ALBERT

Beschreibung: Abstract 1033 Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic and life-threatening hematopoietic stem cell disorder characterized by deficiency of the GPI-anchored complement inhibitory proteins CD55/59. Chronic hemolysis from this deficiency leads to serious clinical morbidities including thromboembolism, chronic kidney disease, and increased mortality. The International Clinical Cytometry Society (ICCS) recommends multiparameter high sensitivity flow cytometry (HSFC) as the method of choice for diagnosing PNH. The ICCS also provides guidance on the clinical indications for testing for PNH, including patients (pts) with bone marrow failure (BMF), unexplained cytopenias, unexplained thrombosis, hemoglobinuria and hemolysis. The aim of this study is to use HSFC with sensitivity up to 0.01% to analyze 6,897 pts who were screened for PNH clones utilizing CD235a/CD59 for RBCs, FLAER/CD24/CD15/CD45 for neutrophils and FLAER/CD14/CD64/CD45 for monocytes. We evaluated the clinical indications for PNH testing with the provided ICD-9 diagnostic (DX) codes and examined the change in PNH clone sizes among pts who had follow-up studies in 3–12 months. Based on a sensitivity of at least 0.01%, 6.1% of all pts (421/6897) were found to be PNH positive. Of those pts, 5,545 pts (80.1%) had ICD-9 DX codes provided. The distribution of PNH clone sizes in these PNH+ pts is shown in Figure 1. Aplastic anemia (AA) and hemolytic anemia comprised the most common reasons for testing. In bone marrow failure syndromes, AA pts had the highest incidence of PNH+ clones, 26.3%, followed by pts with unexplained cytopenia, 5.7%, myelodysplastic syndrome (MDS), 5.5%, and anemia (unspecified or in chronic illness), 3.6% (Table 1). The incidence of PNH+ clones for symptoms such as hemolytic anemia was 22.7%, followed by hemoglobinuria 18.9%, and unspecified hemolysis, 7.9%, unspecified iron deficiency, 2.5%, and thrombosis, 1.4%. Of the 421 PNH positive pts, 89 pts (22%) were identified as having follow-up studies in 3–12 months. These pts were categorized into PNH clone sizes of 0.01% – 0.1% (27 pts, 30%), 0.11% – 1% (7 pts, 8%), 1.1% – 10% (18 pts, 20%) and 10.1% – 100% (37 pts, 42%). Of the 64 pts who had PNH clone sizes of 0.01% – 0.1% or 10.1 – 100%, one patient (0.02%) had a follow-up study that resulted in a change of category. Of the 25 pts with PNH clones sizes between 0.11% – 1% and 1.1% – 10%, 10 pts (40%) had a follow-up study resulting in an increase in category, 6 pts (24%) had a follow-up study resulting in a decrease in category and 9 pts (36%) had a follow-up study resulting in no change in category.Figure 1.Distribution of PNH Clone Sizes based on 421 PNH+ PatientsFigure 1. Distribution of PNH Clone Sizes based on 421 PNH+ PatientsTable 1:Incidence of PNH Clones in Patients with ICD-9 Diagnostic Code at Dahl-Chase Diagnostic ServicesICD-9 Diagnostic CodeGeneral DescriptionIncidence of PNH Clone284, 284.01, 284.8, 284.81, 284.89, 284.9Aplastic Anemia26.3% (94/357)238.7, 238.72, 238.73, 238.74, 238.75, 238.76Myelodysplastic Syndrome (MDS)5.5% (32/585)287.5Unexplained Cytopenia5.7% (13/230)284.1Pancytopenia6.0% (63/1058)285.2, 285.21, 285.29, 285.9Anemia Unspecified3.6% (40/1122)283, 283.1, 283.10, 283.11, 283.19, 283.2, 283.9Hemolytic Anemia22.7% (147/647)791, 791.2Hemoglobinuria18.9% (14/74)790.6, 790.99, 790.4Hemolysis7.9% (18/227)325, 415.1, 415.11, 434, 434.01, 444.22, 451.11, 451.19, 452, 453, 453.0, 453.2, 453.4, 453.41, 453.89, 453.9, 557, 557.1Thrombosis1.4% (14/967)280.9Unspecified Iron Deficiency2.5% (7/278)Other ICD-9 diagnostic codes2.1% (26/1232)Not Provided4.8% (51/1065)Note: Table reflects patients who had more than one ICD9 code associated with their laboratory tests. In this single-laboratory experience, we evaluated the incidence of PNH in these high risk groups. In this study, 26.3% of pts with the diagnosis of BMF had PNH+ clones detected, underscoring the need to test this group of pts. The study confirmed the utility of testing pts with unexplained hemolytic anemia, hemolysis and hemoglobinuria where the combined rate of positivity was 48%. In addition, this study highlights the need to monitor pts with small PNH clones by HSFC analysis as these pts may show significant variation over time. This examination of ICD-9 DX code association with presence of PNH+ clones confirms the need to actively test high risk populations for PNH based on the ICCS recommendations to ensure accurate diagnosis and early intervention. Disclosures: Weitz: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Illingworth:Dahl-Chase: Employment; Alexion: Consultancy, Honoraria, Research Funding.

