Yana Zhang

Maintenance rituximab after autologous stem cell transplant for high-risk B-cell lymphoma induces prolonged and severe hypogammaglobulinemia

Maintenance rituximab after autologous stem cell transplant for high-risk B-cell lymphoma induces prolonged and severe hypogammaglobulinemia

The Spermatozoa Protein, SLLP1, Is a Novel Cancer–Testis Antigen in Hematologic Malignancies

Purpose: Neoplastic cells often aberrantly express normal testicular proteins. Because these proteins have a very restricted normal tissue expression, they may be suitable targets for immunotherapy. SLLP1 is an intra-acrosomal, nonbacteriolytic, c lysozyme–like protein recently isolated from human spermatozoa. In this study, we determined whether SLLP1 is a novel cancer–testis antigen in hematologic malignancies Experimental Design: SLLP1 expression in hematologic tumor cells and normal tissues was determined using a combination of reverse transcription-PCR, real-time PCR, and Western blot analysis. The presence of antibodies against SLLP1 was determined by ELISA analysis. Results: SLLP1 was aberrantly expressed in the tumor cells from 2 of 9 acute myeloid leukemia, 3 of 11 chronic lymphocytic leukemia, 4 of 14 chronic myeloid leukemia, and 6 of 17 multiple myeloma. In contrast, they were not detected in corresponding specimens from any healthy donors. SLLP1 exhibited a very restricted normal tissue expression, being found only in testis/spermatozoa. SLLP1 was expressed in some tumor cells at a level of >25%. High titer IgG antibodies against SLLP1 were also detected in the sera of some of these patients. Conclusions: SLLP1 is a novel cancer–testis antigen in hematologic malignancies and is capable of eliciting B-cell immune responses in vivo in cancer-bearing individuals. Our results, therefore, support SLLP1 as a protein target appropriate for additional in vitro study to define its suitability for immunotherapy.

SPAN-Xb expression in myeloma cells is dependent on promoter hypomethylation and can be upregulated pharmacologically

SPAN‐Xb is a novel cancer‐testis antigen in multiple myeloma (MM). In this study, we determined the mechanisms regulating SPAN‐Xb expression in MM. SPAN‐Xb promoter sequence was first cloned into the CAT‐reporter vector to determine the role of promoter methylation in the regulation of gene expression. Tumor cells were treated with 5‐azacytidine and a panel of cytokines were used to determine their ability to induce SPAN‐Xb expression. Bisulfite conversion with sequence analysis was applied to a panel of tumor cells and normal tissues to correlate the CpG dinucleotide hypomethylation and SPAN‐Xb expression. We found that SPAN‐Xb promoter function could be silenced by methylation. 5‐Azacytidine induced promoter hypomethylation and resulted in SPAN‐Xb expression, at both the transcript and protein levels. Hypomethylation of the CpG dinucleotides at positions −310, −307, −299 and −221 within the SPAN‐Xb promoter strongly predict for SPAN‐Xb expression. Both IL‐7 and GM‐CSF were also able to upregulate the expression of SPAN‐Xb in myeloma cells, but only after the promoter sequence has been hypomethylated. Our results provide the first evidence showing the role of promoter methylation in the primary regulation of SPAN‐Xb and the ability of IL‐7 and GM‐CSF to further enhance SPAN‐Xb gene and protein expression in myeloma cells.

Pattern of gene expression and immune responses to Semenogelin 1 in chronic hematologic malignancies.

