Activity of oral fludarabine phosphate in previously treated chronic lymphocytic leukemia.

PURPOSE: A prospective, multicenter, open-label phase II clinical trial was conducted to assess the efficacy and safety of oral fludarabine phosphate. Reference to an historical group of patients treated with the intravenous (IV) formulation allowed the investigators to compare the two formulations. PATIENTS AND METHODS: Efficacy was assessed using the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) and National Cancer Institute (NCI) criteria for complete remission (CR), partial remission (PR), stable disease, or disease progression. Safety monitoring included World Health Organization (WHO) toxicity grading for all adverse events. RESULTS: Seventy-eight (96.3%) of 81 recruited patients with previously treated B-cell chronic lymphocytic leukemia (CLL) received 10-mg tablets of fludarabine phosphate to a dose of 40 mg/m(2)/d for 5 days, repeated every 4 weeks, for a total of six to eight cycles. According to IWCLL criteria, the overall remission rate was 46.2% (CR, 20.5%; PR, 25.6%). The comparative figures using NCI criteria were 51.3% (CR, 17.9%; PR, 33.3%). Overall, 30 incidents of severe adverse events were reported for 22 patients. WHO grade 3 or grade 4 hematologic toxicities included granulocytopenia (53.8%), leukocytopenia (28.2%), thrombocytopenia (25.6%), and anemia (24.4%). Gastrointestinal adverse events were more common with the oral formulation than previously reported with IV fludarabine phosphate. However, these events were generally mild to moderate. CONCLUSION: This study demonstrates that oral fludarabine phosphate has similar clinical efficacy to the IV formulation and a safety profile that is both predictable and essentially similar to that of the IV formulation.

Randomized comparison of fludarabine, CAP, and ChOP in 938 previously untreated stage B and C chronic lymphocytic leukemia patients.

To comparatively assess first-line treatment with fludarabine and 2 anthracycline-containing regimens, namely CAP (cyclophosphamide, doxorubicin plus prednisone) and ChOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), in advanced stages of chronic lymphocytic leukemia (CLL), previously untreated patients with stage B or C CLL were randomly allocated to receive 6 monthly courses of either ChOP, CAP, or fludarabine (FAMP), stratified based on the Binet stages. End points were overall survival, treatment response, and tolerance. From June 1, 1990 to April 15, 1998, 938 patients (651 stage B and 287 stage C) were randomized in 73 centers. Compared to ChOP and FAMP, CAP induced lower overall remission rates (58.2%; ChOP, 71.5%; FAMP; 71.1%; P <.0001 for each), including lower clinical remission rates (CAP, 15.2%; ChOP, 29.6%; FAMP, 40.1%; P =.003). By contrast, median survival time did not differ significantly according to randomization (67, 70, and 69 months in the ChOP, CAP, and FAMP groups, respectively). Incidences of infections (< 5%) and autoimmune hemolytic anemia (< 2%) during the 6 courses were similar in the randomized groups, whereas fludarabine induced, compared to ChOP and CAP, more frequent protracted thrombocytopenia (P =.003) and less frequent nausea-vomiting (P =.003) and hair loss (P <.0001). For patients with stage B and C CLL first-line fludarabine and ChOP regimens both provided similar overall survival and close response rates, and better results than CAP. However, there was an increase in clinical remission rate and a trend toward a better tolerance of fludarabine over ChOP that may influence the choice between these regimens as front-line treatments in patients with CLL.

Chromosomal DNA and p53 stability, ubiquitin system and apoptosis in B-CLL lymphocytes.