Beschreibung: Recent studies in tumor immunology indicate that malignant cells frequently express normal testicular-specific proteins. Because these proteins show restricted normal tissue distribution, they are usually highly immunogenic and may be potential targets for immunotherapy. In the present study, we have used a pair of sequence-specific primers in reverse transcription–polymerase chain reaction (RT-PCR) and sequence analysis to demonstrate that the X-linked gene encoding SPAN-Xb is expressed in multiple myeloma and other hematologic malignancies such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and acute myeloid leukemia (AML). RT-PCR analysis demonstrates that SPAN-Xb is a cancer/testis antigen and shows a restricted normal tissue expression. It is not expressed in any normal tissue except testis. SPAN-Xb recombinant protein was produced and used in enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. High-titer immunoglobulin G (IgG) antibodies, of IgG3 or IgG2 subclass, against SPAN-Xb were detectable in the sera of these patients. In contrast, SPAN-Xb mRNA or antibodies could not be detected in any of the healthy donors. There was a good correlation betweenSPAN-Xb gene expression and B-cell immune responses. These results suggest the in vivo immunogenicity of the SPAN-Xb protein. The presence of high-titer IgG responses suggests that the B-cell responses are likely to have been generated with CD4 T-cell cognitive help. Based on these data, we conclude that SPAN-Xb is a novel member of the family of cancer/testis antigens aberrantly expressed by, and capable of inducing, immune responses in patients with multiple myeloma and other hematologic malignancies.

Beschreibung: Rituximab, a chimeric CD20 antibody, has been incorporated in various chemotherapy regimen for B-cell lymphoma. It has also been used as maintenance therapy in some of these patients. However, the effects of its long-term use on the immune system has not been previously documented. Here, we present our preliminary analysis of the effect of maintenance rituximab therapy on the B-cell immune repertoire in nine consecutive patients with B-cell lymphoma who were treated with maintenance rituximab after autologous stem cell transplant (ASCT) for high-risk disease. Nine patients (seven men and two women) with high-risk B-cell lymphoma were treated. Their diagnosis were: advanced mantle cell lymphoma in first CR (6), Stage IV DLCL in CR1 (1) and high grade NHL in CR2 (2). Median age was 66 years (range 38–72 years). CR was achieved using R-CHOP (8) or R-DHAP (1). Autologous stem cells were harvested during hematopoietic recovery from the last course of chemo-immunotherapy and G-CSF. Within two weeks after stem cell harvest, each patient received intravenous melphalan (200 mg/m2) following by the infusion of at least 2 x 106/kg CD34+ cells. All patients received a rituximab infusion (375 mg/m2) every three months starting D+100 after ASCT for a total of 2 years or until disease relapse. Unlike patients who underwent ASCT without rituximab, in whom B-cell recovery occured between 3–6 months, we observed severe delays in the immunoglobulin recoveries in these patients. At 9 months after transplant, all patients were IgM and IgG deficient, with only 1/9 patient acheived a normal IgA level. Even at 24 months after transplant, all patients were still IgM deficient and only 20% achieved normal IgA and IgG levels. The severity of immuno-deficiencies was next examined. We observed severe immunoglobulin deficiency (defined by 〈 50% normal levels) in IgM in all patients and in IgG and IgA in more than 20% patients at 24 months, suggesting that the immunoglobulin recovery was both delayed and severely affected. The severe immunoglobulin deficiencies may be clinically relevant. One patient developed two episodes (follow-up of 12+ months), one seven episodes (follow-up 28+ months) and another two episodes of chest infection and a vancomycin-sensitive chronic diarrhea (follow-up 18+ months). All three patient had severe and prolonged immunoglobulin deficiencies. With a median follow-up of 27 months (range 12–31 months), all patients have remained lymphoma-free. These preliminary results, therefore, suggest that rituximab administration every three months after autologous transplant for high-risk B-cell NHL delays immunoglobulin recovery and may be associated with increased risks of infection. Although this approach may reduce lymphoma relapse, careful monitoring of the immunoglobulin recovery and interventional as appropriate should be done routinely in these patients.

