High-dose therapy and autologous stem cell transplant does not result in long-term disease-free survival in patients with recurrent chemotherapy-sensitive ALK-negative anaplastic large-cell lymphoma.

Primary systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large-cell lymphoma (ALCL) has a poor prognosis. This study sought to determine if high-dose therapy and ASCT results in long-term disease-free survival (DFS) in patients with recurrent, chemotherapy-sensitive ALK-negative ALCL. All patients with non-Hodgkin's lymphoma (NHL) who underwent ASCT at Wake Forest University and Upstate Medical University from 1 January 1990 to 12 December 2002 were reviewed to determine if they had T-, B- or null-cell NHL that was CD30+/CD15-/ALK negative. In all, 16 patients were thus identified as having ALK-negative ALCL. All 16 patients underwent ASCT at the time of first relapse and form the basis of this report. Median age of the 16 patients was 51 years. There were 11 males and five females. International prognostic index scores in 12 patients at the time of relapse were: low 3, LI 6 and HI 3. Of 15 patients, 13 relapsed after ASCT; one patient was lost to follow-up. Median progression-free survival for the 15 patients was 12 weeks (3-212+ weeks). Of 15 patients, 10 have died; nine of recurrent disease. Median overall survival for the 15 evaluable patients was 72 weeks. In our experience, high-dose therapy and ASCT does not produce long-term DFS in patients with recurrent chemotherapy-sensitive ALK-negative ALCL.

Fatal hemolysis after high-dose etoposide: is benzyl alcohol to blame?

A 53-year-old African-American man with relapsed non-Hodgkin's lymphoma developed seizures and respiratory arrest 2 hours after an infusion of high-dose etoposide in preparation for an autologous bone marrow transplant. Laboratory tests revealed both rapid hemolysis and severe metabolic acidosis. The patient died the following day. Based on toxicities observed, we suspect that our patient possessed an ethnic polymorphism of the enzyme alcohol dehydrogenase. Further research is required to determine the relationship between the benzyl alcohol metabolic rate and toxicity and genetic polymorphisms of alcohol dehydrogenase in African-Americans.

Epitope mapping of HTLV envelope seroreactivity in LGL leukaemia.

Sera from approximately 50% of patients with large granular lymphocyte (LGL) leukaemia react with a recombinant human T-cell leukaemia/lymphoma virus (HTLV) transmembrane envelope protein, p21e. Two immunodominant epitopes within env p21e have been defined by reactivity against recombinant proteins GD21 and BA21. In this study sera from 41 patients with LGL leukaemia were examined for reactivity against these recombinant HTLV env proteins. Overall, 21/41 (51%) sera reacted to p21e. Only two sera reacted to GD21. The predominant immunoreactivity against p21e was directed against the BA21 epitope, with 19/41 (46%) sera being BA21 positive. Seroconversion to BA21 protein was also documented. PCR analyses confirmed the low incidence of protypical HTLV sequences (2/41, 5%). These data document an association between BA21 seroreactivity and LGL leukaemia. This finding raises the possibility that such BA21 seroreactivity could be due to cross-reactivity to a cellular or retroviral antigen sharing some amino acid homology with the transmembrane glycoprotein of HTLV.

Large granular lymphocyte leukaemia occurring after renal transplantation.

Post-transplantation lymphoproliferative disorders (PTLD) are a clinicopathologically heterogeneous group of lymphoid proliferations. The majority are of B-cell origin and associated with Epstein-Barr virus (EBV) infection. In contrast, the development of T-cell PTLD is much less common and EBV does not appear to be involved in pathogenesis. In this report we describe three patients who developed large granular lymphocyte (LGL) leukaemia after renal transplantation. These patients had clonal expansion of CD3+, CD8+, CD57+, CD56- LGL. We were unable to detect CMV antigen or find evidence for EBV or human T-cell leukaemia/lymphoma virus genome in the LGL from these patients. These data show that LGL leukaemia should be included as one of the types of T-cell proliferations which can occur post transplant.

