Hematopoietic Cell Transplantation After Nonmyeloablative Conditioning for Advanced Chronic Lymphocytic Leukemia
http://www.100md.com
《临床肿瘤学》
the Fred Hutchinson Cancer Research Center
University of Washington
VA Puget Sound Health Care System
Hematologics Inc, Seattle, WA
Stanford University, Stanford, CA
University of Leipzig, Leipzig, Germany
Baylor University, Dallas, TX
Oregon Health & Science University, Portland, OR
University of Utah, Salt Lake City, UT
University of Colorado, Denver, CO
Medical College of Wisconsin, Milwaukee, WI
University of Torino, Torino, Italy
Emory University, Atlanta, GA
ABSTRACT
PURPOSE: Patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) have a short life expectancy. The aim of this study was to analyze the outcome of patients with advanced CLL when treated with nonmyeloablative conditioning and hematopoietic cell transplantation (HCT).
PATIENTS AND METHODS: Sixty-four patients diagnosed with advanced CLL were treated with nonmyeloablative conditioning (2 Gy total-body irradiation with [n = 53] or without [n = 11] fludarabine) and HCT from related (n = 44) or unrelated (n = 20) donors. An adapted form of the Charlson comorbidity index was used to assess pretransplantation comorbidities.
RESULTS: Sixty-one of 64 patients had sustained engraftment, whereas three patients rejected their grafts. The incidences of grades 2, 3, and 4 acute and chronic graft-versus-host disease were 39%, 14%, 2%, and 50%, respectively. Three patients who underwent transplantation in complete remission (CR) remained in CR. The overall response rate among 61 patients with measurable disease was 67% (50% CR), whereas 5% had stable disease. All patients with morphologic CR who were tested by polymerase chain reaction (n = 11) achieved negative molecular results, and one of these patients subsequently experienced disease relapse. The 2-year incidence of relapse/progression was 26%, whereas the 2-year relapse and nonrelapse mortalities were 18% and 22%, respectively. Two-year rates of overall and disease-free survivals were 60% and 52%, respectively. Unrelated HCT resulted in higher CR and lower relapse rates than related HCT, suggesting more effective graft-versus-leukemia activity.
CONCLUSION: CLL is susceptible to graft-versus-leukemia effects, and allogeneic HCT after nonmyeloablative conditioning might prolong median survival for patients with advanced CLL.
INTRODUCTION
Chronic lymphocytic leukemia (CLL), a low-grade lymphoproliferative disorder, is the most common adult hematologic malignancy in Western countries.1 Median age of CLL patients at diagnosis is 72 years,2 and only 10% to 15% are younger than 50 years.3 Fludarabine is the typical first-line therapy for CLL. It has shown better response rates than single alkylating agents or combination chemotherapy, but with no survival benefit.4–6 Twenty percent of CLL patients are refractory to first-line fludarabine therapy, and their median survival is only 12 months,7 whereas all other patients eventually experience disease relapse after response to fludarabine, with a median survival of 21 months.8 The use of alemtuzumab, the drug approved by the US Food and Drug Administration for treatment of patients with fludarabine-refractory CLL, has extended survival to 16 months, with a 2-year survival rate of 40%.9 Treatment with other nucleoside analogs, combination chemotherapy with or without fludarabine, or new biologic agents such as rituximab has not demonstrably extended survival of fludarabine-refractory CLL patients over that achieved with alemtuzumab.7,9–16 In previously treated CLL, some improvement of survival was noted after a combination of fludarabine/cyclophosphamide/rituximab.17
Autologous high-dose hematopoietic cell transplantation (HCT) has been offered to a selected group of young and chemotherapy-sensitive CLL patients with minimal tumor burden and successful collection of hematopoietic progenitor cells. Although nonrelapse mortality (NRM) was less than 10%,18 autologous HCT was associated with relapse rates of 41% to 58%, with no survival plateau.18,19 Fludarabine-refractory CLL has usually been a reason for ineligibility for autologous HCT.20 Myeloablative allogeneic HCT has the advantage of graft-versus-leukemia (GVL) effects, which result in a low risk of relapse (13% to 25%) in younger recipients of unrelated or related grafts.18,19,21 However, myeloablative allogeneic HCT is associated with 35% to 60% NRM.19,21–24 Despite the high NRM, 3-year survival rates among patients with fludarabine-responsive or -nave CLL who underwent myeloablative allogeneic HCT have ranged from 41% to 56%, with a suggestion of a plateau in disease-free survival (DFS).18,19,21 Survival rates for patients with fludarabine-refractory CLL were 36% at 2 years25 and 32% at 5 years.23 On the basis of preclinical canine studies,26 we have developed a nonmyeloablative regimen for allogeneic HCT in patients with hematologic malignancies consisting of conditioning with 2 Gy total-body irradiation (TBI) with or without three doses of fludarabine, and post-transplantation immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP).27–29 Here we describe the outcomes of 64 patients with chemotherapy-refractory CLL who underwent related or unrelated HCT using this regimen.
PATIENTS AND METHODS
Eligibility Criteria
This analysis includes data from 64 patients diagnosed with CLL who underwent allogeneic HCT after nonmyeloablative conditioning on Fred Hutchinson Cancer Research Center (FHCRC; Seattle, WA) multi-institutional protocols for patients with hematologic malignancies between December 16, 1997, and December 11, 2003. The primary differences between protocols were the use of HLA-matched related or unrelated grafts, variations in the duration and intensity of CSP and MMF, and the addition of fludarabine to 2 Gy TBI. These changes were made over time to reduce both the risks of graft-versus-host disease (GVHD) and graft rejection.
Patients were treated at 12 centers with the FHCRC acting as the coordinating center. Protocols were approved by the institutional review boards of the FHCRC and the collaborating sites. All patients signed consent forms approved by the local institutional review boards. Inclusion criteria included diagnoses of CLL,30 small lymphocytic lymphoma, or CLL/prolymphocytic leukemia; age 50 years; younger than age 50 years but at high risk for NRM because of failed prior treatment with high-dose HCT or pre-existing comorbidities; and failure of one or more front-line therapies. Exclusion criteria included pregnancy, cardiac ejection fraction of less than 40% for related recipients and of less than 35% for unrelated recipients, pulmonary diffusion capacity less than 35%, decompensated liver disease (fulminant hepatic failure or liver cirrhosis with portal hypertension), Karnofsky performance score less than 60, and serologic evidence of infection with HIV. No exclusions were made for disease status, chemotherapy sensitivity, renal insufficiency, or active bacterial or fungal infections.
In this study, chemotherapy (and fludarabine) -refractory CLL was defined as either inability to meet National Cancer Institute (NCI) Working Group Criteria for partial remission (PR) or complete remission (CR),30 or relapse/progression within 6 months of completion of chemotherapy. Pretransplantation comorbidities were scored using an adapted form of Charlson Comorbidity Index (CCI),31,32 whereas post-transplantation toxicities were graded using the NCI Common Toxicity Criteria.
HLA Typing and Matching
Patients and their donors were matched for HLA-A, -B, and -C by at least intermediate-resolution DNA typing, and -DRB1 and -DQB1 by high-resolution techniques.33 One unrelated recipient/donor pair had a single HLA-C allele mismatch.
