Human leukocyte antigens and genetic susceptibility to lymphoma.

Familial aggregation, coupled with ethnic variation in incidence, suggests that inherited susceptibility plays a role in the development of lymphoma, and the search for genetic risk factors has highlighted the contribution of the human leukocyte antigen (HLA) complex. In a landmark study published almost 50 years ago, Hodgkin lymphoma (HL) was the first disease to be associated with HLA variation. It is now clear that Epstein-Barr virus (EBV)-positive and -negative HL are strongly associated with specific HLA polymorphisms but these differ by EBV status of the tumours. HLA class I alleles are consistently associated with EBV-positive HL while a polymorphism in HLA class II is the strongest predictor of risk of EBV-negative HL. Recent investigations, particularly genome-wide association studies (GWAS), have also revealed associations between HLA and common types of non-Hodgkin lymphoma (NHL). Follicular lymphoma is strongly associated with two distinct haplotypes in HLA class II whereas diffuse large B-cell lymphoma is most strongly associated with HLA-B*08. Although chronic lymphocytic leukaemia is associated with variation in HLA class II, the strongest signals in GWAS are from non-HLA polymorphisms, suggesting that inherited susceptibility is explained by co-inheritance of multiple low risk variants. Associations between B-cell derived lymphoma and HLA variation suggest that antigen presentation, or lack of, plays an important role in disease pathogenesis but the precise mechanisms have yet to be elucidated.

Configuration and expression of the T cell receptor beta chain gene in human T-lymphotrophic virus I-infected cells.

We studied the configuration and expression of the gene encoding the beta chain of the T cell receptor (TCR beta) in cell lines and primary tumor cells infected by the human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I). Most of the cell lines and all the primary tumor cells showed rearrangement of the TCR beta gene, and in each case the rearrangement was distinct. The majority of cases examined were clonal with respect to a particular TCR beta gene rearrangement. Primary tumor cells from one case (SD) were found to have a tandem duplication of a portion of chromosome 7; this appears to have resulted in the presence of three alleles of the TCR beta gene, each of which is arranged differently. This suggests that the chromosomal abnormality, and possibly infection by HTLV-I, occurred before TCR beta gene rearrangement. Cell lines infected by HTLV-I express levels of TCR beta mRNA similar to PHA stimulated lymphocytes, suggesting that this gene is not transcriptionally activated as a result of infection by HTLV-I. Cloned T cells of known antigen specificity that are infected by HTLV-I in vitro show impairment of immune function, including loss of antigen-specific responsiveness and the acquisition of alloreactivity. Comparison of the configuration of the TCR beta gene before and after infection revealed no changes detectable by Southern blot analysis. Levels of expression of the TCR beta gene at the mRNA level and surface expression of the T3 complex were also not significantly altered, suggesting that changes in immune function cannot be attributed to quantitative changes in the TCR molecule. The configuration of the TCR beta gene in primary tumor cells infected by HTLV-I was compared with that in the derived cell lines. In all pairs examined, the configuration in the primary tumor cells was different from that in the cell lines, strongly suggesting that the cells that grow in culture are not the original neoplastic cells.

The prevalence and characterisation of TRAF3 and POT1 mutations in canine B-cell lymphoma.

The genetic and mutational basis of canine lymphoma remains poorly understood. Several genes, including TRAF3 and POT1, are mutated in canine B-cell lymphoma (cBCL), and are likely involved in the pathogenesis of this disease. The purpose of this study was to assess the prevalence of TRAF3 and POT1 mutations in a cohort of dogs with cBCL, compared to dogs with non-cBCL diseases (including four dogs with T-cell lymphoma [cTCL]). Forty-nine dogs were included (n = 24 cBCL; n = 25 non-cBCL). Eleven dogs had matched non-tumour DNA assessed to determine if mutations were germline or somatic. All dogs had TRAF3 and POT1 assessed by Sanger sequencing. The prevalence of deleterious TRAF3 and POT1 mutations in cBCL was 36% and 17%, respectively. A deleterious TRAF3 mutation was suspected to be germline in 1/5 cases with matched non-tumour DNA available for comparison. Deleterious mutations were not found in specimens from the non-cBCL group. Several synonymous variants were identified in both genes in cBCL and non-cBCL samples, which likely represent polymorphisms. These results indicate TRAF3 and POT1 mutations are common in cBCL. Deleterious TRAF3 and POT1 mutations were only identified in dogs with cBCL, and not in dogs with non-cBCL diseases, suggesting they are important in the pathogenesis of cBCL. Future studies to investigate the prognostic and therapeutic implications of these mutations are required.

