A population based, case control study of non-Hodgkin's lymphoma in patients with rheumatoid arthritis.

OBJECTIVE: Epstein-Barr virus (EBV) associated lymphoproliferative disorders (LPD) similar to those that occur in immunosuppressed solid organ recipients have been reported in patients with rheumatoid arthritis (RA). These LPD cause significant morbidity and/or mortality in a state of sustained immunosuppression, but may spontaneously regress if immunocompetence is restored. We determined the population based frequency of EBV associated LPD relative to all non-Hodgkin's lymphomas (NHL) that occur in the general population of patients with RA. METHODS: Forty-two case patients with NHL and RA and 49 control patients with NHL and no RA were identified in a population based, case control study of NHL that occurred in a 6 county Northern California area during the years 1988-94. The lymphoma tissue specimens were reviewed and the diagnosis of NHL was confirmed. In addition, the specimens were analyzed for NHL grade, histologic subtype, histopathologic features associated with immunosuppression, immunophenotype, and the presence of EBV genome in the tumor cells. RESULTS: No significant differences were identified between NHL in the RA case group and the control group (no RA) with respect to any variables investigated. One patient (2%) in the case group and one (2%) in the control group developed LPD containing EBV. CONCLUSION: Our findings reveal that EBV associated lymphomas represent only a small fraction of all NHL in the general RA patient population. EBV associated LPD should be recognized when they occur because they require a special approach to patient management. However, these data indicate that the majority of NHL that occurs in patients with RA is probably coincidental with RA and not the result of significant immunosuppression.

Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice.

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) sequences are present in primary effusion lymphomas (PEL). KSHV+ cell lines have been established from such lymphomas. Here we report the first description of the establishment of a KSHV+, EBV- cell line (BCP-1) from the peripheral blood of a patient with PEL. Using this cell line and a KSHV+, EBV+ PEL cell line (HBL-6) previously established from ascitic fluid, we investigated whether in nonobese diabetic/severe combined immunodeficiency disease (Nod/SCID) mice tumors representing PEL can be established. When injected intravenously (IV) into Nod/SCID mice, BCP-1 and HBL-6 infiltrated organs, with only occasional macroscopic tumor formation. Intraperitoneal injections (ip) led to the development of ascites and diffuse infiltration of organs, without obviously solid lymphoma formation, resembling the diffuse nature of human PEL. To investigate a possible mechanism for the peculiar phenotype of PEL, we examine the presence of adhesion molecules and homing markers on PEL cells before and after growing in mice. Both BCP-1 and HBL-6 cells lack expression of important cytoadhesion molecules including CD11a and CD18 (LFA1 alpha and beta chains), CD29, CD31, CD44, CD54 (ICAM-1), and CD62L and E (L and E selectins).

Absence of human herpesvirus 8 and Epstein-Barr virus genome sequences in cutaneous epithelial neoplasms arising in immunosuppressed organ-transplant patients.

Recent studies have implicated herpesvirus 8 and Epstein-Barr virus in the development of cutaneous malignancies in immunosuppressed patients. In order to examine the strength of this association, we examined 37 malignant, pre-malignant and benign cutaneous epithelial neoplasms removed from immunosuppressed organ recipients for the presence of human herpesvirus 8 and Epstein-Barr viral genome sequences using polymerase chain reaction (PCR) and in situ hybridization. We examined 2 actinic keratoses, 1 benign keratosis, 11 invasive squamous cell carcinomas, 17 squamous cell carcinomas in situ and 6 basal cell carcinomas. We also examined 4 basal cell carcinomas, 1 invasive squamous cell carcinoma and 3 squamous cell carcinomas in immunocompetent hosts. In contrast to findings reported by other investigators, we were unable to detect viral genome sequences in any of the biopsies examined. Our findings suggest that human herpesvirus 8 and Epstein-Barr virus likely do not play an etiologic role in cutaneous epithelial oncogenesis in immunocompromised patients.

Extranodal head and neck lymphomas in Guatemala: high frequency of Epstein-Barr virus-associated sinonasal lymphomas.