Conditioning Regimen and Post-Transplantation Immunosuppression
Eleven related recipients were conditioned with 2 Gy TBI alone at a rate of 0.07 Gy/min from linear accelerators or opposing 60-cobalt sources on day 0, whereas the remaining related (n = 33) and all unrelated recipients received, in addition, three doses of fludarabine 30 mg/m2/d on days –4 to –2. Postgrafting immunosuppression included CSP and MMF, as described previously.28,34 Six unrelated recipients received MMF 15 mg/kg every 12 hours and 14 received MMF 15 mg/kg every 8 hours. Grading and treatment of GVHD was done as previously described.35,36
Collection of Hematopoietic Cells and Supportive Care
All patients received granulocyte colony-stimulating factor-mobilized peripheral-blood mononuclear cells.28,34 Antimicrobial and cytomegalovirus prophylaxis and blood product support were administered as described.28 Mobilizing growth factors were administered for persistent neutropenia only after day +28.
Analyses of Donor Chimerism, Disease Responses, and Risk Factors
Donor engraftment was confirmed by chimerism analyses.28,34,37,38 Disease responses were assessed using NCI Working Group Criteria30 with the following additions. CR or PR required complete or at least 50% clearance of CD5/CD19-coexpressing CLL cells from both marrow and peripheral blood as detected by flow cytometry (the lowest level of detection was 0.2%) using previously described methods39 and clearance of clonal cytogenetic abnormalities, respectively. Disease progression was defined as development of new lesions or 50% increase in soft tissue masses by clinical assessment or by imaging studies; 50% increase in circulating lymphocytes by morphology or flow cytometry; or 50% increase in bone marrow CLL by morphology or flow cytometry. Failure to respond to discontinuation of immunosuppression or the requirement of additional therapy, such as donor lymphocyte infusion (DLI), another HCT, and/or chemotherapy, was considered transplantation failure.
Beta2-microglobulin was assessed using an immunoglobulin assay Abbott test kit (Abbott Park, IL). The upper limit of the reference range was 2.5 μg/mL, which was the value detected in the 95th percentile of the population at the University of Washington Medical Center (Seattle, WA).
Cytogenetics were assessed in most patients (n = 51) using conventional karyotype G banding in addition to interphase cytogenetics with florescence in situ hybridization. Ten patients were tested only by conventional cytogenetic methods, and three patients were not evaluated for cytogenetic abnormalities before HCT.
Monitoring Minimal Residual Disease
Minimal residual disease was monitored by detection of tumor-specific immunoglobulin heavy chain (IgH) Variable, Diversity, and Joining (VDJ) rearrangements. DNA was extracted from cells collected from peripheral blood and marrow before and after HCT, and used to detect allele-specific complementary-determining region III (CDRIII) sequences. DNA was polymerase chain reaction (PCR) amplified by forward primers from the Variable region (VH) of the IgH gene and a reverse primer derived from the consensus sequence at the 3' end of the Joining region (JH) of the IgH gene. The PCR product was then separated on 2% agarose gels and stained with ethidium bromide (0.5 μg/mL). The PCR product was then separated on 6% polyacrylamide gel. The band was then eluted overnight in water, reamplified, and quantitated using the quantitative standard "Low DNA Mass Ladder" (Invitrogen, Carlsbad, CA). The band was washed using an enzyme mixture of Shrimp alkaline phosphatase (Roche, Basel, Switzerland) and ExoI (NEBiolabs, Beverly, MA), and then subjected to a fluorescence-based automated DNA sequencing in an ABI 3700 sequence detector (Applied Biosystems, Foster City, CA) using corresponding forward and reverse primers. The sequence data were compared with germline sequences of the VH, diversity, and JH regions of the IgH gene to identify the patient-specific CDRIII sequence. Using the patient-specific CDRIII sequence as a forward primer and the reverse JH primer, PCR amplification was performed on the post-HCT samples along with a negative control, a reagent control, and serial log dilutions of the patients' pretreatment samples (1:10 to 1:106). The PCR products were size fractionated in 6% polyacrylamide gel, stained with ethidium bromide, and then photographed. The sensitivity of the test was 1 in 105.
Statistical Methods
Overall survival (OS) and DFS were estimated by the Kaplan-Meier method. Cumulative incidence estimates were calculated for acute and chronic GVHD, relapse, relapse-related mortality, and NRM. Hazard ratios were estimated from Cox regression models. Deaths were treated as competing events in analyses of graft rejection, GVHD, and disease progression. Progression and NRM were the components of DFS and were treated as competing events. Multivariate models were constructed in a stepwise fashion, using a threshold significance level of .05 for inclusion in the model. Multivariate P values for a variable were based on adjustment for all other variables in the model. All P values were derived from likelihood ratio statistics and were two sided.
RESULTS
Patient Characteristics
Forty-four patients received related grafts and 20 patients received unrelated grafts (Table 1). Patients had multiple risk factors: median age was 56 years; median interval between diagnosis and HCT was 4.4 years; CCI scores of 1 for pretransplantation comorbidities were present in 48% of patients (20 patients had a score of 1, seven patients had a score of 2, and four patients had a score of 3; Table 2); chemotherapy resistance to pretransplantation salvage treatment was present in 53% of patients and untreated relapse occurred in 11% of patients; there was a median of four prior treatment regimens; and disease refractoriness to at least one regimen was present in all but two patients (97%). Thirty patients (47%) were refractory to one regimen, 23 patients (36%) were refractory to two regimens, and nine patients (14%) were refractory to three regimens. Eighty-eight percent of patients were refractory to fludarabine, 25% of patients were refractory to rituximab, 30% of patients were refractory to alkylating agents, and 22% of patients were refractory to other miscellaneous regimens. Among fludarabine-refractory patients, 47 patients did not respond or experienced disease progression, whereas nine patients experienced disease relapse within 6 months after a fludarabine-containing regimen. In addition, patients had multiple adverse disease burden characteristics40,41: bulky lymphadenopathy (lymph node diameter 5 cm, 28%), splenomegaly (47%), CD5/CD9 coexpression of CD38 more than 30% (58%), beta2-microglobulin more than 2.5 μg/mL (53%), 50% marrow infiltration with CLL cells (52%), and unfavorable cytogenetics42 (39%). No significant differences in adverse disease burden characteristics were present between related and unrelated recipients.
Favorable cytogenetics included either a normal karyotype or a single abnormality of del(13q), whereas unfavorable cytogenetics included del(11q), del(17p), trisomy 12, other rare abnormalities [t(11:17), t(11:13), t(14:18), t(5:17), and monosomy 7], and complex karyotypic abnormalities (> two abnormalities).
Engraftment Kinetics and Donor Chimerism
Patients had a median neutrophil nadir of 39 cells/μL and a median duration of neutropenia (< 500 cells/μL) of 11 days. Fifty-six percent of patients did not develop severe thrombocytopenia (< 20 x 103 cells/μL). Median levels of peripheral-blood donor CD3, CD33, and whole marrow chimerism by day 28 were 90%, 99%, and 95%, respectively. Sixty-one patients had sustained donor engraftment and three patients experienced graft rejection. One related-donor recipient, conditioned with 2 Gy TBI, rejected his graft at day +86, followed by disease progression and failure to respond to both DLI and a second HCT from the same donor. Two unrelated recipients rejected their grafts on days 28 and 25, respectively; both had received MMF twice daily. One of the two patients died after 2 months as a result of pulmonary hemorrhage and the other had a sustained CR for 2 years and then experienced disease relapse. None of the three patients who rejected their grafts received pre-HCT alemtuzumab or had post-HCT cytomegalovirus infection.
