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: 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: Considering the heterogeneity of genetic aspects related with MM pathogenesis, recent studies have been focused on prognostic factors that could define the best therapeutic approach for each case or to contribute for the development of new therapies. In this way, gene expression profiling analysis can be very helpful to define such prognostic factors. Serial analysis of gene expression (SAGE) allows not only the identification of genes expressed in a given tissue, but also their expression levels. Until now, there was no data of gene expression in normal or neoplastic plasmacytes in public SAGE databases. Purpose: The aim of this study is to create two SAGE libraries using normal and neoplastic plasmacytes and to identify genes differentially expressed in MM. Methods: Normal plasmacytes were obtained from palatine tonsils of children who underwent tonsillectomy. MM library was obtained from bone marrow plasmacytes of two IgGk newly diagnosed MM patients. Purified normal and neoplastic plasmacytes were obtained after magnetic sorting of CD-138-positive cells (MACS - Magnetic Cell Sorting of Human Cells, Miltenyi Biotec). Both SAGE libraries were obtained using the kit I-SAGE (Invitrogen), according to manufacture’s instructions. Results: We generated 4,000 clones from each library and, after automatic sequencing, we got 29,918 tags from normal and 10,340 tags from tumoral libraries (corresponding to 8,241 and 2,359 unique tags, respectively). After comparison of unique tags expression levels, we selected 15 overexpressed (at least 10 times) genes in the MM library (ZFHX1B, XBP1, PIM2, LGALS1, RANBP2, HSPC132, PRDM2, DDX5, ARMCX3, LSM5, JUND, MAPKAPK2, SP140, KLF2 and NTN1). Once these genes are related with transcription, signaling, cell proliferation and/or apoptosis, they can have important role in the tumorigenesis process and may be possible therapeutic targets for MM control