Sinonasal lymphomas of T cell or natural killer cell (T/NK cell) phenotype represent a subset of extranodal head and neck lymphomas. T/NK cell sinonasal lymphomas have been described in diverse geographic settings, including China, Japan, Peru, Northern Europe, and North America. The frequency of these lymphomas is highly dependent on the geographic location in which they occur, their incidence being low in Europe and North America and relatively high in Asian countries and in Peru. Regardless of their geographic location, they are typically associated with the Epstein-Barr virus (EBV). Few studies have addressed the relative frequency of sinonasal lymphoma within the group of extranodal head and neck lymphomas. We investigated the anatomic distribution, immunophenotypical profile, and EBV status of 33 cases of extranodal head and neck lymphoma from patients in Guatemala. The anatomic distribution of these lymphomas is similar to that seen in Asian countries: 17 (52%) in the sinonasal area, five (15%) in the palate, and 11 (33%) in other locations. Fifteen (88%) of the 17 sinonasal lymphomas showed a T or null cell phenotype with a strong association with EBV by in situ hybridization. Most Guatemalan patients with these lymphomas were of Mayan descent. In Guatemala, the relative frequency of sinonasal lymphomas within the group of head and neck lymphomas is significantly higher than that reported for Western countries. In addition, the relative frequency of T/NK versus B cell sinonasal lymphomas is higher than that described in North America and similar to that observed in Asian countries and Peru.

No role for human herpes virus 8 in the etiology of infantile capillary hemangioma.

Infantile capillary hemangiomas are vascular neoplasms that can appear quite infiltrative histologically and that are characterized by cords of cells with areas of marked cellularity. These lesions have been shown to contain a population of rapidly proliferative endothelial cells. Given the recent association between the presence of human herpes virus 8 (HHV8) and another proliferative vascular lesion, Kaposi's sarcoma, we used polymerase chain reaction technology to examine a series of 16 biopsy specimens from 15 infantile capillary hemangiomas for the presence of HHV8 DNA. We were unable to detect HHV8 DNA in any of the lesions studied. All of the cases were examined in parallel with a case of Kaposi's sarcoma that was known to be positive for HHV8 DNA and a series of negative controls. In all of our cases, amplification of beta-globin gene DNA demonstrated adequate preservation of DNA in the tissue studied. These findings suggest that not all endothelial cell proliferations can be attributed to HHV8 and that the etiology of some of these conditions remains unclear.

Epstein-Barr virus strain type and latent membrane protein 1 gene deletions in lymphomas in patients with rheumatic diseases.

OBJECTIVE: Recent studies have shown that immunomodulatory therapy for the treatment of rheumatic diseases can be associated with the development of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. The present study was undertaken to determine the strain type of EBV in lymphoproliferative disorders that occur in patients with rheumatic disease and to investigate EBV latent membrane protein 1 (LMP-1) gene deletions that occur in these lymphoproliferative disorders. METHODS: Ten EBV-associated lymphoid neoplasms in patients with rheumatoid arthritis or dermatomyositis were analyzed by polymerase chain reaction to determine EBV strain type and to investigate for the presence of a previously characterized 30-basepair deletion in the LMP-1 gene. RESULTS: The results indicated that lymphoproliferative disorders in these patients can harbor EBV strain type A or B, with a predominance of type A infection (80%). It was also shown that both wild-type and mutated LMP-1 genes can be found in these neoplasms, with the deleted form of the LMP-1 gene occurring in one-third of cases in this series. CONCLUSION: LMP-1 deletions associated with certain aggressive lymphoid neoplasms are not required for the genesis of lymphoproliferative disorders in patients with rheumatic disease. The relative frequencies of type A and type B EBV strains in these lymphoproliferative disorders show similarities to the frequencies in patients with post-solid organ transplantation immunosuppression-associated lymphoproliferative disorders.

Iatrogenic lymphoproliferative disorders in nontransplantation settings.

Iatrogenic (IAT) immunosuppression-related lymphoproliferative disorders (LPDs) can occur outside the solid organ transplantation setting in patients who receive immunomodulatory therapy for a variety of underlying diseases. Most frequently, these patients suffer from rheumatologic diseases and are receiving one or more therapies during the time that LPDs develop. Specific therapies include methotrexate, azathiaprine, cyclosporine, prednisone, and others as well as combinations of therapies. In a significant subset of patients, these IAT LPDs spontaneously regress when therapy is discontinued, a finding that implicates immunomodulatory therapy, at least in part, in the development of these LPDs. The morphological spectrum of IAT LPDs includes atypical polymorphous LPDs, diffuse aggressive non-Hodgkin's lymphomas, Hodgkin's disease, and lymphoproliferations resembling Hodgkin's disease. The diagnosis requires a combined morphological and immunonphenotypic approach; in situ hybridization studies for Epstein-Barr virus (EBV) provide important information in the evaluation of these lesions. Withdrawal of immunosuppressive therapy and observation for a short period should be considered in the initial treatment of IAT LPDs, especially when they are EBV positive. This approach may obviate the need for unnecessary cytotoxic chemotherapy or radiation therapy in a significant subset of patients. However, some of these IAT LPDs behave in an aggressive manner; therefore, close clinical management is warranted.

