Department of Hematology, Medical University of ód, ód, Poland

Correspondence to: T Robak, Department of Hematology, Medical University of ód Copernicus Memorial Hospital, 93-513 ód, ul Pabianicka 62, Poland; Fax: 4842 689-51-92

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world. The natural clinical course is highly variable and chemotherapy is usually not indicated in early and stable disease. Treatment is needed in the progressive form of this leukemia. Chlorambucil, with or without steroids, has been for many years the drug of choice in the treatment of CLL. More recently, treatment approaches have included nucleoside analogues, (NA) fludarabine (FAMP) and cladribine (2-CdA, 2-chlorodeoxyadenosine), which seem to be the treatment of choice for patients failing standard therapies. Their role as first line therapy is being investigated in randomized trials and the results have recently been published. These studies have shown a higher overall response and complete remission (CR) rate and longer response duration in patients treated initially with NA than with chlorambucil or cyclophosphamide-based combination regimens. In contrast, overall survival is similar in patients treated with NA and alkylating agents. However, the randomized trials were designed as crossover studies which may influence survival. Combined use of NA with other cytotoxic drugs, cytokines, monoclonal antibodies and other agents may increase the CR and prolong survival time. However, the results of randomized trials comparing combination treatment with NA alone are not yet available. In conclusion, alkylating agents still have an important place in the routine management of the majority of CLL patients. NA should be routinely used as second line treatment and possibly as first line therapy in younger patients, who are candidates for potentially curative treatment such as stem cell transplantation and/or monoclonal antibodies.

Leukemia (2002) 16, 1015-1027. DOI: 10.1038/sj/leu/2402531

chlorambucil; nucleoside analogues; fludarabine; cladribine

Introduction

B cell chronic lymphocytic leukemia (CLL) is a clonal hematopoietic disorder characterized by proliferation and accumulation of small lymphocytes. CLL is the most common adult leukemia in Europe and Northern America with an annual incidence rate of three to five cases per 100 000.¹ The median age at diagnosis is about 65 years, with only 10-15% of patients under the age of 55 years.^2,3 The diagnosis of CLL does not imply the need for immediate therapy, and the management of CLL patients is determined by the stage and activity of the disease.⁴

For many years alkylating agents, especially chlorambucil with or without steroids, have been considered the drugs of choice for the first line of treatment for CLL^5,6 and many hematologists still believe that such an approach should be considered as the gold standard.^7,8,9,10 More recently, renewed interest in CLL therapy has been created by the introduction of novel nucleoside analogues (NA), particularly fludarabine (FAMP) and cladribine (2-chlorodeoxyadenosine, 2-CdA), as well as combined chemotherapy.^{11,12,13,14,15,16,17,18}

The place of alkylating agents and NA in the management of CLL patients at the beginning of the new millennium, based on the results of randomized clinical trials, is the subject of this review.

Indications for chemotherapy

The management of CLL is determined by the stage and activity of the disease. There is no evidence that cytotoxic therapy based on alkylating agents has beneficial effects in patients with the indolent form of the disease. Several randomized trials indicate that initiation of chlorambucil-related therapy during the indolent phase of CLL does not prolong the survival time of CLL patients.^10,15,16,17 Dighiero and Binet note that approximately one-third of CLL patients never require treatment and die from causes unrelated to this disease.⁴ Only one-third have aggressive disease at diagnosis and should be immediately treated. The remaining patients have indolent phase of the disease at diagnosis, but with time the disease becomes progressive and they also require treatment.

The French Cooperative Group on CLL conducted two randomized trials on 1535 previously untreated patients with Binet stage A disease.¹⁰ In the first study, patients were randomized either to a group treated daily with oral chlorambucil or to a group which did not receive treatment. In this study there was a survival advantage for the untreated group. However, the difference was not statistically significant. In the second study, patients were randomly assigned to receive either intermittent chlorambucil plus prednisone or no treatment. Again, as in the first study, treatment of indolent CLL did not increase survival time, despite the fact that chlorambucil slowed disease progression. It should be noted that in the first trial there was a relatively high frequency of epithelial neoplasms in the group treated with chlorambucil. Furthermore, there were six cases of acute leukemia in the group of 422 patients treated with chlorambucil as initial or secondary treatment, but no such cases among those patients who were never treated with this agent.¹⁰ Similar conclusions were drawn from the studies performed by the IGCI CLL Cooperative Group,¹⁵ and the Cancer and Leukemia group B,¹⁶ which compared patients with B-CLL who received early treatment with chlorambucil to a group of B-CLL patients with deferred therapy in relation to the time of progression of the disease.

The CLL Trialists Collaborative Group recently performed a meta-analysis of the randomized trials that compared the effects of immediate vs deferred chemotherapy with chlorambucil or chlorambucil with prednisone for early stage CLL.¹⁸ There were 2048 patients with early disease in six such trials. The 10-year survival rate was slightly better in the early treated group, but not significantly different to that of the group with deferred treatment (47% vs 44%).

The results of the studies presented above support the conservative treatment strategy for patients with early-stage CLL, including Rai stage 0 to I and Binet stage A. The majority of patients with these stages should not be treated with alkylating agents until progression. Whether nucleoside-based therapy in early stages of CLL has potential benefit still remains open to question.

