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Novel agents and strategies for treatment of p53-defective chronic lymphocytic leukemia
Best Practice & Research Clinical Haematology Vol. 20, No. 3, pp. 545–556, 2007 doi:10.1016/j.beha.2007.03.005 available online at http://www.sciencedirect.com
14 Novel agents and strategies for treatment of p53-defective chronic lymphocytic leukemia Michael R. Grever *
MD
Chairman & Professor of Medicine
David M. Lucas
PhD
Research Scientist
Amy J. Johnson
PhD
Research Scientist
John C. Byrd
MD
Professor of Medicine & Director of Hematologic Malignancies Division of Hematology–Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
Chronic lymphocytic leukemia (CLL) is a common leukemia with a highly variable natural history. A subset of patients with high-risk CLL rapidly progress to develop symptomatic disease requiring treatment. Over-represented in this group are those who have a deletion of 17p13.1, the chromosomal location of the tumor suppressor gene P53. Of all prognostic factors examined in CLL, del(17p13.1) has a superior predictive value for poor response to conventional therapy. In this article we review the current published data on prognostic factors relevant to treatment in CLL. We next provide therapeutic recommendations for patients with del(17p13.1) that are available to oncologists in general practice. Chemoimmunotherapy, alemtuzumab, or high-dose corticosteroids are all effective as initial therapy for these patients, but progression is generally rapid. If allogeneic immune therapy is to be considered, it should be approached as part of initial or first salvage therapy. The investigational agent flavopiridol has also demonstrated clinical activity in this subset of patients. Identification of small molecules and new treatment approaches for patients with del(17p13.1) is a major focus of several investigators. Selection of therapy based on high-risk genomic features represents an appropriate treatment approach supported by currently available published data. * Corresponding author. Tel.: þ1 614 293 8724, Fax: þ1 614 293 6656. E-mail address: [email protected] (M.R. Grever). 1521-6926/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
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Key words: p53; ATM; interphase cytogenetics; chemoimmunotherapy; alemtuzumab; corticosteroids; stem-cell transplant; CD40; flavopiridol.
B-cell chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia that occurs in adults. Previously identified as a single disease characterized by a typical immunophenotype of CD5þ, CD19þ, CD20þ, CD23þ, and dim sIg, CLL is now recognized to have a varied natural history based predominately on IgVH gene mutational status. As identified by two key reports, patients with CLL cells that use unmutated IgVH generally have a shorter time to initiation of treatment and inferior survival relative to those with CLL cells that use mutated IgVH.1,2 Genetic lesions recognized as best predictors of poor outcome in CLL – including the interphase cytogenetic abnormalities del(17p13.1) and del(11q22.3)3, complex or imbalanced translocations on stimulated karyotype examiniation4, P53 mutations5, ATM mutations6, and other p53 dysfunction (as measured by p21 induction) – occur most frequently in CLL cells that use unmutated IgVH.7 To date, prospective application of these features to predict response to therapy and estimate remission duration has been initiated in several large clinical trials. Herein we provide a review of genetic risk factors relevant to therapy, in which we identify patients with del(17p13.1) as being the group at highest risk of undergoing relapse and dying from CLL following standard therapies. We next provide treatment recommendations and review new agents emerging in preclinical investigations to treat this patient subset. P53 MUTATIONS AND DELETIONS: MOLECULAR ABERRATIONS PREDICTING RESPONSE TO THERAPY IN CLL While abnormalities of P53 occur more frequently in patients with solid tumors, the deletion or mutation of this gene in previously untreated CLL patients is reported to be 10–15%.8–13 P53 dysfunction resulting from inactivation of the ataxia telangiectasia mutated protein (ATM) accounts for an additional abnormality in 15–20% of patients.6,14 Overall, there is a defect in p53-mediated induction of p21 in approximately 20% of CLL patients.6 However, as the disease progresses following initial therapy, the frequency of abnormal p53 function increases to nearly 50% of patients.15 The evolution of increased drug resistance in CLL patients following initial therapy may be explained by the progressive selection of drug-resistant subsets of malignant cells with either deletion or mutation of relevant proteins that induce apoptosis following DNA damage. Several early studies reported that patients with mutated P53 have disease characterized by resistance to chemotherapy.8–13 El Rouby and colleagues10 found P53 gene mutations in 15% of 53 patients with previously untreated CLL. Among the 29 patients with a normal P53 gene who required alkylator drug therapy, 93% achieved a partial remission. In contrast, only one of eight (13%) with a mutated P53 gene responded (P ¼ 0.00009). Sturm and colleagues5 studied 138 patients with CLL, of whom 78% had received prior therapy with an alkylating agent. Mutations in P53 were identified in 16.7%, and survival was only 12.5 months in this group, whereas those with normal P53 had a median survival of 38.3 months (P < 0.0001). P53 mutations were observed in 20 of the 80 patients who had prior alkylator therapy, whereas just three of 58 (6%) of untreated patients had this genetic aberration. While the authors noted that treatment with alkylating agents were associated with P53 dysfunction, suggesting that DNA-damaging therapies may lead to p53 inactivation and subsequent resistance to other commonly used chemotherapies, serial samples were not analyzed to address
Novel agents and strategies for p53-defective CLL 547
this hypothesis. Neither the study by El Rouby nor that by Sturm included examination for deletion of the alternative P53 allele as part of their analysis. Do¨hner and colleagues16 reported a cohort of 100 patients with chronic B-cell leukemias in which del(17p13.1) was noted in 17 patients. Patients with this abnormality had a shorter time to development of symptomatic disease (median 59 days versus 396 days, P < 0.01). Remission was achieved in 20 of 36 patients without del(17p13.1) but, importantly, none of the 12 patients with del(17p) responded to treatment with fludarabine or pentostatin (P < 0.001). Here the impact of P53 mutation was not assessed, preventing independent assessment of the impact of del(17p) versus P53 mutation. A recent CALGB study of chemoimmunotherapy including fludarabine and rituximab reported that neither interphase cytogenetics nor P53 mutation predicted response to therapy.17 However, progression-free survival was significantly improved in patients with relatively low-risk cytogenetics (e.g. del(13q)) compared to patients with highrisk cytogenetics (e.g. del(17p) and del(11q), P ¼ 0.005). The frequency of P53 mutations that occurred independently of del(17p) were limited and prevented assessment of their independent prognostic significance. A second phase-II study of 65 patients treated with pentostatin, cyclophosphamide, and rituximab showed no responses in patients with del(17p13.1), but failed to demonstrate a difference in progression-free survival of del(11q22.3) patients relative to other interphase cytogenetic groups.18 Thus, both of these phase-II studies of chemoimmunotherapy provide support for the idea that del(17p13.1) portends poor outcome with initial treatment. Prospective studies of the US Intergroup, German CLL Study Group (GCLLSG), and UK Leukaemia Research Fund (LRF) examining the importance of del(17p13.1) and del(11q22.3) in treatment response and progression-free survival with fludarabine and/or fludarabine plus cyclophosphamide therapy have also been performed and recently were reported. Oscier and colleagues reported the UK LRF CLL4 trial evaluating the effects of chlorambucil, fludarabine, and fludarabine plus cyclophosphamide in previously untreated CLL patients.19 Regression analysis of age, stage, gender, IgVH gene mutational status, and cytogenetic factors found that stage (P ¼ 0.002), IgVH mutational status (P ¼ 0.05), 17p deletion (P ¼ 0.02), and 11q deletion (P ¼ 0.01) all significantly affected response to therapy. In a multivariate analysis, age, stage, and 17p deletion had a significant impact on survival. p53 mutational analysis was not performed. Eichhorst and the German CLL Study Group (GCLLSG) compared fludarabine to fludarabine plus cyclophosphamide in previously untreated CLL patients.20 Five of the nine patients who did not respond to the FC combination regimen were found to have a 17p deletion. A prospective cytogenetic analysis of this study demonstrated that median overall survival was significantly decreased in patients with del(17p) (P < 0.001).48 More detailed analysis of this study is under way at this time. Grever and colleagues evaluated a comprehensive set of prognostic markers in concert with E2997, a randomized phase-III trial of 278 previously untreated, symptomatic CLL patients who received either fludarabine or fludarabine plus cyclophosphamide.21 The response was not different on the basis of interphase cytogenetics or P53 mutational status. The occurrence of either a del(17p) (hazard ratio 3.428, P ¼ 0.0002) or a del(11q) (hazard ratio 1.904, P ¼ 0.006) was associated with reduced progressionfree survival. The median progression-free survival for patients with del(17p13) and del(11q22) was 10.8 months and 21.5 months, respectively. Patients with a mutation of P53 without associated del(17p) did not experience a poorer outcome. These data demonstrate that high-risk genomic features – including del(17p) and del(11q) – are
