Biology and treatment of chronic lymphocytic leukemia

Annals of Oncology 16 (Supplement 2): ii113 ii123, 2005 doi:10.1093/annonc/mdi731 Biology and treatment of chronic lymphocytic leukemia P. Kokhaei, M. Palma, H. Mellstedt & A. Choudhury Departments of Hematology and Oncology, and Immune and Gene Therapy Laboratory, Cancer Centre Karolinska, Karolinska University Hospital, Stockholm, Sweden Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of a clone of malignant B cells in lymphoid tissues, bone marrow (BM) and peripheral blood [1]. B-CLL is defined by the expansion of B cells expressing CD5, CD19 and CD23, as well as low levels of surface immunoglobulins (IgM, IgD), CD21 and CD22, and CD79b. Close to 99% of B-CLL lymphocytes in peripheral blood are in the G 0 /early G 1 phase of the cell cycle [2]. CLL is the most frequent type of leukemia in the western hemisphere and accounts for 30% of all leukemias. It has an incidence of 2.3 3.3 per 100 000 people [3]. B-CLL more frequently affects elderly people (median age 60 years), but a significant portion of cases (10 15%) is diagnosed in individuals younger than 50 years old [4]. Survival of patients with CLL is highly variable, with some patients living as long as sex- and age-matched healthy individuals and others dying soon after diagnosis. The variability in the prognosis, as well as the different treatment options, makes it necessary to treat patients with CLL according to their individual risk [2, 5]. Prognostic factors Clinical stage The most common and validated prognostic factors are the clinical staging systems proposed by Binet et al. [6] and Rai et al. [7], which define early (Rai 0, Binet A), intermediate (Rai I/II, Binet B) and advanced (Rai III/IV, Binet C) stages of the disease on the basis of the extent of lymphoid areas involvement and on the presence of anemia and thrombocytopenia (Table 1). Estimated median survival times for these stages are >10, 5 7 and 1 3 years, respectively. These systems have, however, two major drawbacks. The first is the fact that BM involvement is inferred by the hemoglobin level (<10 and <11 g/dl for the Binet and Rai systems, respectively), irrespective of the patient s gender. Secondly, the systems do not allow prediction of when and to what extent disease progression will occur in early stage patients, as >60% of CLL patients are at that stage at the time of diagnosis. Some clinical parameters have, however, been indicated as reliable for the identification of Binet stage A patients with smouldering disease [8 10]. These criteria are: lymphocyte count in peripheral blood <30 10 3 /dl; hemoglobin >12 g/dl; non-diffuse (interstitial or nodular) lymphoid infiltration pattern in the BM; long lymphocyte doubling time. Still, in the subgroup of patients fulfilling these criteria, 10% progress within 5 years, indicating that these criteria do not completely allow prediction of the clinical outcome of early-stage disease. This has prompted investigators to look for clinical and biological markers which could be considered as additional prognostic factors. IgVH gene status, CD28 and ZAP-70 expression B-CLL can be subdivided into two subgroups depending upon the presence or the absence of mutations in the variable heavy chain (VH) genes of the immunoglobulin (Ig) locus of malignant B cells. A 2% difference from the corresponding germ-line gene has been taken as a cut-off point to distinguish B-CLL patients with mutated immunoglobulin variable (IgV) genes from those without mutations. This value was chosen to eliminate the influence that allelic variants of an individual s germline VH genes could have in defining mutations [11]. Observations on variable light chain (VL) genes led to similar results and also showed a concordance between the VH and VL gene status in individual patients. Retrospective studies on two relatively large cohorts of B-CLL patients demonstrated that the presence of unmutated IgVH genes correlated with a more rapid progression of the disease and a shorter survival time. At present, IgVH gene mutation status is the most powerful predictor of clinical outcome in B-CLL. The proportion of unmutated patients is close to 30 40%. Molecular, phenotypic and clinical characteristics of the IgV-based defined B-CLL subgroups are summarized in Table 2 [1, 12]. CD38 and ZAP-70 are two surrogate markers for VH mutation analysis. CD38 is expressed on the surface of a proportion of B-CLL patients. CD38 expression is an important independent negative prognostic factor. CD38 expression is a predictor of poor clinical outcome and 30% positivity is the cut-off point to discriminate between CD38+ and CD38 patients [1]. ZAP-70 is a protein associated with the z chain of the T-cell receptor that is selectively expressed by T cells and natural killer cells. Normal and neoplastic B cells do not express ZAP-70 in significant quantities. However, elevated q 2005 European Society for Medical Oncology