Widely accepted guidelines for the initiation of chemotherapy in CLL patients have been proposed by the National Cancer Institute sponsored Working Group.^19,20 According to these guidelines the criteria for the initiation of therapy may not be identical for routine clinical practice and for patients included in clinical trials. Therapy should not be initiated in patients who have smoldering CLL, including those with Rai stage 0 or Binet A until disease progression, or unless disease-related symptoms such as fever, chills, body weight loss or pronounced fatigue are present. Laboratory results supporting deferred therapy include non-diffuse pattern of bone marrow involvement, serum Hb concentration 13 g/dl, peripheral blood lymphocytes less than 30 ´ 10⁹/l and lymphocyte doubling time longer than 12 months.^20,21,22 However, if the disease progression becomes evident treatment should be commenced. The criteria for treatment initiation include disease-related symptoms, especially fever, body weight loss and extreme fatigue, increasing bone marrow failure, autoimmune anemia and/or thrombocytopenia responding poorly to corticosteroid treatment, massive or progressive splenomegaly and/or lymphadenopathy, progressive lymphocytosis and recurrent infections.

First line therapy

Chlorambucil with or without corticosteroids

Chlorambucil (phenylbutyric acid nitrogen mustard) was first synthesized almost 50 years ago and its efficacy in CLL was established soon afterwards.²³ However, the precise mechanism of action of this agent in CLL is still uncertain.²⁴ Chlorambucil can bind to a variety of cellular structures such as membranes, proteins, RNA and DNA. DNA cross-linking is thought to be the most important factor in its antileukemic activity.²⁴ Furthermore, induction of apoptosis may also contribute to the activity of chlorambucil and this may correlate with the clinical effect of this drug.²⁵ There is evidence that chlorambucil, similarly to NA, may induce cell death in CLL by the P53-dependent pathway.²⁴

Chlorambucil, when used alone or with prednisone, produces an initial response rate of between 60% and as high as 90%, with a complete response (CR) in up to 60% of all patients, depending on the doses administered and response criteria.^6,7,8,9

The doses of chlorambucil are very important for its efficacy in CLL. Administration of this drug orally as the first line treatment either at a dose of 4 to 8 mg/m²/day for 4-8 weeks or as a single dose of 15-30 mg/m² every 2 to 4 weeks results in remission in approximately 30 to 70% of patients.^26,27 The highest overall response rates (OR) and CR rates have been achieved when chlorambucil was administered at the fixed dose of 15 mg daily up to CR or grade 3 toxicity, or for a maximum of 6 months.^7,28 In the study performed by Jaksic et al⁷ utilizing this dosage schedule, an 89.5% OR and a 59.5% CR was observed in patients with advanced and/or progressive CLL. Furthermore, this study showed that a maintenance treatment with chlorambucil 5-15 mg twice a week, according to the hematological tolerability, may be an important factor in prolonged response duration and overall survival time. In a previous study, Jaksic et al²⁸ randomized 181 CLL patients to a group treated according to a high dose chlorambucil daily schedule or to a group treated with a weekly dose of chlorambucil (75 mg every week for 6 weeks) plus prednisone. The total dose of chlorambucil in the weekly schedule was six times lower than in the daily regimen. A 70% CR rate was observed in the group treated with the high daily dose of chlorambucil and only a 31% CR in the group treated with lower doses. Survival time of patients treated with higher doses of chlorambucil were also significantly longer.

Corticosteroids are less active than alkylating agents in CLL and they are generally inactive in akylator-refractory patients.^29,30 Nevertheless, Han et al³¹ have shown that therapy based on the combination of prednisone with chlorambucil resulted in a better response rate compared to therapy based on either drug used alone. However, the combination regimen of chlorambucil with prednisone does not lead to longer survival times than chlorambucil in monotherapy. Other alkylating agents have been less extensively investigated in CLL than chlorambucil, primarily in the context of combination therapy. Cyclophosphamide (CY) has a similar activity to chlorambucil.³³ It is usually administered orally as a single agent at a dose of 2 to 3 mg/kg/day or intravenously at 20 mg/kg every 2 to 3 weeks. Cyclophosphamide is occasionally used when chlorambucil is poorly tolerated. It is more frequent that CY is used in combination with vincristine and prednisone (COP, CVP). However, in randomized trials COP leads to inferior or similar results to those obtained after treatment with chlorambucil with prednisone.^34,35,36

Alkylating agents in combination therapy

Several groups have investigated the efficacy and toxicity of the combination of alkylating agents with other cytotoxic drugs as first line therapy of CLL. CHOP (COP + doxorubicin) was the most frequently used combination chemotherapy regimen. However, in three randomized studies comparing intermediate doses of chlorambucil and prednisone with CHOP no advantage in overall survival time for CHOP was shown.^37,38 In contrast, high-dose chlorambucil (15 mg daily up to CR or grade 3 toxicity, or a maximum of 6 months) was shown to have a significant advantage over CHOP in terms of OR (89.5% and 75% respectively, P < 0.001) and median overall survival time (68 months and 47 months, P < 0.005).⁷ Direct comparison of COP and CHOP in a randomized study has shown longer median survival for Binet stage C CLL patients treated with CHOP (62 months) than for patients treated with COP (22 months).³⁹ A meta-analysis of the randomized trials comparing chlorambucil ± prednisone with CY-based combination chemotherapy (COP or CHOP) or chlorambucil + epirubicin has been recently performed by the CLL Trialists Collaborative Group.¹⁸ Ten randomized trials and a total of 2022 patients were taken into account. The 5-year survival time was 48% for both groups. A subgroup of six trials involving an anthracycline-containing regimen was analyzed separately, but again, overall survival was no better for this group of patients than for chlorambucil therapy. There were 325 deaths among 627 patients treated with the anthracycline-based regimen and 306 deaths among 636 patients treated with chlorambucil-based therapy. The death ratio was 1.07 (95% CI = 0.91-1.25) and the difference was not statistically significant.