Absence of human herpes virus 8 DNA sequences in large granular lymphocyte (LGL) leukemia.

The etiology of large granular lymphocyte (LGL) leukemia is uncertain. Recently, a Kaposi's sarcoma-associated herpes virus, denoted as human herpes virus 8 (HHV-8), has been identified. Some data suggest that HHV-8 and Epstein-Barr virus (EBV) may interact to induce malignant transformation. Infection with EBV has been implicated in the pathogenesis of some cases of LGL leukemia. Therefore, we performed PCR analyses for HHV-8 detection in samples from nineteen patients with LGL leukemia; three of these samples contained the EBV genome. We could not detect HHV-8 sequences in any of these patients. Therefore, HHV-8 infection is not involved in the pathogenesis of T-LGL leukemia.

Humoral immune abnormalities in T-cell large granular lymphocyte leukemia.

The prevalence of humoral immune dysfunction has not been defined in a large series of patients with T-cell large granular lymphocyte leukemia (T-LGL) confirmed to be clonal by T-cell receptor analysis. Therefore we evaluated the presence of multiple autoantibodies in 27 patients with this disease. Humoral immune abnormalities included: rheumatoid factor (RF) (15/27 patients), antinuclear antibody (ANA) (13/27 patients), polyclonal hypergammaglobulinemia (15/24 patients), elevated serum immunoglobulins (17/26 patients), immune complex formation (18/25 patients), elevated beta-2 microglobulin (13/18 patients) and neutrophil-reactive IgG (18/20 patients). Disease manifestations in these patients were due to complications of cytopenia or autoimmune abnormalities. Infection was a common finding (21/27 patients) and likely reflected their neutropenia. Rheumatoid arthritis (11/27 patients), anemia (12/27 patients) and thrombocytopenia (10/27 patients) were less common but still frequently observed. This study demonstrates the presence of multiple autoantibodies in a large series of patients with documented clonal T-LGL proliferations.

Resolution of autoimmune hemolytic anemia following splenectomy in CD3+ large granular lymphocyte leukemia.

A 24 year old female with a 4 year history of anemia and absolute lymphocytosis was evaluated and found to have T cell large granular lymphocyte (T-LGL) leukemia associated with autoimmune hemolytic anemia, neutropenia, mild thrombocytopenia and splenomegaly. In an effort to ameliorate her symptomatic cytopenias, she was treated with prednisone and subsequently methotrexate without success. In February 1993, she underwent splenectomy for symptomatic anemia. Splenectomy resulted in an increased hemoglobin concentration to normal levels, resolution of all laboratory evidence of hemolysis, and disappearance of thrombocytopenia. This response has been durable despite persistence of the abnormal LGL clone. We suggest that splenectomy may be an effective treatment for autoimmune hemolytic anemia and/or thrombocytopenia often associated with T-LGL leukemia. As this disease often exhibits a chronic clinical course with morbidity resulting from consequences of resultant cytopenias rather than visceral involvement with leukemic LGL, effective treatment of cytopenias despite persistence of the abnormal LGL clone is beneficial.

Interleukin-12 is a costimulatory cytokine for leukemic CD3+ large granular lymphocytes.

