Alexandra M. Levine

Non-Hodgkin's lymphoma in 90 homosexual men: relation to generalized lymphadenopathy and the acquired immunodeficiency syndrome

We describe the histologic and clinical features of non-Hodgkins lymphoma diagnosed between January 1980 and December 1983 in 90 homosexual men from San Francisco, Los Angeles, Houston, and New York. The median age was 37 years, with an age distribution identical to that for cases of AIDS reported to the Centers for Disease Control. Sixty-two per cent of the patients had high-grade (aggressive) subtypes of lymphoma, 29 per cent had subtypes of intermediate grade, and 7 per cent had low-grade subtypes. Histologic subtypes and malignant cell phenotypes were consistent with a B-cell origin. All but two men had extranodal lymphoma: central-nervous-system, bone-marrow, bowel, and mucocutaneous sites were most commonly involved. Thirty-five of 66 evaluable men (53 per cent) had complete responses to combination chemotherapy or radiotherapy or both, and thus far, 19 (54 per cent) of them have had a relapse. Mortality and morbidity were closely related to prodromal manifestations; death or illness have occurred in 19 (91 per cent) of the 21 men who presented with AIDS, in 26 (79 per cent) of the 33 who presented with generalized lymphadenopathy, and in 5 (42 per cent) of the 12 who had no prodromal manifestations. Mortality rates analyzed according to histologic grade were higher than currently reported rates in other patient populations. Kaposis sarcoma or severe opportunistic infections characteristic of AIDS developed in 14 of 33 men (42 per cent) who presented with generalized lymphadenopathy and in 3 of 12 (33 per cent) without prodromal manifestations. We conclude that non-Hodgkins lymphoma in members of an AIDS risk group is a serious manifestation of AIDS and the AIDS-related complex.

