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Clinical Science

The Natural History and Molecular Heterogeneity of HIV-Associated Primary Malignant Lymphomatous Effusions

Komanduri, Krishna V.*; Luce, Judith A.*; McGrath, Michael S.*†‡; Herndier, Brian G.; Ng, Valerie L.*‡

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Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology: November 1, 1996 - Volume 13 - Issue 3 - p 215-226
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Abstract

A rare subset of lymphomas presenting as primary malignant lymphomatous effusions in body cavities in the absence of tissue-based disease has been recently described for individuals infected with the human immunodeficiency virus, type 1 (HIV-1) (1-7). Recognition of this disease as a distinct clinical entity has been confounded not only by the variety of diagnostic terms used [e.g., “body cavity-based lymphomas” (5,7), “primary lymphomatous effusions” (4), or “anaplastic large cell lymphomas” (2,3)] but also by their inclusion in descriptive series that have combined primary and secondary (i.e., malignant effusions arising in individuals with tissue based disease) HIV-1-associated malignant effusions (1). Furthermore, despite detailed immunophenotypic and molecular analysis of the malignant cells in these effusions, and the definition of this disease as a distinct clinicopathologic entity, the published information on the clinical features of this disease process (i.e., presentation, natural history, and outcome) has been scant.

We have observed eight cases of HIV-associated primary malignant lymphomatous effusions in the absence of tissue-based lymphoma over a 5-year period. Each of these cases generated considerable confusion and controversy regarding diagnosis, extent of clinical staging, and optimal clinical management, and previously published cases lacked sufficient clinical information to guide clinical decisions. To develop clinical guidelines for the diagnosis and management of future cases, we retrospectively reviewed the medical records of these eight patients to identify clinical features at the time of presentation and—because six of these eight patients did not receive cytoreductive therapy because of the unclear clinical significance of malignant cells confined to an effusion without tissue-based disease—to gain insight into the natural history of this disease. We also performed molecular and immunophenotypic studies on the malignant cells and measured effusion associated cytokine levels (i.e., IL-6 and IL-10) to compare with those previously reported and to gain insight into potential pathogenetic factors operative in this unusual disease.

MATERIALS AND METHODS

Case Definition

All HIV-1 infected patients who had malignant lymphomatous effusions and who lacked lymphadenopathy or evidence of tissue-based lymphoma were included.

Specimens

Routine clinical laboratory test values on peripheral blood or primary malignant lymphomatous effusions of these patients were obtained as part of their diagnostic evaluation or for their subsequent clinical management.

Additional molecular and immunophenotypic analyses were performed on two different sources of effusion fluids. The first source was the remainder of the original primary malignant lymphomatous effusions received in the clinical hematology or cytopathology laboratories; within 2 h of receipt and after diagnostic evaluation, the remainder of the effusion fluids were processed as described below. In some cases, additional effusion fluid was obtained by repeat para-, pleuro-, or pericardiocentesis in accordance with guidelines established by the University of California San Francisco Committee on Human Research.

All immunophenotyping and molecular analyses were performed in research laboratories in accordance with the National Institutes of Health Guidelines for Research involving Recombinant DNA Molecules and Biological Agents.

Mononuclear Cell Preparations

Mononuclear cells were prepared from effusion fluids by an initial pelleting of cells (1,000 g, 10 min, room temperature), resuspension of the cell pellet in phosphate buffered saline (PBS), pH 7.5, and standard Ficoll-Hypaque gradient centrifugation. Cells collecting at the interface were either washed twice with PBS and DNA extracted immediately, or resuspended in 10% dimethyl sulfoxide (DMSQ)/90% fetal calf serum and stored in liquid nitrogen. For specimens of limited volume, cells were centrifuged directly onto glass slides (cytospin preparations) and stored at -20°C, or centrifuged into a small but visible cell pellet, embedded in OCT fixative, and stored at -20°C.

Cell-free supernatants from the primary malignant effusions obtained after the initial cell pelleting were saved and stored at -20°C (patients 2 and 8) or -70°C (patients 4, 5, 6, 7). Cell-free supernatant from the primary malignant effusion of patient 3 was not saved. Specimens from patient 1 were obtained at the time of autopsy (performed 14 days after death); only cell pellets were saved.

