Hematopoietic cell transplantation from HLA-identical sibling ... - Nature

Leukemia (2005) 19, 990–997 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu

Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML FR Kerbauy1, R Storb1,2, U Hegenbart3, T Gooley1,2, J Shizuru4, HK Al-Ali3, JP Radich1,2, DG Maloney1,2, E Agura5, B Bruno6, EM Epner7, TR Chauncey1,2,8, KG Blume4, D Niederwieser3,9 and BM Sandmaier1,2,9 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2University of Washington, Seattle, WA, USA; 3University of Leipzig, Leipzig, Germany; 4Stanford University, Stanford, CA, USA; 5Baylor University, Dallas, TX, USA; 6University of Torino, Torino, Italy; 7University of Arizona, Tucson, AZ, USA; and 8VA Medical Center, Seattle, WA, USA

A total of 24 patients (median age 58; range, 27–71 years) with chronic myeloid leukemia (CML) in first chronic (CP1) (n ¼ 14), second chronic (n ¼ 4), or accelerated phase (n ¼ 6) who were not candidates for conventional hematopoietic cell transplantation (HCT), received nonmyeloablative HCT from HLA-matched siblings a median of 28.5 (range, 11–271) months after diagnosis. They were conditioned with 2 Gy total body irradiation (TBI) alone (n ¼ 8) or combined with fludarabine, 90 mg/m2 (n ¼ 16). Postgrafting immunosuppression included cyclosporine and mycophenolate mofetil. All patients initially engrafted. However, 4 of 8 patients not given fludarabine experienced nonfatal rejection while all others had sustained engraftment. With a median follow-up of 36 (range, 4–49) months, 13 of 24 patients (54%) were alive and in complete remission. There were five (21%) deaths from nonrelapse mortality, one (4%) during the first 100 days after transplant. The proportions of grade II, III, and IV acute GVHD were 38, 4, and 8%, respectively. The 2-year estimate of chronic GVHD was 32%. The 2-year survival estimates for patients in CP1 (n ¼ 14) and beyond CP1 (n ¼ 10) were 70 and 56%, respectively. This study shows encouraging remission rates for patients with CML not eligible for conventional allografting. Leukemia (2005) 19, 990–997. doi:10.1038/sj.leu.2403730 Published online 31 March 2005 Keywords: chronic myeloid leukemia; nonmyeloablative; allogeneic hematopoietic cell transplant

Introduction Despite the recent introduction of new therapies, conventional allogeneic hematopoietic cell transplantation (HCT) has remained the only treatment capable of inducing durable molecular remissions and cures for patients with chronic myeloid leukemia (CML).1–3 While remissions following HCT are thought to be the consequence of both the high-dose cytotoxic conditioning regimens and graft-versus-leukemia (GVL) effects, the latter seems to play an important role, in particular, in chronic phase (CP) patients.4,5 Direct evidence for the GVL effect comes from the observation that a high proportion of CP1 patients who relapsed after HCT could be cured with donor lymphocyte infusions (DLI), which induced potent GVL effects against CML cells.6–8 The results of conventional HCT are strongly influenced by disease stage at transplantation, with reports from our Center documenting 86% survival for CP patients, 50% for accelerated phase (AP) patients, and only 10% for blast crisis (BC) patients.1,9–11 In addition, Correspondence: Dr BM Sandmaier, Fred Hutchinson Cancer Research Center and University of Washington School of Medicine, 1100 Fairview Avenue N., D1-100, PO Box 19024, Seattle, USA; Fax: þ 1 206 667 6124; E-mail: [email protected] 9 DN and BMS contributed equally to this paper. Received 16 December 2004; accepted 22 February 2005; Published online 31 March 2005

younger patients who received HCT early after diagnosis have been shown to have better outcomes (Clift et al, Blood 1995; 82: 1954–1956 (abstract)).10,12–14 On the other hand, conservative therapies such as interferon-alpha (IFNa) or, more recently, imatinib have been proven to be safer and easier to administer compared to HCT, but these alternate therapies have induced complete molecular remissions only in a minority of patients. Moreover, the long-term outcomes with imatinib are currently unknown (O’Brien et al, Blood 2001; 98: 846a (abstract); Hughes et al, Blood 2002; 100: 93a (abstract)).15,16 Patients who fail to respond to these therapies rely on allogeneic. HCT as the last and best possible chance to achieve cure of their diseases. A major concern with conventional allogeneic HCT has been the potential of nonrelapse mortality (NRM) due to toxicities of conditioning regimens, which has limited eligibility to patients without comorbidities and those younger than 55–65 years at most centers. Given that the median age at diagnosis of patients with CML ranges from 65 to 70 years (http://www.seer.cancer. gov), most patients with this disease have not been eligible for conventional HCT. In an attempt to overcome this limitation, several nonmyeloablative conditioning regimens have been developed with the goals of reducing NRM and eradicating disease through reliance on GVL effects.17–20 The nonmyeloablative conditioning regimen used in the current report included 2 Gy total body irradiation (TBI) with or without fludarabine and was combined with postgrafting immunosuppression provided by mycophenolate mofetil (MMF) and cyclosporine (CSP). This regimen has been shown to be safe and effective in patients with various hematological malignancies who either had pre-existing comorbidities or were older than 65 years (Sandmaier et al, Blood 2001; 98: 742a (abstract)).17 The present report evaluated the outcomes for patients with CML who were treated with this approach.

