[Diffuse large B cell lymphoma: management in 2012]. / Prise en charge du lymphome B diffus à grandes cellules en 2012.

Diffuse Large B Cells Lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and comprises a large number of different entities with different clinico-pathological characteristics. The role of positron emission tomography is essential during the initial staging and post treatment assessment, and potentially at early- or mid-treatment evaluation of response. First line therapy comprises immuno-chemotherapy with rituximab and different cytotoxic agents that differ for components, dosages and frequency of administration taking worldwide-recognized pre-treatment prognostic variables into account. After relapse, peripheral blood stem cells transplantation remains the only chance of cure. This review attempts to summarize the current state of our knowledge by highlighting the leads pursued to further improve current therapeutic results.

Long-term follow-up of an age-adapted C5R protocol followed by radiotherapy in 99 newly diagnosed primary CNS lymphomas: a prospective multicentric phase II study of the Groupe d'Etude des Lymphomes de l'Adulte (GELA).

BACKGROUND: This prospective multicentric phase II study aimed to confirm the results of the C5R protocol of high-dose methotrexate (MTX)-based chemotherapy (CT) for immunocompetent primary central nervous system lymphoma. PATIENTS AND METHODS: A total of 99 patients received age-adapted CT (C5R protocol) followed by radiotherapy. Patients younger than 61 years (group 1, n = 45) received the full C5R with MTX, doxorubicin, vincristine, cyclophosphamide, and cytarabine. Patients aged 61-70 years (group 2, n = 36) received reduced doses. Patients older than 70 years (group 3, n = 18) received four courses of MTX, cyclophosphamide, and etoposide. RESULTS: Median age was 63 years and 51% of patients had performance status of more than one. Seventeen patients died of toxicity during CT. Complete response was achieved in 56%, 53%, and 28% of patients in groups 1, 2, and 3, respectively. With a median follow-up of 83 months, the 5-year progression-free survival was 31%, 28%, and 11% and the 5-year overall survival 42%, 31%, and 17% for groups 1, 2, and 3, respectively. Leukoencephalopathy occurred in 32% of assessable patients, in both group 1 and groups 2-3. CONCLUSION: The C5R protocol was feasible in the multicentric setting with favorable long-term survival in patients younger than 60 years. Despite dose adaptation, results in older patients were disappointing.

Chromosomal translocations independently predict treatment failure, treatment-free survival and overall survival in B-cell chronic lymphocytic leukemia patients treated with cladribine.

Chromosomal translocations represent an important prognostic indicator in B-cell chronic lymphocytic leukemia (B-CLL). However, their value had been neither determined in homogeneously treated patients nor compared to that of IgV(H) mutational status. Sixty-five B-CLL patients were investigated using cytogenetics, interphase fluorescence in situ hybridization (FISH), analysis of IgV(H) and of TP53 mutational status before treatment with 2-chloro-2'-deoxyadenosine (CdA). Translocations (n=45) were detected in 42% of the patients, including both balanced (n=12) and unbalanced (n=33) types. IgV(H) was mutated in 43% of the patients. Patients with translocations were more heavily pretreated (P=0.05), presented with more complex karyotypes (P<0.001), 17p abnormalities and TP53 mutations, and had a higher failure rate (59 vs 21% in patients without translocations, P=0.004). Patients with unbalanced translocations displayed a shorter median treatment-free survival (TFS, 6.9 vs 35.9 months, log rank 22.72, P<0.001) and overall survival (OS, 13.0 vs 68.0 months, log rank 16.51, P<0.001), as compared to patients without translocation. In multivariate analysis, unbalanced translocations were independently associated with therapeutic failure, short TFS and short OS. IgV(H) mutational status was independently associated with risk of failure and TFS, but not OS. In B-CLL patients treated with CdA, translocations are strong predictors of outcome.

Outcomes of diffuse large B-cell lymphoma patients relapsing after autologous stem cell transplantation: an analysis of patients included in the CORAL study.

