correlation between MAGEC1/CT7 expression and advanced stage has been demonstrated for MM. It has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function of this protein in the pathophysiology of MM is not yet understood. Objectives: (1) To clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle regulation in myeloma cell line SKO-007 and (2) to evaluate the impact of silencing MAGE-C1/CT7 on cells treated with bortezomib. Material and Methods: Short hairpin RNA (shRNA) specific for MAGE-C1/CT7 was inserted in the pRETROSUPER(pRS) retroviral vector. The pRS-shRNA-MAGE-C1/CT7 was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time PCR (RQ-PCR) and western blot. Functional studies included cell proliferation, cell cycle analysis using propidium iodide, and analysis of apoptosis using annexin V staining. Results: SKO-007 MM cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. SKO-007 cells were divided into three derivatives: empty vector (pRS) and ineffective shRNA (antisense strand deleted – GC bases) [both used as controls for all the experiments] and inhibited (shMAGE-C1/CT7). MAGE-C1/CT7 mRNA expression was ∼5 times lower in inhibited cell line than control cells by RQ-PCR. Western blot showed 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. We used empty vector, ineffective shRNA and inhibited cells to determine whether inhibition of MAGE-C1/CT7 was associated with cell cycle dysregulation. We detected differences between inhibited cells and both controls regarding the proportion of myeloma cells in the G2/M phase (p

Description: NF-k B is a transcription factor that controls the expression of a variety of genes involved in different cellular processes, including apoptosis regulation, cell proliferation and expression of adhesion molecules. In multiple myeloma (MM) patients, NF-k B confers longer survival and drug resistance to tumor cells. Therefore, a better understanding of molecular mechanisms involved in NF-k B pathway can contribute to identify new therapeutic targets for this incurable disease. Purpose: To evaluate the expression of NF-k B pathway genes in MM patients by real-time quantitative PCR and its possible correlation with disease clinical features and survival. Patients and methods: Expression of eight genes related to NF-k B pathway (NFKB, IKB, RANK, RANKL, OPG, IL6, VCAM and ICAM) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than the observed in normal samples. These results were compared to clinical variables using Pearson’s Qui-square and T-Test. Overall survival (OS) was estimated using Kaplan-Meier method. Results: 18.9% of MM cases were classified as ISS 1, 34% ISS 2 and 39.6% ISS 3. The percentages of overexpression of the eight genes were: NFKB 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM 10% and ICAM 26%. We found association between IL6 expression level and the International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. We also found association between IL6 overexpression and presence of chromosome 13 deletion (p= 0.035), a well-known marker of worse prognosis in MM. Mean value of ICAM relative expression was also associated with ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normoexpression (p=0.04). Conclusions: IL6 overexpression was found in 39.6% of MM cases and confirms its participation in myeloma progression, regulating growth and survival of tumor cells. Besides, IL6 overexpression was significantly associated with advanced stage disease and del(13) and had impact on MM patient’s survival. ICAM was overexpressed in 26% of cases and its level was associated with ISS scores. Therefore, ICAM seems to have some potential as therapeutic target in MM.