Hepatitis C Virus Infection in Patients with B-Cell Non-Hodgkin Lymphoma

Hepatitis C virus (HCV) is the major etiologic agent of post-transfusion and sporadic non-A, non-B chronic hepatitis [1, 2]. Although HCV is a hepatotrophic virus, the HCV genome and its replicative intermediate have been detected in peripheral blood mononuclear cells in patients with chronic HCV infection [3]. The association between HCV and type II mixed cryoglobulinemia is unequivocal [4-6]. Antibodies to HCV (anti-HCV) and HCV RNA have been found in up to 98% of patients with mixed cryoglobulinemia [4], and approximately 36% of patients with chronic HCV infection have mixed cryoglobulinemia [7]. Of interest, mixed cryoglobulinemia is considered by some investigators [8, 9] to be a variant of low-grade B-cell non-Hodgkin lymphoma. Because HCV RNA can be detected in the peripheral blood mononuclear cells of patients with chronic hepatitis C [10], the persistence of HCV in these cells may chronically stimulate B-lymphocytes. This may cause clonal expansion of these immunoglobulin-secreting cells and eventually results in malignant B-cell lymphoproliferative diseases. Consistent with this hypothesis, chronic infection of B lymphocytes by a DNA parvovirus was shown to induce polyclonal and, later, monoclonal immunoglobulin production in minks [11]. On the basis of these observations, it has been hypothesized that chronic HCV infection, alone or in combination with other factors, may lead to the development of B-cell lymphoma. Several Italian studies [12-15] have reported a high prevalence (9% to 32%) of chronic HCV infection in patients with B-cell non Hodgkin lymphoma. However, data from the United Kingdom have not confirmed these observations [16, 17]. To determine the prevalence of HCV infection among patients with B-cell non-Hodgkin lymphoma in the United States, we did a controlled study of a large cohort of patients. Methods Patients Between October 1994 and May 1996, 120 consecutive patients with B-cell non-Hodgkin lymphoma were evaluated at the outpatient hematology clinic of the Los Angeles County-University of Southern California (LAC-USC) Medical Center. During the same period, we enrolled 268 additional patients as controls: 154 unselected patients with malignant hematologic conditions other than B-cell non-Hodgkin lymphoma who were seen at the same clinic (control group 1, which comprised 44 patients with acute leukemia, 45 with Hodgkin disease, 11 with T-cell lymphoma, 23 with chronic myelogenous leukemia, 20 with multiple myeloma, 10 with chronic lymphocytic leukemia, and 1 with hairy-cell leukemia) and 114 unselected patients without malignant hematologic conditions who were attending the general medicine clinic at LAC-USC (control group 2, which comprised 69 patients with systemic hypertension or ischemic heart disease, 35 with diabetes mellitus, and 10 with primary hypothyroidism). All potential study participants were tested for antibodies to HIV. Markers for HCV infection (anti-HCV and HCV RNA) and hepatitis B virus infection (hepatitis B surface antigen, antibodies to hepatitis B core antigen, and antibodies to hepatitis B surface antigen) were also determined. Patients who tested positive for antibodies to HIV (2 patients) or hepatitis B surface antigen (6 patients) were excluded. We did not exclude patients with known HCV infection or liver disease. Identical inclusion and exclusion criteria were used to enroll patients and controls. All patients were given a questionnaire that asked about demographic characteristics and potential risk factors for viral hepatitis and chronic liver disease. A patients ethnicity was determined on the basis of self-report. A liver panel, which included prothrombin activity and levels of alkaline phosphatase, albumin, globulin, total and direct bilirubin, lactic dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, was obtained for all patients at the time of evaluation. All patients gave written informed consent, and the study was approved by the institutional review board at LAC-USC Medical Center. Pathologic Evaluation of Lymphoma Lymphoma was diagnosed on the basis of morphologic evaluation of lymph node tissue or extranodal tissues, including bone marrow specimens. Immunophenotypic analysis for surface B- and T-lymphocytic markers was performed by using monoclonal antibodies, as described elsewhere [18, 19]. Non-Hodgkin lymphoma was graded by using the Modified Working Formulation classification [20, 21]. All slides were independently rereviewed by two expert hematopathologists who were not aware of the HCV infection status of the patients. Assessment of Hepatitis C Virus Infection We detected HCV antibodies by using a second-generation enzyme-linked immunosorbent assay (Anti-HCV EIA 2, Ortho Diagnostic Systems, Raritan, New Jersey). Blood samples for HCV RNA determination and HCV genotyping were processed and stored under the optimal conditions described by Davis and colleagues [22]. Serum HCV RNA was measured by