Normal testicular-specific proteins are frequently aberrantly expressed by tumor cells. Based on this, we have investigated Semenogelin 1, a major protein of human semen coagulum thought to be highly specific to seminal vesicles, in leukemic cells. Using reverse transcription-polymerase chain reaction, Semenogelin 1 gene was frequently expressed in chronic myeloid leukemia (5 of 8, 62.5%) and chronic lymphocytic leukemia (5 of 12, 41.7%) but rarely in multiple myeloma (2 of 30, 6.7%). The gene was not expressed in bone marrow or peripheral blood from healthy donors. Semenogelin 1 expression is normally confined to the testis, suggesting that it is a novel Cancer-Testis (CT) antigen. Translation of the mRNA to Semenogelin 1 protein was confirmed by Western blot analysis of tumor cell lysates and by immunocytochemistry. The recombinant Semenogelin 1 protein was used with a control Escherichia coli-derived recombinant protein in ELISA and Western blot analysis to show that high titer IgG antibodies against Semenogelin 1 were detected in some patients, suggesting the in vivo immunogenicity of the protein. Immune responses predicted gene expression by the leukemia cells. Semenogelin 1 was also frequently coexpressed with other CT antigens, Sperm protein 17 and SPAN-Xb. These results therefore indicate that Semenogelin 1 is a novel CT antigen capable of inducing B-cell responses in vivo in chronic leukemias.

A yeast two-hybrid system using Sp17 identified Ropporin as a novel cancer-testis antigen in hematologic malignancies.

Since most intracellular proteins are expressed with their ligands, ligands of cancer–testis (CT) antigens may also be CT in their distribution. Applying Sperm protein 17 (Sp17) as the bait in a yeast 2‐hybrid system of a testicular cDNA library, 17 interacting clones were isolated and all encoded Ropporin, a spermatogenic cell‐specific protein that serves as an anchoring protein for the A‐kinase anchoring protein, AKAP110. Ropporin showed a very restricted normal tissue gene expression, detected only in testis and fetal liver. Ropporin mRNA could also be detected in tumor cells from patients with multiple myeloma, chronic lymphocytic leukemia and acute myeloid leukemia. Interestingly, expression of Sp17 did not necessarily predict for the expression of Ropporin suggesting that their coexpression in these tumor cells was random rather than coordinated. Ropporin gene expression in tumor cells is associated with the presence of high titer IgG antibodies against Ropporin, suggesting the in vivo translation of the mRNA into protein and the immunogenicity of the protein to the autologous hosts. Using a CT antigen as the bait in a yeast 2‐hybrid system may, therefore, identify novel tumor antigen. Our results also suggest that Ropporin is a novel CT antigen in hematologic malignancies.

Clofarabine induces hypomethylation of DNA and expression of Cancer-Testis antigens.

In this study, treatment of lymphoid tumor cells with low dose clofarabine upregulated the expression of Sp17 and SPAN-Xb. This was associated with an increase in hypomethylated CpG dinucleotides and a decrease in global DNA methylation, as demonstrated by decreases in the percent of methylated Alu repeats. The most optimal concentration of clofarabine to induce DNA hypomethylation and CT antigen expression was between 1x10(-9) and 1x10(-8)M. Above this, clofarabine resulted in tumor cell growth inhibition and apoptosis. Our results provide the first evidence for the CT antigen-inducing and DNA hypomethylating property of low concentration clofarabine.

SPAN-XB core promoter sequence is regulated in myeloma cells by specific CpG dinucleotides associated with the MeCP2 protein.

SPAN‐Xb 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 shed light on potential therapeutic strategies to upregulate antigen levels for SPAN‐Xb‐based tumor vaccines. Using a panel of truncated promoter constructs, we localize the core sequence of SPAN‐XB promoter to the 73 bp at the 3′ end of the promoter, a region within the full length promoter that lacks CpG dinucleotides. Reporter gene expression assays showed that the core promoter function is significantly modulated by the adjacent CpG sequences. Chromatin immunoprecipitation assays revealed a specific association of MeCP2 with the promoter, and MeCP2 binding strongly correlated with repression of SPAN‐XB gene. 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 core promoter function in myeloma cells is associated with MeCP2 protein binding and regulated by specific CpG dinucleotide sequences.

