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Clonotypic B cells are frequently isolated from the peripheral blood of patients with multiple myeloma (MM).[1][1] These clonotypic B cells may be the clonogenic cells of MM. We have hypothesized that rituximab, a chimeric CD20 monoclonal antibody, may be a useful maintenance therapy in MM after

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.

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.

Sperm protein 17 (Sp17) is thought to promote heparan sulfate–mediated cell-cell adhesion.[1][1],[2][2] Recent works indicated the presence of Sp17 transcripts in tumor cells.[2-4][2] Since Sp17 mRNA is detected only within testis, Sp17 could be an ideal target for tumor vaccine. This notion is

Despite advances in modern chemotherapy, CLL remains incurable. CLL is an indolent disease. It expresses a panel of Cancer-Testis (CT) antigens. CLL leukemia cells are susceptible to the cytotoxicity of T cells. CLL is, therefore, an ideal disease for immunotherapeutic approaches. Immunotherapy, in addition to being less toxic and more specific than chemotherapy, provides a different mode of cytotoxicity that may synergize with that induced by chemotherapeutic agents. Immunotherapy also offers the prospect of inducing immune memory that may be important for long term disease-free survival of patients with CLL. However, there are obstacles that may prevent successful immunotherapy. CLL patients are generally immunosuppressed even before any therapy is given and the immunosuppression increases as the disease progresses. Therefore, any immunotherapeutic approaches for CLL should be aplied in early disease when immunosuppression is least encountered. We previously demonstrated the expression of a CT antigen, SEMG 1, in 3/9 patients with CLL. Furthermore, we also demonstrated that the presence of high titer IgG in the serum of patients expressing SEMG 1, suggesting the in vivo immunogenicity of SEMG 1 in the cancer-bearing autologous host. We have also recently used SEMG 1 as the bait in a yeast two-hybrid system of testicular cDNA library and identified that Protamine 1 is the interacting ligand of SEMG 1 and that Protamine 1 is also a novel CT antigen, suggesting that both SEMG 1 and Protamine 1 may be suitable antigens for tumor vaccine development. However, the expression of SEMG 1 and Protamine 1 in early CLL is unknown. We have in this study set out to determine whether or not SEMG 1 and/or Protamine 1 could be used for the design of tumor vaccine for the targeting of patients with early CLL, in particular, those with poor risk disease, as predicted by Zap 70 expression. Using pairs of sequence-specific primers in RT-PCR on a cohort of CLL (41 Stage 0/I and 6 Stage II/III), we found that SEMG 1 gene is expressed in 24/47 (51%) and Protamine 1 in 16/47 (34%) of CLL patients. Gene expression in most cases was associated with the detection by immunocytochemistry of SEMG 1 and/or Protamine 1 in the CLL cells. The expression frequency of SEMG1 and Protamine 1 in CLL did not appear to differ between early and late stage disease. 19/41 of patients with early stage disease and 5/6 of patients with late disease expressed SEMG 1; 12/41 of patients with early stage disease and 4/6 patients with late disease expressed Protamine 1. Furthermore, the expression of these antigens was equally distributed between Zap 70+ and Zap 70− CLL. SEMG 1 was expressed in 4/6 of Zap 70+ CLL (all 6 had early disease) and 2/9 of Zap 70− CLL (1/8 early disease and 1/1 late disease). Interestingly, although Protamine 1 expression in CLL predicted for SEMG 1 co-expression, only 67% of SEMG 1+ CLL expressed Protamine 1. Our results, therefore, suggest that both SEMG 1 and Protamine 1 are suitable targets for tumor vaccine development for some patients with early CLL, especially those with high risk disease, as predicted by Zap 70 expression.