Patients and methods

Patients and eligibility A total of 24 consecutive patients were treated between October 20, 1998 and January 14, 2003, at the University of Leipzig, Germany (n ¼ 8); Fred Hutchinson Cancer Research Center/ University of Washington and Veterans Affairs Medical Center in Seattle, WA (n ¼ 7); Stanford University, Stanford, CA (n ¼ 5); Baylor University, Dallas, TX (n ¼ 2); University of Torino, Italy (n ¼ 1), and University of Arizona, Tucson, AZ (n ¼ 1). Results were analyzed as of February 2004. Major protocol eligibility requirements were (1) Philadelphiapositive (Ph þ ) CML in CP1, CP2 or AP; (2) ineligibility for conventional HCT because of age (older than 65 years)

Nonmyeloablative HCT for chronic myeloid leukemia FR Kerbauy et al

991 and/or comorbid conditions (coronary artery disease, active hepatitis, presence of fungal infection, and diabetes mellitus with end-organ damage or associated with morbid obesity and/ or hypertension requiring multiple drugs for therapy) (Sorror et al, Blood 2004; 104: 324a (abstract)),21 and/or failed preceding conventional autologous or allogeneic HCT; (3) human leukocyte antigen (HLA)-identical related donors as assessed by intermediate- to high-resolution molecular matching for HLA-A, -B, -C, and allele-level matching for HLA-DRB1 and -DQB1;22,23 (4) discontinuation of IFN-a at least 1 month before HCT and not in partial or complete cytogenetic remission; and (5) Karnofsky score more than 70%, cardiac ejection fraction more than 40%; bilirubin and transaminases less than two times normal; diffusing lung carbon monoxide (DLCO) greater than 50% of predicted; and absence of poorly controlled hypertension. Institutional review boards at each of the participating institutions approved the study protocols, and written informed consents were obtained from all patients and donors. The patients’ characteristics are summarized in Table 1. There were 14 male and 10 female subjects. The median patient age was 58 (range, 27–71) years, and the median donor age was 52 (range, 25–72) years. A total of 14 patients had CML in CP1, 4 in CP2 and 6 in AP. All patients had received preceding therapies, and the median time between diagnosis and HCT was 28.5 (range, 11–271) months. Nine patients were ineligible for conventional HCT because of age above 65 years, 11 because of the presence of comorbidities, and four patients because of both age and the presence of comorbidities.

Treatment and evaluations The first eight patients received conditioning with 2 Gy TBI alone. When four of eight patients experienced nonfatal graft rejections, the subsequent 16 patients were given, in addition to 2 Gy TBI, three doses of fludarabine, 30 mg/m2/day, from days 4 to 2. TBI was given as a single fraction on day 0, delivered at 0.07 Gy/min from dual 60-cobalt sources or linear accelerators, followed by donor hematopoietic cell infusions. In all patients, the source of donor cells was granulocyte colonystimulating factor (G-CSF)-stimulated peripheral blood mononuclear cells containing medians of 7.9 (range, 2.0–18.1) 106 CD34 þ cells/kg, and 3.4 (range, 1.7–4.9) 108 CD3 þ cells/ kg. CSP was administered orally at 6.25 mg/kg twice daily starting on day 3. CSP levels were targeted to the upper therapeutic range (approximately 500 ng/ml; Abbott TDX, Abbot Park, IL, USA) until day 35 and then tapered through day 56 after HCT for patients who received TBI alone. In patients who received TBI plus fludarabine, CSP was tapered from days 56 to 180 after HCT. MMF was given orally twice daily at 15 mg/kg, starting on day 0 to day 27 after HCT. Prophylaxis against Pneumocystis carinii, fungal, toxoplasmosis, and cytomegalovirus (CMV) infections was used (Sullivan et al, Exp Hematol 1983; 11: 193 (abstract)).24–26 The percentage of donor chimerism levels among peripheral blood T cells, granulocytes, and unfractionated marrow were assessed on days 28, 56, 84, 180, and 360 after HCT using fluorescence in situ hybridization (FISH) to detect X and Y chromosomes for recipients of sex-mismatched transplants27 and polymerase chain reaction (PCR)-based analyses of variable number tandem repeats (VNTR) for recipients of sex-matched transplants.28 The primary study objective was establishment of mixed chimerism at day 28 after HCT, defined as the detection