More intensive combination regimens consisting of alkylating agents have also been investigated as front line therapy in CLL patients.^40,41,42 However, despite the fact that these drug combinations usually induced higher CR rates than chlorambucil or COP, they were usually more toxic and their influence on survival was uncertain. As a whole, the results from randomized trials indicate that there is no advantage in using alkylating agent-based combination therapy rather than chlorambucil alone in treatment of patients with progressive or advanced stages CLL. However, the maximal tolerated doses of chlorambucil should be used in preference to lower doses of this agent. The value of combination regimens of alkylating drugs with NA will be discussed later in this article.

Nucleoside analogues

Nucleoside analogues: fludarabine (FAMP), cladribine (2-chlorodeoxyadenosine, 2-CdA) and 2'-deoxycoformycin (Pentostatin, DCF) represent a novel group of cytotoxic agents with high activity in CLL.^11,43,44,45 These three agents share similar chemical structures and mechanisms of action such as induction of apoptosis. The cytotoxic mechanism of FAMP and 2-CdA occurs via their phosphorylated derivatives, causing inhibition of ribonucleotide reductase and DNA polymerase, which results in decreased DNA synthesis.¹¹ FAMP is intracellulary phosphorylated to fludarabine triphosphate and incorporated into DNA, which results in the termination of DNA or RNA synthesis.¹² Several in vitro studies have indicated that FAMP triggers apoptosis.

2-CdA is phosphorylated by deoxycytidine kinase (DK) and accumulated as chlorodeoxyadenosine triphosphate (2-CdATP). High activity of this enzyme in lymphocytes along with their low 5'-nucleotidase (5'-NT) activity is the probable explanation for 2-CdA's relatively high selectivity for these cells.^46,47 This metabolite disrupts cell metabolism by incorporating into the DNA of actively dividing cells, including DNA single-strand breaks, and inhibiting DNA synthesis. In vitro studies have indicated that 2-CdA increases the level of apoptosis of B-CLL cells. Moreover, recent laboratory studies suggest that 2-CdA can induce CLL cell death by direct mitochondrial injury that is different than the intrinsic caspase-9 apoptotic pathway initialized by FAMP.⁴⁷

Nucleoside analogues are routinely administered intravenously. Bioavailability of oral FAMP and 2-CdA is about 50%.^48,49,50 Clinical studies demonstrate that both drugs given orally have a similar clinical efficacy and safety profile to the intravenous formulations.^51,52 Moreover, the bioavailability of oral FAMP is unaffected by food.⁵⁰ An oral formulation of FAMP has recently become commercially available. The conclusions drawn from the recent studies are that an oral dose of approximately 40-50 mg/m²/day would provide systemic exposure equivalent to 25 mg/m²/day given routinely intravenously.⁵⁰ Oral formulation of FAMP is easier to use and can be administered on an outpatient basis.

Nucleoside analogues in front line treatment

The majority of studies with nucleoside analogues in CLL was performed in pre-treated patients. However, in recent years important results concerning the use of these agents as front line therapy have also been published.^{8,9,14,43,53,54,55,56,57,58,59,60,61} Keating et al¹³ have presented a long-term follow-up of 174 patients with progressive or advanced CLL treated with FAMP as single agent (71 patients) or combined with prednisone (103 patients) as front line therapy. The overall response was 78%, including 29% CR and 49% PR, and the median survival time was 63 months. The survival time of patients with CR and PR nodular disease was significantly longer than for the rest of PR patients. Furthermore, responding patients survived longer than non-responding patients. Patients with advanced stage disease (Rai III and IV) had shorter survival than patients with earlier stage. Patients younger than 70 years of age survived longer than older patients.

Important conclusions can be drawn from randomized studies comparing FAMP with chlorambucil, CAP (cyclophosphamide, doxorubicin, prednisone) or CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) in previously untreated CLL patients (Table 1).^9,53,60 In the study conducted by the French Cooperative Group in previously untreated CLL patients with Binet stages B or C, patients were randomly assigned to receive either FAMP or CAP, both for a duration of six courses.⁵³ A higher overall response rate was observed following FAMP therapy (71%) than after CAP therapy (60%). However, the differences were not statistically significant. Despite the fact that FAMP induced significantly longer responses than CAP there was no difference in survival time between groups. These results have been recently confirmed in a larger group of 938 patients.⁶⁰ This study showed that in patients with Binet B and C CLL first line treatment with FAMP or CHOP regimens both provided similar overall responses and survival rates, better results than CAP, while clinical remission rate was higher for FAMP than CHOP. Similar results were obtained in the North America Intergroup trial.⁹ In this trial, previously untreated CLL patients were randomized to arms treated with FAMP or with chlorambucil or FAMP combined with chlorambucil. Assignment of patients to the FAMP + chlorambucil group was halted during the course of the study because of evidence of significant toxicity and the absence of a significantly superior response rate than was evident in the FAMP arm. The overall response rate and the CR were significantly higher in the group of patients treated with FAMP (63% and 20%, respectively) than in the group treated with chlorambucil (37% and 4%, respectively) (P < 0.001). The median duration of remission and median progression-free survival was also longer in the group treated with FAMP-based therapy (25 months and 20 months, respectively) than in chlorambucil-treated patients (14 months each) (P < 0.001). However, as in the French Cooperative Group study, the survival time was not significantly longer in patients treated with FAMP (66 months) compared to the group who were given chlorambucil (56 months) (P = 0.08). The most important message from these randomized studies is that there are no differences in survival times in patients treated with FAMP and chlorambucil, CHOP, or CAP. The choice of the best first line therapy for CLL patients should, therefore, be based on the assessment of the patient's future quality of life and the cost of treatment.