Most intracellular proteins are expressed with their interacting ligands. If a protein shows restricted normal tissue expression, its interacting ligand will likely also follow a restricted normal tissue expression pattern. We hypothesized that protein molecules interacting with CT antigens may also be testicular restricted and potential CT antigens. Identification of these proteins provides the opportunity for their application in polyvalent tumor vaccines to overcome the problems associated with antigen heterogeneity within a tumor specimen. We have applied two known CT antigens, Sperm protein 17 (Sp17) and SEMG1, as baits in yeast two-hybrid systems of a testicular cDNA library to identify the protein interacting with these two antigens and determine whether the interacting protein are also CT antigens. To do so, we first isolated and amplified cDNA encoding Sp17 and SEMG1. Following successful amplification and sequence confirmation, the cDNAs were sub-cloned into pGBKT7 and transformed into yeast strain AH109 and selected on SD/-Trp plates. Mating was performed between AH109-pGBKT7-Sp17 or pGBKT7-SEMG 1 and pre-transformed human testis cDNA library in yeast strain Y187. Following mating, the culture was first selected on SD/-His/-Leu/-Trp plates and then on SD/-Ade/-His/-Leu/-Trp/X-a -Gal plates. A total of 17 positive clones were isolated using Sp17 and 24 positive clones using SEMG 1 as the bait. Following confirmation of interaction, the colonies were expanded and the the plasmids subjected to sequence identification by nucleotide analysis. All 17 clones isolated using Sp17 encoded Ropporin 1 and all 24 clones isolated using SEMG 1 encoded Protamine 1. Using RT-PCR on total RNA derived from a panel of normal tissues, we demonstrated the very restriction normal tissue expression of Protamine 1 and Ropporin 1, being present only in normal testis, indicating that they are also testicular-specific genes. Analysis of a panel of fresh tumor cells, we showed the aberrant expression of both Protamine 1 and Ropporin 1 in a proportion of hematologic malignancies, including acute myeloid leukemia, multiple myeloma and chronic lymphocytic leukemia, supporting the notion that Ropporin 1 and Protamine 1 are both novel CT antigens in hematologic malignancies. Furthermore, these antigens were also able to elicit high titer B-cell responses in vivo in these patients, suggesting their immunogenicity in the autologous host, even in cancer-bearing patients. Interestingly, the expression of one partner CT antigen within an individual tumor specimen does not necessary predict for the co-expression of the interacting CT antigen. In conclusion, we have described a novel approach to the identification of CT antigens that could be used for immune targeting. This approach could be applied using other known CT antigens to identify other tumor antigens. The lack of a good correlation between the expressions of the partner protein with the interacting protein suggests two important points. First, the aberrant expression of these interacting pair of molecules is not a result of coordinated intracellular regulatory mechanisms but likely due to random processes. Second, if the function of one protein is dependent on the presence of its ligand, then these individual molecules expressed within the tumor cells are unlikely to be of any functional significance in the tumor cells from most patients.

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.

Semenogelin (SEMG) 1 is a protein of semen coagulum with limited expression in normal tissues. 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 aberrantly expressed by tumor cells of hematologic malignancies, including multiple myeloma (MM). The aberrant expression of SEMG 1 in tumor cells of hematologic malignancies is associated in vivo with the generation of high titers IgG directed at SEMG 1 protein, suggesting the immunogenicity of the protein in the cancer-bearing patients. The combination of being immunogenic in cancer patients and limited expression in normal tissue expression makes SEMG 1 a potential candidate protein for tumor vaccines. In this study, we have set out to determine the molecular mechanisms associated with SEMG 1 expression in MM. Treatment of SEMG 1-positive MM cells with IL-4 and IL-6 resulted in the upregulation of SEMG 1 expression. In SEMG 1-negative MM cells, SEMG 1 expression could only be upregulated by IL-4 and IL-6 after pre-treatment with 5-azacytidine, suggesting that DNA methylation is likely the primary regulatory mechanism for SEMG 1 expression. Treatment of SEMG 1-negative MM cells induced SEMG 1 gene and protein expression. SEMG 1 promoter only has one CpG dinucleotide, located at position -11 of the gene. Bisulfite conversion and nucleotide sequencing was carried out on the genomic DNA from MM cells. MM cells that did not express SEMG 1 were 100% methylated. In contrast, 100% of the sequences obtained from SEMG 1-positive MM cells were unmethylated at the cytosine residue of the CpG dinucleotide. Induction of SEMG 1 expression by 5-azacytidine was associated with a decrease in the % of methylation of this cytosine residue, from 100% to 20%. These results, therefore, further implicate the role of DNA methylation in the primary regulation of SEMG 1 expression. Applying antibodies directed at MeCP2 in chromatin immunoprecipiation, MeCP2 protein binding to the SEMG 1 promoter sequence of MM cell lines and fresh MM cells was correlated to SEMG 1 gene silencing, suggesting the likely role of the MeCP2 protein in SEMG 1 gene repression. Further analysis by promoter truncation studies indicated the dependence of the promoter function on the sequence spanning the two putative GATA-1 binding sites within the gene. Using a reporter gene expression system, both IL-4 and IL-6 were found to upregulate SEMG 1 via their effect on the hypomethylated promoter gene. The effects of IL-4 and IL-6 on the function of the SEMG 1 promoter were dose dependent. In conclusion, the present study demonstrates that SEMG 1 expression in MM cells is regulated through the interaction between primary regulatory effect of promoter methylation, MeCP2 protein binding and the secondary effect of specific cytokines. Our findings provide insight into the molecular mechanisms affecting SEMG 1 expression and suggest the possible use of hypomethylating agents to upregulate SEMG 1 expression in tumor cells. Obviously, it remains to be determined whether or not there is a differential dose response among the different normal tissues in their sensitivity to the antigen-inducing effect of these agents.