of 45% peripheral blood donor CD3 þ cells. Conversion to full donor chimerism was defined as detection of X95% donor CD3 þ T cells. Patients with decreasing donor chimerism or persistence of disease were eligible to receive DLI if they had X5% of donor CD3 þ T cells, had no evidence of GVHD, and immunosuppression had been discontinued for at least 2 weeks. Patients were scheduled to receive up to three DLI at increasing cell doses at intervals of 28 days if disease progressed or 65 days for persistent disease. Five patients received a total of six DLI (one patient, two infusions and four patients, one infusion).The median number of CD3 þ T cells among six DLI administered to five patients was 1 (range, 1– 6.6) 107 CD3 þ cells/kg. Patients who had rapid progression of disease early after transplantation or graft rejection (o5% donor peripheral blood CD3 þ T cells) were considered treatment failures and were eligible for other therapies not included in this protocol. Acute and chronic GVHD were graded as previously described.29 Complete remissions (CR) were defined as normalization of white blood cell and platelet counts with disappearance of the Ph þ chromosome and VNTR evidence of complete donor myeloid chimerism; partial remission was defined as normalization of white blood cell and platelet counts with reduction by X50% of the pretreatment numbers of Ph þ chromosome positive marrow cells or o35% Ph þ cells in at least 15 metaphases analyzed (whichever was lowest); no response was defined as persistence or progression of disease by morphologic, cytogenetic, and VNTR analyses at 12 weeks after the final DLI. Toxicities were classified according to National Cancer Institute Common Terminology Criteria for adverse events (CTCAE), version 2.0 (National Cancer Institute Bethesda, MD USA). Patients were classified according to European Group for Blood and Marrow Transplantation (EBMT) Risk Score.30,31 Pathology evaluations, cytogenetics studies, and reverse transcriptase polymerase chain reaction (RT-PCR) monitoring were performed on days 28, 56, and 84 after HCT, then at 6month intervals for 2 years, and yearly thereafter. A two-step, ‘nested’ RT-PCR was used to amplify the chimeric bcr-abl mRNA.32

Statistical analyses Overall survival and progression-free survival were estimated by the Kaplan–Meier method. Cause-specific mortalities and chronic GVHD were summarized using cumulative incidence estimates.33 Relapse/disease progression was regarded as a competing risk for relapse-related mortality, relapse a competing risk for NRM, and death without chronic GVHD a competing risk for GVHD.

Results

Engraftment and donor chimerism Donor chimerism levels: All patients had initial engraftment with a median of 37% (range, 10–79) donor peripheral T-cell chimerism on day 28. Median percentages of donor chimerism levels among peripheral blood T cell, granulocyte, and marrow compartments on days 28, 56, and 84 after HCT, based on the presence or absence of fludarabine in the conditioning regimen, are shown in Figure 1. Overall, achievement of full donor peripheral T-cell chimerism was slower to Leukemia

992

Leukemia

Table 1

Characteristics and outcomes of patients with CML given HLA-matched related HCT after 2 Gy TBI

Patient Conditioning with fludarabine

59 M

Disease status

CP1

Months from Dx to HCT 43

Treatment before HCT

Hu

Comorbidities

Age

2

71 M

CP1

23

6 Homoharringtonine+AraC, INF, 9 INF+Ara-C

Age

3

49 F

CP1

30

Hu/INF, Hu+Thioguanine

4 5

70 M 54 M

CP1 AP

31 23

1 2CdA, Hu Hu

6 7

40 M 65 M

AP AP

45 1

Hu Hu

DM, peripheral neuropathy, CAD Age + CAD Previous MI, DM CAD, DM, abn LFT Chronic hepatitis Age

8 9 10 11 12 13 14 15 16

+ + + + + + + +

60 M 55 M 52 F 61 F 48 M 27 M 60 F 56 F 46 M

AP CP1 CP1 CP1 CP1 CP1 CP1 CP1 CP1

34 11 15 7 11 11 96 11 18

1 IC-3 Hu/IFN (1 year), Hu/Anagrelyde INF, 10 Ara-c Hu, INF Hu, INF Hu Ablative HCT, HU Hu, INF INF, Hu/INF, Hu, Bu

17 18 19

+ + +

66 F 60 M 63 F

CP1 CP1 CP2

24 59 50

Hu, Imatinib, INF, Hu Hu, Imatinib Imatinib, 2 Hu, INF/Hu, INF

20 21

+ +

66 F 37 F

CP2 CP2

271 48

22 23

+ +

27 M 53 F

CP2 AP

27 30

24

+

60 M

AP

34

Hu, 3 1 Hu, Hu

INF, Imatinib Hu, 1 FLAG-IDA, Imatinib, Ara-C Imatinib

Hu, INF+Ara-c, Imatinib, Ara-C+anagrelide

Rejection /# DLI

Yes/2

GVHD

Survival (days)