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highly predictive of shortened progression-free survival in treated CLL. This study has sufficient cases with isolated P53 mutations to assess the independent prognostic significance of this marker, and the data suggest that with chemotherapy this mutation does not predict treatment failure. Overall, these data support a risk-stratified therapy approach for the treatment of CLL that predominately includes interphase cytogenetics, but not P53 mutational analysis. In particular, all of these studies identify patients with del(17p13.1) as a very-high-risk group for which treatment must be tailored. THERAPIES FOR DEL(17P13.1) CLL Chemoimmunotherapy Several chemoimmunotherapy-based approaches have been published reporting promising response rates and extended progression-free survival. These treatments include fludarabine/rituximab (FR)22,23, pentostatin/cyclophosphamide/rituximab (PCR)18, and fludarabine/cyclophosphamide/rituximab (FCR).24 Three patients in the FR study had del(17p13.1), and all attained a partial remission that was of short duration.17 None of the 3 patients with del(17p13.1) in the PCR study responded to therapy.18 There are no data with del(17p13.1) relative to FCR response or progression-free survival. It is the authors’ experience that responses with FCR are similar to those with FR, but they too are of short duration. For a young CLL patient with del(17p13.1), treatment with FR or FCR should be considered, with further immediate consideration for alternative clinical trials. Additionally, our approach is to try to identify an allogeneic donor for such patients and to proceed to allogeneic stem-cell transplant (see below), given the short duration of response observed with this subset of patients and the lack of effective salvage therapy available to them at relapse. Alemtuzumab Alemtuzumab (Campath-1H) is a humanized anti-CD52 monoclonal antibody that effectively fixes complement and depletes normal lymphocytes and lymphoma cells.25–27 CD52 is a 21–28 kDa glycopeptide expressed on the surface of nearly all human lymphocytes, monocytes, and macrophages.28–30 The function of CD52 is uncertain. Alemtuzumab was approved for marketing in fludarabine-refractory CLL on the basis of treatment efficacy in 93 heavily pretreated, fludarabine-refractory CLL patients.31 This trial demonstrated an intent-to-treat response rate of 33%. The median time to progression for responders was 9.5 months, and the median overall survival was 16 months for all patients and 32 months for responders. Although the median peripheral-blood CLL cell count decreased by more than 99.9%, alemtuzumab was less effective against nodal disease, with only 12% of patients who had lymph nodes 5 cm responding.31 Since the approval of alemtuzumab, a randomized trial of this agent versus chlorambucil in previously untreated patients has demonstrated superior response rates in the patients receiving alemtuzumab.32 Surprisingly, morbidity due to infection was not dramatically different between the two arms of this study, with exception of asymptomatic cytomegalovirus (CMV) reactivation. This study highlights that much of the infectious morbidity observed with alemtuzumab when applied in the relapsed setting might be abrogated if it is used earlier in the course of treatment. This is particularly relevant to patients with del(17p13.1) who do not appear to respond well to standard fludarabine-based therapies or chemoimmunotherapy.
Novel agents and strategies for p53-defective CLL 549
Stilgenbauer and colleagues were the first to report a complete response in a single patient with del(17p13.1) receiving alemtuzumab.33 Following this intriguing observation, our group sought to determine whether alemtuzumab was an effective therapy in this patient population.34 Thirty-six patients with fludarabine-refractory CLL – 15 (42%) of whom had P53 mutations or deletions – were treated with alemtuzumab. Clinical responses were noted in six of 15 patients (40%) with P53 mutations and/ or deletions, versus four of 21 patients (19%) without P53 abnormalities. The median response duration for this subset of patients was 8 months (range 3–17 months). These data confirm the results of Stilgenbauer and colleagues33 and suggest that alemtuzumab may be an effective therapy for CLL patients with P53 mutations or deletions. Two other retrospective studies have confirmed these responses in high-risk patients with del(17p13.1). More importantly, two studies examining alemtuzumab as initial therapy or in the relapsed setting confirmed the equivalent efficacy of alemtuzumab in CLL patients with del(17p). Currently, significant data exist to support the use of alemtuzumab as initial therapy for this specific subset of CLL patients. Patients with del(17p13.1) who are either elderly or with disease predominately in the blood, marrow, and spleen should be considered for initial treatment with alemtuzumab. Corticosteroids In the laboratory, lymphocytes with p53 dysfunction respond to corticosteroids in a similar manner to those with normal p53 function. This observation prompted one group to examine the efficacy of high-dose solumedrol among patients with relapsed and refractory CLL.35 This report included 25 patients, 11 of whom had p53 dysfunction as assessed by mutational analysis, del(17p13.1) assessed by interphase cytogenetics, or p21 induction as measured by flow cytometry assay. The overall response rate was 77%, with a median duration of 12 months (range 7–23þ). Five of the 11 patients with p53 dysfunction responded, two of whom achieved nodular partial remission. Patients with P53 abnormalities fared worse than those without. The major toxicity observed in this study was infection in seven of the 25 patients. Because of this, it is the authors’ preference to avoid this therapy unless other alternatives are not available. Non-myeloablative stem-cell transplant Allogeneic stem-cell transplant likely represents the only curative therapy for CLL. However, this approach has previously been applicable only to patients under the age of 50, just 10% of patients diagnosed with this disease. Elimination of the aggressive preparative regimens and addition of highly immunosuppressive therapies commonly used in CLL – such as fludarabine, pentostatin, and/or alemtuzumab – has resulted in the ability to engraft allogeneic cells from a related or unrelated HLA-matched donor. This treatment approach has increased the number of CLL patients eligible for allogeneic immune therapy to patients 70 years or younger in most series, with greatly reduced regimen-related toxicities. Nonetheless, chronic graft versus host disease represents a substantial problem. Examination of outcome with this approach in several series demonstrates similar progression-free survival and appearance of a plateau suggestive of cure with 3–5 years follow-up in most series.36–39 However, despite the high frequency of patients with a del(17p) who are appropriate candidates for allogeneic transplant, virtually all of these studies lack patients with del(17p). Only one
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report suggests that this approach may be beneficial for this high-risk group.40 Reasons for this likely include the need to have disease control at the time of transplant, which is rarely achieved with this subset of patients. Additionally, patients with del(17p13.1) have relatively short remission durations, and selection biases introduced by insurance clearance and/or initial denial of such therapy, as well as the time required to identify a related or unrelated donor, make them unlikely to undergo this therapy. To test the efficacy of non-myeloablative stem-cell transplant in CLL, it will be necessary to apply this therapy earlier in the course of the disease. Such a study is currently being planned by the CLL Research Consortium and the Cancer and Leukemia Group B that will incorporate this treatment approach as part of the initial treatment for del(17p13.1) patients as a consolidation approach. Activation via CD40 ligation The CD40 antigen is expressed on normal B cells and in virtually all B-cell malignancies, including CLL. The ligand for CD40 (CD40L or CD154) is expressed on activated CD4þ T cells. The CD40/CD40L interaction stimulates the B cells, dendritic cells, and monocytes to proliferate, to differentiate, to up-regulate co-stimulatory molecules, and to increase antigen presentation. In normal and malignant B cells the activation of CD40 appears to be a major stimulus of B7-1 (CD80) and B7-2 (CD86), which are required for T-cell activation. The first clinical trial to enhance CLL immunogenicity was conducted with CLLtransfected cells with an adenovirus vector to express CD40L as a transgene (AdCD154)41 (see Chapter 15). A one-time injection of Ad-CD154-transduced CLL cells induced up-regulation of immune accessory molecules on bystander tumor cells, high blood levels of interleukin 12 and interferon g, and increased blood T-cell numbers. Clinically, reduction in leukemia cell counts and lymph-node size was demonstrated without signs of autoimmunity. A late observation from this study is that the CD40 activation of CLL cells makes them sensitive to agents that activate the extrinsic pathway of apoptosis. This is believed to occur via the p73 pathway with subsequent up-regulation of BID, DR4, DR5, and CD95.42 Death through this pathway can occur irrespective of P53 status, making this a potentially relevant therapy for patients with p53-dysfunctional disease. Discovery of small molecules with p53-independent activity in CLL Flavopiridol Flavopiridol, introduced as a cyclin-dependent kinase inhibitor, is quite effective in vitro in inducing apoptosis in leukemic cells from patients with CLL.43,44 The concentrations required to achieve this effect are in the 1.5 mM range. These encouraging early data showed that flavopiridol-mediated apoptosis was achieved in CLL patient cells irrespective of previous chemotherapy and of P53 status. Essentially identical conditions for in-vitro exposure also suggested that this agent could be useful for patients with drug-resistant CLL. Because of the assumption that flavopiridol mediates cell death via inhibition of cyclin-dependent kinase, in the initial clinical studies it was administered using prolonged intravenous infusions of over 24–72 hours.45,46 Unfortunately, very little clinical benefit was observed in these initial trials. In contrast, a shorter infusion of flavopiridol over 1 hour in a CALGB phase-II trial produced a response rate of 11%.46 The impact of the