Immunophentoyping

Immunophenotyping was performed on (a) frozen sections of cell pellets, (b) cytospin preparations of cell suspensions, or (c) Ficoll-Hypaque purified mononuclear cells, as previously described (8).

Because of the limited amount of malignant cells in frozen sections of cell pellets or cytospin preparations for patients 1, 2, and 3, only limited immunophenotyping by immunohistochemical staining was performed using antibodies directed against CD3, CD14, CD20, CD30 (Ki-1), CD38, CD45, and HLA-DR, as previously described (9).

Flow cytometric analysis of cell suspensions for patients 4-8 was performed on a gated subpopulation of large mononuclear cells with little side scatter (which morphologically corresponded to the malignant lymphoma cells) on a FACS scan (Becton-Dickinson, San Jose, CA, U.S.A.). For the malignant cells from the effusions obtained from patients 4, 7, and 8, immunophenotypic analysis was performed with antibodies directed against CD3, CD4, CD5, CD8, CD10, CD14, CD15, CD16+56, CD19, CD20, CD21, CD22, CD23, CD25, CD38, CD45, CD54, HLA-DR, Leu 9, and κ, and λ light chains. For the malignant cells obtained from the effusions of patients 5 and 6, immunophenotypic analysis was performed with antibodies directed against CD3, CD4, CD5, CD8, CD14, CD20, CD22, CD38, CD45, and HLA-DR.

Fluorescein-, PerCP-, phycoerythrin-conjugated, or biotinylated antibodies directed against the different cell surface markers and similarly labeled matched isotypic control antibodies were obtained from Becton-Dickinson (Mountain View, CA, U.S.A.). A positive result was recorded if >5% of the gated cells stained positively with antibody directed against a specific cell surface antigen.

Molecular Analysis

Southern Blot Analysis

DNA was extracted from cells pelleted from the body cavity fluids using a standard proteinase K digestion, phenolchloroform extraction, and ethanol precipitation procedure (10). Southern blot analysis was performed with 20 μg of genomic DNA per analysis using digoxigenin-labeled probes and nonradiometric detection of probe hybridization (Genius system, Boehringer-Mannheim, Indianapolis IN, U.S.A.). For patients 1 and 7, insufficient DNA precluded Southern analysis for HHV-8 gene sequences.

Probes used in this study included (a) a JH probe (Oncogene Sciences, San Diego, CA, U.S.A.), (b) an EBV long internal repeat probe, pDK14 (11), labeled with digoxigenin by nick translation according to manufacturer's recommendations (Genius system, Boehringer-Mannheim, Indianapolis, IN, U.S.A.), and (c) a 233 bp HHV-8 probe internal to a 330 bp BamH1 HHV-8 genomic fragment (12) generated by PCR and labeled with digoxigenin by substituting dig-dUTP for 30% of the total dUTP concentration in the initial PCR reaction mixture (see below).

Polymerase Chain Reaction (PCR)

PCR was used to detect the presence of EBV or HHV-8 gene sequences in DNA extracted from cells present in the malignant effusions. For EBV detection, primers specific for a polymorphic region of the EBNA 3c gene that distinguishes type 1 from type 2 EBV (125 bp product versus 245 bp product) and thermal cycling conditions were used as previously described (13). For HHV-8 gene sequence detection, primers predicted to yield a 233 bp fragment and thermal cycling conditions were as previously described (12). Amplification of a portion of the HLA DQ locus (expected product size of 242 bp) in parallel to verify the presence of adequate template DNA was performed as previously described (14).

Positive and negative DNA controls for PCR-based experiments included that isolated from (a) 10C9, a B-cell line lacking EBV, and HHV-8 (8), (b) HS-1, a B-cell line infected with EBV type 1 (15) and (c) 2F7, a B-cell line infected with EBV type 2 (8).

All PCR reactions contained final concentrations of 200-500 ng of genomic DNA, 200 nM dNTPs, 1X Taq polymerase buffer, 500 nM of each primer, and 1 U of Taq polymerase (Roche Molecular Systems, Branchburg, NJ, U.S.A.) in a total volume of 20 μl. Five to ten microliters of the PCR reactions were analyzed on 1% agarose/2% NuSieve gels. Southern transfer and hybridization were performed as previously described (8,10).