Primary low-grade endometrial B-cell lymphoma.

We describe three cases of primary low-grade B-cell lymphoma of the endometrium and contrast the histological, immunohistochemical, and molecular features with two examples of benign endometrial lymphoid infiltrates. The first case was an incidental finding in a curettage specimen, confirmed on a subsequent hysterectomy. The other two cases of lymphoma were incidental findings on hysterectomy procedures performed for prolapse and cervical dysplasia, respectively. All three lymphomas occurred in patients in their sixties; none formed gross tumors. Histologic examination revealed lymphoid nodules adjacent to endometrial glands. The lymphoid cells showed mild nuclear enlargement and slight irregularities of the nuclear contour. None of the three patients had evidence of disease outside the endometrium by physical examination, bone marrow biopsy, or sampling of pelvic lymph nodes. Immunohistochemistry demonstrated a B-cell phenotype of the lymphoid cells (CD20 positive, CD79a positive) with aberrant coexpression of the T-cell-associated marker CD43. Polymerase chain reaction (PCR) amplification of the VDJ region of the immunoglobulin heavy-chain was performed on DNA isolated from paraffin sections. These studies demonstrated a clonal proliferation of B-lymphocytes in two cases. In the third case, a faint band was found superimposed on a background smear, suggesting the presence of a B-cell clone. In contrast, the two examples of histologically benign lymphoid aggregates of the endometrium consisted predominantly of T cells with rare B-lymphocytes; there was no evidence of coexpression of CD43 by B-cells. The PCR amplification from the benign lymphoid aggregates did not support a clonal process. Primary lymphoid neoplasms of the endometrium are rare, and all cases described so far have been high-stage, high-grade neoplasms. To our knowledge, this is the first report of primary low-grade B-cell lymphoma of the endometrium, presumably arising from endometrial lymphoid tissue.

Hodgkin's disease and lymphoproliferations resembling Hodgkin's disease in patients receiving long-term low-dose methotrexate therapy.

Recently, it has been shown that patients with rheumatologic diseases who are treated with methotrexate can develop immunosuppression-associated lymphoproliferative disorders. Although a variety of lymphoproliferations have been described in the setting of methotrexate therapy, only rare cases of Hodgkin's disease (HD) have been reported. In this study, we provide a more complete characterization of the spectrum of lymphoproliferations that resemble HD or show features diagnostic of HD that occur in patients receiving long-term low-dose methotrexate therapy. Eight patients were receiving methotrexate for various disorders. Four cases were considered to represent lymphoproliferations resembling HD; the other four cases were diagnosed as HD because they showed diagnostic morphologic and immunophenotypic features. All three patients with lymphoproliferations resembling HD on whom follow-up was available experienced tumor regression with methotrexate withdrawal or with methotrexate withdrawal and steroids; none of these three patients required further therapy. All three patients with HD on whom follow-up was available are alive and free of disease following chemotherapy or radiation therapy. In two of these patients, the tumor persisted or progressed despite discontinuation of methotrexate with observation; the third patient received chemotherapy at the same time methotrexate was stopped. Our findings indicate that a spectrum of lymphoproliferations resembling HD or diagnostic of HD can occur in patients receiving long-term low-dose methotrexate therapy. Recognition of these lymphoproliferative disorders is clinically important because a subset of these neoplasms will completely resolve with discontinuation of methotrexate, thereby obviating the need for chemotherapy or radiation therapy.

p53 mutations in Hodgkin's disease.

Although numerous studies have demonstrated increased expression of p53 protein in the Reed-Sternberg cells of Hodgkin's disease, little data exist as to whether mutations of the p53 gene is a common occurrence in this neoplasm. Using a microdissection technique coupled with PCR, single-strand conformation analysis, and DNA sequencing, we studied 23 cases of Hodgkin's disease for mutations within exons 5 to 8 of the p53 gene. We found seven mutations within six cases; six were missense mutations. An identical missense mutation was found in three cases (codon 243, methionine to isoleucine), and another identical missense mutation was found in an additional two cases (codon 204, glutamic acid to lysine). Verification of the mutations was accomplished either by direct Southern blotting of PCR-amplified p53 exon products from re-extracted DNA or by hybridization of cloned PCR-amplified p53 exon products from re-extracted DNA with a mutant-specific oligonucleotide. There was no good correlation between the presence of p53 mutations and the level of p53 protein expression, which was found to be overexpressed in all cases, the level of MDM2 protein expression, or the proliferation rate as determined by K-67 antibody. None of the cases with p53 mutation had evidence of Epstein-Barr virus within the Reed-Sternberg cells, as compared with 7 of 17 of the other cases (p < 0.06). These results suggest that p53 mutation may represent an important mechanism in the pathogenesis of Hodgkin's disease, and this mechanism may be independent of Epstein-Barr virus.