GVHD and Toxicities
Grades 2, 3, and 4 acute GVHD were observed in 42%, 17%, and 2% of patients, respectively. Those incidences were 43%, 14%, and 3% for related and 39%, 25%, and 0% for unrelated recipients, respectively. At 2 years, 50% of patients had developed chronic extensive GVHD (46% for related and 69% for unrelated recipients, respectively; P = .56). Alemtuzumab, administered to 10 patients at a median of 3.4 months (range, 0.7 to 23.7 months) before HCT, did not significantly influence the incidences of grades 2 to 4 acute GVHD (30% v 60%; P = .23), grades 3 to 4 acute GVHD (10% v 17%; P = .67), or chronic extensive GVHD (34% v 53%; P = .42) after HCT.
Grade 4 toxicities were infrequent and most commonly included hematologic toxicity (32%; Fig 1), in part explained by the fact that 25% of patients had neutropenia (< 500 cells/μL) and/or thrombocytopenia (< 20 x 103 cells/μL) before HCT.
Disease Responses and Clearance of Molecular Disease
Three patients without measurable disease at HCT have remained in CR. Among 61 patients with measurable disease at HCT, the 2-year cumulative probabilities for achieving CR and PR were 50% and 17%, respectively. Related recipients had lower CR rates (42%) than unrelated recipients (78%; P = .005). This difference remained significant after adjusting for pretransplantation disease burden characteristics (P = .03). Of 54 patients with CD5/CD19-coexpressing CLL cells detected by flow cytometry pre-HCT, 36 cleared their disease post-HCT, whereas 21 of 33 patients cleared previously identified cytogenetic abnormalities (Fig 2A) . All three patients with del(11q) before HCT achieved post-transplantation CR; one of these patients died as a result of NRM, and two are alive at 6 and 49 months, respectively, after HCT. Among the five patients with del(17p) before HCT, two died early as a result of progression, one died as a result of NRM in CR, and two are alive in CR and PR at 12 and 7 months post-transplantation, respectively. Seven patients had complete cytogenetic and flow cytometric responses while clinically they were in PR. Among patients who achieved CR, two experienced disease relapse at 2 and 3 years after HCT, respectively; among patients with PR, two experienced disease progression. IgH VDJ detection before and after HCT was performed in 11 FHCRC patients who were in CR (Fig 2B). Clone-specific IgH VDJ rearrangements in all 11 patients cleared within 3 to 30 months after HCT. Ten of the 11 patients had fludarabine-refractory CLL. Nine of the 11 patients are alive and eight have sustained molecular remissions at a median of 38 months (range, 28 to 63 months), whereas one patient experienced disease relapse 39 months after HCT.
The estimated 2-year rate of relapse/progression was 26%. Rates of relapse/progression were 34% for related and 5% for unrelated recipients (hazard ratio [HR], 0.3; P = .08) at 2 years (Fig 3), and after adjusting for pretransplantation disease burden characteristics, the HR remained 0.3 (P = 0.1). The estimated 2-year rate of relapse-related mortality (Fig 4) was 18% (22% for related and 5% for unrelated recipients; P = 0.16). At a median follow-up of 24 months (range, 3 to 63 months; 31 months [range 3 to 63 months] for related and 12 months [range, 3 to 39 months] for unrelated recipients), 39 patients were living: 25 in CR, five in PR, two with stable disease, and seven with relapse/progressive disease.
Timing of GVHD and GVL Effects
The median times for onset of acute and chronic GVHD were 1.4 and 4.5 months, respectively (Table 3). In comparison, the median times for resolution of cytogenetic abnormalities, disappearance of CD5/CD19-coexpressing CLL cells, disappearance of lymphadenopathy, and achievement of molecular remissions were 3.0, 5.7, 8.6, and 12 months, respectively. Of note, the respective onsets for each of the landmarks occurred earlier among unrelated than related recipients. Two patients experienced disease relapse at 28.2 and 37.9 months, respectively, after achieving initial remissions, whereas 16 patients experienced disease progression after a median of 3.3 months with stable disease. Overall, 15 related recipients and one unrelated recipient experienced disease progression.
DLI
Eight patients received DLI for treatment of disease progression/relapse (n = 6) or graft rejection (n = 1) among related recipients, and for Epstein-Barr virus-associated lymphoproliferative disease (n = 1) arising from unrelated donor cells (the patient's CLL was in CR). Five patients received DLI after chemotherapy (Table 4). Six patients failed to respond to DLI. One patient achieved PR associated with GVHD followed by multiorgan failure and death. One patient experienced CR of his lymphoproliferative disease and is alive 9.3 months after HCT.
NRM and Survival
Rates of day 100 and 2-year NRM were 11% and 22%, respectively, with no significant differences between related and unrelated recipients (Fig 4). The nonrelapse deaths included infections with acute (n = 4) or chronic (n = 6) GVHD, cardiac arrest (n = 2), pulmonary hemorrhage after rejection (n = 1), cerebrovascular stroke (n = 1), and de novo metastatic lung cancer (n = 1). Kaplan-Meier probabilities of DFS and OS at 2-years were 52% and 60%, respectively. Median DFS was 28 months, whereas median OS has not yet been reached (4-year OS, 54%). Two-year OS and DFS of related compared with unrelated recipients were 56% v 75% (P = .33) and 44% v 75% (P = .15), respectively (Fig 4).
Risk Factors Predicting Outcome After HCT
A number of factors were examined in stepwise multivariate analyses for their effects on outcome (Table 5). Bulky lymphadenopathy independently predicted a high incidence of relapse/progression (HR, 3.8; P = .009). Accordingly, estimated 2-year rates of relapse/progression were 52% v 14% among patients with or without bulky lymphadenopathy. CCI scores of 2 independently predicted both a higher risk of NRM (HR, 8.1; P = .008) and decreased survival (HR, 6.4; P = .002). Specifically, patients with CCI scores of 0, 1, and 2 had 2-year NRM rates of 7%, 31%, and 46%, and OS of 78%, 49%, and 33%, respectively. Marrow infiltration with 50% CLL cells was associated with worse survival (HR, 2.4; P = .05), whereas recipient age of 60 years was associated with favorable survival (HR, 0.3; P = .02). Unfavorable cytogenetics had no significant effect on outcome even after restricting the analyses to those 51 patients who had fluorescence in situ hybridization evaluations before HCT.
DISCUSSION
Patients with CLL who are unresponsive to or have early relapse after treatment with fludarabine have median survivals of 12 and 20 months, respectively.7,8 Better treatments are needed. Current treatments include the use of the anti-CD52 monoclonal antibody alemtuzumab,9 and the combination of fludarabine, cyclophosphamide, and rituximab,11 which result in CR rates of 3% and 7%, respectively. Alemtuzumab extended median survival from 12 to 16 months, with a 2-year survival of 40% when compared with historical controls. Myeloablative allogeneic HCT might result in long-term DFS. However, even in younger patients with fludarabine-sensitive/nave CLL, conventional HCT had high early NRM,19,24 and OS rates ranged from 32% to 36%.23,25 Here, we report on the use of a nonmyeloablative conditioning regimen for allogeneic HCT for older patients with chemotherapy-refractory CLL, and show CR and PR rates of 50% and 17%, respectively, with 2-year survival of 60%. Encouragingly, progression/relapse rates have been low, especially for unrelated recipients (2-year relapse rates were 5% and 34%, respectively). Longer follow-up is required. Eighty-eight percent of the current patients had fludarabine-refractory CLL, and 64% had active or chemotherapy-resistant disease at the time of HCT.