ii114 Table 1. Clinical staging systems and the prognostic impact Binet classification Rai classification Median overall survival (years) Stage Definition % of patients Risk group Stage Definition % of patients A <3 lymphoid areas a 60 Low 0 Lymphocytosis only 30 >10 B >3 lymphoid areas 30 Intermediate I Lymphoadenopathy 25 5 7 II Hepato or splenomegaly 25 ± lymphoadenopathy C Hemoglobin <10 g/dl or 10 High III Haemoglobin <11 g/dl b 10 1 3 platelets <10010 3 /dl b IV Platelets <10010 3 /dl b 10 a Lymphoid areas considered are the following five: unilateral or bilateral cervical, axillary and inguinal lymphnodes, spleen and liver. b With exclusion of haemolysis and unrelated causes of anemia or thrombocytopenia. levels of ZAP-70 mrna in gene expression studies, and high levels of ZAP-70 protein were detected by western blotting and flow cytometry in the unmutated B-CLL subgroup. Clinically, ZAP-70 overexpressing patients have a poor clinical outcome. A study by Rassenti et al. [13] showed that 50% of the patients, with <20% of their B-CLL cells expressing ZAP- 70, survived for 10 years or longer, whereas patients with >20% expression of ZAP-70 had a median survival of <5 years. Telomere length A cell s telomeres shorten with each cell division. The length can provide an approximation of the number of replications a cell has undergone [14]. Telomere length is shorter in B-CLL cells than in normal unfractionated or CD5+ B cells, indicating a more extensive replicative history for the leukemic cells. The telomeres of unmutated patients are significantly shorter than those of mutated patients, suggesting a more active proliferation in the former B-CLL subgroup. Finally, cells from unmutated patients have a higher telomerase activity, which presumably reflects a more pronounced activation status and functions to prevent excessive telomere shortening leading to clonal extinction [1]. Activation-induced cytidine deaminase Activation-induced cytidine deaminase (AID) is a novel member of the cytidine deaminase family and is essential for somatic hypermutation (SHM) and class switch recombination (CSR). Expression of AID is normally restricted to B cells in the germinal center, where SHM and CSR occur [15]. In one study, AID mrna expression in 20 CLL patients and five controls was investigated. In peripheral blood B cells of 12 B- CLL patients and five controls no AID mrna was detected. Seven of eight CLL patients that highly expressed AID were unmutated. It is likely that this is a reaction to a blocked pathway. Splice variants of AID in B-CLL might form inactivating heterodimers with the wild-type enzyme [16]. T-cell compartment There is increasing evidence of T-cell dysfunction in B-CLL that may contribute to the etiology and progress of the disease. The absolute number of T cells is increased in B-CLL patients and T-cell subsets are redistributed, with CD4+ T cells predominating in involved BM and lymph nodes. An absolute CD8+ lymphocytosis correlates with disease progression. A low expression of CD4 and CD8 (as found in autoimmune diseases) is seen with abnormal expression of other surface molecules. Although the expression of the T-cell surface activation markers CD25 and CD152 may be increased on culture in Table 2. Molecular, phenotypic and clinical characteristics of the IgV gene-defined B-CLL subgroups Characteristics B-CLL with non-mutated IgVH B-CLL with mutated IgVH V gene mutations Few or none Significant numbers Gender More males Males = females Age at presentation No difference No difference Treatment requirement Common Uncommon Lymphocyte doubling time Frequently <12 months Frequently >12 months Activation markers CD38+/CD69+ CD71+/CD62L+ CD38 expression High Low ZAP-70 expression High Low Telomere length Uniformly short Diverse lengths Telomerase activity High Low Retention of BCR Yes No signaling Frequency of 13q 26 50 deletion (%) Frequency of 11q 27 4 deletion (%) Frequency of 17p or 35 7 11q deletion (%) Frequency of trisomy 12 (%) 19 15 B-CLL, B-cell chronic lymphocytic leukemia; BCR, B-cell receptor.