Pregnancy-associated lymphomas. A clinicopathologic study.

BACKGROUND: The natural histories of Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHL) during pregnancy are not well understood. METHODS: All cases of HD and NHL diagnosed during pregnancy at Stanford University Medical Center since 1987 were reviewed and clinical follow-up was obtained. Various immunohistochemical studies and in situ hybridization for Epstein-Barr virus (EBV) encoded RNA were performed in a subset of cases. RESULTS: Seventeen cases of HD and 12 cases of NHL were accessioned (median age; 27 yrs). The HD cases were classified as 13 nodular sclerosis type, 3 mixed cellularity type, and 1 unclassified. Clinical follow-up revealed most of the patients had Stage II to III disease and were diagnosed on average at 22 weeks gestation. Most of the patients deferred therapy until after delivery and had no evidence of disease at the last follow-up except for one death with disease but not from it. NHL were classified according to the working formulation as high or intermediate grade lymphomas of various types, including both nodal and extranodal sites. Clinical follow-up revealed most had Stage II to IV disease and were diagnosed on average at 23 weeks gestation. Patients with HD tended to survive longer than those with NHL (raw mortality, P = 0.04). In situ hybridization failed to provide support for the presence of EBV in a subset of patients with NHL. CONCLUSIONS: The clinical behavior of these neoplasms during pregnancy does not appear to be significantly different from that outside of the setting of pregnancy. Treatment of selected HD patients apparently may be safely deferred until after delivery. Patients with NHL present with higher stage disease and have a poorer prognosis than those with HD.

True histiocytic lymphoma following therapy for lymphoblastic neoplasms.

True histiocytic lymphomas (THLs) are rare tumors in which the malignant cells show morphologic and immunophenotypic evidence of histiocytic differentiation. We describe THLs that arose after therapy for one case of T-lineage lymphoblastic lymphoma (LyL) and two cases of acute lymphoblastic leukemia (ALL) (both CD10+, one pre-B phenotype). The lymphoblastic neoplasms were not unusual in any way, and responded well to standard therapy. The THLs arose 10 to 20 months after complete remission was achieved for the lymphoblastic neoplasms, at which time there was still no clinical or pathologic evidence of the lymphoblastic neoplasms. All three THLs exhibited clinical and morphologic features of malignancy. Neoplastic cells in the THLs had abundant eosinophilic vacuolated cytoplasm and pleomorphic nuclei, and expressed histiocytic antigens in the absence of lymphocyte-specific lineage markers. Because THLs are rare neoplasms, their occurrence after otherwise successful therapy for lymphoblastic neoplasms in these three cases may constitute a distinct clinicopathologic entity.

Epstein-Barr virus clonality in lymphomas occurring in patients with rheumatoid arthritis.

OBJECTIVE: A causative role for Epstein-Barr virus (EBV) in the development of lymphoma in patients with rheumatoid arthritis (RA) has been proposed. We investigated the molecular features of EBV-positive diffuse large cell lymphomas in 2 patients with RA. METHODS: Southern blot analysis for immunoglobulin gene rearrangements, terminal repeat analysis for clonality of the EBV genome, and double-labeling of the lymphoma cells by in situ hybridization and immunoperoxidase staining were performed. RESULTS: In both cases, double-labeling studies localized the EBV genome to the malignant B cells. Both neoplasms contained clonal immunoglobulin gene rearrangements and clonal EBV genomes. CONCLUSION: Our data indicate that EBV infection was an early step in the development of these neoplasms. The findings further extend knowledge on the similarity of this subset of lymphomas to posttransplantation lymphomas and emphasize the role of immunosuppression in their genesis.

Pathologic features of nodular lymphocyte predominance Hodgkin's disease in extranodal sites.

