Myelomonocytic antigens in B-cell chronic lymphocytic leukemia

The clinical significance of myelomonocytic (MyMo) antigens in B-cell chronic lymphocytic leukemia (B-CLL) is unclear. We have analyzed the expression of MyMo antigens (CD13, CD14 (LeuM3, My4, Mo2), CD15, CD11b, CD11c, CD33 and CD68) on B-lymphocytes (CD19+) in 105 B-CLL patients and in 35 controls. A double direct staining technique and flow cytometric analysis was performed. The expression of MyMo antigens on the control group did not exceed 4% B-lymphocytes. A MyMo antigen was considered as positive when present in > or = 10% of B-lymphocytes. Among the B-CLL patients, 28 (26.7%) were positive for CD11c, 21 (20.0%) for CD11b, nine (8.6%) for CD15, five (4.8%) for CD13, two (1.9%) for Mo2, and one (1.0%) for My4. No patient was positive for LeuM3, CD33 or CD68. CD11c was more frequently expressed in patients with a short lymphocyte doubling time (< 12 months) (P = 0.05) and CD11b in the group with a higher number of lymphoid areas involved (P = 0.02). No correlation was found between lymphoid morphology and MyMo antigen expression. Fourteen of the 80 patients at risk subsequently progressed to a more advanced stage. Multivariate analysis identified hemoglobin (P = 0.004) and CD11b positivity (P = 0.009) as independent variables for disease progression. Fifteen patients died during evolution. Seven out of the 21 CD11b positive patients and eight of the 84 CD11b negative patients died (LR: P = 0.02, BG: P = 0.05). In the multivariate analysis, only CD11b positivity (> or = 10%) added prognostic value to clinical stages.     

The cellular biology of B-cell chronic lymphocytic leukemia

In conclusion, B-CLL cells through their immunophenotype have the functional potential required to interact with cells in what has been called the immunological synapse, i.e. the cognate interactions between T-cells, antigen-presenting cells and B-cells during immunopoiesis. The data reviewed herein provides substantial evidence to suggest that B-CLL cells in fact can interact, not only with T-cells but also with endothelial cells and stromal cells in the bone marrow. These interactions, in particular signaling through CD40, contribute to extended survival and proliferation of B-CLL cells and, thereby, the risk of complete malignant transformation of the clone. Therefore, this review would suggest that the answers to how B-CLL is initiated may be found in molecules responsible for the normal regulation of immunopoiesis. Transformation to malignancy, by contrast, is likely to be caused by loss of control over the G1 restriction in the cell cycle in B-CLL cells.     

Twenty years of research on B-cell chronic lymphocytic leukemia

The typical MRI features of the most common pancreatic diseases, such as pancreatitis and adenocarcinoma of the pancreas, have been established. However, even in these common pancreatic disorders, MRI correlation with the underlying pathology is limited for clinical reasons. We emphasize MR-pathological correlation of inflammatory and neoplastic pancreatic changes, including pancreatitis, adenocarcinoma, acinar cell carcinoma, rare cystic and solid pancreatic neoplasms, and islet cell tumors. By highlighting the correlation of key pathological features with MR findings, a better understanding of the MR appearance of pancreatic pathology can be provided. In addition, MRI may prove a powerful tool in detection and characterization of pancreatic tumors.     

5''-nucleotidase activity in lymphocytes from patients affected by B-cell chronic lymphocytic leukemia

Port site metastasis often occurs after laparoscopic colorectal resection of Dukes B and Dukes C tumors. To evaluate the feasible indication for laparoscopic surgery for the cure of colorectal cancer, we performed a clinicopathologic study with special reference to tumor size. A total of 233 patients who underwent curative resection of colorectal cancer at our department during 15 years were examined. There were 59 Dukes A tumors, and their mean size was 2.86 cm. Tumors < 2 cm, compared with tumors > 2 cm, were characterized by grossly superficial type (68% vs. 9%, p < 0.01), negative serosal invasion (95% vs. 24%, p < 0.01), and absence of lymph node metastasis (91% vs. 60%, p < 0.01). Among tumors < 3 cm, node-negative cases were distinguished by location in the colon (73% vs. 22%, p < 0.05) and by histologically well-differentiated type (75% vs. 11%, p < 0.01) in comparison with node-positive cases. All 14 patients with Dukes A tumor undergoing laparoscopic colectomy were free of recurrence during a mean follow-up period of 34 months. The results indicate that all cancers < 2 cm and well-differentiated colon cancers < 3 cm are good candidates for laparoscopic colorectal surgery for cure.     

