Absence of Kaposi's-sarcoma-associated herpesvirus-like DNA sequences (KSHV) in angiosarcomas developing in body-cavity and other sites.
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DNA, Viral↗
Biomedical subjects
D M Knowles
Publications and source records attributed to D M Knowles.
Etiology and pathogenesis of AIDS-related non--Hodgkin's lymphoma.
The incidence of NHL is greatly increased in HIV-infected individuals. The vast majority are clinically aggressive B cell-derived neoplasms exhibiting BL, IBL, or LCL histology. Approximately 80% arise systemically (nodal and/or extranodal), and the remaining 20% arise as primary CNS lymphomas. A small proportion are body cavity-based lymphomas associated with KSHV infection. Possible factors contributing to lymphoma development include HIV-induced immunosuppression, chronic antigenic stimulation, and cytokine overproduction. These alterations are associated with the development of oligoclonal B-cell expansions. The appearance of NHL is characterized by the presence of a monoclonal B-cell population displaying a variety of genetic lesions, including EBV infection, c-myc gene rearrangement, bcl-6 gene rearrangement, ras gene mutations, and p53 mutations/deletions. The number and type of genetic lesions varies according to the anatomic site and histopathology. In the case of BL, virtually 100% exhibit c-myc gene rearrangements, two thirds display p53 gene mutations, one third contain EBV, and none exhibit bcl-6 gene rearrangements. In contrast, in the case of IBL, virtually 100% contain EBV, 25% display c-myc gene rearrangements, 20% display bcl-6 gene rearrangements, and very few exhibit p53 gene mutations. These findings suggest that more than one pathogenetic mechanism is operational in the development and progression of AIDS-related NHLs. Further work will be necessary to develop a complete understanding of the etiology and pathogenesis of NHL in the setting of HIV infection. AIDS-related NHL remains an important biologic model for investigating the development and progression of high-grade NHLs as well as NHLs that develop in immune-deficient hosts.
B-Lymphocytes↗
Primary body cavity-based AIDS-related lymphomas.
Five patients with advanced AIDS developed a unique type of high grade primary body cavity-based non-Hodgkin's lymphoma (NHL). The lymphomas were exclusively in serous effusions with no detectable mass disease in the body cavities and no lymphadenopathy or organomegaly. All of the lymphomas exhibited virtually identical morphology, which could not be precisely classified, but appeared to bridge features of large cell immunoblastic and anaplastic large cell lymphomas. Immunophenotypically the lymphoma cells lacked expression of any B- or T-lymphocyte antigens, but expressed CD45 and the activation antigens CD30, CD38, CD71, and HLA-DR. Clonally rearranged immunoglobulin heavy chain and kappa light chain genes were identified by Southern blot analysis. Molecular studies also revealed Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) genomes and germline configuration of the c-myc protooncogene. In two cases studied cytogenetically, the lymphoma cells manifested complex chromosome abnormalities. These lymphomas are clinically and biologically unique and found predominantly in patients with advanced AIDS, in many cases with pre-existing Kaposi's sarcoma.
Adult↗
Primary characterization of a herpesvirus agent associated with Kaposi's sarcomae.
Detection of novel DNA sequences in Kaposi's sarcoma (KS) and AIDS-related body cavity-based, non-Hodgkin's lymphomas suggests that these neoplasms are caused by a previously unidentified human herpesvirus. We have characterized this agent using a continuously infected B-lymphocyte cell line derived from an AIDS-related lymphoma and a genomic library made from a KS lesion. In this cell line, the agent has a large episomal genome with an electrophoretic mobility similar to that of 270-kb linear DNA markers during clamped homogeneous electric field gel electrophoresis. A 20.7-kb region of the genome has been completely sequenced, and within this region, 17 partial and complete open reading frames are present; all except one have sequence and positional homology to known gammaherpesvirus genes, including the major capsid protein and thymidine kinase genes. Phylogenetic analyses using both single genes and combined gene sets demonstrated that the agent is a gamma-2 herpesvirus (genus Rhadinovirus) and is the first member of this genus known to infect humans. Evidence for transient viral transmission from infected to uninfected cells is presented, but replication-competent virions have not been identified in infected cell lines. Sera from patients with KS have specific antibodies directed against antigens of infected cell lines, and these antibodies are generally absent in sera from patients with AIDS without KS. These studies define the agent as a new human herpesvirus provisionally assigned the descriptive name KS-associated herpesvirus; its formal designation is likely to be human herpesvirus 8.