Oligonucleotide probes homologous to internal sequences of that predicted to be generated by PCR using EBNA 3c-specific primers were as previously described (11), and 3'-end-labeling with digoxigenin was done according to manufacturer's recommendations (Genius system, Boehringer-Mannheim, Indianapolis, IN, U.S.A.). The HHV-8 probe was the same as that used for Southern analysis of genomic DNA digests (see previous section).

Interleukin 6 and Interleukin 10 (IL-6, IL-10) Determinations

The malignant effusion from patient 1 was obtained at the time of autopsy 14 days after death; interleukin levels were not determined on this fluid because of possible variability introduced by autolysis or in situ “storage” of fluid at 4°C for this length of time. For patient 3, an insufficient volume of malignant effusion was available for interleukin level determinations.

Levels of IL-6 and IL-10 in stored frozen supernatants (obtained after pelleting out cells) or fresh plasma from EDTA anticoagulated blood obtained from four healthy volunteers (3 male, 1 female), separated within 2 h of phlebotomy and at the same time of assaying IL-6 and IL-10 levels in the primary malignant effusions was determined using a commercially available ELISA (Quantikine human IL-10 or IL-6 immunoassay, R&D systems, Inc., Minneapolis, MN, U.S.A.) according to the manufacturer's recommendations. The interassay variability for markedly elevated IL-6 and IL-10 levels in the supernatants was five-fold.

Average IL-6 and IL-10 levels in the fresh plasma obtained from four normal healthy individuals were ≤5 pg/ml (all 4 plasmas had undetectable IL-6 levels) and 48 pg/ml (range 29-66 pg/ml), respectively, consistent with previously published reference ranges for normal healthy individuals values using similar immunoassay systems (16-18). A “normal” IL-6 plasma level of 5 pg/ml was chosen for calculating the malignant fluid to plasma ratio of IL-6.

Establishment of an in vitro Cell Line

A cell line (BCBL-1) was established in vitro from the primary malignant lymphomatous effusion of patient 8 by in vitro culture of the Ficoll-Hypaque purified malignant cells in RPMI 1640/20% autologous ascites/50 μg/ml gentamicin/0.05 mM 2-mercaptoethanol supplemented with 1 mM sodium pyruvate and 2 mM L-glutamine at 37°C in 5% CO2. Weekly or biweekly (depending on the growth of the cells) medium changes were performed until the cells grew well in vitro. The culture was then “weaned” into the same medium with gradual substitution of 10% fetal calf serum for the 20% autologous ascites. The cell line underwent further cloning by limiting dilution.

DNA extracted from this cell line was used for all molecular analyses because insufficient DNA was obtained from the limited amount of malignant cells purified from the original malignant lymphomatous effusion of this patient. The molecular characterization of HHV-8 expressed by this cell line has been recently published (19).

RESULTS

With the exception of patients 4 and 5, both of whom presented in June of 1996 after HIV-associated primary malignant effusions was gradually being recognized as a distinct clinicopathologic entity (1-7), the diagnosis in the other six patients in this study was retrospective and was based on the grossly atypical cytology in their effusion fluid coupled with lack of concomitant tissue-based disease.

Demographic Features

The pertinent demographic features of the eight patients with primary malignant lymphomatous effusions are shown in Table 1. The mean age at the time of diagnosis was 37 years (range 31-47), and homosexuality was the major risk factor for HIV-1 acquisition. Six of the eight patients had total CD4+ lymphocytes counts <100/μl; the other two patients with total CD4+ lymphocyte counts of 181 and 190/μl had these values obtained 196 or 519 days prior to their diagnosis of primary malignant lymphomatous effusions. Three patients had preexisting Kaposi's sarcoma and had undergone systemic treatment for it, six had prior opportunistic infections, three had prior antiviral or antiretroviral therapy, and four were receiving prophylaxis for HIV-associated opportunistic pathogens.