Given the minimal-intensity conditioning, the achievement of CR and PR must be attributed to powerful GVL effects. Although 16 of 64 patients had progressive CLL early after HCT at a median of 3.3 months, only two of 64 patients had late relapses 28 to 38 months after HCT. These relapse/progression rates do not seem to be significantly different from the rates of 13% to 25% at 3 years reported after myeloablative HCT in younger patients with less advanced disease.19,21 Not surprisingly, greater disease burden before HCT, such as bulky lymphadenopathy, predicted the highest relapse/progression rates. Such patients might benefit from earlier referral to nonablative HCT trials after failed treatment with fludarabine; those patients had received a median of five preceding regimens, and 50% of them were refractory to two regimens or the addition of novel biologic agents such as rituximab.43
Current unrelated recipients experienced greater CR rates and less relapse/progression than related recipients. This difference might be due in part to higher disease burdens before HCT among related recipients. In part, it might be due to greater GVL effects owing to greater genetic disparities with their donors for minor histocompatibility antigens on malignant and nonmalignant hematopoietic cells.44–46 This assumption is also supported by more rapid disease responses among unrelated recipients. Clearly, longer follow-up is needed to verify these early observations. The observed improved outcome among patients age 60 years was primarily due to less relapse/progression and less relapse-related mortality. This finding remains unexplained, but justifies further exploration of nonablative HCT in elderly patients.
We also evaluated the effects of known poor prognostic factors on outcome after nonmyeloablative HCT. In contrast to other studies, we found unfavorable cytogenetics47–50 or CD38+ expression51,52 were not associated with worse outcome.53 Although this might be due to small patient numbers, it was possible that GVL effects outweighed adverse effects of these prognostic factors, or perhaps that these factors had less prognostic significance for chemotherapy-refractory CLL than for early-stage or untreated patients receiving standard chemotherapy.54–56 Ongoing studies are examining the effects of adverse prognostic factors including zeta-chain-associated protein 7057 and unmutated immunoglobulin gene54 in larger numbers of patients.
Although our results in patients with advanced CLL have been encouraging, there is room for improvement, given that 22% of patients have died as a result of NRM and 18% have died as a result of relapsed/progressive CLL. Pretransplantation comorbidities, as assessed by increased CCI scores, were an independent strong predictor for NRM, consistent with previous observations in patients with various hematologic malignancies.32,36 Unfortunately, comorbidities remain a problem of this population, given the advanced age of most patients with CLL. One major cause of NRM has been GVHD and associated infections; in particular, invasive mold infections.58,59 Our data suggested that there were more deaths from acute GVHD among patients with CCI scores of more than 0 compared with those with a score of 0 (13% v 0%; P = .1). Studies are under way to reduce the incidence of severe GVHD by lengthening the duration of immunosuppression or adding a third agent such as rapamycin. Other studies have focused on better early diagnosis and prevention of fungal infections.59–64
To address the issue of relapse/disease progression, we examined the effects of DLI in a small number of patients (n = 8). Surprisingly, we found that patients who experienced disease progression generally did not respond to DLI. This may be due to the use of a suboptimal dose of DLI for inducing strong GVL effects. Alternatively, some patients with bulky advanced disease may have tumor cell clones that are resistant to the GVL-mediated killing. Future approaches using more specific targeting of malignant CLL cells by isolation, expansion, and injection of specific cytotoxic donor T lymphocytes may improve tumor control.
Other groups have reported the use of reduced-intensity conditioning regimens followed by HCT for treatment of CLL. Dreger et al65 retrospectively summarized data from 77 patients with CLL who underwent transplantation in 29 European centers, mainly from related donors (82%), using several different reduced-intensity regimens; Schetelig et al66 reported data from 30 patients administered fludarabine, busulfan, and antithymocyteglobulin conditioning and undergoing transplantation from related (50%) or unrelated donors (50%); and Khouri et al43 reported data on 17 patients undergoing transplantation from related donors using fludarabine and cyclophosphamide conditioning. Patients enrolled onto these studies had shown OS of 72%, 72%, and 80% and DFS of 56%, 67%, and 60% at 2 years, respectively. In a comparison of risk factors, 88% of our patients had fludarabine-refractory CLL compared with 82% fludarabine exposure in the study by Dreger et al,65 33% fludarabine-refractoriness in the study by Schetelig et al,66 and 100% refractoriness or relapse after fludarabine in the study by Khouri et al.43 In addition, patients in our study were older (median age, 56 v 50 to 54 years), had received more prior regimens (median, four v three), and had more or comparable chemotherapy-resistant disease at the time of HCT (53% v 35% to 53%, respectively).
The two studies that included unrelated grafts reported worse survivals (HR, 2.3)65 and higher rates of NRM (28% v 0%, respectively)66 among unrelated compared with related recipients. In contrast, in our study we discovered better survival (75% v 56%), lower relapse (5% v 34%), and similar NRM (20% v 22%) rates for unrelated compared with related recipients, respectively. Longer follow-up is required, both to assess risk of disease relapse and toxicity due to GVHD and infections.
Reduced-intensity conditioning regimens containing alemtuzumab have been used to decrease the risk of GVHD after allogeneic HCT. Mackinnon et al67 reported the use of this approach in low-grade lymphoma (including nine patients with CLL) followed by preemptive DLI to control disease progression, relapse, or refractoriness. They reported 3-year rates of OS and DFS of 73% and 65%, respectively, for the entire group of patients with low-grade lymphoma.
We have demonstrated the feasibility of and early outcome results with a nonmyeloablative conditioning regimen to prepare patients with fludarabine-refractory CLL for HLA-matched related or unrelated allogeneic HCT. This approach has a significant NRM (22% at 2 years) but is associated with CR and PR rates of 50% and 17%, respectively, including molecular remissions in all 11 patients tested. Only one of those 11 patients eventually experienced disease relapse. No other treatment option offers this level of disease response in this patient population. Comparisons of nonmyeloablative HCT results with those after conventional therapies including alemtuzumab or the combination of fludarabine, cyclophosphamide, and rituximab are difficult to make because of differences in patient characteristics. However, current 2-year OS and DFS of 60% and 52%, respectively, compared favorably with previously reported results.9 Results support the need for prospective phase III studies comparing the different treatment modalities for patients with fludarabine-refractory CLL.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Employment: Monic J. Stuart, Genentech; Michael R. Loken, HematoLogics. Leadership Position: Michael R. Loken, HematoLogics. Consultant/Advisory Role: Michael R. Loken, HematoLogics. Stock Ownership: Monic J. Stuart, Genentech; Michael R. Loken, HematoLogics. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank the data coordinators Chris Davis and Heather Hildebrant; the study nurses Steve Minor, Mary Hinds, and John Sedgwick; Bonnie Larson and Helen Crawford for manuscript preparation; Paula Ladne for PCR analysis; and all of the transplantation teams at the participating institutions.
NOTES
Supported by grant Nos. CA78902, CA92058, CA18029, CA49605, and CA15704 from the National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Leukemia and Lymphoma Society Specialized Center of Research grant 7040. M.S. was supported in part by a grant from the Oncology Research Faculty Development Program of the Office of International Affairs of the National Cancer Institute. B.B. was supported by a grant from Ministero dell'Istruzione, dell'Università, della Ricerca, Italy.