Novel agents and strategies for p53-defective CLL 551
drug administration schedule of flavopiridol was investigated when a major difference in protein binding between human and non-human plasma became apparent.47 Experiments showed that substitution of human serum for fetal bovine serum (FBS) typically used in laboratory studies resulted in a substantial decrease in free drug levels, from 63–100% to 5–8%. In reviewing the extensive pharmacokinetics data from previous phase-I/II trials that had demonstrated little benefit, it became apparent that the desired plasma concentrations had not been achieved in patients with the prolonged administration schedule. With this knowledge, the pharmacologists were able to model a loading dose (30 minutes) followed by a 4-hour infusion schedule that would more aptly achieve the desired plasma concentrations in patients. The revised phase-I study in patients with advanced CLL included a loading dose of 30 mg/m2 over 30 minutes, followed by a 4-hour intravenous infusion of 30 mg/m2 each week for 4 weeks. This was followed by a 2-week rest.47 Progressive flavopiridol dose escalation was initially hampered by excessive and aggressive hyper-acute tumor lysis syndrome, a complication of highly effective therapy unusual in CLL. Prophylactic hydration, management of hyperkalemia, and liberal use of rasburicase along with careful monitoring were required during the administration of this agent. Completion of the phase-I trial in advanced CLL resulted in a remarkable overall response rate of 42%, with many of these responses being durable (median duration of response in excess of 12 months). Furthermore, the responses were observed in patients with genetically high-risk disease, including five of 12 (42%) with del(17p13.1) and 13 of 18 patients (72%) with del(11q22.3). In reviewing the completed phase-I study47, several important aspects merit attention. Patients with a pre-treatment leukocyte count >200 109/L developed the most dangerous form of hyper-acute tumor lysis. Five of eight of these patients required hemodialysis with the first dose of the drug, as compared to just one of 34 (3%) patients with a pre-treatment leukocyte count <200 109/L. In contrast, massive lymphadenopathy (>5 cm) did not necessarily predict aggressive tumor lysis syndrome. While all patients must receive careful monitoring during flavopiridol treatment, the frequency of dangerous tumor lysis syndrome appears to be related to a very high circulating leukocyte count. Concurrent pharmacokinetic studies indicated that some patients require an increase in the dose of flavopiridol during the 4-hour infusion to achieve and maintain the targeted plasma concentration of 1.5 mM. Consequently, the current phase-II trial in CLL is being executed with the loading dose of 30 mg/m2, followed by a 4-hour intravenous infusion of 30 mg/m2 for the initial dose and 50 mg/m2 for subsequent 4-hour infusions in those patients who do not have excessive tumor lysis and no response at the 30 mg/m2 level. These modifications, coupled with continued close monitoring, have helped mitigate the problems associated with the administration of this new agent. Importantly, no serious opportunistic infections have been observed to date following treatment with in this heavily-pretreated population of patients with advanced CLL. These encouraging clinical findings have been translated into plans for examining the role of flavopiridol in treating patients earlier for minimal residual disease. If the observation of efficacy despite high-risk cytogenetics – e.g. del(17p13.1) and del(11q22.3) – can be confirmed, this agent may be moved forward to treating patients with residual disease following initial cytoreductive induction therapy. This maneuver would potentially increase the numbers of patients achieving a complete remission, and hopefully result in durable and long-lasting responses. Additional studies are ongoing with flavopiridol that incorporate the use of this agent with fludarabine and rituximab as part of the ‘up-front simultaneous’ therapy.48
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The FFR regimen has reportedly achieved a 75% complete response rate in a group of patients with low-grade B-cell malignancy, including CLL. While there will be several methods for incorporating this novel agent, the optimal model based on pharmacokinetics and clinical results will be assessed after the current phase-II trials with the loading dose and 4-hour continuous infusion study have been concluded. Flavopiridol had initially been thought to be most active in rapidly proliferating malignancies. Therefore, the observations that this agent has dramatic effects in patients with neoplastic cells that are not rapidly dividing (mostly in G0) raises interesting questions about its actual mechanism of action. Is this agent really inducing tumor cell death through apoptosis or necrosis? Studies in mitochondrial function have revealed that flavopiridol induces mitochondrial membrane depolarization early during in vitro culture, in the same time frame as clinical effects begin to be observed (i.e. 6–8 hours after exposure to the agent). If the induction of rapid cell death is truly mediated by disruption of mitochondrial function and release of cytochrome C, this could explain the lack of dependence upon an intact P53 system. Other novel agents Several unique molecules have the potential to induce apoptosis in CLL cells independently of p53-mediated pathways. Another novel purported cyclin-dependent kinase inhibitor, R-roscovitine (CYC202), is undergoing evaluation for clinical efficacy.49 This agent shows selectivity for malignant B cells compared to normal blood mononuclear cells, and can impact leukemic cells regardless of the presence of ATM or P53 mutations.49 Thus, the induction of apoptosis mediated by roscovitine appears to be p53-independent. Additional agents in preclinical development include a wide variety with diverse mechanisms of inducing apoptosis in CLL cells. For example, Brefeldin A is a unique molecule that targets the endoplasmic reticulum and protein transport pathways. This agent can have direct effects on the Golgi apparatus, and has been reported to overcome fludarabine resistance in malignant B cells.50 Jasmonates are another group of novel anti-cancer agents that act directly and selectively on human cancer-cell mitochondria.51 Agents that can induce apoptosis by perturbing mitochondrial integrity may be powerful new therapeutic agents in otherwise drug-resistant cells (Table 1). A final example of a novel natural product with unique properties as a potential agent in the treatment of CLL is silvestrol.52,53 This plant-derived molecule isolated from Aglaia foveolata, is currently undergoing preclinical studies as a broad-spectrum Table 1. Novel therapeutic agents which work independently of p53. In clinical use or under clinical investigation: Alemtuzumab (Campath) Flavopiridol (Alvocidib) R-Roscovitine (CYC202) Under preclinical exploration for CLL: representative agents: Acadesinea Silvestrol Brefeldin-A a
Acadesine has apparently been investigated for cardiovascular research. These data may be useful in exploring this agent in patients with CLL.
Novel agents and strategies for p53-defective CLL 553
anti-cancer agent which shows cytotoxicity in the low nanomolar range. Silvestrol has been reported to regulate the G2/M checkpoint gates in LNCaP tumor cells, inducing genes that regulate apoptosis independently of P53. Encouraging preliminary studies have also shown in vivo inhibition of tumor cell growth in small animal models. In studying the in vitro effect of this agent on leukemic cells from CLL patients and lymphocytes from healthy volunteers, it appears that the sensitivity of CLL B cells to silvestrol is far greater than that of normal T cells, potentially a significant advantage in developing new therapies for this disease. Also, silvestrol-mediated apoptosis appears to be P53-independent, in that no differences in cytotoxicity are noted between samples from CLL patients with and without the del(17p) abnormality.54 This important quality may thus confer a use for silvestrol in the future for circumventing mechanisms of resistance in patients with CLL and other B-cell malignancies.
Research agenda continued development of mechanistically novel therapeutic agents. The search for agents that circumvent P53-mediated apoptosis associated with DNA damage will potentially enable killing of drug-resistant cells that emanate from exposure to current chemotherapy prioritization of agents that demonstrate B-cell selectivity. This will be essential in limiting damage to residual normal immune effector cells. Because many of our existing therapeutic agents cause direct damage to T cells, strategies are needed to add effective agents that will not further compromise the ability of patients to withstand the dangers of fatal infection continued optimization of strategic combinations of novel agents in select patient populations. Significant efforts are being made to identify patients who will not respond optimally to conventional treatments prior to therapy. The earlier use of novel agents and combinations in these high-risk patients may produce improved responses while avoiding unnecessary toxicities accompanying suboptimal therapies
ACKNOWLEDGEMENTS This work was supported by the CLL Research Consortium (P01 CA81534-02), The Leukemia and Lymphoma Society of America, and The D. Warren Brown Foundation. REFERENCES 1. Hamblin TJ, Davis Z, Gardiner A et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–1854. 2. Damle RN, Wasil T, Fais F et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94: 1840–1847. *3. Dohner H, Stilgenbauer S, Benner A et al. Genomic aberrations and survival in chronic lymphocytic leukemia. The New England Journal of Medicine 2000; 343: 1910–1916. 4. Mayr C, Speicher MR, Kofler DM et al. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood 2006; 107: 742–751.