2-CdA is structurally similar to FAMP. Unfortunately, the results of a randomized study comparing the activity of both agents in CLL have not been published so far. Moreover, there is less experience with the use of 2-CdA than with FAMP in patients with CLL in the majority of Western countries. In addition, 2-CdA is usually more expensive than FAMP. Similarly to FAMP this agent has been found to be more effective in previously untreated CLL patients than in the patients refractory to, or relapsed after, conventional therapy. In different studies the overall response rate ranged from 56 to 85% and the CR from 10 to 47%.^{9,15,44,68,69,70,79,80,81}

We have compared the efficacy and toxicity of 2-CdA with prednisone vs chlorambucil with prednisone therapy in previously untreated patients with progressive CLL in a prospective randomized multicenter study.⁸ Data obtained from this trial on 229 patients indicate that the OR rate after 2-CdA and prednisone therapy was significantly higher than after chlorambucil and prednisone treatment (Table 1). Moreover, the clinical CR rate after 2-CdA was also significantly higher (47%) than after chlorambucil treatment (12%) (P < 0.001). However, there was no difference in survival time in either group (Figure 1). There was also no difference in the event-free survival time between the patients treated with 2-CdA+P and those treated with chlorambucil and prednisone. It is worth noting that the conclusions from our study are similar to the results of the study performed by Rai et al⁹ comparing the efficacy of FAMP and chlorambucil as a primary therapy. In both studies CR and OR rates were higher and the duration of response longer in patients treated with nucleoside analogues (Table 1) However, the most important finding in both studies is that there is no difference in survival time in patients treated with nucleoside analogues or chlorambucil, although it should be noted that both trials were designed as a cross-over study and most patients in the chlorambucil group were administered FAMP or 2-CdA in refractory cases or early relapse.^8,9 This may have influenced survival time. In the retrospective analysis of patients treated with high-dose chlorambucil as primary therapy and 2-CdA as second line therapy, compared with the historical group never treated with nucleoside analogues, we have found longer survival times in patients with advanced stages of CLL (Rai stage III and IV) in the 2-CdA-treated group.⁶²

Bone marrow suppression with anemia, neutropenia and thrombocytopenia is the dose-limiting factor for NA.^8,9 Prolonged thrombocytopenia, neutropenia and anemia were observed especially after multiple courses of therapy and in heavily pre-treated patients. A randomized study confirmed a strong myelosuppressive effect of 2-CdA, which resulted in a high incidence of neutropenia and infections in patients treated with this nucleoside analogue (23% and 56%, respectively) in comparison to chlorambucil (11% and 40%, respectively) (P = 0.02).⁸ A higher incidence of neutropenia and infections after FAMP administration (27% and 16%, respectively) than chlorambucil therapy (19% and 9%, respectively) was also observed in Rai et al's study, but the differences were not statistically significant.^9,63 In addition, opportunistic infections were observed in patients treated with NA, especially when in combination with prednisone.⁶⁴

Some reports suggest that NA may induce autoimmune hemolytic anemia (AIHA) in patients with CLL, despite the reduction in leukemia clone.^{65,66,67,68,69,70} Di Raimondo et al⁶⁵ performed a retrospective study of 112 patients treated with FAMP and have found AIHA after the administration of this drug in five patients and the deterioration of pre-existing AIHA in four patients, which may correspond to the natural prevalence of AIHA in CLL and may not be related to FAMP. In our cohort of 114 patients with CLL, hemolysis was observed after a relatively large number of 2-CdA courses (ie five or six), and shortly after administration of the drug in only two (1.8%) patients with no previous history of AIHA.⁶⁷ This suggests that in both cases hemolysis might have been triggered by 2-CdA. A higher frequency of this complication has been reported by Chasty et al.⁶⁸ In this study, 19 B-CLL patients were treated with 2-CdA. Four of these patients (21%) developed severe AIHA and two of them died due to a complication of hemolysis. In the randomized study performed by Leporrier et al,⁶⁰ AIHA was observed with similar frequency in CLL patients treated with CHOP, CAP or FAMP. In our randomized study AIHA was noted in seven patients treated with 2-CdA and in two patients treated with chlorambucil, but this difference was not statistically significant (P < 0.3).⁸ A similar frequency of AIHA has also been shown in a retrospective analysis of 1203 patients treated with chlorambucil plus prednisone (1.8%) or FAMP with prednisone (2.5%).⁷⁰ The probability of drug-induced AIHA seems to be higher if hemolysis occurs shortly (ie 1-2 months) after NA administration.⁶⁸ When it occurs later, it is probably not drug-related and appears by chance. On the other hand, some studies have suggested that 2-CdA may be useful in the treatment of AIHA secondary to CLL.^47,67 Despite the fact that the results of the prospective multicenter randomized studies do not support the conclusion that the risk of AIHA is higher in CLL patients treated with NA than in patients treated with chlorambucil or other alkylating agent-based regimens, we suggest that NA should be avoided in CLL patients with a Coombs positive test and/or autoimmune phenomena prior to treatment. For the patients who require transfusions, blood products should be irradiated.⁶⁸