The ubiquitin system regulates diverse biological processes such as DNA replication and repair, biogenesis of ribosome, peroxisome and nucleosome, cell cycle, stress response and signal transduction pathways. Thus, the reported role of the ubiquitin system in apoptotic death control as well the alteration of its control in carcinogenesis should come as no surprise. Indeed, we and other groups have reported that the ubiquitin system is involved in apoptotic cell death of normal human lymphocytes and that this control is altered in B lymphocytes derived from chronic lymphocytic leukemia patients (B-CLL), rendering these malignant cells hypersensitive to specific inhibition of protein degradation/processing through proteasomal function. This approach recently allowed us to demonstrate that the stability of the tumor suppressor and pro-apoptotic protein p53 is differentially regulated in B-CLL versus normal lymphocytes and that this difference might at least partly explain the impaired response of B-CLL lymphocytes to apoptotic death activation. These results strongly suggest an imbalance in p53 regulation in B-CLL cells that leads to a variable response to DNA damage and constitutively expressed chromosomal instability. The question we and others would like to address is whether this alteration, or more likely a subset of alterations of the ubiquitin-proteasome pathway, is specific to B-CLL malignancy or if it is a hallmark of cancer cells in general. In either case, a better understanding of the ubiquitin-dependent control of apoptosis should pave the way towards a methodological approach for in vitro development of discriminating treatments which may be of potential usefulness in clinical trials of B-CLL.

Intensive chemotherapy with hematopoietic cell transplantation after ESHAP therapy for relapsed or refractory non-Hodgkin's lymphoma. Results of a single-centre study of 65 patients.

This study was designed to assess the results of protracted courses of ESHAP (etoposide, cytarabine, cisplatin, methylprednisolone) therapy followed by intensive chemotherapy and hematopoietic cell transplantation (IC+HCT) for relapsed or refractory non-Hodgkin's lymphoma (NHL). Treatment consisted of 3 cycles of ESHAP; responsive patients (pts) then received 3 more cycles, and IC+HCT was used for pts in maintained partial (PR) or complete (CR) remission after the sixth ESHAP. Sixty-five pts entered the study. At enrollment, 27 pts had bone marrow (BM) and/or central nervous system (CNS) lymphomatous infiltration. Disease status was primary refractory lymphoma in 41 pts (63 %), and relapse in 24 pts (37 %). Results showed that two pts were not evaluable for the therapeutic response because of early treatment-related death. Thirty-nine (62 %) pts entered PR or CR after 3 cycles of ESHAP. Eleven pts subsequently had disease progression. Twenty-eight pts were in persistent CR or PR after 6 cycles of ESHAP. Refractory pts did not show a different response rate to relapsing pts (chi2= 1.73). Five pts were excluded from IC+HCT because of an inadequate graft or treatment-related toxicity. Twenty-three (35 %) pts completed the procedure. Five pts (22 %) relapsed after IC+HCT. The overall survival rate of the 39 responsive pts is 45 % at 60 months, with a median survival time of 30 months. Median survival among the 35 pts in whom second-line chemotherapy failed is 7.1 months, with a 4-year survival rate of 3 %. Despite the poor prognostic features of this group, 45% of pts responding to the first 3 cycles of chemotherapy are in prolonged remission, suggesting that rather than to transplant after just 2 cycles of salvage therapy, pursuing second-line chemotherapy may better discriminate between patients more likely to benefit from a subsequent transplant.

Detection of minimal residual disease in B chronic lymphocytic leukemia (CLL).

UNLABELLED: In the absence of specific chromosomal translocations the best method for detecting minimal residual disease (MRD) in B cell malignancies is based on the uniqueness of immunoglobulin (Ig) genes rearrangement. We here report a very sensitive method for assessing MRD in complete hematological remission (CHR) chronic lymphocytic leukemia (CLL) patients as defined by the international workshop on CLL (IWCLL). PATIENTS: Twelve CLL patients in CHR and complete phenotypic remission (CPR) were included in the study. Eight of them received Fludarabine (FDR), one was treated by Chop regimen, and the remaining 3 were rescued by polychemotherapy followed by autologous bone marrow transplantation (ABMT). METHODS: DNA extracted from peripheral blood lymphocytes (PBL) of each patient was amplified with VH family specific and framework 3 primers in 5' and a consensus JH primer in 3', before treatment and sequentially after the CPR completion. When no clonal rearrangement could be detected by this assay, the CDR3 sequence specific probe of the clone was used as the 3' primer, associated to the VH family specific primer in 5'. PCR products were analyzed by classical procedures in agarose and/or acrylamide gels. RESULTS: Mixtures of leukemic cells and normal PBL showed detection of a single leukemic cell among more than 10(5) normal cells. Four out of the 12 patients achieved molecular remission (MR) when employing CDR3 amplification. All 3 autografted patients were in MR, whereas only one out of the 9 patients treated by chemotherapy alone achieved MR. When using a clone specific probe, a clonal signal was observed in all cases but one (ABMT). Results presented here confirm that MR may be achieved in a few cases of B-CLL. Further studies are needed to determine the exact relationship between MRD and clinical outcome.