ii115 B-CLL serum, response to the common mitogens, PHA and PWM, is reduced. This along with the excess of CD8 cells may partly explain the variable cooperation of T cells with B- cell production of immunoglobulin in B-CLL [17]. The abnormal kinetics and levels of CD28 and CD152 expression on T cells in B-CLL may lead to a state of hyporesponsiveness or anergy, and could be one of the mechanisms of immune deficiency in this disease [18]. B-CLL cells purified from involved lymph nodes and BM, but not from peripheral blood, constitutively express mrna for the T-cell attracting chemokines CCL17 and CCL22. CD40-crosslinking of peripheral blood B-CLL cells induces the expression of both chemokines at RNA level. CCL22 is also released and is capable of attracting CD4+/CD40L+ T cells. These findings indicate that the stimulation of malignant cells via a physiological signal present in the tumor microenvironment endows B-CLL cells with the chemoattracting capacity for activated CD4+ T-cells, which in turn can deliver survival signals to tumor cells [19]. Survivin is a prominent member of the family of inhibitor of apoptosis proteins (IAP), which integrates apoptosis and proliferation. The expression of survivin is absent in peripheral blood B-CLL cells but can be induced in vitro by CD40 stimulation. Survivin-positive cells are more resilient to apoptosis and demonstrate an increased rate of proliferation. In vivo, survivin-positive cells are localized in lymph node pseudofollicles and in rare BM clusters of proliferating CD5+ B cells that are interspersed with T cells, essentially of the CD4+ type and frequently CD40L+, indicating the in vivo availability of this signal to leukemic cells [20, 21]. Genetic abnormality A number of genomic aberrations have been described in B-CLL. In one study chromosomal aberrations were detected in 268 of 325 patients (82%) [22]. In another study of 325 patients, the most frequent abnormalities were the deletion involving a 13q14 band (55%) confined with the mutated subgroup, a deletion in 11q (18%) and trisomy of chromosome 12, which was associated with progressive lymphocytosis, resistance to chemotherapy and a fatal clinical course (16%). Deletions in 17p (7%) are confined almost entirely to the unmutated subset. These changes revealed the heterogeneity of the leukemic cell population, and indicated that these chromosomal abnormalities are probably secondary events in CLL leukemogenesis as a consequence of clonal evolution [22, 23]. Disease-related complications About 10 15% of patients undergo disease transformation into large cell lymphoma (Richter s syndrome) or, more rarely, Hodgkin s disease [24]. Signs of suspected disease transformation include exacerbation of lymphadenopathy, increasing serum lactate dehydrogenase levels, hypercalcemia or detection of a serum M component. Diagnosing disease transformation is important, since the treatment in such a situation is that for aggressive lymphoma and, unless a response is achieved, the prognosis is poor with a median survival of <2 years after transformation [5]. Hypogammaglobulinemia is observed in 20 30% of patients and is considered to be the main cause of bacterial infections. Immunoglobulin replacement does not change life expectancy, although it may improve quality of life by decreasing the number of infections [25]. It has been estimated that almost 60% of deaths are caused by bacterial or viral infections [26]. Together with hypogammaglobulinemia, which is present in a significant number of patients, T-cell defects lead to impaired immune responses to intracellular bacteria or viral infections, autoimmune disorders and possibly also secondary neoplasms are associated with CLL [5]. Primary treatment B-CLL is a slowly progressive disease that occurs predominantly after 55 years. Patients often remain asymptomatic for many years after diagnosis and the anticipated survival, even for the group of patients with the worst prognostic factors, is several years. In 1998, the French Cooperative Group on CLL reported the results of two trials comparing the impact on survival of patients with Binet stage A CLL receiving therapy (chlorambucil ± prednisone) either at the time of diagnosis or at the time of disease progression. The results showed that initial treatment slowed the rate of disease progression, but had no impact on overall survival (OS) [27]. Indeed, despite the fact that the only option to cure CLL patients is provided by BM transplantation (BMT), it must be considered that treatment-related side effects may have a detrimental effect on survival of low-risk patients. The decision of initiating treatment, the choice of the therapy and timing must be evaluated carefully by health-care providers. The NCI-sponsored working group on CLL recommends that treatment should be forwarded until the fulfilment of the following criteria of active disease [28]: (i) systemic symptoms without evidence of infection: weight loss >_ 10% within the previous 6 months, extreme fatigue [i.e. Eastern Cooperative Oncology Group (ECOG) performance status 2 or worse; cannot work or unable to perform usual activities], fever >_388C for >_2 weeks, night sweats; (ii) evidence of progressive marrow failure as manifested by the development of, or worsening of, anemia and/or thrombocytopenia; (iii) autoimmune anemia and/or thrombocytopenia poorly responsive to corticosteroid therapy; (iv) massive (i.e. >6 cm below the left costal margin) or progressive splenomegaly; (v) massive nodes or clusters (i.e. >10 cm in longest diameter) or progressive lymphadenopathy; and (vi) progressive lymphocytosis with an increase of >50% over a 2-month period, or an anticipated doubling time of <6 months. As far as the choice of treatment is concerned, many drugs are known to be effective in CLL. The oldest is chlorambucil, which achieves 60 70% partial response (PR) in previously untreated patients, but no significant complete response (CR) rate. Response rates gained by combining anthracyclines and alkylating agents [cyclophosphamide, adriamycine, prednisone