In the CORAL study, 255 chemosensitive relapses with diffuse large B-cell lymphoma (DLBCL) were consolidated with autologous stem cell transplantation (ASCT), and 75 of them relapsed thereafter. The median time between ASCT and progression was 7.1 months. The median age was 56.1 years; tertiary International Prognosis Index (tIPI) observed at relapse was 0-2 in 71.6% of the patients and >2 in 28.4%. The overall response rate to third-line chemotherapy was 44%. The median overall survival (OS) was 10.0 months (median follow-up: 32.8 months). Thirteen patients received an allogeneic SCT, and three a second ASCT. The median OS was shorter among patients who relapsed <6 months (5.7 months) compared with those relapsing ⩾12 months after ASCT (12.6 months, P=0.0221). The median OS in patients achieving CR, PR or no response after the third-line regimen was 37.7 (P<0.0001), 10.0 (P=0.03) and 6.3 months, respectively. The median OS varied according to tIPI: 0-2: 12.6 months and >2: 5.3 months (P=0.0007). In multivariate analysis, tIPI >2, achievement of response and remission lasting <6 months predicted the OS. This report identifies the prognostic factors for DLBCL relapsing after ASCT and thus helps to select patients for experimental therapy.

Inhibition of the ERK pathway promotes apoptosis induced by 2-chloro-2'-deoxyadenosine in the B-cell leukemia cell line EHEB.

2-Chloro-2'-deoxyadenosine (CdA) is a nucleoside analogue active in B-cell chronic lymphocytic leukemia (B-CLL). Although the mechanism of action of CdA has been extensively investigated in leukemic cells, the possibility that this nucleoside analogue interacts with the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway has never been explored. In this study, we show that CdA, at concentrations close to the IC50, activated the ERK pathway in the B-cell line EHEB. Because activation of this pathway is assumed to exert anti-apoptotic effect, we combined CdA with inhibitors of the ERK pathway. The latter were found to enhance CdA-induced apoptosis. These results suggest that the efficacy of CdA could be strengthened by combination with inhibitors of the ERK pathway.

Study of the efficacy of a pronucleotide of 2-chloro-2'-deoxyadenosine in deoxycytidine kinase-deficient lymphoma cells.

2-Chloro-2 '-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 '-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.

Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.

Outcome of patients with relapsed diffuse large B-cell lymphoma who fail second-line salvage regimens in the International CORAL study.

Salvage chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard second-line treatment for relapsed and refractory diffuse large B-cell lymphoma (DLBCL). However, the strategy is less clear in patients who require third-line treatment. Updated outcomes of 203 patients who could not proceed to scheduled ASCT in the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) are herein reviewed. In the intent-to-treat analysis, overall response rate to third-line chemotherapy was 39%, with 27% CR or CR unconfirmed, and 12% PR. Among the 203 patients, 64 (31.5%) were eventually transplanted (ASCT 56, allogeneic SCT 8). Median overall survival (OS) of the entire population was 4.4 months. OS was significantly improved in patients with lower tertiary International Prognostic Index (IPI), patients responding to third-line treatment and patients transplanted with a 1-year OS of 41.6% compared with 16.3% for the not transplanted (P<0.0001). In multivariate analysis, IPI at relapse (hazard ratio (HR) 2.409) and transplantation (HR 0.375) independently predicted OS. Third-line salvage chemotherapy can lead to response followed by transplantation and long-term survival in DLBCL patients. However, improvement of salvage efficacy is an urgent need with new drugs.

Inhibition of RNA transcription: a biochemical mechanism of action against chronic lymphocytic leukemia cells by fludarabine.

Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leukemia (CLL) and other indolent lymphocytic malignancies. Although the incorporation of fludarabine into DNA is a key event in causing cytotoxicity in proliferating leukemia cells, the precise mechanisms by which fludarabine kills CLL cells remain unclear because of the quiescent nature of this malignancy. The present study demonstrated that inhibition of RNA transcription correlated significantly with the cytotoxic action of fludarabine in CLL cells. In contrast, suppression of the low level of DNA synthesis did not affect the survival of the leukemia cells. In addition, inhibition of fludarabine incorporation into cellular DNA through repair synthesis in CLL cells did not alter the cytotoxicity of this drug. Rather, inhibition of RNA synthesis by fludarabine led to a specific diminishment of certain cellular proteins from CLL cells. The combination of fludarabine with another RNA synthesis inhibitor, actinomycin D, or with the protein synthesis inhibitor, puromycin, substantially enhanced the cytotoxic activity against CLL cells. These results suggest that termination of mRNA transcription and the consequent depletion of proteins required for cell survival may be a novel biochemical mechanism of action of fludarabine in CLL cells. Thus, inhibition of RNA/protein synthesis may provide a new therapeutic strategy for the treatment of CLL patients.

