Reduced tocopherol content of B cells from patients with chronic lymphocytic leukemia

The tocopherol content of lymphocytes, erythrocytes, and plasma from patients with chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), and normal subjects was measured by a sensitive high performance liquid chromatographic method. Lymphocytes from patients with CLL had lower values of tocopherol (1.7 +/- 1.0 micrograms/10(9) cells) than lymphocytes from normal subjects (3.8 +/- 0.7 micrograms/10(9) cells). Mononuclear cells from patients with HCL had an increased tocopherol content of 6.2 +/- 1.0 micrograms/10(9) cells. Subfractionation of the lymphocytes from patients with CLL into T- and B-cell subgroups showed that the tocopherol content of T cells was the same as in normal subjects (4.1 +/- 0.5 micrograms/10(9) cells versus 3.5 +/- 1.2), but that the tocopherol content of the B cells was markedly reduced compared to normals (2.6 +/- 1.0 versus 6.0 +/- 1.3).     

Increased ascorbic acid content in chronic lymphocytic leukemia B lymphocytes.

Human lymphocyte extracts analyzed by high-performance liquid chromatography reveal a major UV-absorbing peak that was shown to be ascorbic acid by spectral, chemical, and enzymatic criteria. Because this peak appeared very prominent in the elution profile of chronic lymphocytic leukemia (CLL) lymphocyte extracts, we measured the ascorbic acid content in lymphocytes from the blood of normal subjects and untreated patients with chronic lymphocytic leukemia. A significantly higher concentration of 111 +/- 15.3 nmol per 10(8) cells (mean +/- SEM) was found in CLL lymphocytes than in normal blood lymphocytes, which contained 42.2 +/- 3.3 nmol per 10(8) cells. Selective enrichment with B and T cells showed that this difference was limited to the chronic lymphocytic leukemia B cell, which had a 5- to 15-fold higher content of ascorbic acid than normal B cells had. In contrast, the ascorbic acid level was similar in normal and CLL T cells. The very high ascorbic acid content provides the chronic lymphocytic leukemia B cell with a reducing substance that could react with oxidants or free radicals.     

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia

Summary The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16⁺, CD57⁺ and cytotoxic CD 8⁺) was studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57⁺, CD 16⁺ and cytotoxic CD 8⁺ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3⁺ lymphocytes, which were 1.7–27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4⁺ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57⁺ receptors in comparison with their peripheral blood CD 57⁺ lymphocytes or the CD57⁺ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.     

Decreased 5''-nucleotidase activity in a T lymphocyte subpopulation from patients with B cell chronic lymphocytic leukemia

The activity of the ectoenzyme 5'-nucleotidase (5'N) was determined in the T lymphocyte subpopulations from patients with chronic lymphocytic leukemia (CLL). 5'N could be detected only in the T cells from patients whose B cells also had enzyme activity. The specific activity of CLL T4 cells was 0.17 +/- 0.02 micron/h/mg protein, similar to that of normal T4 cells, which was 0.13 +/- 0.08 micron/h/mg. The CLL T8 cells, however, had a significantly lower 5'N activity (0.17 +/- 0.02 micron/h/mg) than normal T8 cells (0.41 +/- 0.11 micron/h/mg) (P = .003). Normal null cells had very low activity, while much higher levels were found in the null cells of CLL patients whose B cells had activity. These findings document a difference in activity of an enzyme between the T8 cell population of patients with CLL and that of normal subjects.     

Membrane difference in peripheral blood lymphocytes from patients with chronic lymphocytic leukemia and Hodgkin's disease.