ii116 (CAP); cyclophosphamide, adriamycine, vincrinstine, prednisone (CHOP)] are slightly higher, but survival is not improved [29]. Newer therapeutic options are discussed in the following paragraphs. Chemotherapy Purine analogs as single agent or in combination Fludarabine is the purine analog that has been most extensively studied and employed in the treatment of CLL patients. Like all purine analogs, it acts by inhibiting DNA polymerase and ribonucleotide reductase, finally promoting apoptosis [30]. In a large randomized trial [31], fludarabine both as single agent and in combination with chlorambucil was compared with chlorambucil alone in >500 previously untreated patients. After an interim analysis showing that the combined treatment resulted in excessive toxicity without increasing the response rate compared with fludarabine alone, patients were assigned only to the single-agent arms (170 patients in the fludarabine arm and 181 patients in the chlorambucil arm). Overall response (OR), median duration of remission and median progression-free survival (PFS) were significantly higher in the fludarabine arm. Response rate was 63% (20% CR+43% PR) versus 37% (4% CR+33% PR), respectively, while median duration of remission was 25 and 14 months and median PFS 20 and 14 months, respectively (P <0.001 for both comparisons). However, median OS was 66 months for the fludarabine arm versus 56 months for the control arm, which was not significantly different. In another large trial [32], 938 patients (651 stage B and 287 stage C) were randomized to receive fludarabine or an anthracycline-containing regimen, either CAP or CHOP as primary treatment for CLL. Overall remission rates were 58.2%, 71.5% and 71.1% for the CAP, the CHOP and the fludarabine arms respectively (P <0.0001). Accordingly, lower CR were found in the CAP arm (15.2% versus 29.6% for CHOP and 40.1% for fludarabine; P = 0.003), while no significant difference was found in median survival time, which was 67, 70 and 69 months in the CHOP, CAP and fludarabine groups, respectively. In both studies, fludarabine confirmed its high activity, providing the highest response rate. Treatment with fludarabine is generally well tolerated, major toxicities being hematological and immunological. The occurrence of autoimmune hemolytic anemia has also to be taken into account. In both the abovereported studies, no statistically significant advantage in OS was seen in the fludarabine arm, despite the higher OR rate, CR rate and duration of response. However, it should be emphasized that in both studies [31, 32] a crossover design was applied and in the group of patients who had failed CAP, CHOP or chlorambucil, a high response rate with second-line fludarabine was achieved. The activity of fludarabine was further studied in combination with alkylating agents, given the synergic effect of these drugs on DNA. In a multicenter phase II ECOG trial, a 63.9% OR rate (41.7% CR + 22.2% PR) was achieved in 36 patients treated with fludarabine in combination with cyclophosphamide [33]. Similarly, higher remission rates and longer PFS were seen with the same combination compared with fludarabine alone in a phase III trial of the German CLL Study Group. The trial compared six courses of fludarabine with six courses of fludarabine and cyclophosophamide (FC) as primary treatment for CLL patients aged <_65 years, in either stage C asymptomatic disease or stage A/B symptomatic disease. OR rates were 84.1% and 95.3%, and a CR rate of 8.6% and 20.3% for the fludarabine arm and the combination arm, respectively (P <0.01 for both comparisons) was noted. PFS was 21 and 46.7 months for the fludarabine and the FC arms (P = 0.0033) respectively, but no survival benefit has been demonstrated so far (M. Hallek, personal communication). Another purine analog, cladribine (2-CdA) was found to achieve similar responses to fludarabine in both previously treated and untreated patients. In a study reported by Robak et al. [34], 378 patients (194 previously untreated and 184 with relapsed or refractory disease) were treated with cladribine with or without prednisone. OR was obtained in 82.5% (45.4% CR + 37.1% PR) of the patients in the untreated group, with a median survival of 19.4 months. In a phase III study by the same investigators [35], cladribine prednisone was compared with chlorambucil prednisone. Both OR and CR rates were higher in the cladribine arm (87% and 47%, respectively) compared with the chlorambucil arm (57% and 12%, respectively) (P = 