Cyclosporin A in the treatment of refractory B chronic lymphocytic leukemia (B-CLL).

Partial tumoral responses have been described after cyclosporin A treatment in refractory B-chronic lymphocytic leukemia patients. We report the effect of this therapy in five patients prospectively treated with oral conventional dose of cyclosporin A. Antitumoral efficiency was marginal (one sustained partial response among five patients) which, added to a potential worsening of natural and therapy-related immunodepression, limits its use in this instance.

2-Chloro-2'-deoxyadenosine inhibits DNA repair synthesis and potentiates UVC cytotoxicity in chronic lymphocytic leukemia B lymphocytes.

2-Chloro-2'-deoxyadenosine (CdA) is a deoxyadenosine analogue which targets enzymes involved in DNA synthesis, and hence might interfere with the resynthesis step of DNA repair. We tested this hypothesis in resting B cell chronic lymphocytic leukemia (B-CLL) lymphocytes, after firstly characterizing unscheduled DNA synthesis occurring in these cells. We observed that the spontaneous incorporation of [methyl-3H]thymidine (dThd) into DNA of B-CLL cells was not completely inhibitable by hydroxyurea (HU) which blocks DNA replication. In addition, in the presence of HU, dThd incorporation could be upregulated by UVC radiation or DNA alkylation, without re-entry of the cells into S phase. CdA was found to inhibit both spontaneous and upregulated DNA synthesis in B-CLL cells. Phosphorylation of CdA was essential to exert this effect. We finally observed a strong synergistic cytotoxicity between UV light and CdA, which was correlated with activation of caspase-3 and high molecular weight DNA fragmentation, two markers of apoptosis. Taken together, these observations indicate that in B-CLL cells CdA inhibits unscheduled DNA synthesis which represents the polymerizing step of a repair process responsive to DNA aggression. Inhibition of this process by CdA, together with a combined activation of the apoptotic proteolytic cascade by CdA and UV, may explain their synergistic cytotoxicity.

Phase I/II study of 2-chloro-2'-deoxyadenosine with cyclophosphamide in patients with pretreated B cell chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma.

Because of their substantial in vitro synergy, we conducted a dose-escalation study of cyclophosphamide (CP) added to 2-chloro-2'-deoxyadenosine (CdA) in patients with previously treated chronic lymphocytic leukemia and non-Hodgkin's lymphoma. CdA was given at a fixed dose (5.6 mg/m2/day) as a 2-h intravenous (i.v.) infusion, immediately followed by a 1-h i.v. infusion of CP, for 3 days. The initial daily CP dose was 200 mg/m2, and was escalated by 100 mg/m2 increments in successive cohorts of three to six patients to determine the maximum-tolerated dose (MTD). Additional patients were included at the MTD to extend toxicity and response analysis. Twenty-six patients received 68 cycles of chemotherapy. The MTD of CP after CdA 5.6 mg/m2, was 300 mg/m2. Acute neutropenia was the dose-limiting toxicity of this regimen, which was otherwise well tolerated. Delivery of repeated cycles was not feasible in eight patients (31%) because of prolonged thrombocytopenia. Severe infections were seen in three of 68 cycles (4%). The overall response rate was 58% (15 of 26; 95% CI, 36-76%), with 15% complete responses and 42% partial responses. These data show the feasibility of the association of CdA with CP. Given the response rate observed, further studies of this regimen are warranted in untreated patients, in particular with chronic lymphocytic leukemia and with Waldenström macroglobulinemia.

Potentiation of antitumor effects of cyclophosphamide derivatives in B-chronic lymphocytic leukemia cells by 2-chloro-2'-deoxyadenosine.

Because 2-chloro-2'-deoxyadenosine (CdA) is active in B-chronic lymphocytic leukemia (B-CLL), and may interfere with DNA repair, we investigated the potentiating effect of CdA on the cytotoxicity induced in vitro in B-CLL lymphocytes by cyclophosphamide (CP) derivatives, which induce DNA damage by DNA cross-linking. Exposure to CdA at clinically achievable concentrations for 2 h, followed by mafosfamide (MAF) or 4-hydroxycyclophosphamide (4HC) for 22 h, resulted in synergistic cytotoxicity in the majority of B-CLL samples tested. Synergy between CdA and MAF was observed in cell samples of sensitive/untreated patients, as well as in cells of resistant/pretreated patients, particularly at the highest concentrations of MAF. In the cells treated with CdA and MAF, we observed loss in ATP and hallmarks of apoptosis, as evidenced by cellular morphology and high molecular weight DNA fragmentation. The synergy could be explained neither by an influence of MAF on the phosphorylation of CdA, nor by an increase in the incorporation of CdA into DNA in the presence of MAF. The in vitro synergy between CdA and CP derivatives provides a rationale for the use of this association in B-CLL patients.

Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB.

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.

A phase I and pharmacokinetic study of docetaxel administered in combination with continuous intravenous infusion of 5-fluorouracil in patients with advanced solid tumors.

Encouraged by preclinical synergism between docetaxel and 5-fluorouracil (5FU), we conducted a Phase I study of docetaxel in combination with continuous i.v. infusion of 5FU in patients with advanced solid tumors to determine the maximum tolerated dose, the recommended dose for Phase II studies, and the safety and pharmacokinetic profiles of this combination. Forty-two patients with advanced solid tumors, most of whom had been previously treated, received docetaxel on day 1 as a 1-h i.v. infusion, immediately followed by a 5-day continuous i.v. infusion of 5FU, every 3 weeks without hematopoietic growth factor support. All patients were premedicated with methylprednisolone. Dose levels of docetaxel/SFU studied were (daily dose, in mg/m2) 60/300, 75/300, 75/500, 75/750, 85/750, 85/1000, and 75/1000. Forty-one patients were assessable for toxicity. The maximum tolerated dose determined during the first cycle was 1000 mg/m2/day for 5 days of 5FU with either 75 or 85 mg/m2 docetaxel. Dose-limiting toxicities at these dose levels were reversible secretory diarrhea (4 of 12 evaluable patients), stomatitis (2 patients), and febrile neutropenia (2 patients). Overall, grade 3/4 neutropenia and febrile neutropenia were seen in 63.4% and 9.8% of the patients, respectively. Four patients experienced grade 3/4 infection, which led to toxic death in one of them. There were five early deaths: (a) one was clearly treatment related; (b) two others were possibly treatment related or remotely treatment related; and (c) two deaths were not related to the study drugs. Partial responses were documented in 5 of 39 evaluable patients. Pharmacokinetic results of both drugs were consistent with those from single-agent studies. The recommended dose of this combination, which showed acceptable toxicity and antitumoral activity at various dose levels, is 85 mg/m2 docetaxel given as a 1-h i.v. infusion on day 1 immediately followed by a 5-day continuous i.v. infusion of 5FU (750 mg/m2/day). This study has been extended by adding cisplatin on day 1 of the combination of docetaxel and 5FU.

Metabolism and cytotoxic effects of 2-chloroadenine, the major catabolite of 2-chloro-2'-deoxyadenosine.

EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro-2'-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2-3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 microM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an IC(50) of 16 microM in EHEB cells and 5 microM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies.

Second primary tumors and immune phenomena after fludarabine or 2-chloro-2'-deoxyadenosine treatment.

The purine nucleoside analogs fludarabine and 2-chloro-2'-deoxyadenosine display substantial activity in the treatment of various chronic lymphoproliferative disorders. Their major toxicities are primarily immunosuppression and myelosuppression. The profound influence of these drugs on the immune system has raised questions as to the emergence of secondary neoplasms or auto-immune disorders after their use. Based on a literature review and on personal observations, this article reviews the potential clinical importance of these concerns.

Limited activity of mini-dose interferon alpha-2a in the treatment of myelodysplastic syndrome.

Impairment in marrow function often characterizes the evolution of myelodysplastic syndrome. As a differentiating agent, interferon alpha 2a (INF alpha) has been shown to be active in the correction of cytopenias related to myelodysplastic syndromes (MDS). We report the clinical course of 9 patients with MDS treated with low-dose subcutaneous INF alpha (1 x 10(6), 3 times per week). A significant effect on anemia was only demonstrated in one patient (11%). In the other, eight, therapy was totally ineffective and four of them could not receive the complete treatment due to worsening cytopenias or leukemic transformation. In conclusion, in our study, INF alpha had only limited activity in the treatment of myelodysplastic syndrome.

Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in the human leukemia EHEB cell line.

To explain why 2-chloro-2'-deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA-resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA-induced increase in G1 to S phase progression.