Lymphocytes were isolated from the peripheral blood of 21 normal persons and 66 patients with chronic lymphocytic leukemia (CLL), CLL in remission, Hodgkin's disease, Hodgkin's disease in remission, various other tumors, or cardiovascular diseases; The lymphocytes were studied for cap formation and agglutinability by concanavalin A, and for cell attachment to the surface of a petri dish. The frequency of cap formation was lowest in lymphocytes from patients with untreated Hodgkin's disease (2.1 plus or minus 0.8%), next lowest in lymphocytes from patients with CLL who were or were not under treatment (7,0 plus or minus 1;3%), and also low in Hodgkin's disease in remission (10.6 plus or minus 1.2%). The frequencies of cap formation by lymphocytes from patients with various other tumors (19.1 plus or minus 2.5%), with CLL in remission (24.0 plus or minus 0.9%), and with nonmalignant diseases (26.0 plus or minus 2.2%) were more similar to the frequency found in lymphocytes from normal persons (29.4 plus or minus 2.8%). Lymphocytes from all the patients, including those in remission, showed a higher degree of agglutinability by concanavalin A than lymphocytes from normal persons. Cell attachment to a petri dish was highest with CLL, next highest with CLL in remission, and low for normal persons and all the other patients. Lymphocytes from normal persons that consisted predominantly of thymus-derived cells gave similar results to isolated normal bone marrow-derived cells. The results indicate that there were different changes in the surface membrane of lymphocytes from patients with CLL, CLL in remission, Hodgkin's disease, and Hodgkin's disease in remission, and that the patients in clinical remission still showed abnormalities in their lymphocytes.     

Chemosensitivity of lymphocytes from patients with B-cell chronic lymphocytic leukemia to chlorambucil, fludarabine, and camptothecin analogs

Chemosensitivity of B lymphocytes, obtained from 65 patients with B- cell chronic lymphocytic leukemia (B-CLL), Rai stages 0 through IV, was determined using the MTT assay. The results were expressed by the drug concentration required for 50% inhibition of cell viability (IC50). The cytotoxicity of chlorambucil (CLB) was compared with that of fludarabine and the DNA topoisomerase I inhibitors, camptothecin, 9- aminocamptothecin, 10,11-methylenedioxy-20(S)-camptothecin (10,11-MDC) and 9-amino-10,11-methylenedioxy-20(S)-campthothecin (9-A-10,11-MDC), and topotecan. Considerable heterogeneity in sensitivity to CLB was observed, with a median IC50 of 40.5 mumol/L in untreated patients. B- CLL cells from patients treated with CLB had a significantly higher median IC50 of 86.0 mumol/L (P < .01). Untreated as well as CLB-treated patients were divided into two subsets. For the purpose of this study, B-CLL lymphocytes with an IC50 CLB of less than 61.0 mumol/L were designated as "sensitive" and those with an IC50 CLB of > or = 61.0 mumol/L were designated as "resistant." After baseline assays, 15 untreated patients received CLB; after treatment, the IC50 increased in B-CLL lymphocytes from 13 of 15 patients. The response to CLB treatment, determined by its effect on the absolute lymphocyte count and by the Eastern Cooperative Oncology Group clinical criteria, was significantly better in patients whose lymphocytes had an IC50 CLB of less than 61.0 mumol/L before therapy (P < .01). B-CLL lymphocytes also had a variable degree of sensitivity in vitro to each of the other drugs. There was significant cross-resistance between CLB and fludarabine (P < 0.01). Whereas only 29% of CLB-resistant B-lymphocyte specimens obtained from individual patients were sensitive to fludarabine in vitro, 52% and 67% of CLB-resistant lymphocyte samples were sensitive to 10,11-MDC and 9-A-10,11-MDC, respectively. We have previously reported that p53 gene mutations were associated with aggressive B-CLL and a poor prognosis. B lymphocytes from seven patients with these mutations were resistant to CLB, and five of six were resistant to fludarabine. Lymphocytes from four of seven were resistant to 10,11-MDC, and three of four were resistant to 9-A-10,11- MDC. This study implies that the MTT assay may be useful in identifying subsets of CLL patients resistant to conventional chemotherapy. However, definitive conclusions can not be drawn in view of the small number of patients studied prospectively. In addition, these results suggest the potential of camptothecin-based therapy for patients unresponsive to standard treatment.     

Relationship of fluorescence polarization to cell lineage in lymphocytes from normal subjects and patients with chronic lymphocytic leukemia

Previous investigations have shown differences in fluorescence polarization between normal and chronic lymphocytic leukemia lymphocytes following incubation with the probe 1,6-diphenyl-1,3,5-hexatriene. In the present study, we determined the fluorescence polarization of unseparated or enriched subpopulations of T and B lymphocytes from normal subjects and patients with chronic lymphocytic leukemia. As had been observed by others, the mean polarization (P) value at 25 degrees C for unseparated chronic lymphocytic leukemia lymphocytes, .240 +/- .007 (N = 22), was lower than that of unseparated normal lymphocytes, .248 +/- .005 (N = 18), P less than .001 (Student's t-test). The difference was greater when B-enriched populations were compared. The mean P value of B-cell-enriched chronic lymphocytic leukemia lymphocytes, .240 +/- .007 (N = 5), was significantly lower than that of B-cell-enriched normal preparations, .256 +/- .004 (N = 5), P less than .001. In contrast, no significant difference was found between normal and chronic lymphocytic leukemia T cells. The anomalous fluorescence polarization manifested by chronic lymphocytic leukemia lymphocytes of B-cell origin serves to distinguish this lineage from its normal counterpart and from T cells of either source.     