0.001). However, no advantage was seen in OS. The preceding data are summarized in Table 3. These data suggest that cladribine has activity similar to that of fludarabine, but the available evidence is still limited. Cladribine cannot be considered a standard alternative primary therapy for CLL. Sequential use of the two drugs is contraindicated by the known cross-resistance between the two purine analogs suggested by both clinical and in vitro studies. Finally, it should be mentioned that, owing to the risk of transfusion-related graft versus host disease, blood products should always be irradiated before being administered to a patient treated with purine analogs [36]. Monoclonal antibodies as single agents Alemtuzumab is a complementarity determining region (CDR)-grafted, human IgG, monoclonal antibody against the CD52 antigen expressed on normal and leukemic B- and T- lymphocytes, macrophages and monocytes. After a small trial showing that alemtuzumab administered intravenously induced three CRs and five PRs in a group of nine previously untreated patients [37], a phase II trial was started in which alemtuzumab was given subcutaneously at a dose of 30 mg three times per week for up to 18 weeks to 41 patients as primary treatment. An OR rate of 87% (19% CR + 68% PR) was noted. The median time to treatment failure has not been reached (>18 months) [38]. Despite the high OR rate in untreated patients, the short follow-up does not yet allow superiority to be claimed over conventional therapy. In the study, the drug was administered subcutaneously to avoid infusion-related toxicity; however, though results were promising, this approach should still be avoided out of the context of clinical

ii117 Table 3. Fludarabine and cladribine as single agents or in combination as primary treatment for CLL Author Treatment No patients ORR (%) CR (%) Median OS (months) Johnson (1996) [80] Fludarabine versus 52 71 23 Not reached CAP 48 60 17 6.9 Rai (2000) [31] Fludarabine versus 170 63 20 66 chlorambucil 181 37 4 56 Leporrier (2001) [32] Fludarabine versus 341 71.1 40.1 69 CAP versus 240 58.2 15.2 70 CHOP 357 71.5 29.6 67 Eichhorst (2003) [81] Fludarabine/cyclophosphamide versus 185 versus 94.2 20.2 Not reached fludarabine 190 85.7 8.6 Not reached Robak (2000) [34] Cladribine±prednisone 194 82.5 45.4 19.4 Robak (2000) [35] Cladribine/prednisone versus 126 87 47 chlorambucil/prednisone 103 57 12 CLL, chronic lymphocytic leukemia; ORR, overall response rate; CR, complete response; OS, overall survival; CAP, cyclophosphamide, adriamycine, prednisone; CHOP, cyclophosphamide, adriamycine, vincrinstine, prednisone. trial. It should be noted that cytomegalovirus (CMV) reactivation occurred in 10% of patients. Data from published alemtuzumab trials are listed in Table 4. Patients receiving alemtuzumab should be monitored carefully for CMV reactivation both during therapy and for at least 2 months afterwards. They should also receive prophylactic therapy for Pneumocystis carinii and varicella zoster virus. The use of irradiated blood products in patients treated with alemtuzumab is recommended. The major complications of alemtuzumab therapy have restricted its widespread use in clinical practice as a first-line single agent. The CCL4B trial addressed the issue of alemtuzumab efficacy as consolidation therapy after CR or PR had been achieved either with fludarabine or fludarabine cyclophosphamide as front-line therapy. PFS was significantly longer in the alemtuzumab arm and molecular remission rates were higher [39]. Increasing evidence seems to indicate that the achievement of a minimal residual disease negative status has a prognostic impact. Rituximab is a chimeric anti-cd20 monoclonal antibody, which has been successfully employed for the treatment of B-cell lymphomas and has proved to be active in CLL in a number of studies. When administered as primary treatment at the dose of 375 mg/m 2 thrice weekly for 4 weeks to 44 CLL/SLL patients, a 51% OR rate and a 4% CR rate was observed. In case of objective response or stable disease, rituximab was then administered in 4-week cycles at 6-month intervals as maintenance therapy. The efficacy of this approach is still being evaluated. In fact, only 28 patients have received one or more additional cycles of rituximab, with an OR rate of 58% (9% CR) and a median PFS of 18.6 months. The 1- and 2-year PFS rates were 62% and 49%, respectively at a follow-up of 20 months [40]. Data from published rituximab trials are listed in Table 5. Combination chemo- and immunotherapy The activity of the combination of fludarabine, cyclophosphamide and rituximab as first-line therapy for CLL patients is Table 4. Alemtuzumab as single agent or in combination for the treatment of untreated or previously treated CLL patients Author Treatment No. patients Prior therapy ORR (%) CR (%) Osterborg (1996) [37] Alemtuzumab 9 No 89 33 Lundin (2002) [38] Alemtuzumab 41 No 87 19 Osterborg (1997) [64] Alemtuzumab 29 Yes 42 3 Kennedy (2001) [65] Alemtuzumab 77 Yes 44 25 Rai (2001) [66] Alemtuzumab 136 Yes 40 7 Keating (2002) [63] Alemtuzumab 93 Yes 33 2 Kennedy (2002) [67] Alemtuzumab/fludarabine 6 Yes 66 16 Faderl (2003) [68] Alemtuzumab/rituximab 32 Yes 63 1.6 CLL, chronic lymphocytic leukemia; ORR, overall response rate; CR, complete response.