a reverse-transcription polymerase chain reaction (RT-PCR) assay (Amplicor HCV test, Roche Molecular Systems, Somerville, New Jersey), as described elsewhere [23]. Briefly, total RNA was extracted from 100 L of serum with guanidinium thiocyanate and precipitated in isopropanol with poly(A) carrier RNA. An equivalent of 5 L of serum was reverse transcribed and amplified in a master mixture containing rTth DNA polymerase, biotinylated primers KY80 and KY78, buffer salts, unguentum, deoxyadenosine triphosphate, deoxycytidine triphosphate, 2-deoxyguanosine-5-triphosphate, and deoxyuridine triphosphate. Deoxyuridine triphosphate was incorporated into each amplification product to serve as a substrate for unguentum (AmpErase, Roche Molecular Systems); this prevented carryover contamination of previously amplified DNA. The reaction was optimized for the use of rTth that, in the presence of manganese, performs both reverse-transcription and DNA-polymerase functions. After RT-PCR was performed, biotin-labeled PCR products were chemically denatured; captured by a solid-phase, HCV-specific probe that was bound to microwell plates; and detected by using an avidin-horseradish peroxidase system with a conventional microtiter plate reader (450 nm). Optical density readings of more than 0.500 were considered positive for HCV, readings of less than 0.300 were considered negative, and readings of 0.300 to 0.500 were considered equivocal. We did HCV genotyping on all HCV RNA-positive specimens by using genotype-specific primers from the HCV core region under conditions described elsewhere [24]. Two rounds of amplification were done: We used genotype-nonspecific primers during the first round and genotype-specific primers during the second round. The amplification products were resolved on 2% agarose gels stained with ethidium bromide. Hepatitis C virus genotype was determined by genotype-specific bands of complementary DNA. Control samples of each detectable genotype (1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a) were evaluated in parallel. Radioimmunoassay (Ausria II, Abbott Laboratories, North Chicago, Illinois) was used to detect hepatitis B surface antigen and antibodies to hepatitis B surface antigen, and enzyme-linked immuno-assay (Corab, Abbott Laboratories) was used to detect antibodies to hepatitis B core antigen. Statistical Analysis The primary comparisons among groups for categorical variables were performed by using the Fisher exact test. We calculated the relative risk and 95% CIs that patients with HCV infection compared with patients without HCV infection would have extranodal lymphoma. We also computed 95% CIs for the prevalence of HCV infection in the B-cell lymphoma group and the two control groups. All CIs were calculated by using StatXact3 for Windows (Cytel Software Corp., Cambridge, Massachusetts). All other analyses were performed by using SAS 6.08 for Windows (SAS Institute, Inc., Cary, North Carolina). Results Patients in the B-cell lymphoma group and the two control groups were similar with respect to age, sex, ethnicity, and risk factors for viral hepatitis (Table 1). The prevalence of anti-HCV and HCV RNA in the three groups are shown in Table 2. Infection with HCV was detected in 26 of 120 patients with B-cell lymphoma (22% [95% CI, 15% to 30%]) compared with 7 of 154 patients (4.5%) in control group 1 and 6 of 114 patients (5%) in control group 2 (P < 0.001). Table 1. Demographic Characteristics of Patients with B-Cell Non-Hodgkin Lymphoma and Controls Table 2. Serologic and Virologic Markers in Hepatitis C Virus-Positive Patients* In the B-cell non-Hodgkin lymphoma group, both HCV RNA and anti-HCV were detected in 21 patients; HCV RNA was the only detectable marker in 4 patients, and HCV RNA was not detected in 1 anti-HCV-reactive patient. All control patients who were considered to be infected with HCV had detectable HCV RNA, although 3 of these patients were anti-HCV negative (Table 2). The most common HCV genotypes in patients with B-cell non-Hodgkin lymphoma were genotype 1a (12 of 26 patients [46%]), genotype 1b (8 of 26 patients [31%]), mixed genotype 1a and 1b (2 of 26 patients [8%]), and genotype 2b (2 of 26 patients [8%]) This incidence is similar to those reported elsewhere [25, 26] for patients with chronic HCV infection in the United States. Chronic liver disease had been diagnosed in 4 HCV-positive patients with B-cell lymphoma (15%) 2 to 6 years before lymphoma was diagnosed. Although 17 of 26 HCV-positive patients with lymphoma had increased aminotransferase activity, only 4 had levels of alanine aminotransferase or aspartate aminotransferase that exceeded 100 U/L. Similarly, most HCV-positive patients in the control groups had only mildly elevated aminotransferase levels. At least one percutaneous risk factor for HCV exposure was identified in 15 of 26 HCV-positive patients (58%) with B-cell non-Hodgkin lymphoma. The period during which patients were at risk for percutaneous exposure to HCV preceded th