The activation signals leading to proliferation of leukemic CD3+ large granular lymphocytes (LGL) are incompletely understood. In this study, the role of recombinant human interleukin-12 (rhIL-12) alone or in combination with other activation signals was studied in vitro. Anti-CD3 monoclonal antibody (MoAb) alone caused marked stimulation of peripheral blood mononuclear cells (PBMC) from three CD3+ LGL leukemic patients, whereas rhIL-12 alone had less effect as measured in a [3H]thymidine incorporation assay. The combination signals of anti-CD3 MoAb and rhIL-12 produced a proliferative response greater than anti-CD3 MoAb alone or rhIL-12 alone. Leukemic LGL, purified by CD8+ affinity chromatography, showed similar proliferative responses as PBMC from LGL leukemic patients, suggesting that the observed effect was indeed due to direct stimulation of leukemic LGL. Radiolabeled IL-12 binding studies demonstrated that anti-CD3 MoAb upregulated the number of IL-12 receptors per cell on PBMC from these patients. Neutralizing antibody to rhIL-12 partially blocked the proliferative response to anti-CD3 MoAb suggesting involvement of IL-12 in the pathway of activation of leukemic LGL via stimulation of the T cell receptor (TCR) (mimicking activation by antigen). These results show that IL-12 acts as a costimulatory cytokine for proliferation of leukemic LGL activated through the TCR in vitro.

CD3+, CD56+ aggressive variant of large granular lymphocyte leukemia.

Clonal expansions of CD3+ large granular lymphocytes (LGL) have been classified as T-LGL leukemia. The majority of patients with T-LGL leukemia have a chronic disease (years) manifested often by severe neutropenia, rheumatoid arthritis, and mild-to-moderate splenomegaly. The characteristic phenotype of the leukemic LGL is CD3+, CD8+, CD16+, CD57+, and CD56-. In this report we describe an aggressive variant of T-LGL leukemia in which leukemic LGL also expressed CD56, as identified by two-color flow-cytometry analysis. In contrast to the chronic nature typical of T-LGL leukemia, these patients presented with a severe systemic illness that was rapidly progressive and resistant to treatment. Atypical clinical features included rapidly increasing spleen size to massive proportions, extensive lymphadenopathy, and the presence of B symptoms (fever, nightsweats, weight loss). Hematologic and pathologic features were also unusual for T-LGL leukemia. These patients had very high LGL counts at diagnosis (range 11,692 to 26,312 microL), which increased rapidly despite treatment. Histopathologic examination of splenic sections showed extensive infiltration of red pulp cords and sinuses by leukemic cells with atrophy of the white pulp. These clinicopathologic features are similar to those described for patients with natural killer cell (NK)-LGL leukemia, whose cells are also CD56+. However, unlike NK-LGL leukemia, we could not show a direct pathogenic role for Epstein-Barr virus (EBV), as Southern-blot analyses using an EBV-joined termini probe were negative in these patients. Our findings suggest that CD3+, CD56+ LGL leukemia is a distinct clinicopathologic entity separate from the usual CD3+, CD56- T-LGL leukemia. The expression on leukemic LGL of CD56, an adhesion molecule, may determine the aggressive biologic nature of this newly described disease.

Interaction of porcine immunoglobulin M with protein A of Staphylococcus aureus.

Intrigued by reports that the mitogenic effect of protein A on B lymphocytes was due to a direct interaction of cell surface immunoglobulin with protein A, the binding of 19 S, 8 S, and Fab mu fragments of 125I-labeled IgM isolated from porcine serum was investigated. Approx. 60% of purified 19 S porcine IgM interacted specifically with Protein A-Sepharose. Mild reduction and alkylation of 19 S IgM to yield monomeric IgM did not appear to alter its ability to bind to protein A. Elution of either molecular species of IgM from protein A and subsequent repassage over Protein A-Sepharose resulted in nearly quantitative rebinding of the IgM to protein A. Fab mu fragments prepared by digestion of 19 S IgM with pepsin exhibited binding characteristics similar to that observed for intact and monomeric IgM. These results suggest that: (1) there are at least two populations of porcine serum IgM, one that binds to protein A and one that does not; (2) these populations are not interconverting; (3) the ability of IgM to bind to protein A is not dependent on the 19 S pentameric structure extant in sera, but rather is an intrinsic property of some and not all four chain IgM protomers; and (4) a binding site for protein A on porcine IgM is localized in the Fab mu (including the C mu 2 domain) regions of the molecule.