ii118 Table 5. Rituximab as single agent or in combination for the treatment of untreated and previously treated CLL patients Author Treatment No. patients Prior therapy ORR (%) CR (%) Byrd (2003) [42] Rituximab/fludarabine 51 No 90 47 Rituximab! fludarabine 53 No 77 28 Keating (2004) [41] Rituximab/fludarabine/cyclophosphamide 224 No 95 71 Schulz (2002) [82] Rituximab/fludarabine 31 20 no, 11 yes 87 34 O Brien (2001) [60] Rituximab 40 Yes 36 0 Byrd (2001) [61] Rituximab 33 Yes 45 3 Wierda (2003) [62] Rituximab/fludarabine/cyclophosphamide 143 Yes 72 28 Gupta (2001) [83] Rituximab/cyclophosphamide/dexamethasone 22 Yes 77 36 CLL, chronic lymphocytic leukemia; ORR, overall response rate; CR, complete response. currently being investigated, but preliminary results from a single-institution trial are encouraging, showing a 71% CR rate in 224 patients [41]. In a randomized phase II study, patients received either six monthly cycles of fludarabine concurrently with rituximab, followed 2 months later by 4 weekly doses of rituximab for consolidation therapy or fludarabine alone, followed 2 months later by rituximab consolidation therapy. Very high response rates were reported both for the concurrent treatment (90% OR, 47% CR) and for the sequential treatment (77% OR, 28% CR). After a median follow-up of 23 months, median response duration and survival have not yet been reached [42]. The combination of fludarabine and alemtuzumab has also been investigated as primary treatment for CLL patients. In a phase II trial by Rai et al. [43], patients received four cycles of intravenous fludarabine and were evaluated for response 2 months after the last treatment. If they had achieved stable disease or a response, they were treated with a 6-week cycle of alemtuzumab. Among 36 patients who entered the alemtuzumab therapy, response rates of 42% CR and 50% PR were noted. Out of 11 patients with stable disease after fludarabine, three achieved CR and five had PR following alemtuzumab. During therapy or within 4 months after treatment with alemtuzumab, eight patients experienced a CMV infection, one of which was fatal. These early results are encouraging, but an impact of maintenance therapy with alemtuzumab on OS has yet to be determined. In a phase I/II study conducted by O Brien et al. [44], alemtuzumab was administered to 41 patients three times weekly for 4 weeks following maximum response to induction chemotherapy. The OR rate was 46% and a molecular disease remission was achieved in 38% of the patients, but infectious toxicity was significant with 22% of the patients experiencing CMV reactivation. Short- and long-term complications of therapy Infections and hemolytic anemia are the most common complications associated with B-CLL therapy. Additionally, both nucleoside analogs and alemtuzumab may affect cellular immunity for an extended period. The occurrence of infections by P. carinii and herpes zoster has been substantially reduced by prophylactic treatment with antibiotics and antivirals. Prophylaxis for CMV reactivation, on the other hand, is not standardized and at present the recommendation is strict surveillance. Salvage therapy for B-CLL The indications for second-line and subsequent treatment are basically the same as for the initial treatment, that is active disease [28]. More aggressive treatments with curative intent, such as allogenic transplantation, should be taken into consideration for early relapses or in first remission in younger patients. Together with clinical stage, adverse prognostic factors and number of prior therapy lines, poor response to previous therapy is negatively predictive of response to subsequent lines of treatment. Alkylating agents A previous history of response to alkylating agents, such as chlorambucil, usually indicates that these drugs are likely to produce some clinical response when used again, but to a lesser extent and with a shorter PFS. The emergence of drug resistance is quite common. The use of chlorambucil in patients relapsing after initial treatment with purine analogs achieves low response rates. In the trial reported by Rai et al. [31], of the 29 patients who crossed over from fludarabine to chlorambucil only 7% had a response, while 58% of the patients from chlorambucil to fludarabine responded (P <0.001). In a phase III trial comparing cladribine prednisone with chlorambucil prednisone, a response rate of 27% was achieved in the group of patients who were treated with chlorambucil after failing cladribine [34]. Anthracycline-containing combination therapy Results from a multicentre randomized trial by the French Cooperative Group on CLL, in which patients who had already received an alkylating agent were randomized to receive either CAP or fludarabine, showed an OR rate of 27% and 48%, respectively (P = 0.036). In the trial by Leporrier et al. [32] that compared fludarabine, CAP and CHOP as primary treatment for CLL, a 39% OR was seen in the group