Clinical Features at Time of Presentation

Five of the eight patients presented with increased abdominal girth and discomfort due to primary malignant lymphomatous ascites. Patient 7, who presented with a distended abdomen due to a primary malignant lymphomatous ascites, also developed a malignant lymphomatous pleural effusion. Patient 5 presented with acute pericardial tamponade caused by a primary malignant lymphomatous pericardial effusion. Patient 3 developed bilateral pleural effusions during hospitalization to evaluate persistent fevers and progressive anemia and thrombocytopenia. Fever was a presenting symptom in only three patients. Palpable lymphadenopathy was not observed on physical examination of any of the eight patients.

Routine laboratory analysis of the primary malignant lymphomatous effusions and peripheral blood of the eight patients is shown in Table 2. For the primary malignant effusions, there was a wide variability in protein and glucose levels, fluid to serum LDH ratios (although all eight were >0.6), and RBC and WBC counts. Of note, the malignant cells were frequently misidentified on the fluid differential cell count as lymphocytes or monocytes.

The malignant cells in the effusion fluids were morphologically distinct. They were large hyperchromatic cells (20-35 μm in diameter) with deep blue cytoplasm, moderate to high nuclear to cytoplasmic ratios, irregularly shaped nuclei, and frequent mitotic figures. The malignant cells in the effusions of patients 4 and 8 have been previously published (ref. 20, Fig. 2 F and E, respectively) and are representative of the malignant cells observed in the other six malignant effusions in this study. Effusions consisting of smaller lymphoid cells with cytoplasmic vacuoles suggestive of Burkitt's lymphoma were excluded from this study.

In peripheral blood, seven of the eight patients were anemic, all were hypoalbuminemic, six had elevated serum LDH levels, and five were hyponatremic. The peripheral WBC count was highly variable. Two patients (Nos. 4 and 6) had significantly elevated peripheral WBCs that consisted of leftshifted neutrophils and rare nucleated erythrocytes (<5 nucleated RBCs/100 WBCs) but lacked teardrop RBC forms; both patients had normal peripheral WBC counts within 2 weeks after therapeutic measures were instituted (either low-dose m-BA-COD for patient 4, or local radiotherapy for Kaposis's sarcoma (KS)-related stasis to the trunk and lower extremities for patient 6).

Five of the eight patients (Nos. 2, 3, 4, 6, and 8) were thrombocytopenic, and three of these five (patients 2, 3, and 4) underwent bone marrow examination for evaluation of their thrombocytopenia. All three patients had adequate numbers of megakaryocytes detected in their bone marrow; in addition, early and marked fibrosis was detected in the bone marrows of patients 2 and 4, respectively. The remaining two thrombocytopenic patients (Nos. 6 and 8) refused bone marrow examination. Only patient 4 received therapy for his thrombocytopenia, which proved to be refractory to low-dose and high-dose intravenous immunoglobulin therapy and to splenectomy. Of interest, pleural fluid from patient 4 containing the malignant cells was obtained to evaluate postoperative fevers and pleural effusion persisting 10 days after his splenectomy.

Staging

Conventional clinical staging for non-Hodgkin's lymphoma (i.e., CT scans of the chest, abdomen, and pelvis, and bilateral bone marrow biopsies) was not consistently performed because of the uncertain clinical significance of malignant-appearing lymphomatous effusions in the absence of node- or tissue-based lymphoma. The individual staging evaluations undertaken for these eight patients are detailed in Table 3. With the exception of two patients (Nos. 1 and 4) who had abnormalities detected by CT that did not undergo biopsy, there was no evidence of lymphadenopathy or tissue-based lymphoma in the remaining six patients.

Bone marrow specimens obtained from patients 2, 3, 4, and 5 lacked microscopic evidence of lymphoma and, with the exception of early and marked fibrosis for patients 2 and 4, respectively (see above), revealed only mixed hematopoiesis with normal to increased cellularity (i.e., 50-95%).

Survival

The mean survival of the eight patients was 60 days (range 6-133 days). Only two of the eight patients could tolerate and thus received chemotherapy: one (patient 4) received two cycles of low-dose m-BACOD (21), with only mild mucositis noted after the first cycle and no obvious effect on measured parameters in the peripheral blood, and the other (patient 5) received four cycles of low-dose CHOP (cyclophosphamide, vincristine, adriamycin, prednisone).