Anomalous function of vimentin in chronic lymphocytic leukemia lymphocytes

Chronic lymphocytic leukemia (CLL) lymphocytes manifest anomalous motility and cap formation. Since these processes involve cytoskeletal proteins, vimentin from intermediate filaments of normal and CLL lymphocytes was investigated using hetero- and monoclonal antisera. The antisera reacted predominantly with a 60-kD polypeptide, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total lymphocyte proteins. When lymphocytes were stained by indirect immunofluorescence, normal lymphocytes demonstrated well defined cytoplasmic fibrils that capped spontaneously after contact with a glass surface and incubation at 37 degrees C. This capping was dependent on energy and intact microfilaments. Lymphocytes from patients with CLL showed several patterns. In one group, the initial staining was weak, and few capped cells were present after incubation. Lymphocytes from other patients had either normal or aberrantly organized fibrils in which capping was diminished. In another group, a fibrillar pattern with normal or increased capping was seen. In total, 47% +/- 5.1% (mean +/- SE) of normal lymphocytes capped after a 1-hr incubation at 37 degrees C (n = 12) compared to 21% +/- 5.1% for CLL lymphocytes (n = 20, p less than 0.002). Purified subpopulations of normal B and T cells did not differ from unfractionated normal lymphocyte populations. These results demonstrate an anomalous vimentin capping response in CLL lymphocytes. They also show that the arrangement of vimentin in these cells differs from that of normal lymphocytes.     

Improving treatment for patients with chronic lymphocytic leukemia

The last two decades have been time of tremendous progress in treatment for patients with chronic lymphocytic lymphoma (CLL). Chemoimmunotherapy (CIT) combining anti-CD20 monoclonal antibodies with purine nucleoside analogs has been a substantial advance for patients with CLL and results in increased response rates, progression-free survival, and overall survival. Despite these improved outcomes, only ≈ 45% of patients achieve a complete remission with CIT and nearly all patients eventually relapse and their remains a need to improve efficacy. Although new combinations of traditional agents may lead to incremental progress, more substantive improvements are likely to result through therapeutic targeting of novel pathways critical to CLL B-cell survival including targeting: (1) leukemia cell apoptotic resistance; (2) survival signals mediated through the B-cell receptor; and (3) nurturing interactions with the microenvironment. In this mini-review, we summarize Mayo Clinic's recent efforts to improve CIT for patients with CLL.     

B cell chronic lymphocytic leukemia with meningeal infiltration by T lymphocytes

Chronic lymphocytic leukemia (CLL) is a generalized malignancy of the lymphoid tissue characterized by an accumulation of monoclonal lymphocytes, usually of the B cell type. Involvement of the central nervous system is a rare complication, usually seen in T cell leukemias. We report a case of a 78-year-old woman with B cell CLL and meningeal infiltration by both B and T lymphocytes, although predominantly T lymphocytes. Neurological symptoms were the first manifestation of this disease. Computed tomography of the brain was negative. The diagnosis of leukemic meningitis was made on the basis of the examination of the cerebrospinal fluid, and which included cytological and flow cytometry analysis. The patient was given systemic chemotherapy in the form of chlorambucil and intrathecal administration of methotrexate and dexamethasone. After recovery, she had regular follow-up. We assume that this rare case of CLL might have been biclonal, with both B and T cell types.     

Glutathione depletion in chronic lymphocytic leukemia B lymphocytes.