Autologous hematopoietic stem cell transplantation as salvage treatment for advanced B cell chronic lymphocytic leukemia.

Given the generally poor outcome of advanced B cell chronic lymphocytic leukemia, experimental approaches are warranted, especially for younger patients in whom classical treatments have failed. We therefore conducted a prospective single-center study, using polychemotherapy (ESHAP) to prepare patients for hematopoietic stem cell collection and autologous stem cell transplantation as consolidation therapy. Twenty patients entered the study. An adequate response to ESHAP was obtained in 13 patients, and sufficient stem cells for grafting were obtained in eight of the 12 patients who underwent the collection procedure. Six of these grafted patients are alive in complete clinical remission a median of 30 months after transplantation. It should be noted that we were only able to graft 40% of the patients enrolled in this study, either because a new remission could not be obtained or because not enough hematopoietic stem cells could be collected. This argues for stem cell collection as soon as a first remission is obtained, even if the autograft is done later in the course of the disease.

Severe autoimmune hemolytic anemia in eight patients treated with fludarabine.

We have used fludarabine to treat 36 patients with various lymphoid malignancies, including 29 with chronic lymphocytic leukemia (CLL). All these patients were heavily pretreated, and FAMP was prescribed on a compassionate basis. Eight patients (22%) developed severe autoimmune hemolytic anemia (AIHA) during or after treatment, and one died. Five patients had no previous history of hemolysis. These cases confirm the high incidence of AIHA after FAMP and suggest that the use of highly effective lymphocytotoxic agents such as fludarabine in heavily pretreated patients increases the risk of AIHA in CLL and other lymphoproliferative disorders.

Simple, fast method of detection apoptosis in lymphoid cells.

To facilitate the analysis of apoptotic cells, the present study proposes a new quantitative method based on the changes of light scatter properties of lymphoid cells undergoing apoptosis measured with a hematology analyzer. Peripheral blood lymphocytes from 40 chronic B-lymphocytic leukemia samples, five acute T-lymphoblastic leukemia samples, three healthy donors and from T-cell lines Jurkat, SUB-T1 and SUP T8) were cultured during 72 hours in medium alone or in the presence of chlorambucil, fludarabine or theophylline, all compounds known to be apoptosis inducers, with or without adjunction of interleukin 4. Samples were run on a Bayer-H1 system and the percentage of apoptotic cells was evaluated by monitoring the lobularity index corresponding to the polymorphonuclear population. Results compared to the dUTP-fluorescein method by flow cytometry and dUTP-peroxidase labeling on slides (TUNEL) showed an excellent correlation (chi-square test: P < 0.01). This method is reliable and simple and allows one to measure routinely the percentage of apoptotic lymphoid cells at short notice in a laboratory of hematology. This is especially valuable, particularly in testing the predictive value of in vitro drug-induced apoptosis before starting a chemotherapy protocol.

Posttransplant lymphoproliferative disorders not associated with Epstein-Barr virus: a distinct entity?