ii119 of patients who were crossed over to CHOP after failing fludarabine. Taken together, these results seem to indicate that anthracycline-containing regimens are less active than purine analogs when used after chlorambucil, but still effective in patients relapsing after a treatment with purine analogs. Purine analogs Second-line or subsequent treatment with fludarabine was investigated in phase I and II trials, which found an OR rate ranging from 32% to 57%, with CR ranging from 3% to 37% [45 49]. In the study by Leporrier et al. [32], 40% of the patients who had failed to respond to CHOP as primary therapy responded to fludarabine. Factors predictive of response were the number of prior therapy lines, serum albumin and hemoglobin levels, age, and response to the last treatment [48, 50]. According to the National Institute for Clinical Excellence guidelines, fludarabine monotherapy should be offered as second-line treatment to patients who have failed a first-line therapy with an alkylating agent [51]. Patients who have initially responded to fludarabine are likely to respond when retreated with the same agent in contrast to patients who were refractory to initial therapy with fludarabine (OR rate 85% versus 12%) [52]. Response rates to second-line cladribine seem to be similar to those achieved by fludarabine, with a similar toxicity profile. In the study by Robak et al. [34], cladribine induced an OR of 48.4% (12.5% CR) in 184 patients with relapsed or refractory disease. In another study, a 58% response rate (31% CR) with a median PFS of 23 months for CR patients and 16 months for PR patients was reported in 52 patients with previously treated CLL who received cladribine [53]. As far as cladribine s activity in fludarabine-refractory disease is concerned, the CALGB 9211 study noted an OR rate of 32% in 28 fludarabine-refractory patients treated with cladribine [54]. A number of phase II studies have shown that purine analogs combined with other chemotherapeutic agents are more effective than purine analogs alone in treating previously treated patients. In an M. D. Anderson trial [55], 128 patients were treated with fludarabine and cyclophosphamide and OR rates of 80% and 38% were achieved in the non-fludarabine refractory subgroup and fludarabine-refractory patients, respectively. Toxicities were significant. A 94% OR rate in previously treated patients with the same combination was reported by another group [56]. A 78% OR rate was reported for the combination of fludarabine, cyclophosphamide and mitoxantrone [57], 60% for fludarabine epirubicin [58] and 74% for pentostatin cyclophospamide [59]. Monoclonal antibodies as single agents or in combination Response rates to rituximab monotherapy in pretreated patients ranged from 36% to 45% (CR 0 3%) [60, 61] (Table 5). Improved results are seen with combination therapy using rituximab and chemotherapeutic agents, such as fludarabine and cyclophosphamide. In a study from the M. D. Anderson Cancer Center, the combination of the three agents induced an OR rate of 23% in 167 pretreated patients. The response rate was significantly higher in the subgroup of patients who had been sensitive to fludarabine than in the fludarabine-resistant group. A comparison of three groups treated either with fludarabine alone, or fludarabine and cyclophoshamide, or the combination of fludarabine, cyclophosphamide and rituximab, was performed at the same institution and showed OR rates of 62%, 68% and 72% (CR 13%, 12% and 28%), respectively [62]. The activity of alemtuzumab in previously treated patients has been investigated in a number of trials (Table 4). A pivotal study showed a 33% ORR (2% CR) in a group of 93 heavily pretreated, fludarabine-refractory CLL patients, leading to the approval of alemtuzumab for the treatment of fludarabine-refractory CLL. The drug showed high activity in patients with better performance status and few symptoms [63]. Similar OR rates with alemtuzumab as monotherapy were observed in a number of other studies [64 66]. The combination of alemtuzumab with either fludarabine [67] or rituximab [68] has shown promising results. Based on the observation that alemtuzumab may display good activity in patients with mutated p53 who have failed to respond to purine analogs [69], a study was initiated to evaluate whether response to alemtuzumab therapy was significantly different in fludarabine-refractory CLL patients who had p53 mutations compared with those who had not. Clinical responses were achieved in 40% of the patients with mutated p53 versus 19% of the patients with no p53 abnormalities [70]. BM transplantation The indolent nature of B-CLL, the age of most patients and the major treatment-related toxicity of this procedure have restricted the use of high-dose therapy followed by stem cell transplantation. Nevertheless, more prognostic factors are now available for the selection of high-risk patients, mortality rates following transplantation have presently decreased and new management options such as non-myeloablative allogeneic transplants have emerged. The evaluation of this aggressive, yet potentially curative, treatment for B-CLL is ongoing. To date, in fact, allogenic transplantation is the only potentially curative therapy for CLL and its use should be considered for younger patients with poor prognostic factors. Autologous transplantation No randomized trials are available that compare autologous transplantation with standard chemotherapeutic approaches. An overview of published data is provided in Table 6. It should be considered, though, that the trials differ substantially in patient selection criteria, type of conditioning therapy and use of in vitro or in vivo purging, and that survival rates are reported from the time of stem cell mobilization in some of the studies and from the date of transplantation in others. This provides a bias that should be taken into account, given the fact that only 50% of the patients that underwent a conditioning therapy received a transplant afterwards. Overall,

