ALBERT

Description: Follicular lymphoma (FL) is a low-grade B-cell non-Hodgkin's lymphoma (NHL) that frequently transforms into diffuse aggressive NHL. The majority of FLs display a t(14; 18) translocation that places the bcl-2 gene into juxtaposition with the lg heavy-chain (H) gene locus. Morphologically transformed malignant FL cells retain their t(14;18) translocation and may acquire additional genetic abnormalities. We analyzed serial biopsy specimens from eight patients with FL for secondary alterations of the rearranged bcl-2 gene in the breakpoint and open reading frame (ORF) regions. Two cases of FL showed no histologic alteration in the second biopsy, and six cases of FL showed morphologic transformation to diffuse large-cell lymphoma (DLL) in the second biopsy. Polymerase chain reaction (PCR) amplification, cloning, and sequencing of the junctional region of the hybrid bcl-2/IgH genes showed identical nucleotide sequences in multiple biopsy specimens of FL that did not show morphologic transformation. In patients in whom FL cells underwent morphologic transformation, FL and autologous DLL cells displayed identical bcl-2/IgH gene nucleotide sequences in five cases and different sequences in one case. In the case for which FL and DLL cells showed different bcl-2/IgH junctional sequences, DLL cells incorporated larger bcl-2 and Ig-joining (JH) gene fragments than the corresponding FL cells, suggesting that DLL clones developed by a distinct t(14; 18) translocation rather than by alteration of the hybrid bcl-2/IgH gene detected in the FL cells. In all eight cases, neither FL nor DLL cells showed alterations of bcl-2 gene sequences in the breakpoint region, suggesting high conservation of the bcl-2 gene during both t(14; 18) translocation and morphologic transformation of the FL cells. PCR single-strand conformation polymorphism (SSCP) and sequence analyses were performed for identification of structural alterations of the bcl-2 gene in the ORF region corresponding to the 239-amino acid p26-bcl-2 alpha protein. A total of 11 point mutations of the ORF were detected in DLL cells of three transformed NHLs, but no alteration of the ORF was detected in FL cells. Four of 11 mutations, at positions 29, 46, 59, and 106, yielded amino acid replacements. These findings demonstrate that FL and DLL cells may be clonally related or unrelated. They also show that transformation of FL cells may be associated with somatic point mutations of the bcl-2 proto- oncogene ORF sequence resulting in alteration of the p26-bcl-2 alpha gene product.

Description: Two unique DNA fragments were recently identified in over 90% of acquired immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma tissues. Sequence analysis suggests that these fragments belong to a previously unidentified human herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). These fragments have also been identified in a subset of non-Hodgkin's lymphomas in human immunodeficiency virus-positive patients; specifically, in body cavity-based lymphomas (AIDS-BCBLs). We have established two cell lines derived from AIDS-BCBLs, BC-1 and BC-2, which retain the KSHV sequences, and have used them to further characterize this putative viral genome. In this report, we demonstrate that the KSHV sequences represent a portion of a much larger DNA molecule that is located predominantly in the nucleus of the infected cells. In situ hybridization of metaphase spreads made from both of these cell lines show these sequences in episomal structures. Their presence in the cells as large nuclear episomes supports previous sequence homology data suggesting that KSHV belongs to the herpesvirus family. These cell lines provide a continuous source of KSHV sequences. Thus, they represent an important tool for future studies of this recently described human herpesvirus that may be involved in the pathogenesis of Kaposi's sarcoma and some AIDS-related non-Hodgkin's lymphomas.

Description: Sixteen cases of histologic intermediate-grade and high-grade AIDS- associated non-Hodgkin's lymphoma (NHL) were studied for the presence and patterns of c-myc gene and bcl-2 locus rearrangements. The presence of Epstein-Barr virus (EBV) sequences and proteins and HTLV-I sequences were also investigated. c-myc gene rearrangements analogous to those observed in sporadic Burkitt lymphomas were detected in 12 of 16 cases. Six of 16 cases had detectable EBV sequences and proteins. None of the cases displayed bcl-2 rearrangements or contained HTLV-I sequences. These data suggest a frequent role for c-myc activation in the pathogenesis of AIDS-associated NHL, independent of histologic type. Conversely, EBV does not appear to be directly involved in lymphomagenesis in the majority of AIDS-associated NHLs.