Presented in part at the Tandem Bone Marrow Transplantation meeting, February 13-17, 2004, Orlando, FL (for part of the patient population).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Morris E, Thomson K, Craddock C, et al: Outcomes after alemtuzumab-containing reduced-intensity allogeneic transplantation regimen for relapsed and refractory non-Hodgkin lymphoma. Blood 104:3865-3871, 2004(Mohamed L. Sorror, Michae)
University of Washington
VA Puget Sound Health Care System
Hematologics Inc, Seattle, WA
Stanford University, Stanford, CA
University of Leipzig, Leipzig, Germany
Baylor University, Dallas, TX
Oregon Health & Science University, Portland, OR
University of Utah, Salt Lake City, UT
University of Colorado, Denver, CO
Medical College of Wisconsin, Milwaukee, WI
University of Torino, Torino, Italy
Emory University, Atlanta, GA
ABSTRACT
PURPOSE: Patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) have a short life expectancy. The aim of this study was to analyze the outcome of patients with advanced CLL when treated with nonmyeloablative conditioning and hematopoietic cell transplantation (HCT).
PATIENTS AND METHODS: Sixty-four patients diagnosed with advanced CLL were treated with nonmyeloablative conditioning (2 Gy total-body irradiation with [n = 53] or without [n = 11] fludarabine) and HCT from related (n = 44) or unrelated (n = 20) donors. An adapted form of the Charlson comorbidity index was used to assess pretransplantation comorbidities.
RESULTS: Sixty-one of 64 patients had sustained engraftment, whereas three patients rejected their grafts. The incidences of grades 2, 3, and 4 acute and chronic graft-versus-host disease were 39%, 14%, 2%, and 50%, respectively. Three patients who underwent transplantation in complete remission (CR) remained in CR. The overall response rate among 61 patients with measurable disease was 67% (50% CR), whereas 5% had stable disease. All patients with morphologic CR who were tested by polymerase chain reaction (n = 11) achieved negative molecular results, and one of these patients subsequently experienced disease relapse. The 2-year incidence of relapse/progression was 26%, whereas the 2-year relapse and nonrelapse mortalities were 18% and 22%, respectively. Two-year rates of overall and disease-free survivals were 60% and 52%, respectively. Unrelated HCT resulted in higher CR and lower relapse rates than related HCT, suggesting more effective graft-versus-leukemia activity.
CONCLUSION: CLL is susceptible to graft-versus-leukemia effects, and allogeneic HCT after nonmyeloablative conditioning might prolong median survival for patients with advanced CLL.
INTRODUCTION
Chronic lymphocytic leukemia (CLL), a low-grade lymphoproliferative disorder, is the most common adult hematologic malignancy in Western countries.1 Median age of CLL patients at diagnosis is 72 years,2 and only 10% to 15% are younger than 50 years.3 Fludarabine is the typical first-line therapy for CLL. It has shown better response rates than single alkylating agents or combination chemotherapy, but with no survival benefit.4–6 Twenty percent of CLL patients are refractory to first-line fludarabine therapy, and their median survival is only 12 months,7 whereas all other patients eventually experience disease relapse after response to fludarabine, with a median survival of 21 months.8 The use of alemtuzumab, the drug approved by the US Food and Drug Administration for treatment of patients with fludarabine-refractory CLL, has extended survival to 16 months, with a 2-year survival rate of 40%.9 Treatment with other nucleoside analogs, combination chemotherapy with or without fludarabine, or new biologic agents such as rituximab has not demonstrably extended survival of fludarabine-refractory CLL patients over that achieved with alemtuzumab.7,9–16 In previously treated CLL, some improvement of survival was noted after a combination of fludarabine/cyclophosphamide/rituximab.17
Autologous high-dose hematopoietic cell transplantation (HCT) has been offered to a selected group of young and chemotherapy-sensitive CLL patients with minimal tumor burden and successful collection of hematopoietic progenitor cells. Although nonrelapse mortality (NRM) was less than 10%,18 autologous HCT was associated with relapse rates of 41% to 58%, with no survival plateau.18,19 Fludarabine-refractory CLL has usually been a reason for ineligibility for autologous HCT.20 Myeloablative allogeneic HCT has the advantage of graft-versus-leukemia (GVL) effects, which result in a low risk of relapse (13% to 25%) in younger recipients of unrelated or related grafts.18,19,21 However, myeloablative allogeneic HCT is associated with 35% to 60% NRM.19,21–24 Despite the high NRM, 3-year survival rates among patients with fludarabine-responsive or -nave CLL who underwent myeloablative allogeneic HCT have ranged from 41% to 56%, with a suggestion of a plateau in disease-free survival (DFS).18,19,21 Survival rates for patients with fludarabine-refractory CLL were 36% at 2 years25 and 32% at 5 years.23 On the basis of preclinical canine studies,26 we have developed a nonmyeloablative regimen for allogeneic HCT in patients with hematologic malignancies consisting of conditioning with 2 Gy total-body irradiation (TBI) with or without three doses of fludarabine, and post-transplantation immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP).27–29 Here we describe the outcomes of 64 patients with chemotherapy-refractory CLL who underwent related or unrelated HCT using this regimen.
PATIENTS AND METHODS
Eligibility Criteria
This analysis includes data from 64 patients diagnosed with CLL who underwent allogeneic HCT after nonmyeloablative conditioning on Fred Hutchinson Cancer Research Center (FHCRC; Seattle, WA) multi-institutional protocols for patients with hematologic malignancies between December 16, 1997, and December 11, 2003. The primary differences between protocols were the use of HLA-matched related or unrelated grafts, variations in the duration and intensity of CSP and MMF, and the addition of fludarabine to 2 Gy TBI. These changes were made over time to reduce both the risks of graft-versus-host disease (GVHD) and graft rejection.
Patients were treated at 12 centers with the FHCRC acting as the coordinating center. Protocols were approved by the institutional review boards of the FHCRC and the collaborating sites. All patients signed consent forms approved by the local institutional review boards. Inclusion criteria included diagnoses of CLL,30 small lymphocytic lymphoma, or CLL/prolymphocytic leukemia; age 50 years; younger than age 50 years but at high risk for NRM because of failed prior treatment with high-dose HCT or pre-existing comorbidities; and failure of one or more front-line therapies. Exclusion criteria included pregnancy, cardiac ejection fraction of less than 40% for related recipients and of less than 35% for unrelated recipients, pulmonary diffusion capacity less than 35%, decompensated liver disease (fulminant hepatic failure or liver cirrhosis with portal hypertension), Karnofsky performance score less than 60, and serologic evidence of infection with HIV. No exclusions were made for disease status, chemotherapy sensitivity, renal insufficiency, or active bacterial or fungal infections.
In this study, chemotherapy (and fludarabine) -refractory CLL was defined as either inability to meet National Cancer Institute (NCI) Working Group Criteria for partial remission (PR) or complete remission (CR),30 or relapse/progression within 6 months of completion of chemotherapy. Pretransplantation comorbidities were scored using an adapted form of Charlson Comorbidity Index (CCI),31,32 whereas post-transplantation toxicities were graded using the NCI Common Toxicity Criteria.
HLA Typing and Matching
Patients and their donors were matched for HLA-A, -B, and -C by at least intermediate-resolution DNA typing, and -DRB1 and -DQB1 by high-resolution techniques.33 One unrelated recipient/donor pair had a single HLA-C allele mismatch.