Infection of human T lymphotropic virus-I-specific immune T cell clones by human T lymphotropic virus-I.

Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection.

Molecular methods for virus discovery.

The potential use of novel cell substrates from diverse animal species raises concerns about the transmission of hitherto unknown viral agents. Viruses that do not cause a cytopathic effect in cell culture may escape detection by conventional methods and molecular methods may therefore prove useful for screening for hitherto unknown viruses. This review describes currently used molecular methods for virus discovery, including degenerate PCR assays, representational difference analysis and rolling circle amplification, and summarises the advantages and disadvantages of each technique.

Epstein-Barr virus-specific cytotoxic T lymphocyte responses in the blood and tumor site of Hodgkin's disease patients: implications for a T-cell-based therapy.

Approximately 40% of Hodgkin's disease (HD) cases carry EBV in the malignant Hodgkin-Reed Sternberg (H-RS) cells, with expression of viral latent membrane proteins (LMPs) 1 and 2. These viral proteins are targets for CTLs in healthy EBV carriers, and their expression in EBV-associated HD raises the possibility of targeting them for a CTL-based immunotherapy. Here we characterize the CTL response to EBV latent antigens in both the blood and tumor-infiltrating lymphocytes of HD patients using two approaches: (a) in vitro reactivation of CTLs by stimulation with the autologous EBV-transformed lymphoblastoid cell line; and (b) an enzyme-linked immunospot assay to quantify frequencies of CTLs specific for known LMP1/2 epitopes. We detected EBV-specific CTLs in blood and biopsy samples from both EBV-negative and EBV-positive HD patients. However, as in healthy EBV carriers, LMP-specific CTL precursors occurred only at low frequency in the blood of HD patients, and with the exception of one EBV-negative HD case, were undetectable in the tumor. These data give rise to two considerations: (a) they may explain why EBV-positive tumor cells persist in the presence of an existing EBV-specific immune response; and (b) they provide a rationale for selectively boosting/eliciting LMP-specific CTL responses as a therapy for EBV-positive HD.

Mutations in the IkBa gene in Hodgkin's disease suggest a tumour suppressor role for IkappaBalpha.

The NF-kappaB/Rel family of transcription factors regulates a wide variety of genes whose products play a fundamental role in inflammatory and immune responses. The implication of NF-kappaB/Rel proteins and their IkappaB regulatory subunits in the control of cellular growth and oncogenesis, was suggested by the induction of fatal lymphomas in birds by the v-rel oncoprotein, and the rearrangement and amplification of several genes encoding the NF-kappaB/Rel/IkappaB signal transduction factors in human malignancies, primarily of lymphoid origin. Hodgkin's disease (HD) is a lymphoma characterized by a low frequency of malignant Hodgkin and Reed-Sternberg (H/RS) cells in a reactive background of non-neoplastic cells. The peculiar activated phenotype of Hodgkin and Reed-Sternberg cells and their pattern of cytokine secretion are believed to be a consequence of constitutive activation of the NF-kappaB transcription factor. Here, we report the detection of mutations of the IkBa gene, in two HD-derived cell lines and in two out of eight biopsy samples from patients with relapsed Hodgkin's disease. The presence of defective IkappaBalpha is thus likely to explain the constitutive activation of NF-kappaB in these cells and suggests that IkappaBalpha is a tumour suppressor controlling the oncogenic activation of NF-kappaB in Hodgkin and Reed-Sternberg cells.

Viruses and Hodgkin's disease.

Hodgkin's disease (HD) is a heterogeneous condition with distinct histological and epidemiological subgroups. Recent data provide evidence that the Epstein-Barr virus (EBV) is associated with a significant proportion of cases. Clonal EBV genomes have been detected in affected tissues and EBV has been localised to RS cells. The significance of these findings is reinforced by the detection of the EBV latent gene product LMP-1, which has known transforming potential, within RS cells in EBV-associated cases. The age distribution of EBV-associated cases is non-random. Paediatric cases, particularly those aged < 10 years, are likely to be EBV-associated as are older adult cases. In contrast, a smaller proportion of young adult cases is EBV-associated, and nodular sclerosis HD cases within this age group are positive infrequently. The results of our studies provide support for the hypothesis that HD has multiple aetiologies but do not support the polio model. The epidemiological evidence suggesting that HD may have an infectious aetiology is strongest for the young adult age group. EBV was suggested as a candidate virus however it is in these cases that there is least evidence for involvement of EBV. It would seem plausible that another virus, possibly another common childhood infectious agent, is responsible for the incidence peak seen in this age group in developed countries.