PURPOSE: Organ recipients are at a high risk of posttransplant lymphoproliferative disorders (PTLD) as a result of immunosuppressive therapy. Most B-cell lymphomas are associated with Epstein-Barr virus (EBV) infection. We describe a morphologically and clinically distinct group of PTLD in 11 patients that occurred late after organ transplantation and were not associated with EBV. PATIENTS AND METHODS: There were seven kidney, three heart, and one liver transplant recipients (group I). The clinical manifestations, pathologic findings, treatment, and outcome were compared with those in 21 patients with EBV-associated PTLD treated in our institution (group II). EBV was detected with at least two techniques: Epstein-Barr-encoded RNA (EBER) in situ hybridization with EBER 1 + 2 probes, Southern blotting, and detection of latent membrane protein 1 (LMP1) expression by immunohistochemistry. RESULTS: The time between transplantation and the diagnosis of lymphoma ranged from 180 to 10,220 days in group I (mean, 2,234; median, 1,800) and from 60 to 2,100 days in group II (mean, 546; median, 180), and was significantly shorter in group II (P = .02). Among 19 tumors diagnosed within 2 years after the graft, 16 were associated with EBV; among 13 tumors diagnosed after more than 2 years, only five were associated with EBV. All of the B-cell PTLDs in group I were classified as monomorphic, meeting the criteria of B diffuse large-cell lymphoma (B-DLCL) with a component of immunoblasts, and genotyping confirmed their monoclonality. Three tumors were T-cell pleomorphic lymphomas. Tumor sites were mainly bone marrow and lymph nodes. Overall median survival was 1 month in group I and 37 months in group II, with two patients still alive in group I and nine in group II. The survival time was significantly longer in group II (P < .01). CONCLUSION: EBV-negative PTLD may be a late serious complication of organ transplantation. Half the tumors observed after kidney transplantation in our center were not associated with EBV and emerged after more than 5 years, which suggests the number of EBV-negative PTLDs in organ recipients might increase with time.

Chlorambucil in indolent chronic lymphocytic leukemia. French Cooperative Group on Chronic Lymphocytic Leukemia.

BACKGROUND: To determine whether chlorambucil treatment benefits patients with indolent chronic lymphocytic leukemia (CLL), we conducted two randomized trials in 1535 patients with previously untreated stage A CLL. METHODS: In the first trial, 609 patients were randomly assigned to receive either daily chlorambucil or no treatment; in the second trial, 926 patients were randomly assigned to receive either intermittent chlorambucil plus prednisone or no treatment. Median follow-up for the first and second trials exceeded 11 and 6 years, respectively. The end points were overall survival, response to treatment, and disease progression. RESULTS: Treatment of indolent CLL did not increase survival in either trial. In the treated group, as compared with the untreated group, the relative risk of death was 1.14 (95 percent confidence interval, 0.92 to 1.41; P=0.23) in the first trial and 0.96 (95 percent confidence interval, 0.75 to 1.23; P=0.74) in the second trial, with 76 percent and 69 percent of patients, respectively, having a response to therapy. Although chlorambucil slowed disease progression, there was no effect on overall survival. In the untreated group in the first trial, 49 percent of patients did not have progression to more advanced disease and did not need therapy after follow-up of more than 11 years; however, 27 percent of patients with stage A CLL died of causes related to the disease. CONCLUSIONS: Chlorambucil does not prolong survival in patients with stage A CLL. Since deferring therapy until the disease progresses to stage B or C does not compromise survival, treatment of indolent CLL is unnecessary.

The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo- and radioresistant human chronic lymphocytic leukaemia lymphocytes to TNF-alpha-initiated apoptosis.

Apoptosis can be triggered by cytotoxic agents and radiation currently used in cancer treatment. However, the apoptotic response appears to vary between cell types (normal or transformed) and between types of malignancy. Thus, irradiation induces apoptosis in normal human lymphocytes but not in lymphocytes derived from a subset of chronic lymphocytic leukaemia (CLL). Moreover, in this subset, spontaneous apoptosis is inhibited by irradiation. Why irradiation does not allow the initiation of the apoptotic death pathway could be explained, at least in part, and in agreement with recent findings on experimental models, by the activation of the transcriptional factor NF-kappaB, which is able to inhibit apoptotic cell response. Low doses (at which no effect is observed with normal human lymphocytes) of the highly specific proteasome inhibitor lactacystin are sufficient to trigger apoptosis in these malignant cells. Proteasome inhibition by lactacystin prevents the nuclear translocation of both p50 and p65 NF-kappaB subunits and sensitizes these cells to apoptosis by tumour necrosis factor (TNF)-alpha treatment. As this subset of CLL is totally resistant to any treatment, proteasome inhibition by lactacystin provides a new therapeutic approach to be explored, considering the sensitivity of malignant CLL-derived lymphocytes to be quite different from that of normal human lymphocytes.