Acute

Chronic

0

No

487b b

Yes/1

0

No

996

No

III

Limited

41902

No Yes/1

II I

Extensive No

41808 225b

Yes/1 No/1

0 II

No Extensive

407b 1589b

Age, DM, CAD DM, CAD DM, Previous MI Age DM, CAD Steato hepatitis, abn LFT Age, previous HCT Age DM, peripheral neuropathy, hypertension (2 drug therapy) Age, lung aspergilloma Age Age

No No No No No No No No No

I II II II IV II II I 0

Limited Limited Extensive Extensive NE Extensive Limited Extensive Limited

41804 41622 41601 386b 111b 41355 41243 41223 41132

No No No

II 0 I

No No No

252b 4397 41279

Age Pulmonary aspergillosis (right lobe resection) DM, morbid obesity DM, peripheral neuropathy, morbid obesity Age

No No

I 0

No Limited

59b 41132

No No

0 II

No Extensive

4324 813b

No

IV

No

145b

Outcome

SD-rejection-DLI 2-relapse-2nd HCT-rejection-died blast crisis SD-PD, rejection-DLI: no responseimatinib-CP2-died with CNS hemorrhage CR CR SD-rejection-DLI-relapse-2nd HCT-CR-died GVHD/aspergillosis SD-PD, rejection-DLI: no response SD-DLI-CR-died MOF atypical pneumonia CR CR CR CR, died with streptococcal pneumonia CR, died with acute GVHD, fungal infection CR CR CR CR CR-relapse (chloroma in CNS) PR-2nd HCT (off study) PR-PD-CR-relapse (chloroma in CNS)-imatinib+radiotherapy-CR PD, died of toxic epidermal necrolysis CR PR PR-Imatinib-PR died disseminated aspergillosis and pseudomonas SD-Died refractory acute GVHD

2CdA ¼ 2-chlorodeoxyadenosine; Abn LFT ¼abnormal liver function; AP ¼ accelerated phase; ARA-c ¼ cytosine arabinoside; Bu ¼ busulfan; CAD ¼ coronary artery disease; CNS ¼ central nervous system; CP1 ¼ first chronic phase; CP2 ¼ second chronic phase; CR ¼ complete remission; DLI ¼ donor lymphocyte infusion; DM ¼ diabetes mellitus; Dx ¼ diagnosis; FLAG-IDA ¼ fludarabine, idarrubicine, ARA-c; G-CSF ¼ granulocyte cytokine stimulating factor; Flu ¼ fludarabine; GVHD ¼ graft-versus-host disease; HCT ¼ hematopoietic cell transplantation; Hu ¼ hydroxiurea; INF ¼ interferon alpha; IC-3 ¼ idarubicin, ARA-c 3 days; MI ¼ myocardial infarction; MOF ¼ multiorgan failure; NE ¼ not evaluable; NMCT ¼ nonmyeloablative hematopoietic cell transplantation; PD ¼ progressive disease; PR ¼ partial remission; SD ¼ stable disease; TBI ¼ total body irradiation. a Median age ¼ 56 years. b Death.


1

Agea Sex


Toxicities and GVHD

993

None of the 24 patients experienced regimen-related central nervous system (CNS) or pulmonary toxicities, mucositis, de novo alopecia, severe nausea and vomiting, hemorrhagic cystitis, or veno-occlusive disease. Grades one and two reversible renal toxicities related to high CSP levels developed in eight (33%) patients. Grade one cardiac toxicity developed in two (8%) patients. Hepatic toxicity developed in two (8%) patients with pre-existing liver disease. Patients at the European sites (Universities of Leipzig and University of Torino; n ¼ 9) had scheduled hospital admissions for HCT. Among the patients who were eligible for outpatient transplantation (n ¼ 15) in the other institutions, six did not require hospitalization during the first 60 days after HCT. The remaining nine patients were hospitalized for a median of 1 (range, 0–56 days) day. The transfusion requirements were low. One (4%) patient received platelet and five (21%) red blood cell transfusions. The proportions of grade II, III, and IV acute GVHD were 38, 4, and 8%, respectively. Nine (38%) patients developed chronic GHVD (two limited and seven extensive) (Table 1). The 2-year estimate of chronic extensive GVHD was 32%. Infections were the most common toxicity affecting 42% of patients.