Postmortem Findings

Four patients (patients 1, 3, 5, and 6) were examined at autopsy. The primary malignant effusion was the immediate cause of death only in patient 6; the other three patients died of pneumococcal sepsis (patient 1), severe wasting syndrome (patient 3), or adult respiratory distress syndrome (patient 5). Residual malignant effusions were present in three patients (Nos. 1, 3, and 6); no residual malignant pericardial effusion was detected for patient 5.

All four patients had lymphomatous infiltrates in serosal surfaces adjacent to the site of the primary malignant lymphomatous effusion. Two patients (Nos. 1, and 5) had extensive fibrosis containing lymphomatous infiltrates in their pleural, pericardial, and peritoneal cavities. One patient (No. 5) had nodules scattered throughout the parenchyma of both lungs consisting of both Kaposi's sarcoma and high-grade lymphoma. One patient (No. 3) had a small 1-cm focus of lymphoma on the pleural diaphragm adjacent to the site of the primary malignant pleural effusion.

For the four patients who did not undergo postmortem examination (patients 2, 4, 7, and 8), marked abdominal distension was observed ante mortem, and the clinical cause of death was attributed to lymphoma.

Molecular Features

The molecular features of the malignant cells in the primary malignant lymphomatous effusions are shown in Fig. 1 and 2 and summarized in Table 4.

Immunoglobulin (Ig) Gene Rearrangements

A JH probe was used to identify rearranged Ig genes (Fig. 1A). Two cases (patients 3 and 6) had rearranged Ig genes (lanes 3a, 6); for patient 3, in particular, an identical Ig gene rearrangement was observed for DNA extracted from the malignant cells in the effusion and for DNA extracted from the 1-cm focus of lymphoma located on the pleural diaphragm detected at autopsy (lane 3b). One case (patient 4) failed to hybridize with the JH probe. The remaining five cases demonstrated hybridization only at the germline position either at the same time intensity (patient 1) or reduced intensity (patients 2, 5, 7, and 8) compared with the hybridization signal observed for the nonrearranged placental genomic DNA control (Fig. 1A, lane P).

Viral Analysis

Four of the eight cases contained EBV EBNA 3c gene sequences [three (patients 2, 4, and 7) with EBV, type 1, and one (patient 3) with EBV, type 2] as detected by PCR (Fig. 2 A and D); only two of these four cases (patients 3 and 7) were monoclonally infected with EBV (Fig. 1B). For patient 3, the monoclonal EBV infection in the primary malignant lymphomatous pleural effusion (Fig. 1B, lane 3a) was identical to that in the nodule of pleural diaphragmatic lymphoma detected at autopsy (Fig. 1B, lane 3b).

HHV-8 gene sequences were detected by PCR in DNA from the malignant cells in all eight primary malignant lymphomatous effusions (Fig. 2B and E) and verified in six of the eight by Southern analysis (Fig. 1C).

Immunophenotype Analysis

The only cell surface marker consistently expressed was CD38 (Table 4). The malignant cells in seven cases expressed cell surface CD45 (patients 1-5, 7, and 8), five cases expressed cell surface CD19, CD20, and/or CD21 (patients 2-5 and 8), two cases expressed cell surface CD5 (patients 4 and 6), one case expressed cell surface CD14 (patient 5), and one case expressed cell surface CD14 (patient 5), and one case expressed kappa light chains (patient 7).

Only three cases (patients 1-3) were examined for cell surface CD30 (Ki-1) expression; all expressed cell surface CD30.

IL-6 and IL-10 Levels

IL-6 and IL-10 levels in the primary malignant lymphomatous effusion cell free supernatants were highly variable and ranged from 340 to 16,000-fold higher than that present in normal human plasma (Table 4).

DISCUSSION

Although a total of 26 cases of HIV-associated primary malignant lymphomatous effusions have been reported to date, the published information has been typically limited to only one aspect of the abnormal malignant cells (i.e., immunophenotyping, molecular analyses, or viral cofactors) (1-7). To our knowledge, our group of eight patients thus constitutes the largest single series of patients for whom complete clinical information is presented, and for which molecular analyses on the malignant cells was systematically performed. To our knowledge, our series is also the first to report postmortem findings that provide insight into the culmination of this disease process.