The increased frequency of second malignancies in CLL is well known. Moreover, prolonged immunosuppression related to NA treatment may additionally increase the risk of second neoplasms. Some authors observed secondary MDS/AML and other cancers in patients treated with these agents.^71,72,73,74 However, the retrospective analysis performed by Cheson et al⁷⁵ in which they compared secondary tumors in CLL patients treated with FAMP, has not shown any increase of the risk of secondary tumors.⁷⁵ Moreover, in a randomized study comparing 2-CdA plus prednisone with chlorambucil plus prednisone as front line therapy in CLL we have observed secondary malignancy in only two patients treated with 2-CdA and in one patient treated with chlorambucil.⁸ As yet, we have not observed any incidence of secondary MDS/AML. However, these complications may become more evident after prolonged observation of the patients as MDS/AML may occur even 5-10 years after treatment.

Use of nucleoside analogues in combination

In an attempt to further improve treatment results and prolong survival of patients with CLL, NA are combined with corticosteroids, other cytotoxic agents and/or monoclonal antibodies. However, there is still little experience in this area, and there is no final conclusion from the studies performed so far.

Nucleoside analogues and corticosteroids

The combination of corticosteroids with NA is even more controversial than with alkylating agents, due to the prolonged immunosuppressive effects of these agents that may be enhanced by corticosteroids, resulting in increased risk of infections, especially opportunistic in nature.^43,64 O'Brien et al⁴³ have shown that the addition of prednisone to FAMP in patients with CLL does not result in a higher response rate than that seen with FAMP alone. In our previous non-randomized study, we have also shown that the addition of steroids did not influence the response to 2-CdA and the overall response rates in previously untreated CLL.¹⁴ However, in previously treated patients, survival time was longer in the group treated with 2-CdA and prednisone than in the group treated with 2-CdA alone. Routine addition of corticosteroids to NA in the treatment of CLL is not warranted, with the possible exception of pre-treated patients with severe anemia and/or thrombocytopenia. In these patients corticosteroids may improve bone marrow function and suppress autoimmunity.

Nucleoside analogues combined with cytostatic drugs

Combined use of NA with other cytotoxic drugs, cytokines, monoclonal antibodies and other agents may increase the CR rate and possibly suppress minimal residual disease (MRD) and prolong survival time. Some pre-clinical in vitro and in vivo studies, as well as early clinical reports, may support such hypotheses. The reports presenting the results of combined therapy of previously treated and untreated patients with CLL are depicted in Table 2.

Nucleoside analogues combined with alkylators

Alkylating drugs were the first candidates for combined use with NA. The interference by these agents with the repair of DNA raises the possibility that they might produce synergistic antitumour effects if combined with CY or chlorambucil, which act mainly by the cross-linking of DNA strands. Clinical studies combining FAMP or 2-CdA with CY have demonstrated significant activity in previously untreated and pre-treated patients with CLL.^{76,77,78,79,80,81,82,83,84,85,86,87} A dose-escalation study on CY added to 2-CdA in patients with previously treated CLL show the feasibility of this association, with the overall response rate of 58% and 15% CR, respectively.⁸⁰ However, neutropenia was the main dose-relating toxicity. Significant activity of FAMP combined with CY has been recently presented by O'Brien et al.⁷⁸ They treated a total of 128 CLL patients with FAMP at a dose of 30 mg/m² for 3 days and CY at either 500 mg/m², 350 mg/m² or 300 mg/m² daily for 3 days. Some of the patients were previously untreated, other were treated with alkylating agents and/or FAMP. More than 80% of the patients not refractory to FAMP alone responded to the combined treatment and a 38% response rate was observed in patients who were refractory to FAMP used earlier in monotherapy. In previously untreated patients the CR rate (35%) was similar to that observed earlier in CLL patients treated with FAMP alone. However, in previously untreated patients treated with FAMP and CY who obtained CR, residual disease was seen only in 8% and the median time to progression has not been reached after a median observation of 41 months. These results may indicate that a combination therapy of FAMP and CY is superior to FAMP alone.

In order to take advantage of FAMP activity without sacrificing dose intensity Weiss et al⁵⁹ initiated a trial of sequential therapy in which patients received six cycles of FAMP (25 mg/m²/day ´ 5 days every 4 weeks) as induction therapy followed by three cycles of high-dose CY (1.5 g/m² or 3 g/m² every 2 weeks for three doses).⁵⁹ The authors have shown that sequential therapy in previously untreated CLL patients is safe and can improve the quality of response in a large patient population compared to the therapy with FAMP alone. A similar study has been recently performed by Tefferi et al.⁸⁸ They started the treatment with six cycles of intravenous CY (1 g/m²) plus prednisone (100 mg/m²/day for 5 days) followed by 2 to 6 cycles of 2-CdA (5 mg/m²/day for 5 days). The OR and CR rates were 90% and 33%, respectively.