Glutathione (GSH) content may be the major determinant of a cell's sensitivity to cytotoxic alkylating agents. In the present study, the GSH concentration was determined in lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia (CLL). Comparable levels were found in both types of cells. Incubation for 20 hours led to a decrease in GSH to 51% of baseline values in CLL B cells. Under the same conditions, normal B- or T-lymphocyte GSH content remained constant. GSH depletion was shown to be a characteristic of the B-CLL B lymphocyte. It was not found in the T cells of patients with B-CLL or in cells from patients with T-CLL. Chlorambucil (CLB) contributes to the decrease in GSH in B-CLL lymphocytes; after incubation with the drug, lower levels of GSH were found than in the normal B or T lymphocytes, B-CLL T cells, or T-CLL (CD4 or CD8) cells. GSH depletion of CLL B lymphocytes may be related to the greater therapeutic efficacy of CLB in B-CLL than in T-CLL.     

Rituximab therapy of patients with B-cell chronic lymphocytic leukemia

Rituximab (IDEC-C2B8) is a chimeric antibody that binds to the B-cell surface antigen CD20. Rituximab has significant activity in follicular non-Hodgkin lymphomas. Much less is known about the effects in chronic lymphocytic leukemia (CLL). We have initiated a phase II trial to evaluate the efficacy and safety of rituximab in patients with CD20+ pretreated CLL. To avoid the rituximab-associated toxicity, we restricted the tumor cell load, as measured by the number of circulating lymphocytes and the spleen size, in the first 2 cohorts of patients included in the study. Patients received 4 intravenous infusions of 375 mg/m2 once a week over a period of 1 month. Of the 28 patients evaluable for response, 7 patients showed a partial remission (National Cancer Institute criteria) lasting for a median of 20 weeks, with 1 patient still in remission after 71 weeks. Based on lymphocyte counts only, we found at least a 50% reduction of lymphocyte counts lasting for at least 4 weeks in 13 (45%) of 29 patients. Fifteen patients from 3 institutions were monitored for the immunophenotype profile of lymphocyte subsets. The number of CD5+CD20+ cells decreased significantly and remained low until day 28 after therapy. T-cell counts were not affected. With the exception of one rituximab-related death, adverse events in the remaining patients were mild. The results suggest that rituximab has clinical activity in pretreated patients with B-CLL. Toxicity is tolerable. Response duration after withdrawal of rituximab is rather short. Therefore, other modes of application and the combination with other agents need to be tested.     

Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes. We studied 178 CLL patients enrolled in a prospective study at the University of Michigan, of whom 139 and 39 were previously untreated and previously treated, respectively. We obtained unbiased, high-density, genome-wide measurements of subchromosomal copy number changes in highly purified DNA from sorted CD19(+) cells and buccal cells using the Affymetrix 50kXbaI SNP array platform (Santa Clara, CA). Genomic complexity scores were derived and correlated with the surrogate clinical end points time to first therapy (TTFT) and time to subsequent therapy (TTST): measures of disease aggressiveness and/or therapy efficaciousness. In univariate analysis, progressively increasing complexity scores in previously untreated CLL patients identified patients with short TTFT at high significance levels. Similarly, TTST was significantly shorter in pretreated patients with high as opposed to low genomic complexity. In multivariate analysis, genomic complexity emerged as an independent risk factor for short TTFT and TTST. Finally, algorithmic subchromosomal complexity determination was developed, facilitating automation and future routine clinical application of CLL whole-genome analysis.     

Development of acute myeloid leukemia in patients with untreated chronic lymphocytic leukemia

The development of acute myeloid leukemia (AML) in patients with untreated chronic lymphocytic leukemia (CLL) is rare. We experienced a 65-year-old man who developed AML with aberrant CD7 expression and monoallelic CEBPA mutation during watchful waiting for CLL. He failed to achieve complete response (CR) by standard induction therapy for AML. We retrospectively reviewed 27 patients who developed AML with untreated CLL published between 1973 and 2016. The median age at diagnosis of AML was 68 years, and the median duration between the diagnoses of AML and CLL was 4.2 years. Diagnosis of AML and CLL was made simultaneously in 16 patients. The CR rate of AML was 42.9%, and the median survival was only 1.5 months after the diagnosis of AML. Patients who achieved CR tended to survive longer than those who did not. Our results demonstrated that the development of AML in patients with untreated CLL was associated with a poor response to chemotherapy and an extremely poor prognosis.     

Pulmonary infiltration from chronic lymphocytic leukemia

We present 2 patients with chronic lymphocytic leukemia infiltration of the lung resulting in centrilobular nodularity on computed tomography. We present the x-ray and computed tomography patterns with pathological findings in these cases.