Low-dose compared with standard-dose m-BACOD chemotherapy for non-Hodgkin's lymphoma associated with human immunodeficiency virus infection

BACKGROUND Reduced doses of cytotoxic chemotherapy or standard-dose therapy plus a myeloid colony-stimulating factor decreases hematologic toxicity and its complications in patients with non-Hodgkins lymphoma associated with infection with the human immunodeficiency virus (HIV). However, the effect of reducing the doses of cytotoxic chemotherapeutic agents on clinical outcome is not known. METHODS We randomly assigned 198 HIV-seropositive patients with previously untreated, aggressive non-Hodgkins lymphoma to receive standard-dose therapy with methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m-BACOD) along with granulocyte-macrophage colony-stimulating factor (GM-CSF; n=94) or reduced-dose m-BACOD with GM-CSF administered only as indicated (n=98). RESULTS A complete response was achieved in 39 of the 94 assessable patients assigned to low-dose therapy (41 percent) and in 42 of the 81 assessable patients assigned to standard-dose therapy (52 percent, P= 0.56). There were no significant differences in overall or disease-free survival; median survival times were 35 weeks for patients receiving low-dose therapy and 31 weeks for those receiving standard-dose therapy (risk ratio for death in the standard-dose group=1.17; 95 percent confidence interval, 0.84 to 1.63; P=0.25). Toxic effects of chemotherapy rated grade 3 or higher occurred in 66 of 94 patients assigned to standard-dose therapy (70 percent) and 50 of 98 patients assigned to low-dose treatment (51 percent, P=0.008). Hematologic toxicity accounted for the difference. CONCLUSIONS As compared with treatment with standard doses of cytotoxic chemotherapy (m-BACOD), reduced doses caused significantly fewer hematologic toxic effects yet had similar efficacy in patients with HIV-related lymphoma. Dose-modified chemotherapy should be considered for most HIV-infected patients with lymphoma.

Treatment of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine

BACKGROUND Infection with the human T-cell lymphotropic virus type I, a retrovirus, can cause a distinctive cancer, adult T-cell leukemia-lymphoma. The median survival of patients with the acute and lymphomatous forms of the disease is short, despite the use of cytotoxic chemotherapy. METHODS We treated 19 patients with acute or lymphomatous forms of adult T-cell leukemia-lymphoma with oral zidovudine (200 mg five times daily) and interferon alfa (Intron A, 5 to 10 million units subcutaneously each day). Seven of these patients had either relapsed after multiagent cytotoxic chemotherapy or failed to respond to that treatment. RESULTS Major responses were achieved in 58 percent of the patients (11 of 19), including complete remission in 26 percent (5 of 19). Four patients in whom prior cytotoxic therapy had failed had major responses, two of which were complete remissions. Six patients have survived for more than 12 months, with the longest remission since the discontinuation of treatment lasting more than 59 months. CONCLUSIONS The combination of zidovudine and interferon alfa has activity against adult T-cell leukemia-lymphoma, even in patients in whom prior cytotoxic therapy has failed. This regimen should be evaluated further for its role in the treatment of adult T-cell leukemia-lymphoma.

The effect of compliance with treatment on survival among patients with hematologic malignancies.

Ninety-four newly diagnosed patients with hematologic malignancies were monitored for compliance with oral medication and scheduled clinic appointments over a 6-month treatment period. They were assigned at entry either to a control condition or an intervention condition designed to improve compliance. Compliance with medication was assessed objectively with serial serum drug and metabolite levels, as well as with self-report indices obtained on a monthly basis. Medical records were abstracted to obtain data on the number of appointments kept, treatment given, disease characteristics, and survival. On univariate analyses using the log-rank test, five variables were found to be significantly related to survival. These included compliance with allopurinol (probably acting as a surrogate for self-medication with other chemotherapeutic agents) (P = .007), control versus educational program cohort (P less than .001), disease severity (P = .009), Karnofsky at diagnosis (P = .011), sex (P = .084), and clinic appointments kept (P = .043). In hierarchical proportional hazards models, the following variables were associated with decreased risk of death: disease severity (P less than .025), keeping over 60% of appointments (P less than .05), allopurinol/oxipurinol compliance (P less than .01), and educational program cohort (P less than .05). After controlling for all other variables, three variables were associated with increased survival: high disease severity (relative risk [RR] = 2.48), high compliance with allopurinol (RR = .45), and educational program cohort (RR = .39). We conclude that compliance with oral medication has a significant effect on patient survival. In addition, the use of special educational and supportive programs designed to improve patient compliance are associated with significant prolongation of patient survival due to, as well as independent of their effects on compliance.