Amino Acid Sequence↗
Kaposi's sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposi's sarcoma and malignant lymphoma.
A new human herpesvirus was recently identified in all forms of Kaposi's sarcoma (Kaposi's sarcoma-associated herpesvirus [KSHV] or human herpesvirus 8), as well as in primary effusion (body cavity-based) lymphomas (PELs). A 12.3-kb-long KSHV clone was obtained from a PEL genomic library. Sequencing of this clone revealed extensive homology and colinearity with the right end of the herpesvirus saimiri (HVS) genome and more limited homology to the left end of the Epstein-Barr virus genome. Four open reading frames (ORFs) were sequenced and characterized; these are homologous to the following viral and/or cellular genes: (i) Epstein-Barr virus membrane antigen p140 and HVS p160, (ii) HVS and cellular type D cyclins, (iii) HVS and cellular G protein-coupled receptors, and (iv) HVS. Since there is considerable evidence that cyclin D1 and some G protein-coupled receptors contribute to the development of specific cancers, the presence of KSHV homologs of these genes provides support for a role for KSHV in malignant transformation. All ORFs identified are transcribed in PELs and Kaposi's sarcoma tissues, further suggesting an active role for KSHV in these diseases.
Base Sequence↗
Molecular characterization of primary mediastinal B cell lymphoma.
Primary mediastinal B cell lymphoma (PMBL) is a diffuse large B cell lymphoma (DLCL) postulated to arise from noncirculating thymic B lymphocytes. Because of its distinctive clinical and morphological features and putative unique cellular origin, PMBL is generally considered a distinct clinicopathological entity. Little is known, however, about the molecular characteristics of PMBL. Therefore, we analyzed 16 PMBLs for molecular alterations involving the bcl-1, bcl-2, bcl-6, c-myc, H-ras, K-ras, N-ras, and p53 genes and for Epstein-Barr virus infection, which are commonly involved in lymphoid neoplasia. Employing a combination of Southern blotting and/or polymerase chain reaction and single-strand conformation polymorphism assays, we detected genetic alterations in 7 of the 16 (44%) PMBLs. Whereas the bcl-6 gene is rearranged in up to 45% of DLCLs, rearrangement of the bcl-6 gene was detected in only 1 of these 16 (6%) PMBLS. Point mutations of the 5' noncoding region of the c-myc gene were demonstrated in 3 other cases (19%), although c-myc gene rearrangements were not seen by Southern blotting. Missense point mutations of the p53 gene were identified in 3 additional PMBLs (19%). Alterations of the bcl-1, bcl-2, or ras genes and evidence of Epstein-Barr virus infection were not observed. In conclusion, a variety of molecular lesions occur in PMBLs and may be involved in their pathogenesis. This molecular genetic pattern bears little resemblance to that known for other B cell malignancies, including DLCL. In particular, the infrequent occurrence of bcl-6 gene rearrangement in PMBLs distinguishes them from other DLCLs of B cell origin, suggesting that PMBLs do not represent a distinct subtype of DLCL.
Adolescent↗
Establishment of AIDS-related lymphoma cell lines from lymphomatous effusions.
AIDS-related non-Hodgkin lymphomas (AIDS-NHL) are most frequently derived from B cells and include small non-cleaved cell lymphoma (SNCCL) and diffuse large cell lymphoma (DLCL) and less frequently anaplastic large cell lymphoma (ALCL) or body cavity-based lymphoma (BCBL). AIDS-NHL cell lines have proved useful to study AIDS-NHL pathogenesis. In this report, we describe the establishment and molecular characterization of two novel AIDS-NHL cell lines (HBL-4 and HBL-6) derived from lymphomatous effusions. HBL-4 was derived from a patient with SNCCL, whereas HBL-6 was derived from a patient with BCBL. The identity of the cell lines with the original tumor clone was established by immunoglobulin gene rearrangement analysis. Both HBL-4 and HBL-6 carry a monoclonal EBV infection and do not contain HIV. In addition, HBL-6 harbors DNA sequences of the recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), now formally called human herpesvirus 8 (HHV8). Finally, HBL-4, but not HBL-6, harbors a rearranged c-MYC allele, while the BCL-6 gene displayed a germline configurations in both cell lines. These AIDS-NHL cell lines may prove useful in understanding the biologic events contributing to AIDS-NHL development.