Conditioning Regimen and Post-Transplantation Immunosuppression
Eleven related recipients were conditioned with 2 Gy TBI alone at a rate of 0.07 Gy/min from linear accelerators or opposing 60-cobalt sources on day 0, whereas the remaining related (n = 33) and all unrelated recipients received, in addition, three doses of fludarabine 30 mg/m2/d on days –4 to –2. Postgrafting immunosuppression included CSP and MMF, as described previously.28,34 Six unrelated recipients received MMF 15 mg/kg every 12 hours and 14 received MMF 15 mg/kg every 8 hours. Grading and treatment of GVHD was done as previously described.35,36
Collection of Hematopoietic Cells and Supportive Care
All patients received granulocyte colony-stimulating factor-mobilized peripheral-blood mononuclear cells.28,34 Antimicrobial and cytomegalovirus prophylaxis and blood product support were administered as described.28 Mobilizing growth factors were administered for persistent neutropenia only after day +28.
Analyses of Donor Chimerism, Disease Responses, and Risk Factors
Donor engraftment was confirmed by chimerism analyses.28,34,37,38 Disease responses were assessed using NCI Working Group Criteria30 with the following additions. CR or PR required complete or at least 50% clearance of CD5/CD19-coexpressing CLL cells from both marrow and peripheral blood as detected by flow cytometry (the lowest level of detection was 0.2%) using previously described methods39 and clearance of clonal cytogenetic abnormalities, respectively. Disease progression was defined as development of new lesions or 50% increase in soft tissue masses by clinical assessment or by imaging studies; 50% increase in circulating lymphocytes by morphology or flow cytometry; or 50% increase in bone marrow CLL by morphology or flow cytometry. Failure to respond to discontinuation of immunosuppression or the requirement of additional therapy, such as donor lymphocyte infusion (DLI), another HCT, and/or chemotherapy, was considered transplantation failure.
Beta2-microglobulin was assessed using an immunoglobulin assay Abbott test kit (Abbott Park, IL). The upper limit of the reference range was 2.5 μg/mL, which was the value detected in the 95th percentile of the population at the University of Washington Medical Center (Seattle, WA).
Cytogenetics were assessed in most patients (n = 51) using conventional karyotype G banding in addition to interphase cytogenetics with florescence in situ hybridization. Ten patients were tested only by conventional cytogenetic methods, and three patients were not evaluated for cytogenetic abnormalities before HCT.
Monitoring Minimal Residual Disease
Minimal residual disease was monitored by detection of tumor-specific immunoglobulin heavy chain (IgH) Variable, Diversity, and Joining (VDJ) rearrangements. DNA was extracted from cells collected from peripheral blood and marrow before and after HCT, and used to detect allele-specific complementary-determining region III (CDRIII) sequences. DNA was polymerase chain reaction (PCR) amplified by forward primers from the Variable region (VH) of the IgH gene and a reverse primer derived from the consensus sequence at the 3' end of the Joining region (JH) of the IgH gene. The PCR product was then separated on 2% agarose gels and stained with ethidium bromide (0.5 μg/mL). The PCR product was then separated on 6% polyacrylamide gel. The band was then eluted overnight in water, reamplified, and quantitated using the quantitative standard "Low DNA Mass Ladder" (Invitrogen, Carlsbad, CA). The band was washed using an enzyme mixture of Shrimp alkaline phosphatase (Roche, Basel, Switzerland) and ExoI (NEBiolabs, Beverly, MA), and then subjected to a fluorescence-based automated DNA sequencing in an ABI 3700 sequence detector (Applied Biosystems, Foster City, CA) using corresponding forward and reverse primers. The sequence data were compared with germline sequences of the VH, diversity, and JH regions of the IgH gene to identify the patient-specific CDRIII sequence. Using the patient-specific CDRIII sequence as a forward primer and the reverse JH primer, PCR amplification was performed on the post-HCT samples along with a negative control, a reagent control, and serial log dilutions of the patients' pretreatment samples (1:10 to 1:106). The PCR products were size fractionated in 6% polyacrylamide gel, stained with ethidium bromide, and then photographed. The sensitivity of the test was 1 in 105.
Statistical Methods
Overall survival (OS) and DFS were estimated by the Kaplan-Meier method. Cumulative incidence estimates were calculated for acute and chronic GVHD, relapse, relapse-related mortality, and NRM. Hazard ratios were estimated from Cox regression models. Deaths were treated as competing events in analyses of graft rejection, GVHD, and disease progression. Progression and NRM were the components of DFS and were treated as competing events. Multivariate models were constructed in a stepwise fashion, using a threshold significance level of .05 for inclusion in the model. Multivariate P values for a variable were based on adjustment for all other variables in the model. All P values were derived from likelihood ratio statistics and were two sided.
RESULTS
Patient Characteristics
Forty-four patients received related grafts and 20 patients received unrelated grafts (Table 1). Patients had multiple risk factors: median age was 56 years; median interval between diagnosis and HCT was 4.4 years; CCI scores of 1 for pretransplantation comorbidities were present in 48% of patients (20 patients had a score of 1, seven patients had a score of 2, and four patients had a score of 3; Table 2); chemotherapy resistance to pretransplantation salvage treatment was present in 53% of patients and untreated relapse occurred in 11% of patients; there was a median of four prior treatment regimens; and disease refractoriness to at least one regimen was present in all but two patients (97%). Thirty patients (47%) were refractory to one regimen, 23 patients (36%) were refractory to two regimens, and nine patients (14%) were refractory to three regimens. Eighty-eight percent of patients were refractory to fludarabine, 25% of patients were refractory to rituximab, 30% of patients were refractory to alkylating agents, and 22% of patients were refractory to other miscellaneous regimens. Among fludarabine-refractory patients, 47 patients did not respond or experienced disease progression, whereas nine patients experienced disease relapse within 6 months after a fludarabine-containing regimen. In addition, patients had multiple adverse disease burden characteristics40,41: bulky lymphadenopathy (lymph node diameter 5 cm, 28%), splenomegaly (47%), CD5/CD9 coexpression of CD38 more than 30% (58%), beta2-microglobulin more than 2.5 μg/mL (53%), 50% marrow infiltration with CLL cells (52%), and unfavorable cytogenetics42 (39%). No significant differences in adverse disease burden characteristics were present between related and unrelated recipients.
Favorable cytogenetics included either a normal karyotype or a single abnormality of del(13q), whereas unfavorable cytogenetics included del(11q), del(17p), trisomy 12, other rare abnormalities [t(11:17), t(11:13), t(14:18), t(5:17), and monosomy 7], and complex karyotypic abnormalities (> two abnormalities).
Engraftment Kinetics and Donor Chimerism
Patients had a median neutrophil nadir of 39 cells/μL and a median duration of neutropenia (< 500 cells/μL) of 11 days. Fifty-six percent of patients did not develop severe thrombocytopenia (< 20 x 103 cells/μL). Median levels of peripheral-blood donor CD3, CD33, and whole marrow chimerism by day 28 were 90%, 99%, and 95%, respectively. Sixty-one patients had sustained donor engraftment and three patients experienced graft rejection. One related-donor recipient, conditioned with 2 Gy TBI, rejected his graft at day +86, followed by disease progression and failure to respond to both DLI and a second HCT from the same donor. Two unrelated recipients rejected their grafts on days 28 and 25, respectively; both had received MMF twice daily. One of the two patients died after 2 months as a result of pulmonary hemorrhage and the other had a sustained CR for 2 years and then experienced disease relapse. None of the three patients who rejected their grafts received pre-HCT alemtuzumab or had post-HCT cytomegalovirus infection.