554 M. R. Grever et al 5. Sturm I, Bosanquet AG, Hermann S et al. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death and Differentiation 2003; 10: 477–484. 6. Austen B, Powell JE, Alvi A et al. Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. Blood 2005; 106: 3175– 3182. 7. Krober A, Seiler T, Benner A et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002; 100: 1410–1416. 8. Fenaux P, Jonveaux P, Quiquandon I et al. Mutations of the p53 gene in B-cell lymphoblastic acute leukemia: a report on 60 cases. Leukemia 1992; 6: 42–46. 9. Fenaux P, Preudhomme C, Lai JL et al. Mutations of the p53 gene in B-cell chronic lymphocytic leukemia: a report on 39 cases with cytogenetic analysis. Leukemia 1992; 6: 246–250. *10. el Rouby S, Thomas A, Costin D et al. p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood 1993; 82: 3452–3459. *11. Wattel E, Preudhomme C, Hecquet B et al. p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood 1994; 84: 3148–3157. *12. Cordone I, Masi S, Mauro FR et al. p53 expression in B-cell chronic lymphocytic leukemia: a marker of disease progression and poor prognosis. Blood 1998; 91: 4342–4349. 13. Barnabas N, Shurafa M, Van Dyke DL et al. Significance of p53 mutations in patients with chronic lymphocytic leukemia: a sequential study of 30 patients. Cancer 2001; 91: 285–293. 14. Stankovic T, Stewart GS, Fegan C et al. Ataxia telangiectasia mutated-deficient B-cell chronic lymphocytic leukemia occurs in pregerminal center cells and results in defective damage response and unrepaired chromosome damage. Blood 2002; 99: 300–309. 15. Lozanski G, Heerema NA, Flinn IW et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood 2004; 103: 3278–3281. *16. Dohner H, Fischer K, Bentz M et al. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. Blood 1995; 85: 1580–1589. *17. Byrd JC, Gribben JG, Peterson BL et al. Select high-risk genetic features predict earlier progression following chemoimmunotherapy with fludarabine and rituximab in chronic lymphocytic leukemia: justification for risk-adapted therapy. Journal of Clinical Oncology 2006; 24: 437–443. 18. Kay NE, Geyer SM, Call TG et al. Combination chemoimmunotherapy with pentostatin, cyclophosphamide and rituximab shows significant clinical activity with low accompanying toxicity in previously untreated B-chronic lymphocytic leukemia. Blood 2007; 109: 405–411. 19. Oscier DG, Richards S, Orchard J et al. Prognostic factors in the UK LRF CLL4 trial. Blood 2005; 106. Abstract 2099. 20. Eichhorst BF, Busch R, Hopfinger G et al. Fludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy of younger patients with chronic lymphocytic leukemia. Blood 2006; 107: 885–891. *21. Grever MR, Lucas DM, Dewald GW et al. Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from a prospective randomized US Intergroup Phase III Trial (E2997). Journal of Clinical Oncology 2007; 25: 799–804. 22. Byrd JC, Peterson BL, Morrison VA et al. Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: results from Cancer and Leukemia Group B 9712 (CALGB 9712). Blood 2003; 101: 6–14. 23. Byrd JC, Rai K, Peterson BL et al. Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood 2005; 105: 49–53. 24. Keating MJ, O’Brien S, Albitar M et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. Journal of Clinical Oncology 2005; 23: 4079–4088. 25. Flynn JM & Byrd JC. Campath-1H monoclonal antibody therapy. Current Opinion in Oncology 2000; 12: 574–581. 26. Hale G, Bright S, Chumbley G et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983; 62: 873–882.
Novel agents and strategies for p53-defective CLL 555 27. Hale G, Dyer MJ, Clark MR et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet 1988; 2: 1394–1399. 28. Treumann A, Lifely MR, Schneider P & Ferguson MA. Primary structure of CD52. The Journal of Biological Chemistry 1995; 270: 6088–6099. 29. Domagala A & Kurpisz M. CD52 antigen: a review. Medical Science Monitor 2001; 7: 325–331. 30. Rowan W, Tite J, Topley P & Brett SJ. Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells. Immunology 1998; 95: 427–436. 31. Keating MJ, Flinn I, Jain V et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 2002; 99: 3554–3561. 32. Hillmen P, Skotnicki A, Robak T et al. Preliminary phase III efficacy and safety of alemtuzumab vs chlorambucil as front-line therapy for patients with progressive B-cell chronic lymphocytic leukemia (BCLL). Journal of Clinical Oncology 2006; 24: 18S. [abstract#6511]. *33. Stilgenbauer S & Dohner H. Campath-1H-induced complete remission of chronic lymphocytic leukemia despite p53 gene mutation and resistance to chemotherapy. The New England Journal of Medicine 2002; 347: 452–453. 34. Lozanski G, Heerema NA, Flinn IW et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood 2004; 103: 3278–3281. 35. Thornton PD, Matutes E, Bosanquet AG et al. High dose methylprednisolone can induce remissions in CLL patients with p53 abnormalities. Annals of Hematology 2003; 82: 759–765. 36. Brown JR, Kim HT, Li S et al. Predictors of improved progression-free survival after nonmyeloablative allogeneic stem cell transplantation for advanced chronic lymphocytic leukemia. Biology of Blood and Marrow Transplantation 2006; 12: 1056–1064. 37. Sorror ML, Maris MB, Sandmaier BM et al. Hematopoietic cell transplantation after nonmyeloablative conditioning for advanced chronic lymphocytic leukemia. Journal of Clinical Oncology 2005; 23: 3819–3829. 38. Maloney DG, Sandmaier BM, Mackinnon S & Shizuru JA. Non-myeloablative transplantation. Hematology 2002. American society of hematology education program book; 2002. p. 392–421. 39. Delgado J, Thomson K, Russell N et al. Results of alemtuzumab-based reduced-intensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study. Blood 2006; 107: 1724–1730. 40. Caballero D, Garcia-Marco JA, Martino R et al. Allogeneic transplant with reduced intensity conditioning regimens may overcome the poor prognosis of B-cell chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene and chromosomal abnormalities (11q- and 17p-). Clinical Cancer Research 2005; 11: 7757–7763. 41. Wierda WG, Cantwell MJ, Woods SJ et al. CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia. Blood 2000; 96: 2917–2924. 42. Dicker F, Kater AP, Prada CE et al. CD154 induces p73 to overcome the resistance to apoptosis of chronic lymphocytic leukemia cells lacking functional p53. Blood 2006; 108: 3450–3457. *43. Byrd JC, Shinn C, Waselenko JK et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Blood 1998; 92: 3804–3816. 44. Kitada S, Zapata JM, Andreeff M & Reed JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000; 96: 393–397. 45. Flinn IW, Byrd JC, Bartlett N et al. Flavopiridol administered as a 24-hour continuous infusion in chronic lymphocytic leukemia lacks clinical activity. Leukemia Research 2005; 29: 1253–1257. 46. Byrd JC, Peterson BL, Gabrilove J et al. Treatment of relapsed chronic lymphocytic leukemia by 72-hour continuous infusion or 1-hour bolus infusion of flavopiridol: results from Cancer and Leukemia Group B study 19805. Clinical Cancer Research 2005; 11: 4176–4181. *47. Byrd JC, Lin TS, Dalton JT et al. Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia. Blood 2007; 109: 399–404. 48. Lin TS, Fischer B, Moran ME et al. Flavopiridol, fludarabine and rituximab is a highly active regimen in indolent B-cell lymphoproliferative disorders including mantle cell lymphoma. Blood 2005; 106. abstr 944.
556 M. R. Grever et al 49. Alvi AJ, Austen B, Weston VJ et al. A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down-regulation of genes involved in transcription regulation and survival. Blood 2005; 105: 4484–4491. 50. Carew JS, Nawrocki ST, Krupnik YV et al. Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL. Blood 2006; 107: 222–231. 51. Flescher E. Jasmonates—a new family of anti-cancer agents. Anticancer Drugs 2005; 16: 911–916. 52. Hwang BY, Su BN, Chai H et al. Silvestrol and episilvestrol, potential anticancer rocaglate derivatives from Aglaia silvestris. The Journal of Organic Chemistry 2004; 69: 3350–3358. 53. Mi Q, Kim S, Hwang BY et al. Silvestrol regulates G2/M checkpoint genes independent of p53 activity. Anticancer Research 2006; 26: 3349–3356. 54. Edwards RB, Lucas DM, Lozanski G et al. Silvestrol, a rocaglate derivative from the Indonesian plant aglaia foveolata, has significant Bcl-2- and p53-independent anti-tumor activity against chronic lymphocytic leukemia cells. Blood 2006; 108 [abstract 2600].