Beschreibung: SPAN-Xb is a spermatid-specific protein. We recently identified SPAN-Xb as a novel Cancer-Testis (CT) antigen in multiple myeloma (MM) (Blood2003; 101: 955–960) and that it frequently elicited immune responses in vivo in these patients, suggesting that SPAN-Xb may be a suitable target for immunotherapy of MM. SPAN-Xb, however, was only detected in the tumor cells from 20% of MM patients. To increase the applicability of a SPAN-Xb tumor vaccine for MM, we have set out to determine if SPAN-Xb expression could be upregulated by pharmacologics in 8 MM cell lines. Since most CT antigen expression occurs due to promoter demethylation, we first determined the ability of a hypomethylating agent, 5-azacytidine, to upregulate SPAN-Xb expression. Prior to exposure to 5-azacytidine, SPAN-Xb transcripts were only detected by RT-PCR in ARP-1 and RPMI 8226 cells. Following incubation in 5-azacytidine (2 uM) for 96 hours, the levels of SPAN-Xb transcripts were upregulated, as determined by RT-PCR and real time PCR, in all MM cell lines (ARK-B, ARP-1, H929, IM9, MM1-R, RPMI 8226 and U266) except KMS-11 (Table 1). This was associated with a corresponding increase in the level of SPAN-Xb protein expression, as shown by immunocytochemistry using a murine monoclonal antibody directed at SPAN-Xb. We next determined if SPAN-Xb gene expression could be increased by cytokine pre-incubation of these cells. IL-2 (100 IU/ml) and IL-4 (1000 IU/ml) did not affect SPAN-Xb gene expression. On their own, IL-7 (0.1 ng/ml) and GM-CSF (10 U/ml) did not affect SPAN-Xb gene expression significantly. However, when combined, especially with 5-azacytidine, a summative effect of these pharmacologics on SPAN-Xb gene expression, as determined by real time PCR, was observed in IM9 cells (Table 2). Our results indicate the potential to upregulate SPAN-Xb expression in MM for therapeutic purpose using 5-azacytidine, GM-CSF and IL-7 and provide insight into the possible mechanisms of regulation of SPAN-Xb gene expression. Table 1 Cells pre-5-azacytidine post-5-azacytidine SPAN-Xb mRNA copy numbers before and after 5-azacytidine exposure ARK-B 0 456 ARP-1 260 1376 H929 0 116 IM9 0 532 KMS-11 0 0 MM1-R 0 428 RPMI 8226 272 1348 U266 0 984 Table 2 Pharmacologics SPAN-Xb mRNA SPAN-Xb mRNA copy numbers after exposure to combination pharmacologics 5-azacytidine 532 IL-7 4 GM-CSF 9 IL-7 + GM-CSF 36 5-azacytidine + IL-7 1088 5-azacytidine + GM-CSF 952 5-azacytidine + IL-7 + GM-CSF 1632 medium only 0

Beschreibung: Sperm protein 17 (Sp17) is a protein recently identified as a novel cancer-testis (CT) antigen in multiple myeloma (MM). Because this tumor antigen demonstrates a very restricted normal tissue expression, Sp17 may be an excellent target for tumor vaccine of MM. In this study, we determined the ability to generate Sp17-specific HLA class I–restricted cytotoxic T lymphocytes (CTLs) from the peripheral blood of 4 patients with MM, 3 consecutive Sp17+patients, and 1 Sp17− patient. Dendritic cells were generated from monocytes of 4 patients with MM and used to present a recombinant Sp17 protein to autologous T cells. Following 4 rounds of antigen stimulation, the CTLs were tested for their ability to kill autologous targets in an Sp17-dependent and HLA-class I– restricted manner in standard cytotoxicity assays. Despite previous chemotherapy and the immunosuppression so often associated with MM, CTL generation was successful in all 4 patients, irrespective of the Sp17 status of their tumors. Most importantly, the CTLs were able to lyse autologous tumor cells that expressed Sp17. Tumor cell lysis in all cases appeared to be mainly mediated by perforin and could be blocked by concanamycin A. We conclude that Sp17 is a suitable target for immunotherapy of MM. Our findings provide the basis for a clinical study aimed at inducing a cellular immune response directed at Sp17+ MM.