Description: The nature of the Reed Sternberg (RS) cell, the malignant cell of Hodgkin's disease (HD), remains unknown. Cytogenetic studies have yielded ambiguous results regarding the chromosomal profile of this cell. In an attempt to further clarify the ploidy status of the RS cell, we analyzed the DNA content of CD30-positive RS cells and RS cell variants in HD lesions from 32 patients using an image analysis system. A diploid and/or near-diploid (DNA index [DI], 1.0 +/- 0.2) and a tetraploid (2.0 +/- 0.2) RS cell population were identified in 9 and in 11 of the 32 cases examined, respectively. An aneuploid RS cell population was identified in 8 of the 32 cases examined. The remaining four cases contained two RS cell subpopulations with different DNA content, each one representing more than 15% of the total RS cell population. There was no significant correlation between the DNA content of the RS cells and the category of HD. Furthermore, analysis of multiple biopsies of an individual patient taken from different lymphoid organs at the same or different time periods showed a constant DNA profile. Our data indicate that RS cells can express variable DNA content and suggests that multiple subpopulations of RS cells with different DNA content may simultaneously coexist within the same HD lesion in some patients. In addition, the RS cell population within each patient appears to express a specific DNA content profile, possibly representing unique clones. These highly individualized profiles potentially may be useful as markers to follow the clinical course of patients with HD.

Description: The human T-cell lymphotropic virus type I (HTLV-I) is capable of inducing adult T-cell leukemia/lymphoma (ATLL). However, the long latency period between infection and development of ATLL, as well as the small fraction of the infected population that actually develops this disease, suggest that additional factors are involved in its pathogenesis. Therefore, we performed a molecular analysis of 10 cases of ATLL presenting in a nonendemic area that were shown to have HTLV-I sequences by polymerase chain reaction as well as clonal T-cell receptor beta gene rearrangements. We analyzed these cases for alterations in some of the oncogenes and tumor suppressor genes frequently involved in hematopoietic neoplasia. Specifically, we used a single-strand conformation polymorphism assay to determine the presence of mutations in the p53 tumor suppressor gene, as well as the K-RAS, N-RAS, H-RAS, and c-myc oncogenes. In addition, we studied the c-myc gene for rearrangements by Southern blotting and assessed expression of the retinoblastoma (Rb) and p53 genes by immunostaining. Analysis of the c-myc gene and the RAS family of oncogenes did not show any alterations. Also, the Rb gene was expressed in all cases analyzed. However, we found mutations of the p53 gene in 3 of the 10 cases and these results were confirmed by sequence analysis. In two of these cases, we showed by restriction fragment length polymorphism analysis of chromosome 17p sequences that the p53 mutations were accompanied by a loss of heterozygocity. Also, these mutations correlated with an altered pattern of p53 expression. Thus, mutations in the p53 locus may be a cofactor for the development of ATLL in some cases, whereas the c-myc, Rb, and RAS genes do not appear to be involved in these neoplasms.