Ascitic Fluid↗
Kaposi's sarcoma-associated herpesvirus in non-AIDS related lymphomas occurring in body cavities.
DNA sequences belonging to the recently discovered Kaposi's sarcoma-associated herpesvirus (KSHV), now provisionally designated human herpesvirus 8, have been previously identified in an uncommonly occurring subset of AIDS-related lymphomas, referred to as body-cavity-based lymphomas (BCBLs), which present as lymphomatous effusions. Pyothorax-associated lymphomas (PALS) are non-Hodgkin's lymphomas that arise in the pleural cavity after long-standing pleural inflammation resulting from therapeutic artificial pneumothorax or from tuberculosis pleuritis. Although PALs present as solid tumor masses, they are otherwise similar to BCBLs in that they also are B cell lymphomas, usually exhibit immunoblastic morphology, and contain Epstein-Barr virus. We investigated whether KSHV sequences are present in 2 BCBLs in patients without AIDS and 12 in Japanese and 2 French PALs. The 2 BCBLs were positive for KSHV sequences, whereaas all 14 PALs were KSHV negative. This finding strongly suggests that BCBLs and PALs are distinct clinicopathological entities and further strengthens the association between the presence of KSHV and an effusion phenotype. Based on these findings, we propose replacing the term body-cavity-based lymphoma with the term primary effusion lymphoma, which describes these non-Hodgkin's lymphomas more accurately and avoids confusion with other lymphomas that may occur in the body cavities, such as the PALs.
Aged↗
Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma.
The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression.
Alleles↗
Herpes-like DNA sequences in a body-cavity-based lymphoma in an HIV-negative patient.
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Aged↗
In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi's sarcoma-associated herpesvirus-like (KSHV) DNA sequences.
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.
Base Sequence↗
Different clonal origin of B-cell populations of chronic lymphocytic leukemia and large-cell lymphoma in Richter's syndrome.
Richter's syndrome is defined as the morphologic transformation of chronic lymphocytic leukemia (CLL) into diffuse large-cell lymphoma (DLL). To determine the clonal nature of the two malignancies, we microdissected the CLL and DLL cells from a lymph node of Richter's syndrome and analyzed the sequences of the rearranged Ig VH-D-JH genes of the two lymphomas. Using the Ig VH-D-JH sequence as a marker of clonality, we delineated the clonal relationship of the CLL and DLL cells. The microdissected CLL and DLL cells productively rearranged different VH, D, and JH genes, suggesting that these DLL B cells emerge as discrete elements independent of the CLL B-cell population. The productively rearranged Ig V gene sequence of the CLL clone was 100% identical to the VH6 germline gene, but the rearranged Ig VH gene of the DLL clone was somatically point-mutated based on comparison of its sequence with those of reported germline genes. In the DLL clone, the random distribution and nature of the somatic point-mutations suggests a lack of antigen selection; the identity of the somatic point-mutations in multiple independent isolates of the same B-cell clone suggests a lack of intraclonal diversity. Thus, Richter's syndrome DLL B cells are monoclonal and can emerge as discrete elements independent of the preexisting CLL cells; antigen selection and clonal diversification are not necessarily associated with the events leading to this aggressive neoplastic transformation.
Aged↗
Cellular origin, antigen reactivity, and VH segment structure of IgM mAbs from AIDS lymphomas.
