ALBERT

Description: Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.

Description: Arsenic trioxide (ATO) induces apoptosis of plasma cells through a number of mechanisms including inhibiting DNA binding by NF-κB. These results suggest that this agent may be synergistic when combined with other active anti-myeloma drugs. To evaluate this we examined the effect of ATO alone and in combination with anti-myeloma treatments evaluated in vitro with MTT assays and using our severe combined immunodeficient (SCID)-hu murine myeloma models. First, we determined the effects of combining ATO with bortezomib or melphalan on the myeloma cell lines RPMI8226 and U266. Cell proliferation assays demonstrated marked synergistic anti-proliferative effects of ATO at concentrations ranging from 5x10−5M – 5x10−9M and melphalan concentrations ranging from 3x10−5M – 3x10−9M. Similar effects were observed when these cell lines were treated with bortezomib and varying concentrations of ATO (5x10−5 M – 5x10−10 M). We also investigated the potential of ATO to increase the efficacy of anti-myeloma therapies in our SCID-hu murine model LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Mice were treated with ATO alone at 6.0 mg/kg, 1.25 mg/kg, 0.25 mg/kg, and 0.05 mg/kg intraperitoneally (IP) daily x5/week starting 19 days post-implantation. Mice receiving the highest dose of ATO (6.0 mg/kg) showed marked inhibition of tumor growth and reduction of paraprotein levels while there was no effect observed in all other treatment groups. Next, 27 days following implantation of our LAGλ–1 intramuscular (IM) tumor, LAGλ–1 mice were treated with ATO (1.25 mg/kg) IP, bortezomib (0.25 mg/kg), or the combination of both drugs at these doses in the schedules outlined above. ATO or bortezomib treatment alone had no anti-myeloma effects at these low doses consistent with our previous results whereas there was a marked decrease in both tumor volume (57%) and paraprotein levels (53%) in mice receiving the combined therapy. The combination of melphalan and ATO was also evaluated in this model. LAGλ–1 bearing mice received therapy with melphalan IP x1/weekly at 12.0 mg/kg, 6.0 mg/kg, 0.6 mg/kg, and 0.06 mg/kg starting 22 days post-implantation and showed no anti-myeloma effects. Twenty-eight days following implantation of LAGλ–1 tumor, mice received ATO (1.25 mg/kg) or melphalan (0.6 mg/kg) alone at doses without anti-myeloma effects, or the combination of these agents at these doses. The animals treated with these drugs alone showed a similar growth and increase in paraprotein levels to control mice whereas the combination of ATO and melphalan at these low doses markedly suppressed the growth of the tumor by 〉50% and significantly reduced serum paraprotein levels. These in vitro and in vivo studies suggest that the addition of ATO to other anti-myeloma agents is likely to result in improved outcomes for patients with drug resistant myeloma. Based on these results, these combinations are now in clinical trials with promising early results for patients with drug resistant myeloma.

Description: Allelotype analysis of adult T-cell leukemia (ATL) was undertaken for the first time to identify chromosomal loci relevant to the development of acute/lymphomatous ATL. Loss of heterozygosity (LOH) was screened using 94 highly polymorphic microsatellite markers, distributed among all nonacrocentric, autosomal chromosomes. In each of the 22 cases, DNA obtained from their leukemic cells in acute/lymphomatous phase was compared with their constitutional DNA from mononuclear cells in chronic or remission phase. Allelic losses of at least on one chromosome arm occurred in 91% of the cases (20 individuals). Among 39 chromosome arms, allelic losses were observed on 31 arms at least for one sample. A high frequency of allelic loss (〉30%) was seen on chromosome arms 6q (41%) and 17p (48%). The mean fractional allelic loss (FAL) was 0.109. These findings suggest that a novel tumor suppressor gene on chromosome arm 6q, as well as the p53 gene on chromosome arm 17p, probably have an important role in the development of acute/lymphomatous ATL. © 1998 by The American Society of Hematology.

Description: A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay (ELISA) with a whole virus lysate as antigen was developed and used to measure the seroprevalence rate and levels of IgG antibodies to HHV-8 in sera/plasma of various patient groups and blood donors. The virus antigen was prepared from the KS-1 cell line, which produces lytic virus, and therefore contains a broad array of viral proteins. Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of 72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency virus (HIV) patients were positive with an average titer of 4,000. A study on a very limited number of serial serum samples from patients before and after diagnosis with KS showed highly elevated antibody titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1+ homosexual patients contain IgG antibodies to HHV-8 suggesting that this population is at high risk for developing KS. Antibody results correlated well with the confirmatory immunofluorescent assays (IFA) using KS-1 cells as the substrate. This HHV-8 IgG antibody detection ELISA is sensitive and specific and does not cross-react with Epstein-Barr virus (EBV) or other human herpesviruses. The results of this HHV-8 antibody survey suggest that this rapid ELISA assay can be used to screen large numbers of sera to find those at risk for developing KS.