Description: Anaplastic large-cell lymphoma (ALCL) represents a morphologically distinct type of non-Hodgkin's lymphoma (NHL) characterized phenotypically by the expression of the CD30 antigen, a new member of the nerve growth factor gene family. The lymphoid origin of ALCL has been documented using immunohistochemical and molecular genetic analyses. However, very little is known so far regarding the precise pathogenetic mechanisms involved in its development and progression. Therefore, we investigated bcl-2, p53, and retinoblastoma gene (Rb) expression immunohistochemically; the occurrence of bcl-2, c-myc, and Rb gene rearrangements using Southern blotting; and the presence of ras and p53 gene somatic mutations by single-strand conformation polymorphism assay in a panel of 18 well-characterized ALCLs. In addition, the presence of Epstein-Barr (EBV) and human T-cell lymphotropic virus type I (HTLV-I) genomes were investigated using polymerase chain reaction. We identified abnormal c-myc gene products in 6 of 18 cases (33%) of ALCL. On the other hand, the bcl-2 and Rb genes were not rearranged and K-, N-, and H-ras gene somatic mutations were not found. Significant levels of p53 protein expression were found in more than 60% of ALCLs, but only a single ALCL carried a p53 gene mutation (exon 5). Only 3 ALCL cases, all occurring in human immunodeficiency virus-infected patients, were positive for EBV genomes. On the other hand, contrary to previous findings, no HTLV-I products could be identified. Despite the fact that the c-myc proto- oncogene appears to be frequently altered in ALCL, no pathognomonic abnormality could be identified and therefore additional studies and new strategies should be designed to identify the pathogenetic mechanisms involved in the development of ALCL.

Description: Each of three individuals with acquired immunodeficiency syndrome (AIDS) developed a pleomorphic malignant neoplasm in which a precise histopathologic diagnosis could not be rendered. In each case, the tumor cells expressed leukocyte common antigen and a variable constellation of antigens associated with B- and T-cell activation (HLA- DR, T9, T10, BL2, BL3, Ki-24, BLAST-2). They lacked all B cell, T cell, myeloid, and monocyte lineage-restricted antigens, resulting in their classification as hematopoietic neoplasms of uncertain lineage. However, antigen receptor gene rearrangement analysis demonstrated that each of these three neoplasms exhibited clonal immunoglobulin heavy chain and kappa light chain gene rearrangements and lacked T-cell receptor beta chain gene rearrangements and therefore were B cell- derived non-Hodgkin's lymphomas (NHL) representative of an equivalent, relatively mature stage of B-cell differentiation. In contrast with most AIDS-associated NHLs, each of these three neoplasms lacked c-myc gene rearrangements and contained Epstein-Barr virus (EBV) proteins and/or sequences. These studies demonstrate that these three AIDS- associated neoplasms of uncertain lineage exhibit a strikingly similar constellation of distinctly uncommon morphologic, immunophenotypic, and molecular genetic characteristics that distinguishes them substantially from the vast majority of NHLs that have been reported to occur in association with AIDS. The consistent presence of EBV proteins and/or DNA sequences suggests that the Epstein-Barr virus played a pathogenetic role in the development of these three AIDS-associated neoplasms. Finally, these studies further illustrate the utility of antigen receptor gene rearrangement analysis in the diagnosis and classification of hematopoietic neoplasms of uncertain lineage.

Description: We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4–21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4–21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4–21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4–21 gene in the human population, the nature of the nucleotide differences in the expressed VH4–21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4–21 gene. Thus, the expression of VH4–21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.

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 pathogenesis of non-Hodgkin's lymphoma (NHL) with a large cell component (DLLC, including diffuse large cell, DLCL; diffuse mixed cell, MX-D; and immunoblastic, IMB) is unknown. A novel candidate proto- oncogene, BCL6, that is involved in chromosome band 3q27 aberrations in NHL has been recently identified. We have investigated the incidence and disease-specificity of BCL6 rearrangements in a large panel of lymphoid tumors, including acute and chronic lymphoid leukemias (96 cases), various NHL types (125 cases), and multiple myelomas (23 cases). BCL6 rearrangements were found in 16/45 (35.5%) DLLC, more frequently in DLCL (15/33, 45%) than in MX-D (1/10, 10%), in 2/31 (6.4%) follicular NHL, and in no other tumor types. BCL6 rearrangements represent the first genetic lesion specifically and recurrently associated with DLLC and should prove useful for understanding the pathogenesis as well as for the clinical monitoring of these tumors.