14 Novel agents and strategies for treatment of p53-defective chronic lymphocytic leukemia Michael R. Grever *
MD
Chairman & Professor of Medicine
David M. Lucas
PhD
Research Scientist
Amy J. Johnson
PhD
Research Scientist
John C. Byrd
MD
Professor of Medicine & Director of Hematologic Malignancies Division of Hematology–Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
Chronic lymphocytic leukemia (CLL) is a common leukemia with a highly variable natural history. A subset of patients with high-risk CLL rapidly progress to develop symptomatic disease requiring treatment. Over-represented in this group are those who have a deletion of 17p13.1, the chromosomal location of the tumor suppressor gene P53. Of all prognostic factors examined in CLL, del(17p13.1) has a superior predictive value for poor response to conventional therapy. In this article we review the current published data on prognostic factors relevant to treatment in CLL. We next provide therapeutic recommendations for patients with del(17p13.1) that are available to oncologists in general practice. Chemoimmunotherapy, alemtuzumab, or high-dose corticosteroids are all effective as initial therapy for these patients, but progression is generally rapid. If allogeneic immune therapy is to be considered, it should be approached as part of initial or first salvage therapy. The investigational agent flavopiridol has also demonstrated clinical activity in this subset of patients. Identification of small molecules and new treatment approaches for patients with del(17p13.1) is a major focus of several investigators. Selection of therapy based on high-risk genomic features represents an appropriate treatment approach supported by currently available published data. * Corresponding author. Tel.: þ1 614 293 8724, Fax: þ1 614 293 6656. E-mail address: [email protected] (M.R. Grever). 1521-6926/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
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Key words: p53; ATM; interphase cytogenetics; chemoimmunotherapy; alemtuzumab; corticosteroids; stem-cell transplant; CD40; flavopiridol.
B-cell chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia that occurs in adults. Previously identified as a single disease characterized by a typical immunophenotype of CD5þ, CD19þ, CD20þ, CD23þ, and dim sIg, CLL is now recognized to have a varied natural history based predominately on IgVH gene mutational status. As identified by two key reports, patients with CLL cells that use unmutated IgVH generally have a shorter time to initiation of treatment and inferior survival relative to those with CLL cells that use mutated IgVH.1,2 Genetic lesions recognized as best predictors of poor outcome in CLL – including the interphase cytogenetic abnormalities del(17p13.1) and del(11q22.3)3, complex or imbalanced translocations on stimulated karyotype examiniation4, P53 mutations5, ATM mutations6, and other p53 dysfunction (as measured by p21 induction) – occur most frequently in CLL cells that use unmutated IgVH.7 To date, prospective application of these features to predict response to therapy and estimate remission duration has been initiated in several large clinical trials. Herein we provide a review of genetic risk factors relevant to therapy, in which we identify patients with del(17p13.1) as being the group at highest risk of undergoing relapse and dying from CLL following standard therapies. We next provide treatment recommendations and review new agents emerging in preclinical investigations to treat this patient subset. P53 MUTATIONS AND DELETIONS: MOLECULAR ABERRATIONS PREDICTING RESPONSE TO THERAPY IN CLL While abnormalities of P53 occur more frequently in patients with solid tumors, the deletion or mutation of this gene in previously untreated CLL patients is reported to be 10–15%.8–13 P53 dysfunction resulting from inactivation of the ataxia telangiectasia mutated protein (ATM) accounts for an additional abnormality in 15–20% of patients.6,14 Overall, there is a defect in p53-mediated induction of p21 in approximately 20% of CLL patients.6 However, as the disease progresses following initial therapy, the frequency of abnormal p53 function increases to nearly 50% of patients.15 The evolution of increased drug resistance in CLL patients following initial therapy may be explained by the progressive selection of drug-resistant subsets of malignant cells with either deletion or mutation of relevant proteins that induce apoptosis following DNA damage. Several early studies reported that patients with mutated P53 have disease characterized by resistance to chemotherapy.8–13 El Rouby and colleagues10 found P53 gene mutations in 15% of 53 patients with previously untreated CLL. Among the 29 patients with a normal P53 gene who required alkylator drug therapy, 93% achieved a partial remission. In contrast, only one of eight (13%) with a mutated P53 gene responded (P ¼ 0.00009). Sturm and colleagues5 studied 138 patients with CLL, of whom 78% had received prior therapy with an alkylating agent. Mutations in P53 were identified in 16.7%, and survival was only 12.5 months in this group, whereas those with normal P53 had a median survival of 38.3 months (P < 0.0001). P53 mutations were observed in 20 of the 80 patients who had prior alkylator therapy, whereas just three of 58 (6%) of untreated patients had this genetic aberration. While the authors noted that treatment with alkylating agents were associated with P53 dysfunction, suggesting that DNA-damaging therapies may lead to p53 inactivation and subsequent resistance to other commonly used chemotherapies, serial samples were not analyzed to address
Novel agents and strategies for p53-defective CLL 547
this hypothesis. Neither the study by El Rouby nor that by Sturm included examination for deletion of the alternative P53 allele as part of their analysis. Do¨hner and colleagues16 reported a cohort of 100 patients with chronic B-cell leukemias in which del(17p13.1) was noted in 17 patients. Patients with this abnormality had a shorter time to development of symptomatic disease (median 59 days versus 396 days, P < 0.01). Remission was achieved in 20 of 36 patients without del(17p13.1) but, importantly, none of the 12 patients with del(17p) responded to treatment with fludarabine or pentostatin (P < 0.001). Here the impact of P53 mutation was not assessed, preventing independent assessment of the impact of del(17p) versus P53 mutation. A recent CALGB study of chemoimmunotherapy including fludarabine and rituximab reported that neither interphase cytogenetics nor P53 mutation predicted response to therapy.17 However, progression-free survival was significantly improved in patients with relatively low-risk cytogenetics (e.g. del(13q)) compared to patients with highrisk cytogenetics (e.g. del(17p) and del(11q), P ¼ 0.005). The frequency of P53 mutations that occurred independently of del(17p) were limited and prevented assessment of their independent prognostic significance. A second phase-II study of 65 patients treated with pentostatin, cyclophosphamide, and rituximab showed no responses in patients with del(17p13.1), but failed to demonstrate a difference in progression-free survival of del(11q22.3) patients relative to other interphase cytogenetic groups.18 Thus, both of these phase-II studies of chemoimmunotherapy provide support for the idea that del(17p13.1) portends poor outcome with initial treatment. Prospective studies of the US Intergroup, German CLL Study Group (GCLLSG), and UK Leukaemia Research Fund (LRF) examining the importance of del(17p13.1) and del(11q22.3) in treatment response and progression-free survival with fludarabine and/or fludarabine plus cyclophosphamide therapy have also been performed and recently were reported. Oscier and colleagues reported the UK LRF CLL4 trial evaluating the effects of chlorambucil, fludarabine, and fludarabine plus cyclophosphamide in previously untreated CLL patients.19 Regression analysis of age, stage, gender, IgVH gene mutational status, and cytogenetic factors found that stage (P ¼ 0.002), IgVH mutational status (P ¼ 0.05), 17p deletion (P ¼ 0.02), and 11q deletion (P ¼ 0.01) all significantly affected response to therapy. In a multivariate analysis, age, stage, and 17p deletion had a significant impact on survival. p53 mutational analysis was not performed. Eichhorst and the German CLL Study Group (GCLLSG) compared fludarabine to fludarabine plus cyclophosphamide in previously untreated CLL patients.20 Five of the nine patients who did not respond to the FC combination regimen were found to have a 17p deletion. A prospective cytogenetic analysis of this study demonstrated that median overall survival was significantly decreased in patients with del(17p) (P < 0.001).48 More detailed analysis of this study is under way at this time. Grever and colleagues evaluated a comprehensive set of prognostic markers in concert with E2997, a randomized phase-III trial of 278 previously untreated, symptomatic CLL patients who received either fludarabine or fludarabine plus cyclophosphamide.21 The response was not different on the basis of interphase cytogenetics or P53 mutational status. The occurrence of either a del(17p) (hazard ratio 3.428, P ¼ 0.0002) or a del(11q) (hazard ratio 1.904, P ¼ 0.006) was associated with reduced progressionfree survival. The median progression-free survival for patients with del(17p13) and del(11q22) was 10.8 months and 21.5 months, respectively. Patients with a mutation of P53 without associated del(17p) did not experience a poorer outcome. These data demonstrate that high-risk genomic features – including del(17p) and del(11q) – are