Clinical and biological relevance of flow cytometric determination of P-glycoprotein expression in acute non-lymphocytic leukemia

OBJECTIVE: To evaluate the characteristics of P-glycoprotein (P-gp) expression of acute non-lymphocytic leukemia (ANLL) at different status and the prognostic and biological features in ANLL at diagnosis. METHODS: Monoclonal antibody UIC2 and indirect immunofluorescence assay by flow cytometry were used to determine P-gp expression of 169 patients with ANLL, including 152 previously untreated, 7 refractory and 10 at remission. RESULTS: P-gp was expressed in 28.9% of the previously untreated ANLL cases and P-gp was lower than that in 71.4% of the refractory cases (P < 0.05). No P-gp expression was found in the patients at remission. For previously untreated AN-LL, P-gp was highly expressed in hybrid acute leukemia (66.7%) and acute monoblastic leukemia (47.4%). P-gp expression was highly associated with surface markers [cluster of differentiation (CD) 34, CD7, CD14, CD42b and CD61] and unfavorable cytogenetic abnormalities. About 23% of P-gp-ANLL obtained complete remission, which was significantly lower than that (76%) in P-gp-cases. CONCLUSIONS: P-gp expression is higher in refractory ANLL cases than that in cases at diagnosis or at remission. P-gp is an index of poor prognosis in adults with ANLL. P-gp+ ANLL cases have unique clinical and biological characteristics.     

Simultaneous occurrence of B-cell chronic lymphocytic leukemia and chronic myeloid leukemia with further evolution to lymphoid blast crisis

The coexistence of chronic myeloid leukemia (CML) and B-cell chronic lymphocytic leukemia (CLL) in the same patient is rare. A 71-year-old woman developed a B-lineage lymphoid blast crisis at 18 months after diagnosis of Ph-positive CML. At this time, a lymphoid cell population with morphologic and immunophenotypic features of CLL was demonstrated. The retrospective review of the tests performed at diagnosis and thereafter disclosed the presence of lymphoid nodules in the initial bone marrow biopsy in the absence of lymphocytosis. Subsequently, there was an appearance of moderate lymphocytosis in the following months. Therefore, diagnosis of CML and coexistent CLL was established. Although a transient remission of blast crisis was achieved, blast cells reappeared two months later and the patient died shortly afterwards. Molecular studies of the immunoglobulin heavy chain gene (IH) rearrangement pattern point to the origin of the diseases in two different cell clones. In addition, previously published cases of simultaneous CLL and CML are reviewed.     

High incidence of relapse after autologous stem-cell transplantation for B-cell chronic lymphocytic leukemia or small lymphocytic lymphoma

BACKGROUND: High-dose therapy followed by autologous stem-cell transplantation (autoSCT) induces complete remissions in the majority of patients with advanced B-cell chronic lymphocytic leukemia or small lymphocytic lymphoma (B-CLL). However, the long-term utility of this therapy for B-CLL is unknown. PATIENTS AND METHODS: Sixteen previously treated patients with B-CLL were transplanted using autologous blood (n = 13) or bone marrow (n = 3). The median age of the patients was 49 f1p4s (range 44-60 years), and the median number of prior chemotherapy regimens was two. Patients were eligible for transplantation if they had chemosensitive disease and no morphologic evidence of malignant cells in the graft. Preparative regimens included cyclophosphamide and total-body-irradiation, with or without cytarabine, or BEAC. RESULTS: All patients engrafted and achieved a complete remission posttransplant. Ten patients were alive at a median of 41 months (range 22-125 months), and five were disease-free. Eight patients have relapsed and six have died (three from progressive malignancy). The projected three-year overall survival, failure-free survival and relapse rates were 68%, 37%, and 56%, respectively. CONCLUSIONS: AutoSCT for advanced B-CLL is associated with a high relapse rate. Whether this therapy can prolong life or produce cures is uncertain.     

p53 expression in B-cell chronic lymphocytic leukemia: a marker of disease progression and poor prognosis

We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.     