Outcomes Disease responses according to disease status at transplant: A total of 14 patients were transplanted in CP1. Figure 1 Median percentages (95% CI) of donor T cells, granulocytes, and bone marrow chimerism levels 28, 56, and 84 days after hematopoietic cell transplantation (HCT). (a) Patients treated with TBI þ fludarabine (n ¼ 16) and (b) patients treated with TBI only (n ¼ 8).

occur than complete granulocyte and marrow chimerism. The addition of fludarabine to the conditioning regimen accelerated donor T-cell chimerism.

Patients conditioned with TBI alone (n ¼ 8): The median number of days in which the absolute neutrophil count (ANC) was less than 0.5 109/l was 0 (range, 0–18), and five (63%) patients maintained ANCs40.5 109/l after HCT. Only one patient experienced decline of platelet counts to o20 109/l. Four of the eight patients rejected their grafts between 3 and 6 months after HCT despite DLI infusions. Patients conditioned with fludarabine plus TBI (n ¼ 16): The median number of days in which the ANC

was o0.5 109/l was 1 (range, 0–16), and six (38%) patients maintained ANCs 40.5 109/l after HCT. Only one patient experienced a decline of platelet counts to o20 109/l. On day 28 after HCT, four patients (25%) had achieved full donor and 12 (75%) mixed donor/host peripheral T-cell chimerism. Subsequently, 15 patients (94%) achieved full donor chimerism in a median of 84 (range, 28–360) days and one patient had 33% donor T-cell chimerism on day 84 before receiving a second myeloablative HCT because of concerns about low donor chimerism. There were no graft rejections in this group of patients.

Two patients (both conditioned with TBI only) rejected their grafts and did not achieve CR. Of the remaining 12 patients, 11 achieved cytogenetic CR. The remaining patient achieved a partial remission but was unevaluable for further response after receiving a second HCT from the same donor. One of the 11 who achieved CR died subsequently from relapse and two died from nonrelapse causes. With a median follow-up of 43 (range, 29–49) months, eight patients were alive and in CR. Four patients were transplanted in CP2. Three were alive, one in CR and one in partial remission, 1132 and 324 days after HCT, respectively. The third patient had initial partial remission followed by progression of disease 237 days after HCT (increased from 1 to 81% of Ph þ chromosome cells by FISH and appearance of 1% of blasts in peripheral blood). By tapering the CSP, the patient achieved complete molecular remission and full donor T-cell chimerism but subsequently relapsed in the CNS (chloroma) 668 days after HCT. She was treated with imatinib and radiotherapy, achieved CR of her chloroma and was in cytogenetic CR when last evaluated 1279 days after HCT. A fourth patient progressed to blast crisis and died from toxic epidermal necrolysis 59 days after HCT. Six patients were transplanted in AP. Two patients achieved CR; one of the two was alive in CR 1804 days after HCT and the second achieved CR after DLI but developed extensive chronic GVHD and died from atypical pneumonia on day 1589 after HCT. One patient had partial remission, was treated with imatinib remaining in CP and died from disseminated infections (pseudomonas and aspergillosis) 813 days after HCT. One patient had stable disease and died 145 days after HCT from acute GVHD. Two patients (conditioned with TBI only) rejected their grafts and died from disease progression on days 225 and 407 after HCT, respectively.

Molecular responses: Among the 15 patients who achieved cytogenetic remission, 13 achieved molecular remission. Two achieved only cytogenic remissions; both were in CP1 at the Leukemia


994 time of HCT. One of the two relapsed in the CNS with a chloroma and died on day 252 after HCT. The second patient achieved a complete cytogenetic remission 1 month after HCT and died from GVHD on day 111 after HCT. The patient was PCR negative for bcr-abl in the peripheral blood but positive in the marrow. Among patients who achieved molecular remissions (n ¼ 13), 10 became PCR negative for bcr-abl after conversion to full donor T-cell chimerism; one patient became PCR negative concurrent with achieving complete donor T-cell chimerism; and two patients achieved molecular remissions 270 and 90 days before conversion to full donor T-cell chimerism, respectively. The median times for achieving cytogenetic and PCR negativities were 3 (range, 1–24) and 7 (range, 3–24) months, respectively.