VH gene expression in hairy cell leukaemia.

Hairy cells are characterized by their typical morphology and expression of specific surface antigens. Although their B-cell origin is now confirmed, their exact position in B-cell development remains unclear. To better define the origin of hairy cells, we analysed the immunophenotype and the Ig VH nucleotide sequence of seven cases of hairy cell leukaemia (HCL). Six of them were typical HCL and the remaining case corresponded to a variant HCL. Analysis of sequenced VH genes revealed that the VH1 family was used in one case, VH2 in one, VH3 in two, VH4 in two and VH5 in one. No preferential usage of VH genes was observed in this small series. In five cases high rates of somatic mutations were observed, with a predominance of mutations and replacements in CDR regions for three. indicating that these cells originate from cells that have been exposed to the hypermutation mechanism. The distribution of mutations in our small series provides some evidence of a selective mutational process.

Variations of teicoplanin concentrations in neutropenic patients.

The aim of this study was to measure the trough plasma concentrations of teicoplanin in neutropenic patients with a view to optimizing the loading dose. Teicoplanin trough plasma concentrations were followed in 11 neutropenic patients after repeated administration of a 6 mg/kg i.v. bolus. The first three injections were given at 12-h intervals, and the rest every 24 h. Trough plasma concentrations at 48 h varied from 5.6 to 13.1 mg/I. The mean trough plasma concentration-time curve indicated a trend towards accumulation. In conclusion, the loading dose of teicoplanin should be tailored to individual neutropenic patients.

Long-term clinical outcome of B-cell chronic lymphocytic leukaemia patients in clinical remission phase evaluated at phenotypic level.

This retrospective study aimed to evaluate the long-term prognostic impact of phenotypic remission in B-cell chronic lymphocytic leukaemia (CLL) patients who have achieved clinical, haematological and bone-marrow complete remission (CR) after conventional chemotherapy. The clinical and phenotypic data of 77 CLL patients in CR with a median follow-up from CR achievement of 54 months (range 5-127) were analysed. 32 patients (42%) displayed a normalized phenotype as evaluated by k:lambda ratio or by CD5+/CD19+ cell numbers. Patients with normalized phenotype demonstrated a significantly higher incidence of female sex, a lower relapse rate, a trend for higher prevalence of stage A and a lower occurrence of CLL-related deaths. The relapse-free survival of patients with normalized phenotype was significantly longer (P = 0.02), whereas no difference in overall survival was found between the two groups. Interestingly, Binet's stage at diagnosis was highly predictive of the overall survival following CR achievement. From the results of the present study we conclude that a phenotype normalization at CR obtained with conventional chemotherapy indicates a higher probability of a longer CR but it does not translate into prolonged survival. Clinical features at diagnosis, such as stage distribution, are apparently stronger predictors of the final outcome. These results emphasize, however, the need for a routine assessment of the quality of response since this information could be crucial in designing therapeutic strategies for young patients suffering from advanced CLL.

VH gene expression in CD5 positive and CD5 negative B cell chronic lymphoid malignancies.

In this review, we report analyses of VH genes in mature B cell malignancies generally or occasionally bearing CD5 antigen such as B CLL, MCL, SLVL and PLL. In the majority of cases, B CLL and MCL use VH genes in germline configuration. However in some cases a higher rate of random mutations is observed. These differences are not related to CD5 expression but are accounted by Ig phenotype, since less mutations are observed in CLL cases expressing membrane mu delta, when compared to forms exclusively expressing membrane mu. PLL and SLVL cases display mutated V genes independently of CD5 expression. Although there is some evidence that CD5+ B cells constitute a separate lineage, the possibility that CD5 constitutes an activation marker cannot be ruled out. Indeed, CD5- B cells can be induced to differentiate into CD5+ B cells and VH gene analyses showed no significative differences between CD5+ and CD5- B cell lymphoproliferative disorders. In this review we have tried to examine B cell chronic malignancies on the basis of phenotype and VH gene usage. Thus we propose a tentative classification where these disorders are allocated according to these characteristics.