Combined real time PCR and immunohistochemical evaluation of sperm protein 17 as a cancer-testis antigen

Abstract:  We previously identified sperm protein 17 (Sp17) as a normal testicular protein aberrantly expressed in a proportion of multiple myeloma (MM). However, recent studies have generated controversies on the normal tissue expression of Sp17 and whether or not it is a suitable target for immunotherapy. In this study, we have used a combination of real time polymerase chain reaction and immunohistochemistry on a large panel of normal tissues. Although Sp17 transcripts could be detected in some normal tissues, the levels of expression were <2% of those in normal testis. In contrast, Sp17+ myeloma cells expressed 3–18% of normal testis levels of Sp17 transcript. Immunohistochemistry using two Sp17 murine monoclonal antibodies, each directed at a non‐overlapping B‐cell epitope, showed Sp17 protein to be expressed only in testis and not any other normal tissues. Specificity of binding of the antibodies to testis was also confirmed in competitive binding assays. Our results therefore further suggest Sp17 as a cancer‐testis antigen in MM and support its suitability as a target for immunotherapy.

A phase II study of low dose intravenous clofarabine for elderly patients with myelodysplastic syndrome who have failed 5-azacytidine

The treatment options for patients with myelodysplastic syndrome (MDS) who have failed DNA hypomethylating agents are limited. In this study, we set out to investigate the efficacy of low dose clofarabine in 10 patients with MDS (four intermediate-2/high risk disease) who had failed 5-azacytidine. The median age was 73 years (range 65–78) and median cycles of clofarabine received were 2 (range 1–4). Nine patients were evaluable for response. An overall response rate of 44% was observed (one CR, one PR, and two HI). All responders had low risk disease. The median duration of response was 12 months (range 6.5–15.5). Although the doses of clofarabine administered were only 12.5–25% of that used in other studies, significant hematologic toxicities were observed. Severe and prolonged pancytopenia occurred in all 10 patients. One patient who had a history of thrombocytopenic gastrointestinal bleed died due to an intracranial bleed despite aggressive platelet support. Low dose clofarabine may, therefore, induce response, but with significant toxicities, in patients with low risk MDS who fail 5-azacytidine. Future work involving a larger patient population is needed to establish the role of low dose clofarabine in low risk MDS.