Potentiation of chlorambucil cytotoxicity in B-cell chronic lymphocytic leukemia by inhibition of DNA-dependent protein kinase activity using wortmannin

In this study, we examined the ability of wortmannin to modulate chlorambucil (CLB) cytotoxicity in lymphocyte samples from patients with B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested previously that enhanced cross-link repair is a primary mechanism of resistance to nitrogen mustards (NMs) in B-CLL. DNA-dependent protein kinase (DNA-PK) is involved in the repair of double-strand breaks and in rejoining steps in recombination mechanisms. Mutants defective in this process are hypersensitive to alkylating agents. We have recently demonstrated that the activity of DNA-PK is a determinant in the cellular response of B-CLL to CLB. The DNA-PK gene has homology to the P110 phosphatidylinositol 3-kinase (PI 3-K). Wortmannin, an inhibitor of P110 PI 3-K, also inhibits DNA-PK activity in vitro. We investigated the effect of wortmannin on DNA-PK activity and CLB toxicity in the lymphocytes from 11 patients with B-CLL. Our results demonstrate that DNA-PK activity is decreased after exposure to wortmannin in a dose-dependent manner. Wortmannin, at nontoxic concentrations, synergistically sensitized B-CLL lymphocytes to the effects of CLB. Moreover, we observed a significant correlation when we compared the fold decrease in DNA-PK activity and the synergistic value (I), obtained when wortmannin was used at 0.1 microM. In the resistant B-CLL lymphocyte samples, there was a highly significant correlation between the ability of wortmannin at 0.1 and 0.25 microM to decrease the level of DNA-PK activity and to increase CLB sensitivity. In a model of primary human tumor cells, our findings suggest that the inhibition of DNA-PK activity may be a powerful way to overcome resistance to NMs such as CLB and point to new possibilities to improve the effectiveness of NM therapy.     

Chlorambucil synergizes with purine analogs in inducing in vitro cytotoxicity in B-cell chronic lymphocytic leukemia

Combinations of different drug concentrations of CLB + FAMP and CLB + 2-CDA were synergistic in, respectively, 42.9% and 34.8%. At leukemic cell survival < or = 50%, 16.4% and 23.4% of all combinations were synergistic in the 2-CDA and FAMP groups, respectively. A significantly higher mean value of antagonistic interactions was observed in the 2-CDA group (p = 0.037).     

Aspirin and salicylate induce apoptosis and activation of caspases in B-cell chronic lymphocytic leukemia cells

We analyzed the effect of aspirin, salicylate, and other nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 for cells from 5 patients was 5.9 +/- 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced an important cytotoxic effect after 4 days of incubation. No effect was observed with other NSAIDs, at concentrations that inhibit cyclooxygenase, such as ketorolac (10 micromol/mL), NS-398 (100 micromol/mL), or indomethacin (20 micromol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 +/- 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and salicylate induced DNA fragmentation and the proteolytic cleavage of poly(ADP(adenosine 5'-diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by analysis of cell viability. B and T lymphocytes from normal donors and T lymphocytes from CLL patients are more resistant to aspirin-induced apoptosis, as determined by analysis of phosphatidylserine exposure. These results indicate that aspirin and salicylate induce apoptosis of B-CLL cells by activation of caspases and that this activation involves cyclooxygenase-independent mechanisms.     

Intussuception as a complication of chronic lymphocytic leukemia

The case of a 58-year-old man with chronic lymphocytic leukemia (B-cell type) who later developed an intussuception of the small intestine due to a tumor is described. The histopathological findings of the removed tumor were compatible with those of diffuse small lymphocytic lymphoma (B-cell type). The residual tumor became smaller with CHOP therapy. It is considered that CLL cell infiltration into the small intestine resulted in intussuception. Since many tumors and lymphomas can form polypoid lesions causing an intussuception. This is a possible complication of CLL and it could occur even when the WBC count is well controlled.     

Deficiency of the ATM protein expression defines an aggressive subgroup of B-cell chronic lymphocytic leukemia

The gene mutated in ataxia telangiectasia, ATM, on human chromosome 11q22-q23 is implicated in cell cycle control and DNA repair. Ataxia telangiectasia patients as well as ATM-deficient mice are immune deficient and develop lymphoproliferative disease. Abnormalities in 11q22.3-q23.1 have also been described in B-cell chronic lymphocytic leukemia (B-CLL). We analyzed B-CLL samples for loss of heterozygosity (LOH) using microsatellite markers located at the ATM (D11S2179), mixed-lineage leukemia (MLL; D11S1356), and BCL1 (D11S987) loci, all of which are located around 11q23. Five (14%) of 36 informative cases showed LOH at the ATM gene, and two of these five cases had LOH at the MLL gene. No LOH was detected at the BCL1 locus, and none of the cases showed LOH at the MLL gene without LOH at the ATM gene. Four of these five cases with LOH at the ATM gene were studied for ATM protein expression by Western blot analysis. All four cases lacked ATM protein. An additional 111 cases of B-CLL were studied for expression of ATM protein by Western blot analysis and RIA. Thirty-eight (34%) of these cases showed ATM levels <50% of that seen in normal lymphoid cells. No morphological or immunophenotypic difference was observed between ATM-deficient B-CLL cases and cases with normal ATM expression. However, patients with ATM deficiency had significantly shorter survival times (35.66 versus 97.3 months; P = 0.003) and more aggressive disease, suggesting that ATM is involved in the leukemogenesis of B-CLL. These data also suggest that the ATM gene may play a role in the reported 11q23 abnormality in B-CLL, which also characterizes an aggressive disease.     