Beschreibung: Sperm protein 17 (Sp17) is a spermatozoa protein that we have previously identified to be aberrantly expressed in myeloma cells. Using a combination of RT-PCR and Northern blot analysis, we demonstrated that Sp17 showed a very restricted normal tissue expression, being detected only in normal testis, suggesting that Sp17 is a novel Cancer-Testis antigen in multiple myeloma. We subsequently showed that Sp17 expression in myeloma cell lines was regulated through promoter methylation and could be upregulated by demethylating agent such as 5-azacytidine. In the present study, we have used a combination of real time PCR and immunohistochemistry on a large panel of normal tissues to determine the pattern of differential expression of Sp17 in normal tissues and in myeloma cells. We also investigated, using restriction digest/PCR, whether or not any differential expression of Sp17 in these normal tissues is also regulated by promoter methylation. Using real time PCR, we found that Sp17 transcripts could be detected in some normal tissues. However, the levels of expression were less than 2% of those in normal testis. In contrast, Sp17+ myeloma cells expressed 3–18% of normal testis levels of Sp17 transcripts. This results, therefore, are similar to those obtained by real time PCR in some other CT antigens in which levels of

Beschreibung: SEMG 1 is major protein of semen coagulum shown to inhibit human sperm capacitation. It plays an important role in sperm clotting and is normally degraded into smaller fragments by prostate-specific antigen. The gene encoding SEMG 1 has been localized to the long arm of chromosome 20, a region of chromosome 20 that is frequently deleted in myeloproliferative diseases and myelodysplastic syndrome. We previously found SEMG 1 to be a Cancer-Testis (CT) antigen that was immunogenic in the cancer-bearing patients, supporting its potential as a target for tumor immunotherapy. CLL patients are generally immunosuppressed even before any therapy is given. The immunosuppression increases as the disease progresses and may prevent successful immunotherapy unless the therapeutic approach is used to patients with early stage disease (Stage 0 or I), even prior to use of any immunosuppressive chemotherapy. However, most CT antigens are expressed in low frequency in early stage cancer. Accordingly, we set out to determine the expression frequency, variant and pattern of expression of SEMG 1 in patients with early CLL. Using a pair of sequence-specific primers in RT-PCR on total RNA derived from patients with early CLL, we found that SEMG 1 transcripts could be detected in 19/41 (46%) patients. The high frequency at which SEMG 1 is expressed in early CLL is in contrast to the generally low frequency of expression of other CT antigens in early malignancies. SEMG 1 expression not only occurred at the transcript level but also protein level, as determined by immunocytochemistry using SEMG 1 MoAbs. These results indicate that SEMG 1 could potentially be a suitable target for the design of tumor vaccine that could be applicable to a large proportion of patients with early CLL. Of the 19 patients expressing SEMG 1, eight expressed Zap 70 protein and 11 did not (p = 0.4; N.S.). Therefore, any SEMG 1-based tumor vaccine could be applicable even to patients with early poor risk CLL (as determined by Zap 70 expression). However, SEMG 1 expression, as determined by immunocytochemistry, within individual CLL patients was heterogeneous, suggesting that tumor vaccine targeting SEMG 1 may have to be administered with therapeutic agents that upregulate SEMG 1 expression to overcome the potential problem of tumor antigen heterogeneity that could prevent the success of the tumor vaccine. Since SEMG 1 exists in two variants, we next determined which SEMG 1 variant was expressed in CLL cells. Using a primer pairs in PCR to amplify across the 180 bp transcript that is deleted in SEMG 143, we showed that in all cases of SEMG 1-positive CLL specimens, only the non-truncated transcripts were detected, indicating the expression of SEMG 150 variant in CLL. Finally, applying diluted patient serum to an ELISA system, we found that high titers SEMG 1 IgG antibodies could be detected in six of these 41 patients but not in any of the 20 healthy donors. Leukemia cells from three of these patients expressed SEMG 1. These results, therefore, suggest that SEMG 1-reactive lymphocytes are present in the immune repertoire of patients with early CLL, irrespective of whether the leukemia cells express SEMG 1, and further support the feasibility of applying a SEMG 1-based tumor vaccine to these patients. In conclusion, SEMG 150 is expressed by the leukemia cells in a high proportion of patients with early CLL, irrespective of the Zap 70 expression status. SEMG 1 may be an immunological target for immunotherapy of nearly half of early CLL patients, especially when SEMG 1-reactive lymphocytes are present in the immune repertoire of these patients.