Identification of human herpesvirus 6-specific DNA sequences in two patients with non-Hodgkin's lymphoma.

Human herpesvirus 6 (HHV-6) is a recently discovered virus which has not been causally linked to any particular disease. In order to investigate the possible role of this virus in the pathogenesis of lymphoid malignancies, we examined tissue samples from 117 patients for the presence of HHV-6-specific DNA sequences. Two cases of non-Hodgkin's lymphoma were found to be positive. One patient had a T cell lymphoma and a preceding history of angioimmunoblastic lymphadenopathy; the other had a B cell lymphoma occurring in the context of Sjögren's syndrome. HHV-6 has been isolated previously from a patient with angioimmunoblastic lymphadenopathy, and viral sequences have been identified in another patient with Sjögren's syndrome and B cell lymphoma. The relationship between HHV-6 and these conditions therefore warrants further investigation.

Epstein-Barr virus and Hodgkin's disease: further evidence for the three disease hypothesis.

The epidemiology of Hodgkin's disease suggests that it is a heterogeneous condition comprising more than one disease entity. The Epstein-Barr virus (EBV) is present in the Reed-Sternberg cells of a proportion of cases and is likely to play a role in the pathogenesis of these cases. In this study we show that EBV association rates vary with age at diagnosis. We suggest that Hodgkin's disease can be divided into three disease entities on the basis of EBV association and age, thereby providing biological support for the multiple aetiology hypothesis proposed by MacMahon (Cancer Res 1966; 26: 1189-1290).

Screening for herpesvirus genomes in common acute lymphoblastic leukemia.

There is epidemiological evidence that infection may play a role in the etiology of childhood leukemia in particular common B cell precursor acute lymphoblastic leukemia. A panel of 20 leukemic samples (panel 1) was examined for the presence of four lymphotropic herpesviruses using conventional molecular techniques. A second independent panel of 27 leukemic samples (panel 2), along with 28 control peripheral blood samples from children with other forms of cancer, was tested for the presence of the same four viruses using sensitive real-time quantitative PCR. While herpesvirus genomes were detected, they were present at very low levels; detection rates and levels were similar in the leukemic and control panels. In addition we surveyed 18 leukemic samples (five from panel 1, six from panel 2 and a further seven samples not previously analyzed) using a degenerate PCR assay capable of detecting the genomes of known herpesviruses plus putative new members of the family. No novel herpesvirus genomes were detected suggesting that a herpesvirus is unlikely to be etiologically involved as a transforming agent in common acute lymphoblastic leukemia.

Epstein-Barr virus and other candidate viruses in the pathogenesis of Hodgkin's disease.

The epidemiologic and clinicopathologic features of Hodgkin's disease (HD) suggest that an infectious agent is involved in the etiology. Over the last 12 years, evidence has accumulated suggesting that Epstein-Barr virus (EBV) is associated with a proportion of cases: EBV genomes are present in Reed-Sternberg (HRS) cells and viral proteins including LMP1, which has oncogenic potential, are expressed. HD has a complex epidemiology and EBV-associated cases are not randomly distributed. Disease occurring in early childhood and older adult age groups is more likely to be EBV-associated than for young adult cases. Paradoxically, there is more evidence supporting an infectious etiology in the latter group of younger patients. Defective EBV genomes and "hit and run" mechanisms involving EBV cannot account for all cases, and the direct involvement of known viral agents, including other lymphotropic herpesviruses, has largely been excluded. Hitherto unknown virus may be responsible for the peak incidence in young adults, which is a feature of HD in developed countries.

Epstein-Barr Virus and HLA-DPB1-*0301 in young adult Hodgkin's disease: evidence for inherited susceptibility to Epstein-Barr Virus in cases that are EBV(+ve).