We describe the gross and histologic features of nodular lymphocyte predominance Hodgkin's disease (NLPHD) occurring in extranodal sites. Fifty-one specimens of NLPHD from 16 patients were studied. The sites of involvement were the spleen, liver, tonsil, salivary glands, skin, colon, soft tissue, and bone marrow, and the morphologic features were similar to those described in node-based NLPHD, including characteristic lymphocytic and/or histiocytic (L&H) cells that were easily identified in a background of a nodular proliferation of small lymphocytes and histiocytes. In the spleen, the normal architecture was generally preserved, and the tumor was found predominantly in the white pulp; the red pulp was rarely involved. In the liver, the tumor involved both the portal and parenchymal areas. In the tonsil, the lympohproliferation closely resembled the typical appearance of NLPHD in a lymph node. In all specimens with materials available for immunohistochemical studies, there were demonstrable L&H cells with an immunophenotype similar to node-based NLPHD, that is, CD45-positive, CD20-positive, and CD15-negative. The unique morphologic and immunologic characteristics of NLPHD are preserved in extranodal sites and allow its distinction from classic Hodgkin's disease and other lymphoproliferative malignancies that may occur in extranodal sites.

Detection of Epstein-Barr virus in multiple sites involved by Hodgkin's disease.

Tissues obtained from 14 patients with multiple anatomic sites involved by Hodgkin's disease were studied for Epstein-Barr virus (EBV) using in situ hybridization for EBV-encoded RNA (EBER) 1 and immunohistochemical methods for EBV latent membrane protein (LMP) expression. Each patient in this study had two to five separately involved anatomic sites, and all biopsy sites, a total of 43 specimens, were analyzed for EBV. EBV was detected in 6 of 14 (42.8%) patients with Hodgkin's disease, including 5 of 11 (45.4%) with nodular sclerosis and 1 of 3 (33%) with mixed cellularity. In these six patients, all biopsy sites were positive for both EBER1 and LMP. In the EBV-positive cases were analyzed the 3'-end of the EBV LMP1 gene in al sites of disease using polymerase chain reaction. In three patients all sites of disease had a 30-base pair deletion. In two patients, there was discordance between sites of disease, with LMP1 gene deletions in some sites and other sites with the LMP1 gene in the germline configuration. The results of this study demonstrate that EBV, when found in Hodgkin's disease, is detectable in all anatomic sites involved. The presence of the same 30-base pair deletion in the EBV LMP1 gene in all sites of disease in three patients suggests that the deletion occurred before dissemination and that all sites are clonally related. However, the discordance between anatomic sites in two patients suggests that LMP1 gene deletion may also occur as a later event, after dissemination. These results lend further support to the hypothesis that EBV plays a role in the pathogenesis of a subset of cases of Hodgkin's disease.

Frequent presence of the Epstein-Barr virus in inflammatory pseudotumor.

Inflammatory pseudotumor is a presumably nonneoplastic, hematopoietic, and spindled fibrous proliferation that may occur at a variety of anatomic sites. The origin of these proliferations is generally unknown. To evaluate the role of the Epstein-Barr virus (EBV) in inflammatory pseudotumor, 18 specimens from 17 patients were studied by in situ hybridization for EBV ribonucleic acid (RNA), and the morphological and immunologic characteristics of the infected cells were evaluated. These specimens included 10 lymph nodes, six splenic masses, and two hepatic masses. Overall, EBV RNA was detected in 41.2% (seven of 18) of the cases. These included two of 10 (20%) lymph nodes, four of six (66.7%) splenic pseudotumors, and one of two (50%) hepatic lesions. The degree of EBV infection was significantly greater within the tumors in comparison with the surrounding, uninvolved tissue. Two morphologically different EBV-positive cell types, spindled and round cells, were evident, and the infected cell type differed significantly when the nodal and extranodal cases were compared. All of the positive extranodal cases shown, numerous EBV-positive spindled cells, whereas no positive spindle cells (only positive round cells, morphologically consistent with lymphocytes) were noted in the two EBV-positive lymph node pseudotumors. Double-labeling immunohistochemical and in situ hybridization studies in some cases identified rare EBV-positive B cells and rare EBV positive T cells in four and three cases, respectively. Most EBV-positive cells in all cases failed to immunoreact with any B- or T-cell markers. Three of five cases studied, however, did show a subpopulation of smooth muscle actin/EBV-positive spindled cells, five of seven cases showed vimentin/EBV-positive spindled cells, and one of four cases had EBV-positive spindled cells that immunoreacted as follicular dendritic cells. These results suggest that EBV plays a role in a significant number of cases of inflammatory pseudotumor with differences in the incidence of EBV infection and the cell type (spindled vs round cell) infected when extranodal and nodal cases are compared, suggesting a difference in pathogenesis. The cell type infected in extranodal cases seemed to be of mesenchymal origin but could not be clearly defined.