Cellular proteins are usually expressed with their interacting counterpart proteins. If the interacting proteins for Cancer-Testis (CT) antigens could be identified, these proteins could also be CT antigens that may be suitable targets for tumor vaccine. We have applied Sperm protein 17 (Sp17), a CT antigen identified in our laboratory, as the bait in a yeast two-hybrid system to screen a testicular cDNA library constructed in yeast strain Y187 to identify the protein interacting with Sp17. A total of 17 positive colonies were isolated, 11 encoded for the AKAP-binding protein, Ropporin. Ropporin is a spermatogenic cell-specific protein that serves as an anchoring protein for the A-kinase anchoring protein, AKAP3. Ropporin has never been reported to be expressed in tumor cells. Using a pair of sequence-specific primers in PCR, we determined the expression of Ropporin in hematologic malignancies. Ropporin mRNA was aberrantly expressed in 6/16 (37.5%) multiple myeloma (MM), 6/14 (43%) chronic lymphocytic leukemia (CLL) and 2/11 (18%) acute myeloid leukemia (AML). Ropporin is another novel CT antigen because the mRNA could not be detected in normal tissues except in testis, fetal brain and fetal liver. The coding sequence of human Ropporin was then cloned into the pQE30 vector to generate a recombinant human Ropporin fusion protein with an N-terminal 6-His tag from E coli. Successful generation of the fusion protein was confirmed on Western blot analysis using an antibody directed at the 6-His tag and also rabbit anti-mouse Ropporin antisera that cross react with the human Ropporin. We then used the recombinant Ropporin protein to determine the immunogenicity of Ropporin in patients with hematologic malignancies by detecting for high titer (1:1000) anti-Ropproin antibodies using sera from patients in an ELISA system. We established the basal level of positivity in 31 healthy individuals. A very low level of signal was obtained (mean OD450nm ± SD = 0.0736 ± 0.0147). Using mean ± 2SD as the cut off signal intensity, high titer anti-Ropporin antibodies were detected in 8/30 (26.7%) MM, 7/24 (29%) AML and 18/31 (58%) CLL. Signals obtained from the two sets of control wells consisting of either another E coli-derived recombinant protein or bovine serum albumin were consistently < 0.01. The specificity of the signals was confirmed in Western blot analysis. Not surprisingly, due to the high sensitivity of the ELISA system, not every ELISA-positive specimen produced a positive Western blot signal. However, all positive Western blot signals were ELISA positive specimens. All ELISA negative specimens did not produce positivity in Western blot analysis. Finally, we carried out a correlative study on paired specimens. Fourteen paired specimens with both tumor RNA and patient sera were available. The detection of positive Ropporin antibodies predicted for the expression of Ropporin gene in the tumor cells, suggesting the translation of the Ropporin mRNA into protein in vivo and that the B-cell immunity in these patients are likely due to the tumor Ropporin. Conclusions: The yeast two-hybrid system may be used to identify protein interacting with CT antigens and these proteins may themselves also be CT antigens;Ropporin is a novel CT antigen in hematologic malignancies;Ropporin is immunogenic in these patients. Further work is warranted to determine the suitability of Ropporin as a candidate antigen for tumor vaccine.