Cases of Hodgkin's disease (HD) may be distinguished by whether they do [EBV-positive ((+ve)) cases] or do not [EBV-negative ((-ve)) cases] have evidence of EBV DNA in the Reed-Sternberg cells. Only one study has attempted to distinguish epidemiological risk factors for EBV(+ve) and EBV(-ve) HD, and none have compared inherited susceptibility. The present study involves a population-based case series of HD, diagnosed in patients between 16-24 years of age in the United Kingdom (n = 118), of whom 87% were classified by EBV status (EBV(+ve), 19, EBV(-ve), 84). History of infectious illness, EBV antibody titers, and HLA-DPB1 type have been compared in EBV(+ve) and EBV(-ve) cases. Reported infectious mononucleosis was more frequent in EBV(+ve) cases (odds ratio (OR), 5.10; 95% confidence interval (CI), 1.12-24.4). EBV antibody titers to viral capsid antigen were significantly higher in EBV(+ve) cases (P for trend = 0.02). Higher proportions of EBV(+ve) (43%) than EBV(-ve) (31%) cases typed positive for HLA-DPB1*0301, but this was not statistically significant; the association of infectious mononucleosis with EBV(+ve) cases was stronger in this HLA subgroup (OR, 17.1; 95%CI, 1.06-1177) than in other cases (OR, 1.24; 95% CI, 0.02-15.4). Although these results are based on small numbers of HD cases, they provide suggestive evidence that the etiology of EBV(+ve) HD may involve inherited susceptibility to EBV.

Case clustering, Epstein-Barr virus Reed-Sternberg cell status and herpes virus serology in Hodgkin's disease: results of a case-control study.

The Leukaemia Research Fund Data Collection Study (DCS) is a specialist registry of leukaemias and lymphomas. The present study involves 494 cases of Hodgkin's disease (HD) registered with the DCS between 1985 and 1989. This entire data set has been tested for localised spatial clustering using an established nearest neighbour method with 18% of all cases in young people classified as clustered (P < 0.05). No clustering was found in older cases. Subsamples were selected from the registered cases for a pilot study in which case clustering, herpes virus antibody titres and Epstein-Barr virus (EBV) presence within the Reed-Sternberg (RS) cells (EBV-RS status) were investigated together. Firstly, a case-control study of HD in young people or nodular sclerosing (NS) subtype (39 HD cases and 26 healthy controls) found significant elevation of antibody titres to EBV-viral capsid antigen (VCA), EBV-early antigen (EA) and human herpes virus 6 (HHV-6) in HD cases compared with controls. EBV viral genome was present in 5 cases and 4 of these were in clusters of HD in young people. Elevation of antibody titres to the EBV antigens was not associated with case clustering or EBV-RS status. Antibody titres to HHV-6 differed significantly between EBV-RS+ and EBV-RS- cases (P = 0.04). Geometric mean titres for HHV-6 for EBV-RS+ and EBV-RS- cases were 11.5 and 73.7, respectively, with the former lower than the control value of 20.5. Secondly, a cluster study included all other cases (n = 14) in clusters containing known EBV-RS+ cases. 3 further cases were EBV-RS+ positive but no cluster consisted entirely of positive cases. Overall, 5/16 clustered, 2/12 peripheral and 1/25 random cases in these studies were EBV-RS+ (P = 0.017). The interpretation of these results in terms of shared aetiological exposures of cases within clusters and the roles of EBV and HHV-6 is discussed, and hypotheses for testing in future studies proposed.

Sensitive ELISA for the gp120 and gp160 surface glycoproteins of HIV-1.

We have used a panel of polyclonal and monoclonal antibodies against gp120 and gp160, the envelope glycoproteins of human immunodeficiency virus type 1, to create rapid, simple, and sensitive twin-site sandwich ELISA specific for gp120 and gp160 or for gp160 alone. These assays can detect 500 COS cells in a population transiently transfected with a construct encoding gp120 and gp160, or 50 pg of recombinant gp160. We estimate that the mean amount of gp120 + gp160 in the transfected population is equivalent to 2.5 x 10(6) molecules per cell, 40-50% of which can be recovered from the culture medium as gp120 after 24 hours. The ELISA can be adapted to assess whether gp120 is detectable in the sera of HIV-1-infected persons: we show that gp120/gp160 is completely stable in normal human serum for at least 24 hours at 37 degrees C.

A model to study viral and cytokine involvement in Sjögren's syndrome.

To investigate mechanisms that may be important in the pathogenesis of Sjögren's syndrome (SS) we developed a protocol for the growth of salivary gland epithelial cells in culture. We examined the effect that viral infection has on the cellular location of the autoantigen La. Autoantibodies to La are common in SS and it has been proposed that viral infection may result in cell membrane expression of La. Co-expression of MHC class II molecules in infected cells could lead to the presentation of La peptides to the immune system. Advenovirus infection of salivary gland epithelial cells resulted in an altered nuclear staining of La. Treatment with interferon-gamma resulted in the expression of La in the cell cytoplasm and HLA-DR molecules at the cell surface. These findings suggest that a cytokine-driven mechanism may generate an autoimmune response to La in SS. Using the polymerase chain reaction (PCR) we tested salivary gland epithelial cell cultures for the presence of human herpesvirus-6 (HHV-6) and Epstein-Barr virus (EBV). Only HHV-6 was detected in 2 of 10 salivary gland epithelial cell cultures although the presence of HHV-6 was not associated with SS. Primary salivary gland cultures may prove useful as an in vitro model to study mechanisms of autoimmunity in SS.