Some of the confusion surrounding this disease entity is attributable to the previous reporting of these cases in association with malignant lymphomatous effusions that have arisen subsequent to tissue-based lymphoma (i.e., secondary malignant lymphomatous effusions) (1,6). Two of our cases might have been initially considered secondary malignant effusions, since in one case (patient 1) there was evidence of mediastinal thickening and in the other (patient 4) there was an atrial mass. The postmortem examination of patient 1, however, revealed extensive mediastinal fibrosis that in retrospect was the most likely cause of the mediastinal widening detected ante mortem; failure to detect mediastinal lymphadenopathy in this postmortem examination supports our diagnosis of a primary malignant effusion. The atrial mass in the other patient did not undergo biopsy and might have been a tissue-based lymphoma. Of note, recent evidence suggests that secondary malignant lymphomatous effusions are morphologically and molecularly distinct from primary malignant effusions (i.e., small noncleaved cells containing c-myc rearrangement and lacking HHV-8) (6). The morphologic and molecular features of the malignant effusions for patient 4 were in fact consistent with those currently thought to be unique to HIV-associated primary malignant lymphomatous effusions (i.e., large anaplastic cells containing HHV-8) (6).

Our retrospective review revealed clinical features common to these eight patients. First, all eight patients had peripheral total CD4+ lymphocytes <200/μl, indicating that this disease occurs in individuals with advanced HIV-1 associated disease. This finding is in contrast with the observation that ≈40% of HIV-associated tissue-based lymphomas at our institution occur in individuals with total peripheral CD4+ lymphocytes >200/μl (21). Second, all patients presented with symptoms related to distension of the relevant body cavity. Third, survival was poor (median of 60 days), consistent with 13 of the 26 previously described cases for which such survival information was included [range 10 days-14 months (1-5,7)]. Fourth, routine clinical laboratory data obtained at the time of presentation were nondiagnostic and of limited clinical utility. The hypoalbuminemia observed in all eight patients most likely reflected poor nutritional status; the hyponatremia observed in six of the eight patients was most likely multifactorial in etiology. Two patients with normal serum LDH levels had disease courses indistinguishable from those with elevated serum LDH levels. Of interest, the three patients with the most marked thrombocytopenia deteriorated the most rapidly. Clinical laboratory values obtained for the effusion fluids were most consistent for those expected for exudative processes.

We have concluded on the basis of our review that clinical staging for this disease is dependent on whether the patient can tolerate and benefit from chemotherapy; six of our patients were so severely debilitated at the time of presentation that chemotherapy was not a treatment option. If chemotherapy is to be considered, further staging evaluation may be necessary to evaluate for occult tissue-based disease, the detection of which may affect selection of an appropriate chemotherapeutic regimen (21). In this regard, we would recommend thoracic, abdominal, and pelvic CT to identify occult nodal disease only if therapy were a serious consideration. Our inability to detect lymphoma in the bone marrow biopsy specimens of three patients, coupled with the fact that such detection would not have affected clinical stage or management, suggests that such examinations may be unnecessary for staging the extent of disease. (Bone marrow examination, however, was useful in eliminating hypoproduction as a cause for the peripheral thrombocytopenia observed in three of the five thrombocytopenic patients.) Similarly, given the rapid decline of these patients and the lack of development of CNS disease before death, examination of cerebrospinal fluid as part of the routine staging evaluation may not be necessary.

While no optimal chemotherapeutic regimen has been defined for this rare subset of lymphomas, it is noteworthy that two patients (patients 6 and 8) who had received extensive prior systemic cytotoxic therapy for their Kaposi's sarcoma were diagnosed with primary malignant lymphomatous effusions during concurrent treatment with etoposide and paclitaxel, respectively, which suggests that previous and concurrent cytotoxic therapy was not protective. Also, given the association of HHV-8 with this rare subset of lymphomas, it is noteworthy that prior or concurrent therapy with antiretroviral and/or antiviral agents provided no apparent protective effect for four of our eight patients.