GVHD and Toxicities
Grades 2, 3, and 4 acute GVHD were observed in 42%, 17%, and 2% of patients, respectively. Those incidences were 43%, 14%, and 3% for related and 39%, 25%, and 0% for unrelated recipients, respectively. At 2 years, 50% of patients had developed chronic extensive GVHD (46% for related and 69% for unrelated recipients, respectively; P = .56). Alemtuzumab, administered to 10 patients at a median of 3.4 months (range, 0.7 to 23.7 months) before HCT, did not significantly influence the incidences of grades 2 to 4 acute GVHD (30% v 60%; P = .23), grades 3 to 4 acute GVHD (10% v 17%; P = .67), or chronic extensive GVHD (34% v 53%; P = .42) after HCT.
Grade 4 toxicities were infrequent and most commonly included hematologic toxicity (32%; Fig 1), in part explained by the fact that 25% of patients had neutropenia (< 500 cells/μL) and/or thrombocytopenia (< 20 x 103 cells/μL) before HCT.
Disease Responses and Clearance of Molecular Disease
Three patients without measurable disease at HCT have remained in CR. Among 61 patients with measurable disease at HCT, the 2-year cumulative probabilities for achieving CR and PR were 50% and 17%, respectively. Related recipients had lower CR rates (42%) than unrelated recipients (78%; P = .005). This difference remained significant after adjusting for pretransplantation disease burden characteristics (P = .03). Of 54 patients with CD5/CD19-coexpressing CLL cells detected by flow cytometry pre-HCT, 36 cleared their disease post-HCT, whereas 21 of 33 patients cleared previously identified cytogenetic abnormalities (Fig 2A) . All three patients with del(11q) before HCT achieved post-transplantation CR; one of these patients died as a result of NRM, and two are alive at 6 and 49 months, respectively, after HCT. Among the five patients with del(17p) before HCT, two died early as a result of progression, one died as a result of NRM in CR, and two are alive in CR and PR at 12 and 7 months post-transplantation, respectively. Seven patients had complete cytogenetic and flow cytometric responses while clinically they were in PR. Among patients who achieved CR, two experienced disease relapse at 2 and 3 years after HCT, respectively; among patients with PR, two experienced disease progression. IgH VDJ detection before and after HCT was performed in 11 FHCRC patients who were in CR (Fig 2B). Clone-specific IgH VDJ rearrangements in all 11 patients cleared within 3 to 30 months after HCT. Ten of the 11 patients had fludarabine-refractory CLL. Nine of the 11 patients are alive and eight have sustained molecular remissions at a median of 38 months (range, 28 to 63 months), whereas one patient experienced disease relapse 39 months after HCT.
The estimated 2-year rate of relapse/progression was 26%. Rates of relapse/progression were 34% for related and 5% for unrelated recipients (hazard ratio [HR], 0.3; P = .08) at 2 years (Fig 3), and after adjusting for pretransplantation disease burden characteristics, the HR remained 0.3 (P = 0.1). The estimated 2-year rate of relapse-related mortality (Fig 4) was 18% (22% for related and 5% for unrelated recipients; P = 0.16). At a median follow-up of 24 months (range, 3 to 63 months; 31 months [range 3 to 63 months] for related and 12 months [range, 3 to 39 months] for unrelated recipients), 39 patients were living: 25 in CR, five in PR, two with stable disease, and seven with relapse/progressive disease.
Timing of GVHD and GVL Effects
The median times for onset of acute and chronic GVHD were 1.4 and 4.5 months, respectively (Table 3). In comparison, the median times for resolution of cytogenetic abnormalities, disappearance of CD5/CD19-coexpressing CLL cells, disappearance of lymphadenopathy, and achievement of molecular remissions were 3.0, 5.7, 8.6, and 12 months, respectively. Of note, the respective onsets for each of the landmarks occurred earlier among unrelated than related recipients. Two patients experienced disease relapse at 28.2 and 37.9 months, respectively, after achieving initial remissions, whereas 16 patients experienced disease progression after a median of 3.3 months with stable disease. Overall, 15 related recipients and one unrelated recipient experienced disease progression.
DLI
Eight patients received DLI for treatment of disease progression/relapse (n = 6) or graft rejection (n = 1) among related recipients, and for Epstein-Barr virus-associated lymphoproliferative disease (n = 1) arising from unrelated donor cells (the patient's CLL was in CR). Five patients received DLI after chemotherapy (Table 4). Six patients failed to respond to DLI. One patient achieved PR associated with GVHD followed by multiorgan failure and death. One patient experienced CR of his lymphoproliferative disease and is alive 9.3 months after HCT.
NRM and Survival
Rates of day 100 and 2-year NRM were 11% and 22%, respectively, with no significant differences between related and unrelated recipients (Fig 4). The nonrelapse deaths included infections with acute (n = 4) or chronic (n = 6) GVHD, cardiac arrest (n = 2), pulmonary hemorrhage after rejection (n = 1), cerebrovascular stroke (n = 1), and de novo metastatic lung cancer (n = 1). Kaplan-Meier probabilities of DFS and OS at 2-years were 52% and 60%, respectively. Median DFS was 28 months, whereas median OS has not yet been reached (4-year OS, 54%). Two-year OS and DFS of related compared with unrelated recipients were 56% v 75% (P = .33) and 44% v 75% (P = .15), respectively (Fig 4).
Risk Factors Predicting Outcome After HCT
A number of factors were examined in stepwise multivariate analyses for their effects on outcome (Table 5). Bulky lymphadenopathy independently predicted a high incidence of relapse/progression (HR, 3.8; P = .009). Accordingly, estimated 2-year rates of relapse/progression were 52% v 14% among patients with or without bulky lymphadenopathy. CCI scores of 2 independently predicted both a higher risk of NRM (HR, 8.1; P = .008) and decreased survival (HR, 6.4; P = .002). Specifically, patients with CCI scores of 0, 1, and 2 had 2-year NRM rates of 7%, 31%, and 46%, and OS of 78%, 49%, and 33%, respectively. Marrow infiltration with 50% CLL cells was associated with worse survival (HR, 2.4; P = .05), whereas recipient age of 60 years was associated with favorable survival (HR, 0.3; P = .02). Unfavorable cytogenetics had no significant effect on outcome even after restricting the analyses to those 51 patients who had fluorescence in situ hybridization evaluations before HCT.
DISCUSSION
Patients with CLL who are unresponsive to or have early relapse after treatment with fludarabine have median survivals of 12 and 20 months, respectively.7,8 Better treatments are needed. Current treatments include the use of the anti-CD52 monoclonal antibody alemtuzumab,9 and the combination of fludarabine, cyclophosphamide, and rituximab,11 which result in CR rates of 3% and 7%, respectively. Alemtuzumab extended median survival from 12 to 16 months, with a 2-year survival of 40% when compared with historical controls. Myeloablative allogeneic HCT might result in long-term DFS. However, even in younger patients with fludarabine-sensitive/nave CLL, conventional HCT had high early NRM,19,24 and OS rates ranged from 32% to 36%.23,25 Here, we report on the use of a nonmyeloablative conditioning regimen for allogeneic HCT for older patients with chemotherapy-refractory CLL, and show CR and PR rates of 50% and 17%, respectively, with 2-year survival of 60%. Encouragingly, progression/relapse rates have been low, especially for unrelated recipients (2-year relapse rates were 5% and 34%, respectively). Longer follow-up is required. Eighty-eight percent of the current patients had fludarabine-refractory CLL, and 64% had active or chemotherapy-resistant disease at the time of HCT.