Interdigitating dendritic cell sarcoma of salivary gland associated lymphoid tissue not associated with HHV-8 or EBV infection.

Interdigitating dendritic cell sarcoma (IDCS) is an extremely rare malignancy derived from professional antigen presenting cells. This report describes a case of IDCS arising in the salivary gland associated lymphoid tissue of the parotid gland of a 51 year woman, presenting with a painless neck swelling. Histologically, sheets of S100(+)/Ccd68(+)/CD45(+)/CD34(-)/CD1a(-) spindle cells were surrounded with an inflammatory infiltrate with no evidence of B or T cell clonal proliferations. No evidence of either human herpesvirus 8 or Epstein-Barr virus could be detected by quantitative polymerase chain reaction in the tumour cells with serological evidence of previous Epstein-Barr virus infection. The patient remains well and disease free 24 months after presentation without specific treatment.

Detection of Epstein-Barr virus genomes in Hodgkin's disease: relation to age.

An investigation as to whether any particular subgroup of patients with Hodgkin's disease was particularly likely to be Epstein-Barr virus (EBV) genome positive was made on samples from 95 patients. These were grouped according to age and Hodgkin's disease subtype, and analysed using Southern blot analysis. Most samples from children or adults aged 50 years or over contained detectable EBV genomes; samples from young adults were only rarely positive. The differences in EBV positivity by age were highly significant, but there was no significant association between EBV and histological subtype after allowing for the effect of age. The results support the hypothesis that Hodgkin's disease in different age groups may have different aetiologies, and suggest that EBV does have a pathogenetic role in Hodgkin's disease in children and older age groups.

Primary T cell lymphoma of salivary gland: a report of a case and review of the literature.

Lymphoma of the salivary gland accounts for 5% of cases of extranodal lymphoma and 10% of malignant salivary gland tumours. Most primary salivary gland lymphomas are B marginal zone lymphomas arising on a background of sialadenitis associated with autoimmune disorders such as Sjorgen's syndrome. Primary T cell lymphoma of the salivary gland is rare. This report describes a case of primary T cell lymphoma arising in the parotid gland of an elderly white man, which was notable for its striking resemblance to a B cell extranodal marginal zone lymphoma. Immunohistochemistry and gene rearrangement studies confirmed the clonal T cell nature of the tumour. There was no molecular evidence of Epstein-Barr virus (EBV) infection of neoplastic or surroundings cells. Only 14 cases of primary T cell lymphoma of the salivary glands have been recorded in the literature, most being from the Orient and having extremely variable prognosis. Those with a T/natural killer cell phenotype are associated with EBV infection. This case highlights the fact that T cell lymphoma in the salivary gland can mimic closely the morphological features of B cell extranodal marginal zone lymphoma.

The Scotland and Newcastle epidemiological study of Hodgkin's disease: impact of histopathological review and EBV status on incidence estimates.

AIMS: The epidemiological and pathological features of Hodgkin lymphoma (HL) are complex. The Epstein-Barr virus (EBV) is consistently associated with a proportion of cases, and these cases are thought to represent a distinct aetiological subgroup of HL. The aim of the present analysis was to determine the age and sex specific incidence of EBV associated and non-associated HL, analysed separately, using data derived from a population based study-the Scotland and Newcastle epidemiological study of Hodgkin's disease (SNEHD). This study also provided a unique opportunity to evaluate accuracy in the current diagnosis and classification of HL. METHODS: SNEHD analysed consecutive cases of HL diagnosed in the study area between 1993 and 1997. Diagnostic biopsy material was retrieved, EBV status of tumours was determined, and histological review was performed. RESULTS: In total, 622 cases were eligible for the study, and EBV studies and histopathological review were performed on biopsy material from 537 and 549 cases, respectively. Accuracy in the overall diagnosis of HL and classification of nodular sclerosis HL was good, but diagnosis of HL in the elderly and classification of other subtypes was less reliable. One third of classic HL cases were EBV associated, and age specific incidence curves for EBV associated and non-associated cases were distinct. CONCLUSIONS: Comparison of age specific incidence curves for EBV associated and non-associated HL supports the hypothesis that these are two distinct aetiological entities. Accuracy in the diagnosis of HL is generally good, but certain subgroups of cases continue to present diagnostic difficulties.