The published immunophenotype of the malignant cells in this disorder has been highly variable, with CD38 being the only cell surface marker consistently expressed (1-7); our eight cases displayed a similar heterogeneity in expression of cell surface markers. In contrast, the molecular features of the malignant cells in previously reported cases suggested a consistent molecular phenotype: 22 of 23 cases had monoclonal Ig gene rearrangements, 21 of 23 patients were monoclonally infected with EBV, and 20 of 20 patients were infected with HHV-8 (1-7). The malignant cells in our eight cases, however, displayed a dramatic heterogeneity when subjected to similar molecular analysis—although we too detected HHV-8 infection in all eight cases, monoclonality could be demonstrated or inferred for only four of the cases and monoclonal EBV infection for only two of the four. For four of our eight cases that failed to demonstrate an Ig gene rearrangement, the interpretation of the results would be consistent with either a polyclonal process or with the existence of a monoclonal population(s) in which both Ig alleles had been excluded. It is unlikely that the methodology employed for Ig gene rearrangement detection lacked sensitivity, since the lymphoma cells in these eight primary malignant lymphomatous effusions constituted at least 50% of the cellular elements present, and we are consistently able to detect a monoclonal population if it constitutes at least 5-10% of the specimen (22). Of note, none of the molecular or immunophenotyping test results added diagnostic value to the initial morphologic assessment of the malignant cells or provided additional information to aid with immediate clinical management; in fact, three of the patients had died prior to completion of immunophenotyping and molecular studies.

When molecular studies for all published cases are combined with the data presented herein, the only consistent finding the malignant cells comprising this rare subset of HIV-associated lymphomas is cell surface expression of CD38 and universal association with HHV-8. The fact that half of our patients in this series lacked EBV but were infected with HHV-8 now suggests that HHV-8 infection alone, in the absence of EBV-derived cofactors as previously hypothesized (5), may be a significant pathogenetic factor in this disease process. In this regard, the establishment of a cell line from patient 8 (19), infected with HHV-8 but lacking EBV, has provided a resource to pursue studies of HHV-8 mediated pathogenic mechanisms. As a related issue, although HHV-8 infection has been highly associated with endemic and HIV-associated Kaposi's sarcoma (12,23), the majority of patients reported to date with HIV-associated primary malignant lymphomatous effusions did not have Kaposi's sarcoma.

Autopsies performed in four of the eight patients in our series revealed that the lymphomatous effusions were not the immediate cause of death in two. For the third patient (patient 6), the massive volume of effusion most likely caused the respiratory distress that ultimately led to the immediate cause of death. For the fourth patient (patient 5), the replacement of lung parenchyma by distinctly unusual nodules consisting of Kaposi's sarcoma and lymphoma probably contributed to his death from adult respiratory distress syndrome. Of interest, a postmortem finding common to all four cases was lymphomatous infiltration of serosal surfaces adjacent to the site of the effusion, which suggests either that the malignant cells were capable of local invasion or that the effusions arose from exfoliation of infiltrating malignant cells. Of note, patient 5, who had received four cycles of low-dose CHOP, had no residual pericardial effusion detected at autopsy; even if the disappearance of his effusion could be attributed to the chemotherapy, lymphomatous infiltration of the lungs and adjacent serosal surfaces was still present.

Two of the four patients examined post mortem had marked pleural, pericardial, peritoneal, and retroperitoneal fibrosis, and two of four patients who underwent bone marrow biopsy had early or marked fibrosis detected. The significance of this fibrosis with HIV-associated primary malignant effusions is unclear, especially given that bone marrow fibrosis—as detected by increased reticulin deposition—has been observed in 21-93% of bone marrows obtained from AIDS patients (24).

Pathogenetic mechanisms previously proposed for this rare subset of HIV-associated lymphomas have been based on the supposition that the malignant cell was of B-cell origin; this theory was supported by the vast majority of cases having a combination of monoclonal Ig gene rearrangements, cell surface expression of CD38 (an activation marker, often expressed in isolation on fully differentiated B-cells, or plasma cells), and monoclonal infection with EBV (1-7). Other HIV-associated B-cell lymphomas have been shown to express transcripts for IL-6, and IL-10, both cytokines involved in B-cell proliferation and differentiation that may be involved in a proposed multistep pathogenesis model for HIV-associated lymphomagenesis (20). IL-6 and IL-10 levels in the primary malignant lymphomatous effusion fluids in this study were markedly elevated, and although the levels were comparable to those observed in a variety of benign and malignant nonlymphomatous effusions (25-28), increased levels of these cytokines may still be relevant to this disease process in the following manner. Additional studies on the malignant cells of patient 4 (20,29) demonstrated expression of both IL-6 and IL-6 receptor (IL-6R) transcripts, suggesting a paracrine mechanism for continued B-cell proliferation. The malignant cells also coexpressed IL-10 transcripts; IL-10 expression in turn could cause terminal differentiation of premalignant B-cells (e.g., plasma cell expressing cell surface CD38 only). Continued proliferation, either through a paracrine mechanism or by continued antigenic stimulus (e.g., HIV-1 antigens, self antigens, HHV-8 antigens) could thus provide a population at increased risk for additional as yet undefined genetic events leading to transformation.