Given the minimal-intensity conditioning, the achievement of CR and PR must be attributed to powerful GVL effects. Although 16 of 64 patients had progressive CLL early after HCT at a median of 3.3 months, only two of 64 patients had late relapses 28 to 38 months after HCT. These relapse/progression rates do not seem to be significantly different from the rates of 13% to 25% at 3 years reported after myeloablative HCT in younger patients with less advanced disease.19,21 Not surprisingly, greater disease burden before HCT, such as bulky lymphadenopathy, predicted the highest relapse/progression rates. Such patients might benefit from earlier referral to nonablative HCT trials after failed treatment with fludarabine; those patients had received a median of five preceding regimens, and 50% of them were refractory to two regimens or the addition of novel biologic agents such as rituximab.43
Current unrelated recipients experienced greater CR rates and less relapse/progression than related recipients. This difference might be due in part to higher disease burdens before HCT among related recipients. In part, it might be due to greater GVL effects owing to greater genetic disparities with their donors for minor histocompatibility antigens on malignant and nonmalignant hematopoietic cells.44–46 This assumption is also supported by more rapid disease responses among unrelated recipients. Clearly, longer follow-up is needed to verify these early observations. The observed improved outcome among patients age 60 years was primarily due to less relapse/progression and less relapse-related mortality. This finding remains unexplained, but justifies further exploration of nonablative HCT in elderly patients.
We also evaluated the effects of known poor prognostic factors on outcome after nonmyeloablative HCT. In contrast to other studies, we found unfavorable cytogenetics47–50 or CD38+ expression51,52 were not associated with worse outcome.53 Although this might be due to small patient numbers, it was possible that GVL effects outweighed adverse effects of these prognostic factors, or perhaps that these factors had less prognostic significance for chemotherapy-refractory CLL than for early-stage or untreated patients receiving standard chemotherapy.54–56 Ongoing studies are examining the effects of adverse prognostic factors including zeta-chain-associated protein 7057 and unmutated immunoglobulin gene54 in larger numbers of patients.
Although our results in patients with advanced CLL have been encouraging, there is room for improvement, given that 22% of patients have died as a result of NRM and 18% have died as a result of relapsed/progressive CLL. Pretransplantation comorbidities, as assessed by increased CCI scores, were an independent strong predictor for NRM, consistent with previous observations in patients with various hematologic malignancies.32,36 Unfortunately, comorbidities remain a problem of this population, given the advanced age of most patients with CLL. One major cause of NRM has been GVHD and associated infections; in particular, invasive mold infections.58,59 Our data suggested that there were more deaths from acute GVHD among patients with CCI scores of more than 0 compared with those with a score of 0 (13% v 0%; P = .1). Studies are under way to reduce the incidence of severe GVHD by lengthening the duration of immunosuppression or adding a third agent such as rapamycin. Other studies have focused on better early diagnosis and prevention of fungal infections.59–64
To address the issue of relapse/disease progression, we examined the effects of DLI in a small number of patients (n = 8). Surprisingly, we found that patients who experienced disease progression generally did not respond to DLI. This may be due to the use of a suboptimal dose of DLI for inducing strong GVL effects. Alternatively, some patients with bulky advanced disease may have tumor cell clones that are resistant to the GVL-mediated killing. Future approaches using more specific targeting of malignant CLL cells by isolation, expansion, and injection of specific cytotoxic donor T lymphocytes may improve tumor control.
Other groups have reported the use of reduced-intensity conditioning regimens followed by HCT for treatment of CLL. Dreger et al65 retrospectively summarized data from 77 patients with CLL who underwent transplantation in 29 European centers, mainly from related donors (82%), using several different reduced-intensity regimens; Schetelig et al66 reported data from 30 patients administered fludarabine, busulfan, and antithymocyteglobulin conditioning and undergoing transplantation from related (50%) or unrelated donors (50%); and Khouri et al43 reported data on 17 patients undergoing transplantation from related donors using fludarabine and cyclophosphamide conditioning. Patients enrolled onto these studies had shown OS of 72%, 72%, and 80% and DFS of 56%, 67%, and 60% at 2 years, respectively. In a comparison of risk factors, 88% of our patients had fludarabine-refractory CLL compared with 82% fludarabine exposure in the study by Dreger et al,65 33% fludarabine-refractoriness in the study by Schetelig et al,66 and 100% refractoriness or relapse after fludarabine in the study by Khouri et al.43 In addition, patients in our study were older (median age, 56 v 50 to 54 years), had received more prior regimens (median, four v three), and had more or comparable chemotherapy-resistant disease at the time of HCT (53% v 35% to 53%, respectively).
The two studies that included unrelated grafts reported worse survivals (HR, 2.3)65 and higher rates of NRM (28% v 0%, respectively)66 among unrelated compared with related recipients. In contrast, in our study we discovered better survival (75% v 56%), lower relapse (5% v 34%), and similar NRM (20% v 22%) rates for unrelated compared with related recipients, respectively. Longer follow-up is required, both to assess risk of disease relapse and toxicity due to GVHD and infections.
Reduced-intensity conditioning regimens containing alemtuzumab have been used to decrease the risk of GVHD after allogeneic HCT. Mackinnon et al67 reported the use of this approach in low-grade lymphoma (including nine patients with CLL) followed by preemptive DLI to control disease progression, relapse, or refractoriness. They reported 3-year rates of OS and DFS of 73% and 65%, respectively, for the entire group of patients with low-grade lymphoma.
We have demonstrated the feasibility of and early outcome results with a nonmyeloablative conditioning regimen to prepare patients with fludarabine-refractory CLL for HLA-matched related or unrelated allogeneic HCT. This approach has a significant NRM (22% at 2 years) but is associated with CR and PR rates of 50% and 17%, respectively, including molecular remissions in all 11 patients tested. Only one of those 11 patients eventually experienced disease relapse. No other treatment option offers this level of disease response in this patient population. Comparisons of nonmyeloablative HCT results with those after conventional therapies including alemtuzumab or the combination of fludarabine, cyclophosphamide, and rituximab are difficult to make because of differences in patient characteristics. However, current 2-year OS and DFS of 60% and 52%, respectively, compared favorably with previously reported results.9 Results support the need for prospective phase III studies comparing the different treatment modalities for patients with fludarabine-refractory CLL.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Employment: Monic J. Stuart, Genentech; Michael R. Loken, HematoLogics. Leadership Position: Michael R. Loken, HematoLogics. Consultant/Advisory Role: Michael R. Loken, HematoLogics. Stock Ownership: Monic J. Stuart, Genentech; Michael R. Loken, HematoLogics. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank the data coordinators Chris Davis and Heather Hildebrant; the study nurses Steve Minor, Mary Hinds, and John Sedgwick; Bonnie Larson and Helen Crawford for manuscript preparation; Paula Ladne for PCR analysis; and all of the transplantation teams at the participating institutions.
NOTES
Supported by grant Nos. CA78902, CA92058, CA18029, CA49605, and CA15704 from the National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Leukemia and Lymphoma Society Specialized Center of Research grant 7040. M.S. was supported in part by a grant from the Oncology Research Faculty Development Program of the Office of International Affairs of the National Cancer Institute. B.B. was supported by a grant from Ministero dell'Istruzione, dell'Università, della Ricerca, Italy.
Presented in part at the Tandem Bone Marrow Transplantation meeting, February 13-17, 2004, Orlando, FL (for part of the patient population).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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