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highly predictive of shortened progression-free survival in treated CLL. This study has sufficient cases with isolated P53 mutations to assess the independent prognostic significance of this marker, and the data suggest that with chemotherapy this mutation does not predict treatment failure. Overall, these data support a risk-stratified therapy approach for the treatment of CLL that predominately includes interphase cytogenetics, but not P53 mutational analysis. In particular, all of these studies identify patients with del(17p13.1) as a very-high-risk group for which treatment must be tailored. THERAPIES FOR DEL(17P13.1) CLL Chemoimmunotherapy Several chemoimmunotherapy-based approaches have been published reporting promising response rates and extended progression-free survival. These treatments include fludarabine/rituximab (FR)22,23, pentostatin/cyclophosphamide/rituximab (PCR)18, and fludarabine/cyclophosphamide/rituximab (FCR).24 Three patients in the FR study had del(17p13.1), and all attained a partial remission that was of short duration.17 None of the 3 patients with del(17p13.1) in the PCR study responded to therapy.18 There are no data with del(17p13.1) relative to FCR response or progression-free survival. It is the authors’ experience that responses with FCR are similar to those with FR, but they too are of short duration. For a young CLL patient with del(17p13.1), treatment with FR or FCR should be considered, with further immediate consideration for alternative clinical trials. Additionally, our approach is to try to identify an allogeneic donor for such patients and to proceed to allogeneic stem-cell transplant (see below), given the short duration of response observed with this subset of patients and the lack of effective salvage therapy available to them at relapse. Alemtuzumab Alemtuzumab (Campath-1H) is a humanized anti-CD52 monoclonal antibody that effectively fixes complement and depletes normal lymphocytes and lymphoma cells.25–27 CD52 is a 21–28 kDa glycopeptide expressed on the surface of nearly all human lymphocytes, monocytes, and macrophages.28–30 The function of CD52 is uncertain. Alemtuzumab was approved for marketing in fludarabine-refractory CLL on the basis of treatment efficacy in 93 heavily pretreated, fludarabine-refractory CLL patients.31 This trial demonstrated an intent-to-treat response rate of 33%. The median time to progression for responders was 9.5 months, and the median overall survival was 16 months for all patients and 32 months for responders. Although the median peripheral-blood CLL cell count decreased by more than 99.9%, alemtuzumab was less effective against nodal disease, with only 12% of patients who had lymph nodes 5 cm responding.31 Since the approval of alemtuzumab, a randomized trial of this agent versus chlorambucil in previously untreated patients has demonstrated superior response rates in the patients receiving alemtuzumab.32 Surprisingly, morbidity due to infection was not dramatically different between the two arms of this study, with exception of asymptomatic cytomegalovirus (CMV) reactivation. This study highlights that much of the infectious morbidity observed with alemtuzumab when applied in the relapsed setting might be abrogated if it is used earlier in the course of treatment. This is particularly relevant to patients with del(17p13.1) who do not appear to respond well to standard fludarabine-based therapies or chemoimmunotherapy.
Novel agents and strategies for p53-defective CLL 549
Stilgenbauer and colleagues were the first to report a complete response in a single patient with del(17p13.1) receiving alemtuzumab.33 Following this intriguing observation, our group sought to determine whether alemtuzumab was an effective therapy in this patient population.34 Thirty-six patients with fludarabine-refractory CLL – 15 (42%) of whom had P53 mutations or deletions – were treated with alemtuzumab. Clinical responses were noted in six of 15 patients (40%) with P53 mutations and/ or deletions, versus four of 21 patients (19%) without P53 abnormalities. The median response duration for this subset of patients was 8 months (range 3–17 months). These data confirm the results of Stilgenbauer and colleagues33 and suggest that alemtuzumab may be an effective therapy for CLL patients with P53 mutations or deletions. Two other retrospective studies have confirmed these responses in high-risk patients with del(17p13.1). More importantly, two studies examining alemtuzumab as initial therapy or in the relapsed setting confirmed the equivalent efficacy of alemtuzumab in CLL patients with del(17p). Currently, significant data exist to support the use of alemtuzumab as initial therapy for this specific subset of CLL patients. Patients with del(17p13.1) who are either elderly or with disease predominately in the blood, marrow, and spleen should be considered for initial treatment with alemtuzumab. Corticosteroids In the laboratory, lymphocytes with p53 dysfunction respond to corticosteroids in a similar manner to those with normal p53 function. This observation prompted one group to examine the efficacy of high-dose solumedrol among patients with relapsed and refractory CLL.35 This report included 25 patients, 11 of whom had p53 dysfunction as assessed by mutational analysis, del(17p13.1) assessed by interphase cytogenetics, or p21 induction as measured by flow cytometry assay. The overall response rate was 77%, with a median duration of 12 months (range 7–23þ). Five of the 11 patients with p53 dysfunction responded, two of whom achieved nodular partial remission. Patients with P53 abnormalities fared worse than those without. The major toxicity observed in this study was infection in seven of the 25 patients. Because of this, it is the authors’ preference to avoid this therapy unless other alternatives are not available. Non-myeloablative stem-cell transplant Allogeneic stem-cell transplant likely represents the only curative therapy for CLL. However, this approach has previously been applicable only to patients under the age of 50, just 10% of patients diagnosed with this disease. Elimination of the aggressive preparative regimens and addition of highly immunosuppressive therapies commonly used in CLL – such as fludarabine, pentostatin, and/or alemtuzumab – has resulted in the ability to engraft allogeneic cells from a related or unrelated HLA-matched donor. This treatment approach has increased the number of CLL patients eligible for allogeneic immune therapy to patients 70 years or younger in most series, with greatly reduced regimen-related toxicities. Nonetheless, chronic graft versus host disease represents a substantial problem. Examination of outcome with this approach in several series demonstrates similar progression-free survival and appearance of a plateau suggestive of cure with 3–5 years follow-up in most series.36–39 However, despite the high frequency of patients with a del(17p) who are appropriate candidates for allogeneic transplant, virtually all of these studies lack patients with del(17p). Only one
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report suggests that this approach may be beneficial for this high-risk group.40 Reasons for this likely include the need to have disease control at the time of transplant, which is rarely achieved with this subset of patients. Additionally, patients with del(17p13.1) have relatively short remission durations, and selection biases introduced by insurance clearance and/or initial denial of such therapy, as well as the time required to identify a related or unrelated donor, make them unlikely to undergo this therapy. To test the efficacy of non-myeloablative stem-cell transplant in CLL, it will be necessary to apply this therapy earlier in the course of the disease. Such a study is currently being planned by the CLL Research Consortium and the Cancer and Leukemia Group B that will incorporate this treatment approach as part of the initial treatment for del(17p13.1) patients as a consolidation approach. Activation via CD40 ligation The CD40 antigen is expressed on normal B cells and in virtually all B-cell malignancies, including CLL. The ligand for CD40 (CD40L or CD154) is expressed on activated CD4þ T cells. The CD40/CD40L interaction stimulates the B cells, dendritic cells, and monocytes to proliferate, to differentiate, to up-regulate co-stimulatory molecules, and to increase antigen presentation. In normal and malignant B cells the activation of CD40 appears to be a major stimulus of B7-1 (CD80) and B7-2 (CD86), which are required for T-cell activation. The first clinical trial to enhance CLL immunogenicity was conducted with CLLtransfected cells with an adenovirus vector to express CD40L as a transgene (AdCD154)41 (see Chapter 15). A one-time injection of Ad-CD154-transduced CLL cells induced up-regulation of immune accessory molecules on bystander tumor cells, high blood levels of interleukin 12 and interferon g, and increased blood T-cell numbers. Clinically, reduction in leukemia cell counts and lymph-node size was demonstrated without signs of autoimmunity. A late observation from this study is that the CD40 activation of CLL cells makes them sensitive to agents that activate the extrinsic pathway of apoptosis. This is believed to occur via the p73 pathway with subsequent up-regulation of BID, DR4, DR5, and CD95.42 Death through this pathway can occur irrespective of P53 status, making this a potentially relevant therapy for patients with p53-dysfunctional disease. Discovery of small molecules with p53-independent activity in CLL Flavopiridol Flavopiridol, introduced as a cyclin-dependent kinase inhibitor, is quite effective in vitro in inducing apoptosis in leukemic cells from patients with CLL.43,44 The concentrations required to achieve this effect are in the 1.5 mM range. These encouraging early data showed that flavopiridol-mediated apoptosis was achieved in CLL patient cells irrespective of previous chemotherapy and of P53 status. Essentially identical conditions for in-vitro exposure also suggested that this agent could be useful for patients with drug-resistant CLL. Because of the assumption that flavopiridol mediates cell death via inhibition of cyclin-dependent kinase, in the initial clinical studies it was administered using prolonged intravenous infusions of over 24–72 hours.45,46 Unfortunately, very little clinical benefit was observed in these initial trials. In contrast, a shorter infusion of flavopiridol over 1 hour in a CALGB phase-II trial produced a response rate of 11%.46 The impact of the