SLLP1 is a unique non-bacteriolytic c-lysozyme-like protein isolated from human spermatozoa. Antisera to SLLP1 blocks binding in the hamster egg penetration assay, suggesting that SLLP1 may be involved in sperm/egg adhesion. A recent study by dot blot analysis on RNA showed that SLLP1 was expressed only in the testis and in Burkitt lymphoma Raji cell line, suggesting that further studies are warranted to determine and characterize SLLP1 expression in tumor cells, in particular, fresh tumor specimens. Using a pair of sequence-specific primers in RT-PCR, we found that SLLP1 transcripts could be detected in 5/8 myeloma cell lines, suggesting that SLLP1 may be expressed in tumor cells from some hematologic malignancies. When we applied the investigations to 52 primary hematologic malignant specimens, SLLP1 transcripts were detected in 6/17 myeloma, 4/14 CML, 3/11 CLL, 2/9 AML and 0/1 hairy cell leukemia. In contrast, SLLP1 transcripts were not detected in the peripheral blood (n=12) or bone marrow (n=3) from any healthy donors. The specificity of the PCR products was confirmed by either sequence analysis or restriction digest with Pvu II. SLLP1 transcripts were translated into its corresponding protein in these tumor cells. Using tumor cell lysate in Western blot analysis, we detected SLLP1 protein in the myeloma cell lines and also in fresh malignant specimens, although positivities were only observed in specimens with high RT-PCR signals. All PCR-negative specimens were also negative in Western blot analysis. The specificity of the Western blot signals were confirmed in all cases by blocking assays with a high concentration of recombinant SLLP1 protein. We next investigated the expression of SLLP1 in a large panel of normal tissues using RT-PCR and real time quantitative PCR. Both approaches showed that SLLP1 is a novel Cancer-Testis antigen in hematologic malignancies. SLLP1 was detected, at a level of 8206 copies/0.25 mcg total RNA, only in normal testis. We also found that the SLLP1 mRNA copy numbers in fresh hematologic tumor specimens were up to 2316 copies/0.25 mcg total RNA, i.e. more than 25% of the level found in normal testis. We cloned and generated SLLP1 recombinant protein from E coli and used the purified recombinant SLLP1 in an ELISA system to detect anti-SLLP1 antibodies. Using sera from 24 healthy donors and the mean + 2SD as the cut-off signal intensities, we found that high titer IgG antibodies directed at SLLP1 could be detected in the sera from 2/9 AML, 5/23 CLL, 6/27 CML and 14/51 myeloma patients. The specificity of the antibodies was confirmed in Western blot analysis. Probably due to the decreased sensitivity of the detection system in Western blot analysis, only 1/2 AML, 3/5 CLL, 4/6 CML and 7/14 myeloma SLLP1 antibody+ sera produced a signal in the Western blot analysis. Interesting, IgG2 was by far the commonest SLLP1 antibodies in these patients. There was a good correlation between SLLP1 gene expression and immune responses. In summary, SLLP1 is a novel CT antigen in hematologic malignancies and is capable of eliciting B-cell immune responses in vivo in cancer-bearing patients. Our results support SLLP1 as a protein target appropriate for further in vitro study to define its suitability for immunotherapy.

SPAN-Xb is a spermatid protein that we have recently identified as a novel Cancer-Testis (CT) antigen in hematologic malignancies. We have also shown that SPAN-Xb expression in tumor cell lines could be upregulated by 5-azacytidine, GM-CSF and IL-7. The ability of 5-azacytidine to increase SPAN-Xb expression suggests that SPAN-Xb gene expression, like the other CT antigens, may be regulated through promoter methylation. On the other hand, the ability of GM-CSF and IL-7 to increase SPAN-Xb expression remained to be determined. In this study, we set out to determine whether or not promoter methylation regulates SPAN-Xb gene expression and the effects of GM-CSF and IL-7 on SPAN-Xb expression is due to their action on the promoter activity. We first isolated and cloned the SPAN-Xb promoter gene into the CAT (chloramphenicol acetyl transferase) reporter system, pCAT*3-Enhancer vector. In vitro methylation was achieved using SssI methylase and the recombinant vectors were transfected into the myeloma cell line, RPMI 8226 cells. CAT activity was assayed in the lysate of the transfectants 48–72 hours after gene transfer. We observed that CAT activitiy in transfectants containining demethylated recombinant pCAT*3-SPAN-Xb promoter vector. In contrast, CAT activity was abrogated once the recombinant vector was methylated in vitro, supporting the role of DNA methylation in the regulation of SPAN-Xb gene expression. CAT activity in the transfectants containing the demethylated vector could be further increased by GM-CSF and IL-7, suggesting that the increase in SPAN-Xb expression we have observed in cells treated with GM-CSF and IL-7 may be the actions of these cytokines on the SPAN-Xb promoter. These cytokines alone, however, were unable to induce CAT activity since transfectants containing the methylated promoter sequence remained negative for the CAT activity even with the addition of GM-CSF or IL-7. To further evaluate the role of DNA methylation on the expression of SPAN-Xb, we carried out the bisulfite conversion assays using genomic DNA from tumor cell lines, normal testis, blood, kidney, pancreas and spleen. Following bisulfite conversion, the modified genomic DNA was subjected to PCR amplification, cloning and sequence analysis. Five clones from each tissues were randomly picked for sequence analysis. A total of 11 CpG islands were identified within the promoter sequence. They were put together into 7 groups according to their positions in the sequence: Group I: −502; Group II: −474; Group III: −450; Group IV: −341; Group V: −311 to −300; Group VI: −226 to −222; Group VII: −184 to −181. Following sequence analysis, we observed that SPAN-Xb expressor (normal testis) was consistently demethylated within Groups V and VI CpG islands. In contrast, SPAN-Xb-negative tissues were consistently methylated at these two CpG islands, localizing the promoter activity of the sequence to these two areas of the promoter. The methylation status at the other CpG island did not predict SPAN-Xb expression. We therefore conclude that: 1. SPAN-Xb expression is regulated by promoter methylation; 2. GM-CSF and IL-7 increase SPAN-Xb expression through their action on the SPAN-Xb promoter, and; 3. The CpG islands between −311 and −300 and −226 and −222 are the regions within the SPAN-Xb promoter sequence that control gene expression.