Chemotherapy for Human Immunodeficiency Virus–Associated Non-Hodgkin’s Lymphoma in Combination With Highly Active Antiretroviral Therapy

PURPOSE This study investigated the efficacy, toxicity, and pharmacokinetic interactions resulting from simultaneous combination chemotherapy and highly active antiretroviral therapy (HAART) for patients with human immunodeficiency virus (HIV)-associated non-Hodgkins lymphoma (NHL). In addition, the effects on viral load, CD4 counts, and opportunistic infections were examined with the use of combination chemotherapy combined with HAART. PATIENTS AND METHODS Sixty-five patients with previously untreated and measurable disease at any stage of HIV-associated NHL of intermediate or high grade were entered onto this study at 17 different centers. The first 40 patients entered onto the study received reduced doses of cyclophosphamide and doxorubicin, combined with vincristine and prednisone (modified CHOP [mCHOP]), whereas the subsequent 25 patients entered onto the study received full doses of CHOP combined with granulocyte colony-stimulating factor (G-CSF). All patients also received stavudine, lamivudine, and indinavir. RESULTS The complete response rates were 30% and 48% among patients who received mCHOP and full-dose CHOP combined with HAART, respectively. Grade 3 or 4 neutropenia occurred in 25% of patients receiving mCHOP and 12% of those receiving full-dose CHOP combined with G-CSF (25% v 12%). There were similar numbers of patients with grade 3 or 4 hyperbilirubinemia (12% and 17%), constipation and abdominal pain (18% and 17%), and transaminase elevation (48% and 52%) on the modified and full-dose arms of the study, respectively. Doxorubicin clearance and indinavir concentration curves were similar among patients on this study and historical controls, whereas cyclophosphamide clearance was 1.5-fold reduced as compared with control values. Human immunodeficiency virus (HIV) load declined from a median baseline value of 29,000 copies/mL to a median minimum value on therapy of 500 copies/mL. CONCLUSION Either modified-dose or full-dose CHOP chemotherapy for HIV-NHL, delivered with HAART, is effective and tolerable.

Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkins B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patients spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Development of B-cell lymphoma in homosexual men. Clinical and immunologic findings.

Serious infections, neoplasms, and immunologic abnormalities have been found in homosexual men. We describe the development of malignant lymphoma in six such patients, three of whom had persistent, generalized lymphadenopathy. In biopsies done before the lymphoma developed, the lymphadenopathy was characterized morphologically by a distinctive pattern of B-cell follicular hyperplasia. All lymphomas were of B-lymphocytic origin, including B-cell immunoblastic sarcoma; small noncleaved, Burkitt-like lymphoma; and plasmacytoid lymphocytic lymphoma. Extranodal presentation with B symptoms occurred in five patients. Median age of our patients was 33 years. Three patients had histories of repeated systemic infections. The peripheral blood lymphocyte count was depressed in four, with depression of OKT 4+ (helper phenotype) cell levels and reversal of the T-helper: T-suppressor ratio in all. We conclude that these patients are at risk for the development of abnormalities of the B-lymphocytic system, manifested by abnormal hyper-B-cell response in enlarged reactive lymph nodes and aggressive, extranodal B-cell lymphomas.