Description: Somatic hypermutation (SHM) targets primarily the immunoglobulin variable region (IgV) genes in germinal center (GC) B cells, thereby allowing antibody affinity maturation. A malfunction of SHM, termed aberrant somatic hypermutation (ASHM), was found in about 50% of diffuse large B-cell lymphomas (DLBCLs), leading to mutations in the 5′ sequences of multiple genes, including oncogenes. Although the SHM mechanism is largely unknown, it was shown to require the activation-induced cytidine deaminase (AID) gene. AID mRNA is expressed in GC B cells and GC-derived lymphomas, but the pattern of expression of the AID protein is not known. Using 2 specific antibodies, here we show that the AID protein can be detected in GC centroblasts and their transformed counterpart (Burkitt lymphoma) but not in pre-GC B cells and post-GC neoplasms, including B-cell chronic lymphocytic leukemia and multiple myeloma. DLBCLs displayed variable levels of AID expression, which did not correlate with IgV ongoing hypermutation, ASHM, or disease subtype. Finally, both in normal and malignant B cells the AID protein appeared predominantly localized in the cytoplasm. These results indicate that the AID protein is specifically expressed in normal and transformed GC B cells; nonetheless, its predominantly cytoplasmic localization suggests that additional mechanisms may regulate its function and may be altered during lymphomagenesis. (Blood. 2004;104:3318-3325)

Description: Allelotype analysis of adult T-cell leukemia (ATL) was undertaken for the first time to identify chromosomal loci relevant to the development of acute/lymphomatous ATL. Loss of heterozygosity (LOH) was screened using 94 highly polymorphic microsatellite markers, distributed among all nonacrocentric, autosomal chromosomes. In each of the 22 cases, DNA obtained from their leukemic cells in acute/lymphomatous phase was compared with their constitutional DNA from mononuclear cells in chronic or remission phase. Allelic losses of at least on one chromosome arm occurred in 91% of the cases (20 individuals). Among 39 chromosome arms, allelic losses were observed on 31 arms at least for one sample. A high frequency of allelic loss (〉30%) was seen on chromosome arms 6q (41%) and 17p (48%). The mean fractional allelic loss (FAL) was 0.109. These findings suggest that a novel tumor suppressor gene on chromosome arm 6q, as well as the p53 gene on chromosome arm 17p, probably have an important role in the development of acute/lymphomatous ATL. © 1998 by The American Society of Hematology.

Description: Glutathione levels have previously been shown to be associated with the development of resistance to a variety of anti-myeloma therapies. Ascorbic acid (AA) depletes intracellular glutathione levels which, in turn, should increase the sensitivity of tumor cells to anti-myeloma agents such as arsenic trioxide (ATO) and melphalan. To determine the synergistic effects of combining AA, with ATO and/or melphalan, we evaluated the effects of these combinations with MTT assays on myeloma cell lines in vitro and using our severe combined immunodeficient (SCID)-hu murine myeloma models. We determined the synergistic effects of combining AA with ATO and/or melphalan on the myeloma cell lines RPMI8226, 8226/dox, U266, and U266/dox in vitro. MTT assays demonstrated marked synergistic anti-proliferative effects of AA at 10 mM when added to these cell lines in the presence of ATO concentrations ranging from 5x10−5 M – 5x10−9 M, and melphalan concentrations ranging from 3x10−5 M – 3x10−9 M. In order to provide further evidence for the clinical relevance of these synergistic effects of AA, we investigated the potential of AA to increase the efficacy of current anti-myeloma therapies in our SCID-hu murine model of human myeloma LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Twenty-eight days following implantation, mice then received treatment intraperitoneally (IP) with either AA (300 mg/kg) daily x5/week, ATO (1.25 mg/kg) daily x5/week, or melphalan (3.0 mg/kg) x1/week, or the combination of these agents. AA, ATO, and melphalan alone have no anti-myeloma effects at these doses, whereas AA+melphalan results in significantly decreased tumor burden and paraprotein levels. The most profound anti-myeloma effects were observed in animals treated with all three drugs together. These data show not only the additional synergistic anti-myeloma effects of AA on both ATO and melphalan in vitro but for the first time suggest that these effects are also present in vivo. This provides the rationale for combining AA with these agents in myeloma patients with resistant disease. In support of this, early results of clinical trials using the combination of AA, ATO and low doses of oral melphalan are promising.