Umbilical cord blood transplant in hepatitis C-associated severe aplastic anemia

Although umbilical cord blood (UCB) transplantation has been practiced for nearly one decade, it has largely been applied to children and small adults due to the general misconception that the number of hematopoietic stem cells in each unit of UCB would not be adequate for a full adult transplant. In the last few years, there has been an increase in the number of adults transplanted using UCB. With a recent report of the successful use of hematopoietic stem cells pooled from two different units of unrelated UCB that may not necessarily be HLA-matched, it is expected that UCB transplantation will become a more widely applicable therapeutic option for adult patients. Severe aplastic anemia (SAA) is a recognized complication of viral hepatitis. Although it usually occurs shortly after the acute phase of the hepatitis, it has also been reported during treatment for chronic hepatitis. Therapeutic options for patients with hepatitis-associated SAA may be difficult. Although UCB transplants have been offered to adult patients with SAA, the clinical outcome has ranged from graft failure to the establishment of mixed chimerism. Its use for adult patients with SAA associated with hepatitis C has, however, never been described. We report the successful use of a single HLA-DRB1-mismatched UCB for transplantation of an adult patient who developed SAA during treatment for hepatitis C. A 45-year-old man was diagnosed with hepatitis C in November 2000 when his routine physical examination showed abnormal liver function tests: total protein 7.7 g/dl, albumin 4.5 g/dl, bilirubin 1.2mg/dl, alkaline phosphatase 95 IU/l, AST 59 IU/l and ALT 124 IU/l. He had a very high viral load of 2 460 100 copies/ml, and the hepatitis C virus was subtyped as 1a. He did not have any significant past medical history or family history of bone marrow diseases. He also had no history of alcohol or intravenous drug abuse, or exposure to myelotoxic agents. The source of the viral infection was unclear but may have been related to the tattoos he had received many years previously. Liver biopsy confirmed chronic active hepatitis. He was treated with a combination of ribavirin and interferon-a (IFNa). His pretreatment complete blood counts were normal. He tolerated the combination antiviral therapy with reduction in his hepatitis C viral load. However, he developed a progressive pancytopenia that persisted despite discontinuation of the ribavirin and IFNa. By June 2002, 4 months after stopping the antiviral therapy, complete blood counts showed: hemoglobin 9.2 g/dl (reticulocyte o1%), white blood count 0.9 10/l (4% neutrophils, 90% lymphocytes) and platelet counts of 9 10/l. Bilateral bone marrow examinations on two separate occasions 2 weeks apart confirmed the diagnosis of SAA, with a bone marrow cellularity uniformly less than 5%. There was no excess of blast cells in the bone marrow. Flow cytometry and cytogenetic analyses of the bone marrow were normal. In particular, the immunophenotypic features of paroxysmal nocturnal hemaglobinuria were absent. He became symptomatic from the severe thrombocytopenia with hemorrhagic complications of mucosal bleeding, necessitating platelet transfusions twice a day, and anemia requiring blood transfusions. Although the first-line treatment for SAA is immunosuppression in this age group, the time to response from the treatment can be as long as 6–8 weeks. During this period, patients who are heavily transfusion-dependent may require a very high level of blood product support and so are at risk of the adverse effects of blood transfusion. Furthermore, patients continue to be at risk of infection that could result in impaired patient performance status. Both these factors could influence the outcome of the treatment if the patient eventually needs a stem cell transplant. Based on these factors and due to the profound hemorrhagic complications the patient was experiencing, it was decided to treat his SAA with a hematopoietic stem cell transplant. At that time, his liver function tests had normalized and the hepatitis C viral load had fallen to below the PCR detectable limit of 10 IU/ml. An unrelated UCB transplant was offered because he did not have any siblings. He weighed 73 kg and his full HLA types were: HLA-A 01, 26; B 08, B 3801; BW-4/6 4; BW-4/6 6; DRB1 0301, 1501; DQB1 02AB; DQB1 06CYU; DRB3 01GM. His blood group was blood group O-positive. He received Busulfan 4mg/kg/day 4 days and Cyclophosphamide 60mg/kg/day 2 days. In vivo T-cell depletion was achieved with antithymocyte globulin 15mg/kg/12 h 6 doses, starting from day 3 to day 1. He was infused with a unit of previously cryopreserved blood group A-positive unrelated UCB. The full HLA types of the infused cord blood were: A 01, A 26; B 08; B 38; BW-4/6 4; BW-4/6 6; DRB1 0301, 1401; DQB1 02AB; 05031; DRB3 01GM; DRB3 02AVK. Therefore, there was a mismatch at one of the HLA-DRB1 loci. A total of 1.77 10/kg nucleated cells, 2.3 10/kg CD34þ cells and 1 10/kg colony-forming units were infused. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A starting on day 2 and methylprednisone 1mg/kg twice a day starting on day þ 9. Grade II skin GVHD developed by day 10, but resolved spontaneously. He engrafted, achieving an absolute neutrophil count of 40.5 10/l on day þ 18, and became platelet transfusion independent by day þ 26. The methylprednisone was tapered starting on day þ 23 and discontinued by day þ 36. The engraftment was documented by variable number of tandem repeats (VNTR) analysis (Figure 1). Serological analysis of the red blood cells also showed full erythroid engraftment with the conversion of his blood group from O-positive to A-positive. He received prophylactic oral Cotrimoxazole and gancyclovir for 6 months after the transplant. His peripheral blood counts normalized by day þ 84 (hemoBone Marrow Transplantation (2004) 33, 565–567 & 2004 Nature Publishing Group All rights reserved 0268-3369/04