Novel agents and strategies for p53-defective CLL 551
drug administration schedule of flavopiridol was investigated when a major difference in protein binding between human and non-human plasma became apparent.47 Experiments showed that substitution of human serum for fetal bovine serum (FBS) typically used in laboratory studies resulted in a substantial decrease in free drug levels, from 63–100% to 5–8%. In reviewing the extensive pharmacokinetics data from previous phase-I/II trials that had demonstrated little benefit, it became apparent that the desired plasma concentrations had not been achieved in patients with the prolonged administration schedule. With this knowledge, the pharmacologists were able to model a loading dose (30 minutes) followed by a 4-hour infusion schedule that would more aptly achieve the desired plasma concentrations in patients. The revised phase-I study in patients with advanced CLL included a loading dose of 30 mg/m2 over 30 minutes, followed by a 4-hour intravenous infusion of 30 mg/m2 each week for 4 weeks. This was followed by a 2-week rest.47 Progressive flavopiridol dose escalation was initially hampered by excessive and aggressive hyper-acute tumor lysis syndrome, a complication of highly effective therapy unusual in CLL. Prophylactic hydration, management of hyperkalemia, and liberal use of rasburicase along with careful monitoring were required during the administration of this agent. Completion of the phase-I trial in advanced CLL resulted in a remarkable overall response rate of 42%, with many of these responses being durable (median duration of response in excess of 12 months). Furthermore, the responses were observed in patients with genetically high-risk disease, including five of 12 (42%) with del(17p13.1) and 13 of 18 patients (72%) with del(11q22.3). In reviewing the completed phase-I study47, several important aspects merit attention. Patients with a pre-treatment leukocyte count >200 109/L developed the most dangerous form of hyper-acute tumor lysis. Five of eight of these patients required hemodialysis with the first dose of the drug, as compared to just one of 34 (3%) patients with a pre-treatment leukocyte count <200 109/L. In contrast, massive lymphadenopathy (>5 cm) did not necessarily predict aggressive tumor lysis syndrome. While all patients must receive careful monitoring during flavopiridol treatment, the frequency of dangerous tumor lysis syndrome appears to be related to a very high circulating leukocyte count. Concurrent pharmacokinetic studies indicated that some patients require an increase in the dose of flavopiridol during the 4-hour infusion to achieve and maintain the targeted plasma concentration of 1.5 mM. Consequently, the current phase-II trial in CLL is being executed with the loading dose of 30 mg/m2, followed by a 4-hour intravenous infusion of 30 mg/m2 for the initial dose and 50 mg/m2 for subsequent 4-hour infusions in those patients who do not have excessive tumor lysis and no response at the 30 mg/m2 level. These modifications, coupled with continued close monitoring, have helped mitigate the problems associated with the administration of this new agent. Importantly, no serious opportunistic infections have been observed to date following treatment with in this heavily-pretreated population of patients with advanced CLL. These encouraging clinical findings have been translated into plans for examining the role of flavopiridol in treating patients earlier for minimal residual disease. If the observation of efficacy despite high-risk cytogenetics – e.g. del(17p13.1) and del(11q22.3) – can be confirmed, this agent may be moved forward to treating patients with residual disease following initial cytoreductive induction therapy. This maneuver would potentially increase the numbers of patients achieving a complete remission, and hopefully result in durable and long-lasting responses. Additional studies are ongoing with flavopiridol that incorporate the use of this agent with fludarabine and rituximab as part of the ‘up-front simultaneous’ therapy.48
552 M. R. Grever et al
The FFR regimen has reportedly achieved a 75% complete response rate in a group of patients with low-grade B-cell malignancy, including CLL. While there will be several methods for incorporating this novel agent, the optimal model based on pharmacokinetics and clinical results will be assessed after the current phase-II trials with the loading dose and 4-hour continuous infusion study have been concluded. Flavopiridol had initially been thought to be most active in rapidly proliferating malignancies. Therefore, the observations that this agent has dramatic effects in patients with neoplastic cells that are not rapidly dividing (mostly in G0) raises interesting questions about its actual mechanism of action. Is this agent really inducing tumor cell death through apoptosis or necrosis? Studies in mitochondrial function have revealed that flavopiridol induces mitochondrial membrane depolarization early during in vitro culture, in the same time frame as clinical effects begin to be observed (i.e. 6–8 hours after exposure to the agent). If the induction of rapid cell death is truly mediated by disruption of mitochondrial function and release of cytochrome C, this could explain the lack of dependence upon an intact P53 system. Other novel agents Several unique molecules have the potential to induce apoptosis in CLL cells independently of p53-mediated pathways. Another novel purported cyclin-dependent kinase inhibitor, R-roscovitine (CYC202), is undergoing evaluation for clinical efficacy.49 This agent shows selectivity for malignant B cells compared to normal blood mononuclear cells, and can impact leukemic cells regardless of the presence of ATM or P53 mutations.49 Thus, the induction of apoptosis mediated by roscovitine appears to be p53-independent. Additional agents in preclinical development include a wide variety with diverse mechanisms of inducing apoptosis in CLL cells. For example, Brefeldin A is a unique molecule that targets the endoplasmic reticulum and protein transport pathways. This agent can have direct effects on the Golgi apparatus, and has been reported to overcome fludarabine resistance in malignant B cells.50 Jasmonates are another group of novel anti-cancer agents that act directly and selectively on human cancer-cell mitochondria.51 Agents that can induce apoptosis by perturbing mitochondrial integrity may be powerful new therapeutic agents in otherwise drug-resistant cells (Table 1). A final example of a novel natural product with unique properties as a potential agent in the treatment of CLL is silvestrol.52,53 This plant-derived molecule isolated from Aglaia foveolata, is currently undergoing preclinical studies as a broad-spectrum Table 1. Novel therapeutic agents which work independently of p53. In clinical use or under clinical investigation: Alemtuzumab (Campath) Flavopiridol (Alvocidib) R-Roscovitine (CYC202) Under preclinical exploration for CLL: representative agents: Acadesinea Silvestrol Brefeldin-A a
Acadesine has apparently been investigated for cardiovascular research. These data may be useful in exploring this agent in patients with CLL.
Novel agents and strategies for p53-defective CLL 553
anti-cancer agent which shows cytotoxicity in the low nanomolar range. Silvestrol has been reported to regulate the G2/M checkpoint gates in LNCaP tumor cells, inducing genes that regulate apoptosis independently of P53. Encouraging preliminary studies have also shown in vivo inhibition of tumor cell growth in small animal models. In studying the in vitro effect of this agent on leukemic cells from CLL patients and lymphocytes from healthy volunteers, it appears that the sensitivity of CLL B cells to silvestrol is far greater than that of normal T cells, potentially a significant advantage in developing new therapies for this disease. Also, silvestrol-mediated apoptosis appears to be P53-independent, in that no differences in cytotoxicity are noted between samples from CLL patients with and without the del(17p) abnormality.54 This important quality may thus confer a use for silvestrol in the future for circumventing mechanisms of resistance in patients with CLL and other B-cell malignancies.
Research agenda continued development of mechanistically novel therapeutic agents. The search for agents that circumvent P53-mediated apoptosis associated with DNA damage will potentially enable killing of drug-resistant cells that emanate from exposure to current chemotherapy prioritization of agents that demonstrate B-cell selectivity. This will be essential in limiting damage to residual normal immune effector cells. Because many of our existing therapeutic agents cause direct damage to T cells, strategies are needed to add effective agents that will not further compromise the ability of patients to withstand the dangers of fatal infection continued optimization of strategic combinations of novel agents in select patient populations. Significant efforts are being made to identify patients who will not respond optimally to conventional treatments prior to therapy. The earlier use of novel agents and combinations in these high-risk patients may produce improved responses while avoiding unnecessary toxicities accompanying suboptimal therapies
ACKNOWLEDGEMENTS This work was supported by the CLL Research Consortium (P01 CA81534-02), The Leukemia and Lymphoma Society of America, and The D. Warren Brown Foundation. REFERENCES 1. Hamblin TJ, Davis Z, Gardiner A et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–1854. 2. Damle RN, Wasil T, Fais F et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94: 1840–1847. *3. Dohner H, Stilgenbauer S, Benner A et al. Genomic aberrations and survival in chronic lymphocytic leukemia. The New England Journal of Medicine 2000; 343: 1910–1916. 4. Mayr C, Speicher MR, Kofler DM et al. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood 2006; 107: 742–751.