ii120 Table 6. Autologous transplantation in CLL Author No. patients reported transplant-related mortality rates range from 4% to 10% and 4-year OS ranges from 65% to 94%. The optimal timing for autologous transplantation and the optimal mobilization and conditioning therapies have yet to be defined. In a study reported by Gribben and colleagues [71, 72], patients were transplanted after a good response had been achieved with chemotherapy. They received a conditioning regimen of cyclophosphamide with total body irradiation, and BM purged in vivo with a cocktail of monoclonal antibodies (B5, anti-cd10, anti-cd20) was used as stem cell source. Treatment-related mortality was 10%, but disease-free survival was 63% at 4 years with an OS of 85%. Transplant mortality seemed to be related to the status of the disease and with the number of lines of prior therapy, while the outcome appeared to be better for patients with mutated IgVH genes [73], patients who were in CR at the time of transplantation and in those who were treated with total body irradiation as part of the conditioning regimen [74]. Optimal timing, mobilization and conditioning regimens are not yet well defined and are the focus of investigation. Available data indicate that autologous transplantation is not curative in CLL. Allogenic transplantation TRM (%) 4-year DFS (%) Gribben (1998) [72] 81 10 63 85 Dreger (2000) [74] 370 10 NI 69 Esteve (2001) [84] 124 6 37 65 Dreger (2000) [85] 77 4 69 94 Dreger (2002) [86] 105 5 NI NI 4-year OS (%) CLL, chronic lymphocytic leukemia; TRM, treatment-related mortality; DFS, disease-free survival; OS, overall survival; NI, no information. Allogenic transplantation is known to achieve better results in terms of OS. The survival curves plateau off at 40% indicating a potentially curative treatment with CLL, but with a treatment-related mortality ranging from 22% to 52% (Table 7). Reduced intensity conditioning, which was introduced after it became clear that the efficacy of allogenic transplantation is partly due to graft-versus-leukemia effect, is likely to reduce the procedure-related mortality, acquiring an important role in the CLL setting, as suggested by early studies [75, 76]. This approach, together with others currently under investigation such as the use of peripheral stem cells and of non-myeloabaltive approaches (mini-bmt), will hopefully improve the outcome of transplanted patients, both in terms of responses and treatment tolerability, extending the applicability of this treatment to a wider population of CLL patients. New perspectives Ongoing clinical trials aim either at investigating the activity of new drugs or at defining the optimal timing for treatment. The MRC CCL5 trial compares immediate versus deferred autologous transplantation in patients who have achieved CR, or very good or nodular PR to first- or second-line treatment. New drugs include the cyclin-dependent kinase inhibitor flavopiridol, the interleukin-2 receptor ligand immunotoxin denileukin diftitox (Ontak) and a number of monoclonal antibodies, such as engineered anti-cd20 antibodies, anti-hla- DR antibodies, anti-cd40 antibodies, TRAIL receptor, DR4- and DR5-directed antibodies, and antibody-like molecules targeting CD37. With the exception of denileukin diftitox, which is already in a phase II trial stage, the others are still entering phase I trials. Hopefully the efforts of many investigators will finally lead to a substantial improvement in the clinical management of CLL patients. Vaccination therapy for B-CLL The generation of an anti-leukemic immune response against the malignant B-cell clone in B-CLL is an attractive approach to therapy. A large body of evidence gathered from in vitro studies and animal experiments, as well as clinical trials in a variety of other malignancies, have established that vaccination therapy in general has negligible toxicity. The indolent nature of B-CLL and the advanced age of the patients make this disease eminently suited for vaccination therapy. There exist very few studies aimed at developing vaccine-based treatment options for B-CLL. Most vaccine approaches have utilized modified leukemia cells or derivatives as Table 7. Allogenic transplantation in CLL Author No. patients TRM (%) 3-year OS (%) 4-year OS (%) 5-year OS (%) 10-year OS (%) Michallet (1996) [87] 54 (85% chemo-refractory) 48 NI NI NI 41 Gribben (1998) [72] 23 22 NI 50 NI NI Michallet (1999) [88] 134 40 54 NI NI NI Horowitz (2000) [89] 242 25 GVHD 45 NI NI NI 27 TR Dreger (2002) [86] 38 39 41 NI NI NI Khouri (2002) [90] 28 10 NI NI 45 NI CLL, chronic lymphocytic leukemia; TRM, treatment-related mortality; OS, overall survival; NI, no information; GVHD, graft-versus-host disease; TR, treatment related.

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