Human immunodeficiency virus‐related lymphoma. Prognostic factors predictive of survival

In an attempt to determine factors predictive of survival in patients seropositive for human immunodeficiency virus (HIV) with acquired immune deficiency syndrome (AIDS)‐related lymphoma, the authors studied 60 such patients, all of whom were treated with curative intent. Eleven patients presented with lymphoma primary to the brain (P‐CNS); the remaining 49 had systemic AIDS‐related lymphoma. Patients with P‐CNS lymphoma had more severe underlying HIV‐related disease than did patients with systemic lymphoma as evidenced by a higher incidence of AIDS before the diagnosis of lymphoma (73% versus 37%; P = 0.04), and lower median number of CD‐4–positive lymphocytes in peripheral blood at diagnosis of lymphoma (30/dl versus 189/dl; P = 0.005). Median survival of such patients was 2.5 months versus 6.0 months for patients with systemic lymphoma (P = 0.04). Forty patients with systemic AIDS‐related lymphoma have died; three factors were strongly associated with shorter survival: (1) Karnofsky performance status (KPS) of less than 70% (multivariate relative survival risk [RSR] = 3.1); (2) history of AIDS before the diagnosis of lymphoma (multivariate RSR = 3.0 for opportunistic infection plus Kaposis sarcoma); and (3) bone marrow involvement (RSR = 3.1)). All three factors (KPS of less than 70%, prior AIDS diagnosis, and marrow involvement) were associated with early demise attributed to AIDS, whereas death attributed to lymphoma per se was associated with only two factors (KPS of less than 70% and marrow involvement). In the absence of all three risk factors, a “good prognosis” group of 17 patients was defined, with a median survival of 11.3 months; the median survival of the remaining patients (“poor prognosis”) was 4.0 months (P = 0.0002). Attainment of complete response to therapy (CR) was strongly related to prolonged survival in the patients in the good prognosis group (17.8 months in patients with CR versus 5.0 months in those with less than CR); however, such meaningful prolongation of survival was not seen in patients with poor prognosis who attained CR (6.3 months versus 3.4 months). The patients with poor prognosis may be unable to tolerate the insult of multiagent chemotherapy, experiencing low CR rates (25%) and death caused by lymphoma and AIDS. However, patients in either prognostic category who attained CR remained at risk for dying of AIDS while the lymphoma was in remission. Thus, it is apparent that meaningful prolongation of survival in the patient with AIDS‐related lymphoma will require not only effective antineoplastic intervention, but also control of the underlying HIV infection. In addition, future therapeutic trials should stratify patients based upon the prognostic factors defined here in an attempt to clarify the results obtained. 68:2466‐2472, 1991.

Determinants of HIV-1 shedding in the genital tract of women

BACKGROUND Plasma HIV-1 RNA concentration has been the best predictor for risk of heterosexual and perinatal transmission. However, direct contact with HIV-1 present locally in the genital tract might be necessary for transmission. We aimed to assess the relation between HIV-1 shedding (RNA or culturable virus) in female genital secretions and other factors that might affect HIV-1 shedding. METHODS This was a cross-sectional study within the Womens Interagency HIV Study (WIHS), a prospective longitudinal cohort study of HIV-infected women. We enrolled 311 HIV positive women from Jan 30, 1997 to July 1, 1998. We did clinical assessments, cultured HIV-1, and measured RNA in peripheral blood mononuclear cells (PBMC) and genital secretions. We compared the results with univariate and multivariate analyses. Presence of HIV-1 RNA or culturable virus in genital secretions was defined as HIV-1 shedding. FINDINGS HIV-1 RNA was present in genital secretions of 57% (152/268) of women whereas infectious virus was detected only in 6% (17/271). Genital tract HIV-1 shedding was found in 80% (130/163) of women with detectable plasma RNA and 78% (116/148) of women with positive PBMC cultures. 33% (27/83) of women with less than 500 copies/mL plasma RNA and 39% (35/90) of those with negative PBMC cultures also had genital tract shedding. INTERPRETATION Plasma RNA concentration, both qualitatively and quantitatively, was the most important factor in predicting genital HIV-1 shedding, even among women receiving potent antiretroviral therapy. However, HIV-1 shedding did occur in women with less than 500 copies/mL plasma HIV-1 RNA. This finding suggests that a separate reservoir of HIV-1 replication may exist in some women.