Chlorambucil is another alkylating agent active in CLL which is a candidate for possible combination with 2-CdA and FAMP. A synergistic antitumour effect of chlorambucil was seen in CLL cells in vitro with DCF and 2-CdA.^88,89 The early clinical trials investigating the combination of chlorambucil with 2-CdA or FAMP in heavily pre-treated as well as previously untreated patients with CLL provided evidence that marrow suppression and infections were dose-limiting factors and that the doses of NA had to be lower in combination than in monotherapy.^89,90

Nucleoside analogues in combination with other agents

In vitro experiments have shown that mitoxantrone and anthracycline antibiotics act synergistically with NA.^91,92 Recent clinical observations have demonstrated that the combinations of FAMP or 2-CdA with mitoxantrone, doxorubicin or epirubicin are effective in CLL.^{56,93,94,95,96} Combined treatment with NA, mitoxantrone and CY has been undertaken both in previously treated and untreated patients with B-CLL.^93,97,98 However, the efficacy of these regimens seem to be similar to that observed after treatment with FAMP or 2-CdA alone. In addition, the toxicity, especially myelosuppression and infections of such combined therapy is high and reduction of NA doses is essential. Prospective, randomized studies are necessary to show any advantage of such combined treatment over the use of NA alone.

The studies involving other cytotoxic agents were performed mainly as pre-clinical phase studies. Promising interaction has been discovered between 2-CdA and bryostain 1, a macrocyclic lactone which is a protein kinase C activator that has a differentiating activity in CLL cells in excess of that found in their normal counterparts.⁹⁹ Mohammad et al¹⁰⁰ has shown recently that the sequential exposure to bryostain 1 followed by 2-CdA results in significantly higher rates of growth inhibition than treatments based on either agent alone. The results of this study encourage further evaluation of bryostain 1 and 2-CdA sequential regimens in clinical trials in CLL.

Hoffman et al¹⁰¹ studied 2-CdA cytotoxicity when combined with five other antineoplastic drugs against two human-derived lymphoma cell lines at the concentrations that have been reported using standard tested therapy with these drugs in patients. Of these agents (cis-platinum, daunorubicin, chlorambucil paclitaxel or etoposide), only the combination of 2-CdA with cis-platinum has shown significantly more enhanced cytotoxicity than either agent alone.

Giles et al¹⁰² tested the combination of FAMP and cis-platinum, with or without cytarabine (ara-C) in patients with CLL who were refractory to FAMP alone or had relapsed prior to FAMP therapy. Eight of 41 (19%) patients achieved a PR but no CR was observed, which indicates that this combination has rather modest activity in patients with progressive, pre-treated CLL. Their findings also indicated that ara-C did not add to the activity of the cis-platinum + FAMP combination.

Management of refractory and relapsed patients

According to NCI-sponsored Working Group guidelines, patients who have relapsed may be observed without therapy until evident progression of the disease-related symptoms.²⁰ In our policy, the indications for re-treatment or second or third line treatment are the same as for first line therapy.⁸ Nucleoside analogues are frequently active agents in patients with CLL refractory to chlorambucil or other alkylating drug-based regimens.^{103,104,105,106,107,108} In refractory or relapsed patients FAMP induces between 1 and 73% of overall responses, including CR in the range of 0-25%.^11,12,13 2-CdA has also been found to be quite effective in the treatment of patients with CLL resistant to conventional treatment. Saven et al¹⁰⁴ reported a response rate of 44% in 90 patients with advanced and previously treated CLL. However, only 4% of the patients achieved CR. In our group of 184 patients with a relapse or refractory disease following prior therapy with chlorambucil and prednisone, some of them also with COP and/or CHOP, 2-CdA induced clinical CR in 12.5% and overall response (OR) in 48.4%.¹⁴

Chlorambucil is less active as a second line treatment in patients refractory to NA. In patients primarily resistant to 2-CdA, the OR rate after treatment with chlorambucil and prednisone as an alternative regimen was only 33%.⁸ In the same study 2-CdA was used as a second line treatment in patients refractory to chlorambucil and the OR rate was 67%. Similar results were obtained by Rai et al.⁹ Of the 79 patients who crossed over from chlorambucil to FAMP, the OR rate was 46%. In contrast, of the 29 patients who crossed over from FAMP to chlorambucil, response was observed in only 7%. The prognostic factors for response and survival time in patients treated with nucleoside analogues as second line therapy are similar to those used in previously untreated patients.^14,105 The factors most strongly associated with response are Rai or Binet stage, hemoglobin levels, serum albumin and the number of prior treatments.

Cross-resistance of nucleoside analogues is a matter of debate. 2-CdA and FAMP are similar in structure and mechanism of action. An in vitro study performed by Morabito et al¹⁰⁶ indicated that CLL cells sensitive to FAMP generally responded to 2-CdA. However, out of 61 samples resistant to 2-CdA only 29.5% were sensitive to FAMP. Conversely 13.9% of 143 samples resistant to FAMP were sensitive to 2-CdA. In the study of Begleiter et al,¹⁰⁷ 10% of cell samples from CLL patients showed a marked difference between in vitro sensitivity to 2-CdA and FAMP. The majority of clinical studies indicate the existence of cross resistance between 2-CdA and FAMP in CLL patients. Although Juliusson et al¹⁰⁸ obtained response to 2-CdA in four patients previously non-responsive to FAMP, O'Brien et al¹⁰⁹ found cross-resistance between these two agents in a larger group of patients. Our studies also indicated cross-resistance when 2-CdA was used as first line therapy and FAMP as a second line therapy.¹⁴ There were only two partial responses to FAMP therapy in 10 CLL patients refractory to 2-CdA and eight patients were non-responding.