Survival and causes of death: Of 24 patients, 13 (54%) were alive and in CR at a median of 36 (range, 4–49) months. Five patients (21%) died from NRM: one on day 111 from fungal infection and acute GVHD (while in CR), one on day 145 from acute GVHD (with stable disease – AP), one on day 386 from strep pneumonia (while in CR), one on day 1589 from infection with extensive chronic GVHD (while in CR), and one on day 813 after HCT from pseudomonas and aspergillosis infection (while in partial remission – CP). Six patients (25%) died from disease relapse or progression. One patient transplanted in CP2 progressed to blast crisis and died on day 59 after HCT and one relapsed in the CNS and died 252 days after HCT. The other four patients were conditioned only with TBI and rejected their grafts. One of four patients, who was in AP at the time of HCT, died from progression of disease 407 days after HCT. A second patient in CP1 at the time of HCT progressed to AP on day 100, was treated with imatinib, and achieved CP2. He died 996 days after HCT secondary to CNS hemorrhage. A third patient, transplanted in AP, progressed to blast crisis and received a second HCT 142 days after the first HCT from a different HLAmatched sibling after conditioning with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg); he developed acute GVHD and died from aspergillosis 83 days later in CR. The fourth patient transplanted in CP1, rejected a second HCT from the same donor after conditioning with 2 Gy TBI and fludarabine, and died in blast crisis 487 days after the first HCT. Outcomes are summarized in Figures 2 and 3 according to stage of disease. Nine patients older than 55 years were transplanted in CP1, and five of the nine died at days 252, 386, 487, 825, and 996. The four surviving patients have been followed for 182, 1072, 1469, and 1477 days. According to EBMT Risk Score, patients were classified as: 0 with score 0–1; 4 with score 2; 8 with score 3; 9 with score 4 and 3 with score 5. One of four (25%) patients with a score of 2 have died, three of eight (38%) with a score of 3 have died, six of nine (67%) with a score of 4 have died, and one of three (33%) with a score of 5 have died.

Figure 2 Estimated probabilities of overall survival, relapse, and NRM after HCT for patients in first chronic phase (CP1, n ¼ 14). The 2year estimates of overall survival, relapse, and NRM were 70, 22, and 15%, respectively.

Figure 3 Estimated probabilities of overall survival, relapse, and NRM after HCT for patients beyond first chronic phase (CP2, n ¼ 10). The 2-year estimates of overall survival, relapse, and NRM were 56, 64, and 12%, respectively.

Outcomes among patients receiving TBI þ fludarabine (n ¼ 16): With the addition of fludarabine to TBI, no rejections occurred. In all, 10 patients were transplanted in CP1. Seven were alive (six in CR) with a median follow-up of 36 (range, 6–49) months. One of the seven patients had achieved partial remission after the nonmyeloablative HCT, but received a second myeloablative HCT because of concerns about low donor T-cell chimerism. There were three deaths, one secondary to relapse of disease and two NRM. The estimated 2-year overall and progression-free survivals were 68% (Figure 4). Six patients were transplanted beyond CP1 (four in CP2 and two in AP). Leukemia

Figure 4 Estimated probabilities of overall survival, relapse, and NRM after HCT for patients in first chronic phase (CP1) conditioned with TBI plus fludarabine (n ¼ 10). Progression-free survival estimates are identical to those for overall survival (68%).

Three patients were alive, one in CR and one in partial remission 1132 and 324 days after HCT, respectively. The third patient was in cytogenetic remissions 1279 days after HCT following hematologic and CNS relapse, as described above. One patient


995 died from toxic epidermal necrolysis 59 days after HCT with stable disease. The other two patients were transplanted in AP, achieved stable disease and died from nonrelapse causes.