In the present studies we analyzed the Ag specificity, VH gene structure, and cellular origin of three IgM mAb-producing cell lines established in vitro from bioptic specimens of three AIDS patients with BL. We found that (i) both HBL-2 and HBL-3 IgM mAbs were cold agglutinins highly specific for the i blood group determinant, a self Ag the expression of which is dominant in the early stages of life, and both mAbs used somatically point-mutated VH 4-21 segments; (ii) HBL-1 IgM mAb, the Ag-specificity of which has not been determined, used a putatively mutated member of the VHIII family; and (iii) both HBL-1 and HBL-2, but not HBL-3, cells expressed CD5 mRNA, suggesting a B-1 cell origin. The utilization of VH4-21 by the HBL-2 and HBL-3 cold agglutinins is consistent with the usage of this gene segment by all the reported pathogenic except the naturally occurring cold agglutinins. This restricted VH gene usage may reflect an inherent affinity of the germline VH4-21 gene product for the i/I carbohydrate structure, and, perhaps, an overrepresentation of VH4-21 in the human early and late B-cell repertoire. Consistent with both an early and late developmental expression of the VH4-21 gene is the B-1 and B-2 cellular origin of the two VH4-21+ cold agglutinins reported here. Thus, the two cold agglutinin autoantibodies possibly emerged at different stages of the natural history of the B-cell repertoires of these patients and might display a different temporal relationship, as discussed below, to the time of emergence of the respective tumoral cells. The somatically mutated status of the HBL-2 and HBL-3 mAb VH segments was suggested by the monomorphism of the human VH4-21 gene, the extension of the nucleotide differences to the, in general, highly conserved JH segment; and it was formally proved in HBL-3 mAb. Positive selection by Ag of the R mutations in the HBL-2 and HBL-3 mAb VH segments was suggested by the differential R:S mutation ratios in the CDRs and FRs (HBL-2 mAb, 5.0 and 1.1, respectively; HBL-3 mAb, 2.2 and 0.3, respectively) but not substantiated by appropriate statistical analysis according to the binomial distribution model.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Molecular genetic analysis demonstrates that multiple posttransplantation lymphoproliferative disorders occurring in one anatomic site in a single patient represent distinct primary lymphoid neoplasms.
BACKGROUND: Posttransplantation lymphoproliferative disorders (PT-LPDs) are a clinicopathologically heterogeneous group of lymphoid proliferations of varied clonal composition, the majority of which are associated with Epstein-Barr virus (EBV) infection. The clonal content and clonal relatedness of 24 separate PT-LPD lesions occurring synchronously in one organ in a single patient were investigated. METHODS: Twenty-four separate PT-LPD lesions from the colon and mesentery of a 15-year-old male, developing 4 months after cardiac transplantation, were studied for clonality based on immunoglobulin heavy chain (IgH) gene rearrangements for the presence, clonality, and type of EBV infection and for the presence of c-myc, ras, and p53 gene alterations. Southern blot hybridization, polymerase chain reaction, and single strand conformation polymorphism assays were employed. RESULTS: All 24 lesions were histologically similar (polymorphic B-cell lymphomas) but exhibited varied clonality and were clonally distinct with respect to both IgH gene rearrangements and EBV infection. All lesions were infected with EBV type A. Structural alterations of oncogenes or tumor suppressor genes were not identified. CONCLUSIONS: Separate PT-LPD lesions occurring synchronously in a single organ or patient may be clonally distinct, suggesting that they represent multiple distinct primary lymphoid proliferations rather than metastatic disease as in conventional malignant lymphomas. This may explain partially the rapid development in some patients of a large PT-LPD tumor burden that may regress rapidly after reduction of immunosuppression.
Adolescent↗
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.
BACKGROUND: DNA fragments that appeared to belong to an unidentified human herpesvirus were recently found in more than 90 percent of Kaposi's sarcoma lesions associated with the acquired immunodeficiency syndrome (AIDS). These fragments were also found in 6 of 39 tissue samples without Kaposi's sarcoma, including 3 malignant lymphomas, from patients with AIDS, but not in samples from patients without AIDS. METHODS: We examined the DNA of 193 lymphomas from 42 patients with AIDS and 151 patients who did not have AIDS. We searched the DNA for sequences of Kaposi's sarcoma-associated herpesvirus (KSHV) by Southern blot hybridization, the polymerase chain reaction (PCR), or both. The PCR products in the positive samples were sequences and compared with the KSHV sequences in Kaposi's sarcoma tissues from patients with AIDS. RESULTS: KSHV sequences were identified in eight lymphomas in patients infected with the human immunodeficiency virus. All eight, and only these eight, were body-cavity-based lymphomas--that is, they were characterized by pleural, pericardial, or peritoneal lymphomatous effusions. All eight lymphomas also contained the Epstein-Barr viral genome. KSHV sequences were not found in the other 185 lymphomas. KSHV sequences were 40 to 80 times more abundant in the body-cavity-based lymphomas than in the Kaposi's sarcoma lesions. A high degree of conservation of KSHV sequences in Kaposi's sarcoma and in the eight lymphomas suggests the presence of the same agent in both lesions. CONCLUSIONS: The recently discovered KSHV DNA sequences occur in an unusual subgroup of AIDS-related B-cell lymphomas, but not in any other lymphoid neoplasm studied thus far. Our finding strongly suggests that a novel herpesvirus has a pathogenic role in AIDS-related body-cavity-based lymphomas.