Description: Abstract 4887 Introduction In recent years, the expression of CT antigens has been studied in various malignant neoplasias. Based on their tumor-associated expression and due to their ability to elicit an immune response in the autologous host, CT antigens are potential targets for vaccine-based immunotherapy of cancer. Since different CT antigens can be expressed simultaneously in the same tumor, CT antigens are possible targets for polyvalent vaccines. Immunotherapy trials employing MAGE-A3 and NY-ESO-1 in MM patients are in progress. We previously reported frequent expression of LAGE-1 on mRNA level in MM patients (∼50%). Due to the high similarity between NY-ESO-1 and LAGE-1 (94% in mRNA and 84% in protein sequences) and the more prevalent presence of the latter, it is interesting to speculate if anti-NY-ESO-1 vaccine might elicit LAGE-1 immunity and hence may be efficient in patients with LAGE-1-positive tumors. Objectives To evaluate mRNA and protein expression of NY-ESO-1 and LAGE-1 in MM patients and to explore the possibility of an anti-NY-ESO-1 vaccine eliciting immunity to LAGE-1. Materials and methods The expression of NY-ESO-1 and LAGE-1 was studied by RT-PCR in 18 normal tissues, 28 bone marrow MM samples and U266 cell line. NY-ESO-1 and LAGE-1 protein expression were analyzed by immunohistochemistry in the MM specimens using monoclonal antibody (mAb) E978 and mAb 219-510-23 respectively. The protein extracts from U266 (NY-ESO-1+/LAGE-1+), H1299 (only NY-ESO-1+), SKBR3 (only LAGE-1+) and HaCat (double negative) cell lines were analyzed by Western Blot with mAb E978 (1:5,000). Results RT-PCR was positive for both genes in testis, placenta and U266 cell line. All other normal tissues were negative. In MM, LAGE-1 was positive in 36% (10/28) and NY-ESO-1 in 18% (5/28) of total bone marrow samples. However, only 2 patients (7%) were positive for protein expression by immunohistochemistry (both cases were also positive by RT-PCR). Both NY-ESO-1 mRNA-positive cell lines U266 and H1299 were positive by Western Blot for mAb E978, but this anti-NY-ESO-1 mAb was unable to identify LAGE-1 protein in U266 and SKBR3 cell line extracts. Conclusions In this small group of MM patients, NY-ESO-1 and LAGE-1 were expressed on mRNA level in 18% and 36% respectively, while both antigens were present on protein level in only 7%. Discrepancies between RNA and protein expression has been described in other tumors for CT antigens previously. The incidence of up to 36% LAGE-1 positive cases suggest that multiple myeloma might be susceptible to LAGE-1 and/or NY-ESO-1 vaccine-based immunotherapy. Also it is may be possible that, due to the high similarity between the proteins, anti-NY-ESO-1 vaccine might elicit LAGE-1 immunity and hence may be efficient in patients with LAGE-1-positive tumors. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4888 Introduction MAGEC1/CT7 gene encodes for a cancer testis (CT) antigen frequently expressed in multiple myeloma (MM) and may be a potential target for immunotherapy in this still incurable disease. This CT gene expression is restricted to malignant plasma cells and may be related with tumor progression, since it seems to play a role as a “gatekeeper” gene for other CT antigens and can be associated with a more aggressive phenotype. However, the exact function of this protein for tumor biology is not yet understood. Objective The aim of this study is to elucidate the role of MAGEC1/CT7 in the control of cellular proliferation, DNA synthesis and invasive potential of Sko-007 myeloma cell line. Material and Methods We used a short hairpin RNA (shRNA) specific for MAGEC1/CT7 gene that was previously inserted in the pRS (pRETROSUPER) retroviral vector. The pRS-shRNA-MAGEC1/CT7 construct was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced and selected for stable expression of shRNA-MAGEC1/CT7. Expression of MAGEC1/CT7 was analyzed by Western Blot and Real Time PCR (RQ-PCR). Functional studies included cell proliferation, 3H thymidine incorporation and invasion potential, using matrigel-coated transwell filters. Results Sko-007 was chosen to perform the initial functional studies since the basal MAGEC1/CT7 gene expression level was higher in this cell line when compared with the other myeloma cell lines (U266 and SK-MM-2). Sko-007wt (wild type) cell was divided into three derivatives, Sko-007ev (empty vector), Sko-007shMAGEC1/CT7Δ (antisense strand deleted – GC bases) and Sko-007shMAGEC1/CT7. MAGEC1/CT7 expression was approximately five times down regulated in Sko-007shMAGEC1/CT7 when compared with Sko-007wt, Sko-007ev, and Sko-007shMAGEC1/CT7Δ cells by RQ-PCR. Sko-007shMAGE1/CT7 had an 80% decrease in MAGEC1/CT7 protein expression when compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ by Western Blot. Our preliminary functional assays did not show any change in cell proliferation and DNA synthesis when Sko-007shMAGEC1/CT7 was compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ. Indeed, Sko-007 cell line has invasive potential, but no change was observed in Sko-007shMAGEC1/CT7 or other cells (Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ). Conclusions Expression of MAGEC1/CT7 mRNA could be inhibited by the shRNA strategy, but the concomitant reduction of its protein level did not seem to have impact in proliferation, DNA synthesis or in invasive potential of Sko-007 MM cell line (Supported CNPq and LICR São Paulo Branch). Disclosures No relevant conflicts of interest to declare.