Discussion Nonmyeloablative conditioning regimens have decreased the occurrence of transplant-related toxicities and made allogeneic HCT available to older patients and those with comorbid conditions who were ineligible for conventional HCT.21 The current regimen appeared to be safe for patients with various stages of CML. Most did not require red blood cell or platelet transfusions, and only half of the patients experienced shortlasting neutropenias. Only five patients had episodes of fever of unknown origin with no associated mortality. There were none of the side effects commonly seen with myeloablative regimens, such as prolonged neutropenia, high incidences of infections, severe mucositis, veno-occlusive disease, diarrhea, alopecia, prolonged hospitalization, and others. Overall, NRM was 21%, which we view as encouraging considering the patients’ ages and/or comorbid conditions. The EBMT risk score validated for patients with CML undergoing myeloablative HCT31 might also be useful for nonmyeloablative HCT but will require validation in a larger cohort of patients. The initial use of 2 Gy TBI as conditioning resulted in early engraftment in all patients. However, four of eight patients so treated experienced nonfatal graft rejections. Previous reports showed incidences of rejections between 13 and 20% when 2 Gy TBI was used to condition patients with other hematological malignancies (Sandmaier et al, Blood 2001; 98: 742a (abstract)).17,34 While the number of patients conditioned with 2 Gy TBI only is small, the higher observed rejection rate could be explained, in part, by the absence of preceding intensive chemotherapy in CML patients.17 In fact, three of the current four patients with rejection were treated only with hydroxyurea. In an effort to overcome the problem of graft rejection, three doses of fludarabine were added to 2 Gy TBI for additional immunosuppression,35 and no rejections were observed in subsequent patients.34 The proportion of grades II to IV acute GVHD was 46% but only three patients developed grade III–IV acute GVHD, which was fatal in two. Chronic extensive GVHD requiring immunosuppressive treatment was seen in seven (29%) patients, and three of these died from complications related to chronic GVHD. Despite the apparent safety and low toxicity of this conditioning regimen, the rate of NRM could be attributed to complications of both acute and chronic GVHD. GVHD has continued to be a problem after allogeneic HCT. However, recent results have suggested that grade III–IV acute GVHD can be substantially reduced by extending CSP to 180 days (3% in unpublished work). Other investigators have suggested that the presence of full donor chimerism was not required for GVL effects.36,37 In the current study, however, none of the patients lacking full donor T-cell chimerism achieved a molecular remission. Three patients who achieved full donor chimerism without achieving a molecular response died of nonrelapse causes. The current regimen appeared to be less effective in patients transplanted in AP compared to those in CP1 and CP2, although the number of patients treated was too small to allow for definitive conclusions. This suggests that GVL effects were not powerful enough to cure patients with more advanced phases of CML, either because the tumor burden was too large or because tumor cells had lost the ability to trigger GVL reactions or serve

as targets for such reactions. The use of preemptive therapy, such as imatinib in the peritransplant period, might reduce the disease burden and result in better disease control in patients with CML-AP.38 That such an approach might be successful is supported by the current observation that three of four patients with CML-CP2 achieved CR. Despite the relatively short follow-up and small number of patients, the current findings suggest that patients given nonmyeloablative HCT can benefit from GVL effects and achieve complete remissions.37 Lange et al39 reported that CML patients who achieved complete molecular remissions after nonmyeloablative HCT continued to remain PCR negative for the duration of follow-up, demonstrating the durability of response. In a study conducted by Slavin and co-workers, 24 patients with CML in CP1 received HLA-matched related and unrelated HCT after reduced-intensity conditioning using fludarabine and busulfan þ /anti-thymocyte globulin (ATG).40 An overall survival of 85% was reported with a median follow-up of 37 months. Although the results were impressive, they were obtained in a much younger group of patients who had a median age of 35 (range, 3–63) years and were transplanted at an earlier stage of their disease (a median of 14.1 (range, 1–54) months from diagnosis). These good results contrast with those in an even younger group of 17 patients with CML in CP1 (median age 32.1; range, 17–46 years) reported by Das et al41 with a similar conditioning regimen of fludarabine and busulfan plus either 2 Gy TBI or ATG, where an overall survival of only 35% was seen after a median follow-up of 30 months. Finally, a retrospective study of the outcome of transplants with reduced-intensity conditioning for CML was conducted by the EBMT and presented at the American Society of Hematology Meeting in 2002. A total of 223 patients with CML CP1 (62% of patients), CP2 (30% of patients), and AP (7.5% of patients) with a median age of 49 years received a variety of reduced-intensity conditioning regimens for both HLA-matched and -mismatched related and unrelated HCT. The overall survivals at 2 years were 68, 59, and 32% for patients in CP1, CP2, and AP, respectively (Crawley et al, Blood 2002; 100: 781a (abstract)). In summary, the present study shows encouraging results for patients with CML in first CP who were ineligible to receive conventional allogeneic HCT and were conditioned with a nonmyeloablative regimen from HLA-matched related donors. This approach should be considered for patients not eligible for conventional HCT.

Acknowledgements We thank the physicians, nurses, and support staff of the collaborating centers for their dedicated patient care. We are also grateful to the study nurses Steve Minor, John Sedgwick, Mary Hinds, and the data coordinators Heather Hildebrant, Debbie Bassuk and Lucy Fetzko who made this analysis possible. We are grateful to Bonnie Larson and Helen Crawford for help with manuscript preparation. This work is supported in part by NIH Grants: CA78902, CA49605, CA18029, CA15704, Bethesda, MD, the European Leukemia Net, the Kompetenznetzwerk Akute und chronische Leuka¨mien, and from a grant from Ministero dell’Istruzione, dell’Universita`, della Ricerca (MIUR), Italy. FR Kerbauy is supported by a grant from FAPESP/Brazil and an award from the Oncology Research Faculty Development Program from the Department of Health and Human Services, NIH, Bethesda, MD. Leukemia