554 M. R. Grever et al 5. Sturm I, Bosanquet AG, Hermann S et al. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death and Differentiation 2003; 10: 477–484. 6. Austen B, Powell JE, Alvi A et al. Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. Blood 2005; 106: 3175– 3182. 7. Krober A, Seiler T, Benner A et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002; 100: 1410–1416. 8. Fenaux P, Jonveaux P, Quiquandon I et al. Mutations of the p53 gene in B-cell lymphoblastic acute leukemia: a report on 60 cases. Leukemia 1992; 6: 42–46. 9. Fenaux P, Preudhomme C, Lai JL et al. Mutations of the p53 gene in B-cell chronic lymphocytic leukemia: a report on 39 cases with cytogenetic analysis. Leukemia 1992; 6: 246–250. *10. el Rouby S, Thomas A, Costin D et al. p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood 1993; 82: 3452–3459. *11. Wattel E, Preudhomme C, Hecquet B et al. p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood 1994; 84: 3148–3157. *12. Cordone I, Masi S, Mauro FR et al. p53 expression in B-cell chronic lymphocytic leukemia: a marker of disease progression and poor prognosis. Blood 1998; 91: 4342–4349. 13. Barnabas N, Shurafa M, Van Dyke DL et al. Significance of p53 mutations in patients with chronic lymphocytic leukemia: a sequential study of 30 patients. Cancer 2001; 91: 285–293. 14. Stankovic T, Stewart GS, Fegan C et al. Ataxia telangiectasia mutated-deficient B-cell chronic lymphocytic leukemia occurs in pregerminal center cells and results in defective damage response and unrepaired chromosome damage. Blood 2002; 99: 300–309. 15. Lozanski G, Heerema NA, Flinn IW et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood 2004; 103: 3278–3281. *16. Dohner H, Fischer K, Bentz M et al. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. Blood 1995; 85: 1580–1589. *17. Byrd JC, Gribben JG, Peterson BL et al. Select high-risk genetic features predict earlier progression following chemoimmunotherapy with fludarabine and rituximab in chronic lymphocytic leukemia: justification for risk-adapted therapy. Journal of Clinical Oncology 2006; 24: 437–443. 18. Kay NE, Geyer SM, Call TG et al. Combination chemoimmunotherapy with pentostatin, cyclophosphamide and rituximab shows significant clinical activity with low accompanying toxicity in previously untreated B-chronic lymphocytic leukemia. Blood 2007; 109: 405–411. 19. Oscier DG, Richards S, Orchard J et al. Prognostic factors in the UK LRF CLL4 trial. Blood 2005; 106. Abstract 2099. 20. Eichhorst BF, Busch R, Hopfinger G et al. Fludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy of younger patients with chronic lymphocytic leukemia. Blood 2006; 107: 885–891. *21. Grever MR, Lucas DM, Dewald GW et al. Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from a prospective randomized US Intergroup Phase III Trial (E2997). Journal of Clinical Oncology 2007; 25: 799–804. 22. Byrd JC, Peterson BL, Morrison VA et al. Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: results from Cancer and Leukemia Group B 9712 (CALGB 9712). Blood 2003; 101: 6–14. 23. Byrd JC, Rai K, Peterson BL et al. Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood 2005; 105: 49–53. 24. Keating MJ, O’Brien S, Albitar M et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. Journal of Clinical Oncology 2005; 23: 4079–4088. 25. Flynn JM & Byrd JC. Campath-1H monoclonal antibody therapy. Current Opinion in Oncology 2000; 12: 574–581. 26. Hale G, Bright S, Chumbley G et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983; 62: 873–882.
Novel agents and strategies for p53-defective CLL 555 27. Hale G, Dyer MJ, Clark MR et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet 1988; 2: 1394–1399. 28. Treumann A, Lifely MR, Schneider P & Ferguson MA. Primary structure of CD52. The Journal of Biological Chemistry 1995; 270: 6088–6099. 29. Domagala A & Kurpisz M. CD52 antigen: a review. Medical Science Monitor 2001; 7: 325–331. 30. Rowan W, Tite J, Topley P & Brett SJ. Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells. Immunology 1998; 95: 427–436. 31. Keating MJ, Flinn I, Jain V et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 2002; 99: 3554–3561. 32. Hillmen P, Skotnicki A, Robak T et al. Preliminary phase III efficacy and safety of alemtuzumab vs chlorambucil as front-line therapy for patients with progressive B-cell chronic lymphocytic leukemia (BCLL). Journal of Clinical Oncology 2006; 24: 18S. [abstract#6511]. *33. Stilgenbauer S & Dohner H. Campath-1H-induced complete remission of chronic lymphocytic leukemia despite p53 gene mutation and resistance to chemotherapy. The New England Journal of Medicine 2002; 347: 452–453. 34. Lozanski G, Heerema NA, Flinn IW et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood 2004; 103: 3278–3281. 35. Thornton PD, Matutes E, Bosanquet AG et al. High dose methylprednisolone can induce remissions in CLL patients with p53 abnormalities. Annals of Hematology 2003; 82: 759–765. 36. Brown JR, Kim HT, Li S et al. Predictors of improved progression-free survival after nonmyeloablative allogeneic stem cell transplantation for advanced chronic lymphocytic leukemia. Biology of Blood and Marrow Transplantation 2006; 12: 1056–1064. 37. Sorror ML, Maris MB, Sandmaier BM et al. Hematopoietic cell transplantation after nonmyeloablative conditioning for advanced chronic lymphocytic leukemia. Journal of Clinical Oncology 2005; 23: 3819–3829. 38. Maloney DG, Sandmaier BM, Mackinnon S & Shizuru JA. Non-myeloablative transplantation. Hematology 2002. American society of hematology education program book; 2002. p. 392–421. 39. Delgado J, Thomson K, Russell N et al. Results of alemtuzumab-based reduced-intensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study. Blood 2006; 107: 1724–1730. 40. Caballero D, Garcia-Marco JA, Martino R et al. Allogeneic transplant with reduced intensity conditioning regimens may overcome the poor prognosis of B-cell chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene and chromosomal abnormalities (11q- and 17p-). Clinical Cancer Research 2005; 11: 7757–7763. 41. Wierda WG, Cantwell MJ, Woods SJ et al. CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia. Blood 2000; 96: 2917–2924. 42. Dicker F, Kater AP, Prada CE et al. CD154 induces p73 to overcome the resistance to apoptosis of chronic lymphocytic leukemia cells lacking functional p53. Blood 2006; 108: 3450–3457. *43. Byrd JC, Shinn C, Waselenko JK et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Blood 1998; 92: 3804–3816. 44. Kitada S, Zapata JM, Andreeff M & Reed JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000; 96: 393–397. 45. Flinn IW, Byrd JC, Bartlett N et al. Flavopiridol administered as a 24-hour continuous infusion in chronic lymphocytic leukemia lacks clinical activity. Leukemia Research 2005; 29: 1253–1257. 46. Byrd JC, Peterson BL, Gabrilove J et al. Treatment of relapsed chronic lymphocytic leukemia by 72-hour continuous infusion or 1-hour bolus infusion of flavopiridol: results from Cancer and Leukemia Group B study 19805. Clinical Cancer Research 2005; 11: 4176–4181. *47. Byrd JC, Lin TS, Dalton JT et al. Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia. Blood 2007; 109: 399–404. 48. Lin TS, Fischer B, Moran ME et al. Flavopiridol, fludarabine and rituximab is a highly active regimen in indolent B-cell lymphoproliferative disorders including mantle cell lymphoma. Blood 2005; 106. abstr 944.
556 M. R. Grever et al 49. Alvi AJ, Austen B, Weston VJ et al. A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down-regulation of genes involved in transcription regulation and survival. Blood 2005; 105: 4484–4491. 50. Carew JS, Nawrocki ST, Krupnik YV et al. Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL. Blood 2006; 107: 222–231. 51. Flescher E. Jasmonates—a new family of anti-cancer agents. Anticancer Drugs 2005; 16: 911–916. 52. Hwang BY, Su BN, Chai H et al. Silvestrol and episilvestrol, potential anticancer rocaglate derivatives from Aglaia silvestris. The Journal of Organic Chemistry 2004; 69: 3350–3358. 53. Mi Q, Kim S, Hwang BY et al. Silvestrol regulates G2/M checkpoint genes independent of p53 activity. Anticancer Research 2006; 26: 3349–3356. 54. Edwards RB, Lucas DM, Lozanski G et al. Silvestrol, a rocaglate derivative from the Indonesian plant aglaia foveolata, has significant Bcl-2- and p53-independent anti-tumor activity against chronic lymphocytic leukemia cells. Blood 2006; 108 [abstract 2600].