Alkylating agent-based regimens, such as COP or CHOP, were the most frequently used second line therapy in patients with advanced CLL refractory to chlorambucil before the nucleoside analogues era. However, these regimens, especially COP, seem to have low efficacy in the treatment of disease refractory to chlorambucil. Itala and Remes¹¹⁰ analyzed the effectiveness of COP therapy in 24 patients previously treated with alkylating agents. The overall response rate to COP was 25%. The main cause of death in all nine patients who died during the COP treatment was sepsis due to neutropenia. This study indicates that COP regimen has low efficacy in the treatment of refractory CLL and that the toxicity of this regimen is unacceptable. A similar conclusion can be drawn from our retrospective analysis of 52 patients who received standard doses of chlorambucil as first line treatment and COP or CHOP as second line.⁶² The overall response rate was only 13.5%.

Patients who received FAMP or 2-CdA as their initial therapy and experienced response lasting for a minimum period of a year may be successfully retreated with the same agents. Keating et al¹³ examined the efficacy of FAMP in retreatment of 63 patients and 41 (67%) have responded. Retreatment with 2-CdA may also be successful. Juliusson and Liliemark¹¹¹ retreated six patients with 2-CdA; eight patients relapsed within 28 months after the initial treatment with this agent. They found similar efficacy of this agent used in initial therapy and in retreatment. However, drug-related cytopenia was more severe during retreatment. In our study, retreatment with at least three cycles of 2-CdA plus prednisone was administered to 12 patients who had relapsed 12 months or later from first remission and six (50%) of them responded.⁸

Newer therapeutical approaches

B cell CLL remains incurable with conventional chemotherapy with alkylating agents and even with NA. Thus, particularly for younger patients, new treatment modalities are needed that have the potential to completely eradicate the disease. In recent years preclinical and clinical studies have been undertaken with selected biological factors, especially monoclonal antibodies and high-dose radiochemotherapy with stem cell reinfusion.

Two monoclonal antibodies directed against the CD52 antigen (CAMPATH-1H, alemtuzumab) and CD20 antigen (Rituximab) demonstrate significant activity in patients with CLL.^{112,113,114,115,116,117,118} CAMPATH-1H is especially active in the treatment of T cell prolymphocytic leukemia (T-PLL).¹¹⁹ A response rate of 73% (11 of 15 patients) with nine (60%) entering complete response was observed in chemotherapy-resistant T-PLL patients following treatment with this antibody. Studies of CAMPATH-1H in CLL have been conducted in previously untreated patients and in patients pretreated with FAMP or alkylating agents.^116,117,118 In the vast majority of CLL patients CAMPATH-1H causes constant reduction of abnormal blood lymphocytosis, usually in less than 4 weeks, and disappearance of CD5/CD19 co-expression cells from blood. The regression of lymphoid infiltration from other sites, especially from lymph nodes, is less clear. In the multicenter study performed by Osterborg et al¹¹⁵ 29 previously treated chemotherapy-resistant patients were treated with CAMPATH-1H, administered as a 30 mg, 2-h intravenous infusion thrice weekly for a maximum period of 12 weeks. Eleven patients (38%) achieved a partial response and one (4%) a complete response. Hematological toxicity, especially long-lasting lymphocytopenia, was noted in the majority of patients. The most important clinical side effects of CAMPATH-1H treatment were infections, mainly localized herpes simplex virus reactivation, oral candidiasis and Pneumocystis carinii pneumonia. Similar results were obtained by other authors.¹¹⁸ A multicenter prospective randomized study comparing CAMPATH-1H and chlorambucil in previously untreated patients with B-CLL has been recently started.

Rituximab is a chimeric monoclonal antibody which specifically binds to the CD20 antigen on normal and malignant B lymphocytes. This agent is also active in CLL and the results of several studies have been recently published.^112,113,114 The activity of CAMPATH-1H and Rituximab in CLL patients resistant to FAMP and their synergistic interactions with some cytotoxic drugs, especially NA, in pre-clinical studies suggests that a combination of these agents may lead to further progress in the treatment of this disease.

Recently, several novel promising compounds besides monoclonal antibodies have been investigated both in pre-clinical and early clinical studies as more specific treatment for CLL than conventional agents: depsipeptide - FR 90 1228, bryostatin, flavopiridol, and biologic factors: vaccines, antisense oligonucleotides.¹²⁰ However, the real value of these factors and their possible clinical applications in the treatment of B-CLL patients needs further laboratory and clinical evaluation.