Finally, the finding of HHV-8 sequences (in the absence of EBV) in half of these lymphomas further implicates HHV-8 as a true human pathogen. The existence of the BCBL-1 cell line that contains only HHV-8 (19) should provide a pure source of HHV-8 for use in developing a diagnostic test to identify individuals who have been infected with HHV-8. Considering the increasing incidence of large cell lymphoma in non-HIV infected individuals, it will be important to determine whether this new virus contributes even indirectly to lymphomagenesis in general.

Acknowledgment: Supported in part by NIH RO1 CA54742 (MSM) and the California Universitywide AIDS Research Program (R94-SF-045, VLN; R93-SF-055, MSM). The authors gratefully acknowledge Vivek Bhargava for assistance with acquisition of the malignant effusion of patient 7, Tracy George and Bruce Shiramizu for helpful comments and suggestions, and Nancy Abbey, Ahmed Elbegarri, Ron Gascon, Farzad Khayam-Bashi, Jane Marsh, and Mark Weinstein for expert technical assistance.

FIG. 1
FIG. 1:
. Southern blot analysis for Ig gene rearrangements, monoclonal EBV infection, and HHV-8 infection. Genomic DNA was isolated from the malignant cells in the primary malignant lymphomatous effusions, digested with Hind III/Bam H1 (A, lanes 1, 2, 3, 4, 5, 6, P) or Bam H1 (A, lanes 3a, 3b, 3c, 7, 8; B and C) and subjected to Southern analysis with probes directed against the JH gene (A), EBV internal repeat (B) or HHV-8 (C). 20 μg of digested DNA was applied to each lane. Lane numbers correspond to patient number as defined in the text and tables; different sources of tissue were analyzed for patients 3 and 4 (3a, malignant cells from the primary effusion; 3b, malignant cells from the 1-cm lymphoma on the pleural diaphragm detected at autopsy; 3c, normal spleen obtained at autopsy; 4a, malignant cells from the primary effusion; 4b, normal spleen from the same patient). Analysis of DNA extracted from normal tissue of the same patients (i.e., lanes 3c, 4b) or from placental DNA (P) was performed in parallel to demonstrate the location of the germline unrearranged JH gene in DNA digested with different restriction enzymes (A, lanes P and 3c) or as a negative DNA control for EBV (B, lanes 3c and 4b) or HHV-8 probes (C, lane P). Molecular weights, in kilobases (A and B) or base pairs (C), are indicated on the ordinate.
FIG. 2
FIG. 2:
. PCR detection of EBV or HHV-8 in the malignant cells of the HIV-associated primary malignant lymphomatous effusions. PCR using primers specific for the EBV EBNA 3c gene (A), HHV-8 (B), or HLA-DQ (C) was performed on genomic DNA isolated from the malignant cells as described in “Materials and Methods.” D and E represent Southern blot analysis of the PCR products shown in panels A and B with probes specific for EBV (D) or HHV-8 (E). Lane numbers correspond to patient numbers as defined in the text and tables. Lane a: water control (no DNA); lane b: 10C9 DNA (negative DNA control for EBV and HHV-8 gene amplification); lane c: HS-1 DNA (positive control for amplification of EBV type 1 EBNA 3c gene); lane d: 2F7 DNA (positive control for amplification of EBV type 2 EBNA 3c gene).

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Keywords:

HHV-8; HIV; Epstein-Barr virus; Primary malignant effusions; Lymphoma; Body cavity; CD38

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