ALBERT

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

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: An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6(DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain(RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL.

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: We have recently demonstrated the presence of Kaposi's sarcoma–associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells from all patients with myeloma studied. To show that these findings were not an artifact of tissue culture, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) to detect KSHV in BM core biopsies. Using ISH to open reading frame-72 (ORF 72), we localized KSHV to BM dendritic cells in 17 of 20 patients with myeloma, 2 patients with plasmacytosis associated with the acquired immunodeficiency syndrome, and 1 case of aplastic anemia. In contrast, BM from normal subjects (n = 4) and patients with lymphoma and leukemia (n = 21) did not contain KSHV. PCR amplification with KSHV primers demonstrated product in fresh BM biopsy samples from 6 of 7 myeloma patients, whereas three normal marrows contained no amplified product. These findings suggest that KSHV, possibly through alterations in the BM microenvironment and production of viral interleukin-6 (vIL-6), may stimulate and maintain abnormal plasma cell proliferation in myeloma and related disorders.

Description: Recent molecular evidence suggests an association with a new herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), and primary effusion lymphomas (PELs). PELs have a characteristic morphology, phenotype, and clinical presentation, with malignant effusions in the absence of a contiguous solid tumor mass. We have established a cell line (KS-1) from a KSHV-positive human immunodeficiency virus (HIV)-negative patient with pleural cavity-based lymphoma that was passaged into triple-immunodeficient BNX mice. In contrast to cell lines from body cavity-based lymphomas derived from HIV-positive individuals that contain both KSHV and Epstein Barr viral genome, these cells contain only KSHV, allowing for uncontaminated virologic studies. Ultrastructural examination identified malignant cells with features of late differentiating B cells (immunoblasts). Cells with viral cytopathic effect contained typical 110-nm intranuclear herpesvirus nucleocapsids and complete cytoplasmic virions, confirming the association of PEL with KSHV.

Description: The clonality of nodular lymphocyte-predominant Hodgkin's disease (NLPHD) and the relationship to composite or sequential large-cell lymphomas (LCLs) is poorly understood. Clonal Ig heavy-chain gene rearrangements (lgHGR) have infrequently been observed in NLPHD by Southern hybridization. The goals of this study were (1) to determine if IgHGR could be identified by polymerase chain reaction (PCR) techniques in the LCL associated with NLPHD; (2) to determine if the lgHGR identified in the LCL could also be found in the associated NLPHD; and (3) to determine if Epstein-Barr virus (EBV) played a role a role in histologic progression to LCL. Using consensus primers to conserved regions in the lgH variable (V) and joining (J) region genes, we analyzed formalin-fixed paraffin-embedded sections from the biopsies of 25 patients referred to the National Cancer Institute (NCI) registry for NLPHD and LCL using both single-step and seminested V-J PCR. The histologically aggressive component was further subclassified as frank LCL or as L&H-cell-rich, but not fulfilling criteria for LCL. Matched samples representing both NLPHD and aggressive components were available in 13 cases. In 12 cases, only one component was available (aggressive, n = 8; NLPHD, n = 4). In addition, we also amplified, with 32P labeling, 12 cases of NLPHD without associated LCL. Two clonal IgHGR were identified in 29 cases (7%) of typical NLPHD, both of which were associated with LCL containing a similar sized band by PCR. The clonal identity of the bands in the NLPHD and associated LCL was confirmed by sequencing the products in these two cases. Eight of 10 cases (80%) of LCL associated with NLPHD contained a clonal band by this technique. By contrast, none of the cases classified as L&H-cell- rich contained an IgHGR. The single-step and seminested PCR methods produced identical results. All clonal LCLs were studied for EBV sequences by in situ hybridization using the EBER1 probe, and were negative. We conclude that the LCLs associated with NLPHD are clonal B- cell malignancies. However, by these methods, the same clone can be identified in only a minority of cases of NLPHD and LCL. EBV does not appear to play a role in histologic progression. Moreover, our results suggest that many cases suspected of being LCL may actually represent NLPHD with increased numbers of L&H cells. In histologically equivocal cases, the diagnosis of LCL should be reserved for those cases in which a clonal B-cell neoplasm can be demonstrated.

Description: Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40.9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.

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.