Beschreibung: Abstract 4955 Based on the observations of changes in DNA methylation status of various tumor suppressor genes, epigenetic targeting of myelodysplastic syndrome (MDS) with DNA hypomethylating agents are currently used for these patients. However, only up to 40% of the patients respond to these treatments. Therapeutic options for non-responders or responders whose disease progresses while on therapy are very limited. Based on our recent findings that low concentration clofarabine induced DNA hypomethylation, we carried out a Phase II study of elderly MDS patients who had failed 5-azacytidine (5-aza) therapy to determine the efficacy and toxicities of low dose intravenous clofarabine (ClinicalTrials.gov Identifier NCT00700011). Patients in Cohort 1 received intravenous clofarabine at 10 mg/m2/day and patients in Cohort 2 5 mg/m2/day for 5 days, each infusion given over 2 hours and each cycle repeated every 4 to 8 weeks, depending on the rate of bone marrow recovery. Eight patients were planned for each of the treatment dose. The patients were treated until disease progression or intolerable toxicities. The International Working Group response criteria were used. The study was closed after 10 patients were treated. Of the 10 patients (6 males and 4 females) treated, ei8 received 10 mg/m2/day (Cohort 1) of clofarabine and 2 patients 5 mg/m2/day (Cohort 2). The median age was 73 years (range 65–78). Five patients had only received 5-aza prior to being treated with clofarabine while the other 5 patients had also received other therapy (lenolidomide and decitabine). Three patients received 1 cycle, 4 patients received 2 cycles, 1 patient received 3 cycles and 2 patients received 4 cycles of clofarabine. The following responses were observed in the 9 evaluable patients: 1 CR (11%), 1 PR (11%), 2 HI (22%), 3 SD and 2 PD. The overall response rate was 44% in this group of elderly patients. All responses were observed in patients who received clofarabine 10 mg/m2/day. Response rate of 67% (4/6) was observed in patients with low risk MDS (IPSS 〈 1). In contrast, all 3 evaluable patients with high risk/Int.-2 MDS showed disease progression. The median time to response was 6 weeks (range 4–6 weeks) and 1 cycle of clofarabine. The patient who achieved a CR remained in CR for 14 months. The 2 patients who achieved HI both restored the erythroid-responsiveness to recombinant erythropoietin and became transfusion-independent, both for 10 months (1 patient received only 1 cycle and the other patient 2 cycles of clofarabine). As of July 31, 2010, diseases in all four responders have relapsed/progressed. The median duration of response was 10 months (range 5–14). Despite using low doses of clofarabine, severe and prolonged pancytopenia was observed in all 10 patients. All 10 patients needed considerable blood and platelet transfusions. Eight of these ten patients also needed hospitalization for neutropenic fever. Of the 22 cycles of clofarabine administered, there were a total of 13 hospital admissions with 100 hospital days. A total of 169 units of irradiated packed red blood cells (median 10 units/cycle; range 1.5–14 units/cycle) and 211 units of irradiated single donor platelets (median 10 units/cycle; range 2–30.5 units/cycle) were used. One patient died, within 2 weeks of completing cycle 1 of clofarabine, from intracranial bleed despite intensive support of severe thrombocytopenia with aggressive platelet transfusion. This patient was already thrombocytopenic prior to starting clofarabine and had a history of severe thrombocytopenic gastrointestinal bleed prior to starting on clofarabine. Nonhematologic toxicities occurred very infrequently and were mainly Grade 2 or lower. In all cases, clofarabine was discontinued due to either disease progression (2 patients) or toxicities (8 patients). With a median follow-up of 7.5 months (range 0.5–22), the overall survival for the whole group was 50%. In conclusion, elderly MDS patients, especially those with low risk disease, who have failed 5-aza may respond to low dose clofarabine. However, even at the low doses used in this study, bone marrow toxicity was significant. It remains to be determined whether the dose of clofarabine can be reduced further to minimize toxicities without compromising efficacy in this group of patients. Future studies should investigate the role of low dose clofarabine in low risk MDS patients. Study supported by Genzyme Corporation, Cambridge, MA, USA Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Although various studies supported the notion that leukemia cells in chronic myeloid leukemia (CML) may be recognized by the immune system, direct evidence showing the immunogenicity in vivo