Acquired Immunodeficiency Syndrome↗
Posttransplantation lymphoproliferative disorders frequently contain type A and not type B Epstein-Barr virus.
Two families of Epstein-Barr virus (EBV), type A and type B, have been defined on the basis of sequence divergence in the EBNA-2 gene. Type A EBV immortalizes B cells more efficiently in vitro and infects immunocompetent individuals more commonly than type B EBV. However, increased rates of infection by type B EBV are seen in immunocompromised hosts and in many lymphoid neoplasms associated with immunocompromise. The posttransplantation lymphoproliferative disorders (PT-LPDs) are a heterogeneous group of B-cell neoplasms that arise in the setting of immunosuppressive therapy, and are associated with EBV infection. Whether type A and/or type B EBV are associated with PT-LPDs is unknown. Therefore, we investigated 27 PT-LPD lesions from 22 solid-organ transplant recipients by polymerase chain reaction (PCR) at the EBNA-2 and EBNA-3c loci to detect sequence deletions that distinguish the two EBV families. Another locus, EBER, was examined by single-strand conformation polymorphism analysis (SSCP), in conjunction with direct sequencing in selected cases. Type A EBV was found in 24 of 27 cases (89%) as seen by amplification of the EBNA-2 and EBNA-3c regions. Four different EBER polymorphisms were detected, confirming the presence of different type A EBV isolates among these cases. Three cases were negative for infection by EBV. Surprisingly, despite the immunocompromised state of the hosts, none of the 27 PT-LPD lesions harbored type B EBV. Thus, although type B EBV may commonly infect peripheral blood lymphocytes in immunocompromised individuals, they do not appear to induce readily PT-LPD formation.
Antigens, Viral↗
Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders.
The posttransplantation lymphoproliferative disorders (PT-LPDs) are a morphologically heterogeneous group of Epstein-Barr virus (EBV)-driven lymphoid proliferations of varying clonal composition. Some PT-LPDs regress after a reduction in immunosuppression, while others progress in spite of aggressive therapy. Previously defined morphologic categories do not correlate with clonality, and neither morphology nor clonality has reliably predicted the clinical behavior of PT-LPDs. We investigated 28 PT-LPD lesions occurring in 22 patients for activating alterations involving the bcl-1, bcl-2, c-myc, and H-, K- and N-ras proto-oncogenes and for mutations involving the p53 tumor suppressor gene. We correlated the results of these studies with the morphology of the lesions, their clonality based on Ig heavy and light chain gene rearrangement analysis, and the presence and clonality of EBV infection. We found that the PT-LPDs are divisible into three distinct categories as follows: (1) plasmacytic hyperplasia: most commonly arise in the oropharynx or lymph nodes, are nearly always polyclonal, usually contain multiple EBV infection events or only a minor cell population infected by a single form of EBV, and lack oncogene and tumor suppressor gene alterations; (2) polymorphic B-cell hyperplasia and polymorphic B-cell lymphoma: may arise in lymph nodes or various extranodal sites, are nearly always monoclonal, usually contain a single form of EBV, and lack oncogene and tumor suppressor gene alterations; and (3) immunoblastic lymphoma or multiple myeloma: present with widely disseminated disease, are monoclonal, contain a single form of EBV, and contain alterations of one or more oncogene or tumor suppressor genes (N-ras gene codon 61 point mutation, p53 gene mutation, or c-myc gene rearrangement). The PT-LPDs are divisible into three categories exhibiting distinct morphologic and molecular genetic characteristics. Alterations involving the N-ras and c-myc proto-oncogenes and the p53 tumor suppressor gene may play an important role in the development and/or progression of the PT-LPDs.
Base Sequence↗
Alterations of the BCL-6 gene in diffuse large-cell lymphoma.
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B-Lymphocytes↗