The use of stem cell transplantation (SCT) for patients with CLL is a relatively new approach and is the subject of recent extensive review.¹²² Briefly, the interpretation of available data are hampered by the heterogeneity of patients and their prior therapy. However, several phase II studies have demonstrated the feasibility of both allogeneic and autologous SCT in this disease.^123,124,125 In allogeneic SCT there is survival plateau of 40-60% and a relapse rate of only 10-25%. This observation suggests that some of the transplanted patients are cured. Unfortunately, despite the fact that allogeneic transplantation has produced prolonged remission in younger patients, the high toxicity of the procedure and high rate of early mortality (25-50%) is rather unacceptable. In this context, an interesting option for patients with CLL is created by non-myeloablative allogenic SCT. The transplant-related mortality of this procedure (less than 20% after 1 year) is significantly lower than after conventional allogeneic SCT. However, long-term results are still uncertain.

Recent studies have shown that autologous SCT produces prolonged molecular remission in high risk CLL patients.^121,123 Unfortunately, the high relapse rate and the absence of a plateau in disease-free survival curves indicate that autologous SCT does not cure CLL. However, to determine the real role of this procedure in the management of CLL further investigation is necessary. It is especially important to evaluate the clinical benefit of autologous SCT in comparison to conventional chemotherapy. Further studies are also necessary to evaluate optimal timing of transplantation, best conditioning regimen, role of purging and clinical significance of minimal residual disease (MRD). Assessment of the residual disease using a sensitive flow cytometric assay or PCR methods in patients treated with CAMPATH-1H and/or undergoing stem cell transplantation has shown that those patients who became MRD negative after this procedure may experience longer disease-free survival time.^123,124 In this context the evaluation of the quality of CR by the estimation of minimal residual disease using flow cytometry or molecular techniques before and after the stem cell transplantation procedure may have important value.^{123,124,125,126}

Conclusions

This review presents the state of the art in treatment of B cell CLL and the current understanding of the role of alkylating agents and NA within this treatment, emerging primarily from the results of randomized trials recently published. The management of CLL is determined by the stage and activity of the disease, assessed by the evaluation of the prognostic factors in evidence. One-third of CLL patients have aggressive disease at diagnosis and should be immediately treated, while in others, only a wait and watch policy is necessary. The results of the clinical trials indicate that NA yield significantly higher CR and overall response rates than alkylating agents in previously untreated patients, but that this does not translate into prolonged survival time. Combination therapy does not appear, as yet, to yield superior results in regard to response rates or survival time to those obtained from single agent therapy in previously untreated patients. Nucleoside analogues, FAMP and 2-CdA, are effective in the majority of patients relapsing after an initial treatment with chlorambucil.

The current policy in our center, which is outlined in Figure 2, is the consequence of available clinical data, as described earlier in this review. After diagnosis and staging, the patients in early stage and stable disease are not treated until disease progression. In patients with active disease therapy is commenced. As the majority of patients are older and since there is no survival time advantage for either alkylating agents or NA therapies, regardless of quality of response, we recommend chlorambucil as first line treatment, with NA for consideration as an option in younger patients. Chlorambucil is safe, has a low incidence of therapy-related toxicity, can be administered on an out-patient basis, and most importantly, the cost involved in administering such treatment is low. In patients refractory to treatment or with an early relapse, the second line treatment recommended is NA, FAMP or 2-CdA. In the majority of Western countries, especially in the USA, FAMP is the preferable NA as it has been the subject of more extensive investigations in CLL patients and is probably less toxic.

The approach to younger patients who may be candidates for SCT may differ. In this subgroup of patients the rate and quality of CR evaluated by the estimation of residual disease may be of special importance. This favors the early use of NA, either in monotherapy or in combination therapy, during the course of the disease which would enable the harvest of stem cells from those patients who achieve complete remission. However, the exact role of SCT either autologous or allogeneic in the standard management of younger CLL patients is still undefined. The published results of preliminary clinical studies seem to be encouraging but the results of randomized studies comparing this method of management with conventional treatment are not available yet.

It is important to recognize that significant differences exist in biology and clinical behavior of CLL patients. The clinical and biological heterogeneity of the disease should be taken into account in selection of the most appropriate treatment. In the future, stratified risk-adapted therapy will be required similarly to current management in acute lymphoblastic leukemia.

The cure of disease remains the ultimate goal in CLL and will be the subject of clinical research in the years to come. Nucleoside analogues and monoclonal antibodies will, probably, present the foundation for innovative therapeutic strategies. Those patients who require treatment should preferably be entered into clinical trials in which they should be stratified according to prognostic factors.

Editor's note

We are very indebted to Dr Peter Daniel who recruited and evaluated all the Reviews published in this Spotlight. Authors who are interested in contributing a Review for this Spotlight are invited to contact the Editor-in-Chief, Dr Muller Bérat.

Acknowledgements

This work was supported in part by grant 4P05B0619 from KBN, and by the Foundation for the Development of Diagnostics and Therapy, Warsaw, Poland. We thank Mr Shane Gollop for the correction of the English version of the manuscript.

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Figure 1 Overall survival time calculated from the first day of treatment to the last day of follow-up or death for patients treated with cladribine (2-CdA) + prednisone (P) (continuous line) or chlorambucil (Chl) + P (dotted line) as front line therapy. (Adapted with kind permission of the publisher from Robak et al.⁸)

Figure 2 Proposed flow-chart of therapeutic decisions in the treatment of chronic lymphocytic leukaemia based on current evidence.

Table 1 Purine nucleoside analogues vs standard chemotherapy as first line therapy in advanced and progressive CLL

Table 2 Combination of purine nucleoside analogues with other cytotoxic agents in patients with CLL