of proteins derived from the leukemia cells is lacking. In this study, we have constructed an expression cDNA library from the leukemia cells of a patient with CML and used the autologous serum to screen for high-titer IgG antibodies directed at the leukemia-derived proteins. We isolated eight distinct clones from the library, suggesting that multiple immune responses were elicited in the autologous host. Sequence analysis showed high degrees of homology to known gene sequences in six of the eight clones. Neither bcr-abl nor proteinase 3 sequences were isolated. Using Northern blot analysis, seven of the eight clones showed ubiquitous expression in normal bone marrow, leukemia cell lines, fresh leukemia cells, and normal tissues. However, clone no. 4 showed restricted mRNA expression, being only detected in some fresh leukemia cells, K562 cells, and normal testicular RNA. Using bacterial lysates in dot blot analysis, a panel of sera from normal individuals and patients with CML and other hematological malignancies were screened for high-titer antibodies against these eight clones. There were, among the CML patients, signficantly higher prevalence of antibodies against seven of the eight clones. They were observed even after omitting from the analysis patients with multiple myeloma whose associated immune paresis may impair immune responses to these proteins. Interestingly, antibodies against these proteins were also detected in a small number of normal individuals. Although the precise clinical significance of our findings remains to be determined, this study provides evidence in support of the potential immunogenicity of leukemia-derived proteins in the autologous host. It also provides basis for further investigations to characterize these proteins, especially clone no. 4, and determine their potential for immune targeting in CML.

Beschreibung: SPAN-Xb is a spermatid-specific protein encoded by the SPAN-XB gene on chromosome Xq27.1. It is a novel Cancer-Testis antigen in multiple myeloma. We recently demonstrated that SPAN-Xb expression in myeloma cells is regulated through promoter methylation and could be upregulated by IL-7 and GM-CSF. In this present study, we set out to investigate the mechanism of SPAN-XB expression and the promoter association with the methyl-CpG binding protein, MeCP2. Elucidation of these interactions is likely to shed light on potential therapeutic strategies to upregulate antigen levels that could be used to improve the outcome of SPAN-Xb-based tumor vaccines. We previously showed that the putative SPAN-XB promoter resides within exon 1 and contains 433 bp, starting from position −546 to position −104 upstream of the translational start site. Using a panel of truncated promoter constructs generated from the 3′ and 5′ ends of the promoter gene, we localized the core sequence of SPAN-XB promoter to the 73 base pairs at the 3-end of the promoter, a region that lacks CpG dinucleotides within the full length promoter. There are 11 CpG dinucleotides within the putative SPAN-XB promoter. We previously found that DNA methylation provides the primary regulatory mechanism for SPAN-XB gene expression. We also identified that hypomethylation at positions −310, −307, −299 and −221 strongly predicted for SPAN-XB expression, suggesting the involvement of these four CpG dinucleotides in the regulation of SPAN-XB gene expression through DNA methylation. In the present study, using reporter gene expression assays, we found that the core promoter function is significantly modulated by these adjacent CpG sequences so that mutation of the CpG dinucleotides outside the core sequence resulted in changes in the promoter function. We also previously demonstrated by bisulfite conversion and sequence analysis the association between methylation at specific CpG dinucleotides with repression of SPAN-XB gene. Here, we extended our study to determine whether or not the methylated cytosine binding protein, MeCP2, interacts with the SPAN-XB promoter gene in myeloma cells. Chromatin immunoprecipitation assays revealed a specific association of the MeCP2 with SPAN-XB promoter, and MeCP2 binding strongly correlated with repression of the SPAN-XB gene in myeloma cell lines and CD138-enriched fresh myeloma cells. Reactivation of the SPAN-XB gene by 5-azacytidine treatment resulted in the loss of MeCP2 from this site. We, therefore, conclude that SPAN-XB promoter consists of a core sequence and a regulatory element; the core element resides within the 73 base pairs at the 3′ end of the full length promoter. SPAN-XB gene expression by the core sequence is regulated in myeloma cells by specific CpG nucleotides and associated with MeCP2 binding to the promoter sequence.