Prognostic significance of the cell cycle inhibitor p27Kip1 in chronic B-cell lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes. The identification of p27(kip1), a cyclin-dependent kinase inhibitor that contributes to cell cycle arrest and represents a link between extracellular signals and cell cycle, prompted us to study p27 protein in the lymphocytes from 88 patients with B-CLL and 32 patients with other chronic B-lymphoproliferative disorders. The expression of p27 protein was higher in B-CLL samples with variations among them. In B-CLL, p27 levels were independent of absolute number of circulating lymphocytes, but strongly correlated with both lymphocyte and total tumor mass (TTM) doubling time. High p27 expression was associated with a poorer overall prognosis. In vitro, there was an increased spontaneous survival of B-CLL cells expressing high p27 levels. Interleukin-4 (IL-4) upregulated p27 levels in B-CLL cells, while fludarabine decreased p27 levels. Thus, our results indicate that p27 may be a valuable kinetic marker in B-CLL by providing instantaneous estimation of the disease doubling time. In addition, these results suggest that there is a link between p27 expression and the ability of CLL cells to undergo apoptosis.     

Common expression of the multidrug resistance marker P-glycoprotein in B-cell chronic lymphocytic leukaemia and correlation with in vitro drug resistance

The expression of P-glycoprotein (Pgp), which is associated with multidrug resistance (MDR), was investigated in 20 B-cell chronic lymphocytic leukaemia (B-CLL) patients by flow cytometry using two Pgp-specific monoclonal antibodies (mAb), MRK-16 which recognizes an extracellular epitope, and JSB-1 which recognizes an intracellular epitope. Sixteen (80%) patients were positive with MRK-16 whereas all patients were positive with JSB-1. The proportion of Pgp-positive lymphocytes from each patient sample varied from 2-94% for MRK-16 and 20-93% for JSB-1. There was no correlation between the level of positivity and disease stage or treatment history. In vitro drug resistance to vincristine (VCR) and doxorubicin (DOX) was determined by the colorimetric MTT assay. All patients were resistant to one or both drugs being consistent with the expression of Pgp. There was no correlation between the level of resistance and disease stage or drug treatment. We investigated the expression of Pgp in the normal counterpart of the B-CLL cells, CD5+CD19+ B-lymphocytes. A minor subpopulation (3%) of CD5+CD19+ lymphocytes isolated from normal controls expressed Pgp suggesting that these cells may be the potential precursors to the B-CLL cell. We conclude that Pgp expression and drug resistance are inherent characteristics of the B-CLL lymphocyte.     

Sudden death in a patient with chronic lymphocytic leukemia

The authors describe a 51-year-old man with chronic lymphocytic leukemia who presented with respiratory distress and then died suddenly while in hospital. Autopsy revealed pulmonary leukostasis and a large intracardiac mass containing mostly mature lymphocytes and fibrin. Although leukostasis and lymphocyte thrombi have been described (albeit rarely) in chronic lymphocytic leukemia, an intracardiac "clot" has not. It seems plausible that this intracardiac mass caused the patient's death.     

Increased bcl-2/bax ratio in B-cell chronic lymphocytic leukemia is associated with a progressive pattern of disease

In order to evaluate clinical implications of altered expression of bcl-2 and bax proteins in B-cell chronic lymphocytic leukemia (CLL) we studied 27 patients with this disease. Cytofluorometric levels of bcl-2 did not reflect the status of disease. In contrast bax expression was lower in progressive than in non-progressive disease, therefore leading to a higher bcl-2/bax ratio in patients of the former group. If confirmed in longitudinal studies, quantitative cytofluorometric evaluation of bcl-2 and bax protein might help to identify patients with progressive disease who could possibly benefit from early therapy.