Description: Primary effusion lymphoma (PEL) is a distinct clinicopathologic entity associated with Kaposi's sarcoma-associated herpes virus (KSHV). Several cytokines, including interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) may be important for survival of KS cells. However, little is known about the interaction of cytokines with KSHV-infected lymphocytes from PEL. Therefore, we investigated what cytokines were produced by KSHV-infected PEL cell lines (KS-1, BC-1, BC-2), what cytokine receptors were expressed by these cells, what response these cells had to selected cytokines, and what was the effect of IL-6 antisense phosphorothioated oligonucleotides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and protein studies showed that these three cell lines produced IL-10, IL-6, and the receptors for IL-6. The granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, IL-12, bFGF, PDGF, and c-kit transcripts were not detected in the cell lines. High levels (0.7 to 5 ng/mL/106cells/48 hours) of IL-6 protein were consistently detected in supernatants of the cell lines by enzyme-linked immunosorbent assay (ELISA) tests. In clonogenic assays, interferon-α (IFN-α) and IFN-γ suppressed the clonal growth of the PEL cells, but GM-CSF, IL-4, IL-6, IL-8, IL-10, and oncostatin M did not change it. We examined for several autocrine loops that have been suggested to occur in KS. Experiments using antisense oligonucleotides showed that the clonal growth of KS-1 and BC-1 was nearly 100% inhibited by IL-6 antisense oligonucleotides (10 μmol/L), but not at all by either oligonucleotides (≤10 μmol/L) to IL-6 sense, IL-6 scrambled, viral IL-6 (vIL-6) antisense, or IL-10 antisense. Furthermore, the IL-6 antisense oligonucleotides had no effect on two B-cell lymphoma cell lines, which were not infected with KSHV. Addition of IL-6 antibody did not inhibit clonal growth of any of the cell lines. Taken together, we have defined the cytokines and their receptors expressed on PEL cells and have found that these cells synthesized IL-6 and IL-6 receptors; interruption of this pathway by IL-6 antisense oligonucleotides specifically prevented the growth of these cells. These findings will offer potential new therapeutic strategies for PEL.

Description: Primary effusion lymphoma (PEL) is a distinct clinicopathologic entity associated with Kaposi's sarcoma-associated herpes virus (KSHV). Several cytokines, including interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) may be important for survival of KS cells. However, little is known about the interaction of cytokines with KSHV-infected lymphocytes from PEL. Therefore, we investigated what cytokines were produced by KSHV-infected PEL cell lines (KS-1, BC-1, BC-2), what cytokine receptors were expressed by these cells, what response these cells had to selected cytokines, and what was the effect of IL-6 antisense phosphorothioated oligonucleotides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and protein studies showed that these three cell lines produced IL-10, IL-6, and the receptors for IL-6. The granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, IL-12, bFGF, PDGF, and c-kit transcripts were not detected in the cell lines. High levels (0.7 to 5 ng/mL/106cells/48 hours) of IL-6 protein were consistently detected in supernatants of the cell lines by enzyme-linked immunosorbent assay (ELISA) tests. In clonogenic assays, interferon-α (IFN-α) and IFN-γ suppressed the clonal growth of the PEL cells, but GM-CSF, IL-4, IL-6, IL-8, IL-10, and oncostatin M did not change it. We examined for several autocrine loops that have been suggested to occur in KS. Experiments using antisense oligonucleotides showed that the clonal growth of KS-1 and BC-1 was nearly 100% inhibited by IL-6 antisense oligonucleotides (10 μmol/L), but not at all by either oligonucleotides (≤10 μmol/L) to IL-6 sense, IL-6 scrambled, viral IL-6 (vIL-6) antisense, or IL-10 antisense. Furthermore, the IL-6 antisense oligonucleotides had no effect on two B-cell lymphoma cell lines, which were not infected with KSHV. Addition of IL-6 antibody did not inhibit clonal growth of any of the cell lines. Taken together, we have defined the cytokines and their receptors expressed on PEL cells and have found that these cells synthesized IL-6 and IL-6 receptors; interruption of this pathway by IL-6 antisense oligonucleotides specifically prevented the growth of these cells. These findings will offer potential new therapeutic strategies for PEL.