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 We next determined if the differential expression of Sp17 in normal tissue was in fact regulated through promoted methylation. We previously identified that the HpaII sites at -359 and −350 of the Sp17 gene were involved in the regulation of Sp17 gene expression. Demethylation at these sites resulted in Sp17 gene expression. We have, therefore, used HpaII digest/PCR across these restriction sites in six normal tissues, i.e. kidney, blood, pancreas, skeletal muscle, spleen and testis. Normal testis consistently failed to produce any PCR products when amplified across the HpaII sites after restriction digest, suggesting that these HpaII sites were demethylated. In contrast, the corresponding HpaII sites in kidney, blood, pancreas, skeletal muscles and spleen consistently produced PCR products of the expected size, indicating methylation and insensitivity to HpaII restriction digest of the gene sequence within the Sp17 promoter. Control amplification across another gene segments in all normal tissues were positive. These results therefore provide evidence that the myeloma CT antigen, Sp17, exhibit a differential normal tissue expression that is regulated through promoter methylation.

Clonotypic B cells are frequently isolated from the peripheral blood of patients with multiple myeloma (MM). These clonotypic B cells may be clonogenic cells of MM. We hypothesized that rituximab may be a useful maintenance therapy in MM after autologous stem cell transplant (ASCT). The rationale was that CD20 antibody would deplete the clonotypic and, hence, clonogenic B cells to reduce the risk of disease relapse. ASC were mobilized with Cytoxan (3g/m2) and G-CSF from patients with MM. Two weeks after ASC collection, high dose IV melphalan (200 mg/m2) was administered followed 24 hours later by the infusion of at least 2x106/kg CD34+ cryopreserved ASC. Rituximab infusion (375 mg/m2) was started on day +30. Each patient received one antibody infusion every 3 months for 2 years or until disease progressed. All patients continued on monthly zoledronate and did not receive any other antimyeloma treatment. A total of 10 patients have been treated. Seven patients who have had post-transplant follow-up periods of >12 months were evaluated. The immunoglobulin recovery and incidence of infections in this group of patients were compared to 6 patients with MM who have undergone an ASCT without rituximab maintenance. The total normal IgM level in all 7 patients was severely depressed following rituximab administration. IgG and IgA were variably affected in these patients. The IgM immunosuppression was prolonged and consistent, being seen in all patients, regardless of the disease status after transplantation. In contrast, the control group showed normalization of the total IgM levels by 3 months after transplant. Two patients treated with rituximab received pneumococcal vaccines 12 months after transplant and neither developed any IgG response to the vaccines. The data indicate that rituximab infusion following ASCT for MM severely impaired B-cell immune reconstitution. Six of the 7 patients developed moderate to severe infections during the first 12 months after initiation of rituximab infusion. There were a total of 23 episodes of infections: 21 pneumonia and 2 septicemia (one pneumococcus and one Pseudomonas). A patient died in CR due to pneumonia. In contrast, only one episode of pneumonia was observed in the control group during the same follow-up period. Therefore, the IgM deficiency probably predisposed the patients to infection. Of the 7 patients who have had more than 12 months of follow-up periods, 4 had disease refractory to standard induction chemotherapy. Of all the 10 patients treated, 6 achieved CR (2 were in CR before treatment, 2 achieved CR 3 months and 2 achieved CR 6 months after starting rituximab). All 4 patients with refractory MM (all had a follow-up of more than 12 months) achieved CR, one before and 3 after starting rituximab. One of the refractory patients has since relapsed, one died of pneumonia in CR 12 months and the other 2 have remained in CR 12+ and 18+ months after ASCT. With a follow-up of 29 months after transplant, the progression-free survival was 56.5% and the overall survival 71.4%. Rituximab infusion after ASCT for MM is therefore associated with severe IgM deficiency and an increased risk of infection. Further works are needed to determine the antitumor activities of rituximab in MM in the setting of minimal residual disease, but this should only be carried out with special attention to the prevention of infection.

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