996 References 1 Thomas ED, Clift RA, Fefer A, Appelbaum FR, Beatty P, Bensinger WI et al. Marrow transplantation for the treatment of chronic myelogenous leukemia. Ann Intern Med 1986; 104: 155–163. 2 Cross NCP, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM. Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood 1993; 82: 1929–1936. 3 Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: 1423–1432. 4 Weiden PL, Flournoy N, Thomas ED, Prentice R, Fefer A, Buckner CD et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300: 1068–1073. 5 Weiden PL, Flournoy N, Sanders JE, Sullivan KM, Thomas ED. Antileukemic effect of graft-versus-host disease contributes to improved survival after allogeneic marrow transplantation. Transplant Proc 1981; 13: 248–251. 6 Dazzi F, Szydlo RM, Cross NC, Craddock C, Kaeda J, Kanfer E et al. Durability of responses following donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2000; 96: 2712–2716. 7 Kolb HJ, Mittermu¨ller J, Clemm CH, Holler G, Ledderose G, Brehm G et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462–2465. 8 Collins RHJ, Shpilberg O, Drobyski WR, Porter DL, Giralt S, Champlin R et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997; 15: 433–444. 9 Clift RA, Radich J, Appelbaum FR, Martin P, Flowers MED, Deeg HJ et al. Long-term follow-up of a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide for patients receiving allogeneic marrow transplants during chronic phase of chronic myeloid leukemia (Letter to Editor). Blood 1999; 94: 3960–3962. 10 Clift RA, Buckner CD, Thomas ED, Bryant E, Anasetti C, Bensinger WI et al. Marrow transplantation for patients in accelerated phase of chronic myeloid leukemia. Blood 1994; 84: 4368–4373. 11 Radich JP, Gooley T, Bensinger W, Chauncey T, Clift R, Flowers M et al. HLA-matched related hematopoietic cell transplantation for chronic-phase CML using a targeted busulfan and cyclophosphamide preparative regimen. Blood 2003; 102: 31–35. 12 Appelbaum FR, Clift R, Radich J, Anasetti C, Buckner CD. Bone marrow transplantation for chronic myelogenous leukemia (Review). Semin Oncol 1995; 22: 405–411. 13 Goldman JM, Szydlo R, Horowitz MM, Gale RP, Ash RC, Atkinson K et al. Choice of pretransplant treatment and timing of transplants for chronic myelogenous leukemia in chronic phase. Blood 1993; 82: 2235–2238. 14 Clift RA, Appelbaum FR, Thomas ED. Treatment of chronic myeloid leukemia by marrow transplantation (Review). Blood 1993; 82: 1954–1956. 15 Talpaz M, Kantarjian HM, McCredie K, Trujillo JM, Keating MJ, Gutterman JU. Hematologic remission and cytogenetic improvement induced by recombinant human interferon alphaA in chronic myelogenous leukemia. N Engl J Med 1986; 314: 1065–1069. 16 Bonifazi F, de Vivo A, Rosti G, Guilhot F, Guilhot J, Trabacchi E, et al., Europena Study Group on Interferon in Chronic Myeloid Leukemia, Italian Cooperative Study Group on CML, France IoC, German CML, UK Medical Research Council Working Party on CML, Spanish CML, Australian CML, Swedish CML. Chronic myeloid leukemia and interferon-alpha: a study of complete cytogenetic responders (Review). Blood 2001; 98: 3074–3081. 17 McSweeney PA, Niederwieser D, Shizuru JA, Sandmaier BM, Molina AJ, Maloney DG et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing highdose cytotoxic therapy with graft-versus-tumor effects. Blood 2001; 97: 3390–3400. 18 Sandmaier BM, McSweeney P, Yu C, Storb R. Nonmyeloablative transplants: preclinical and clinical results. Semin Oncol 2000; 27 (Suppl. 5): 78–81. Leukemia

19 Giralt S, Estey E, Albitar M, van Besien K, Rondo´n G, Anderlini P et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graftversus-leukemia without myeloablative therapy. Blood 1997; 89: 4531–4536. 20 Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998; 91: 756–763. 21 Diaconescu R, Flowers CR, Storer B, Sorror ML, Maris MB, Maloney DG et al. Morbidity and mortality with nonmyeloablative compared to myeloablative conditioning before hematopoietic cell transplantation from HLA matched related donors. Blood 2004; 104: 1550–1558. 22 Mickelson E, Smith A, McKinney S, Anderson G, Hansen JA. A comparative study of HLA-DRB1 typing by standard serology and hybridization of non-radioactive sequence-specific oligonucleotide probes to PCR-amplified DNA. Tissue Antigens 1993; 41: 86–93. 23 Petersdorf EW, Smith AG, Mickelson EM, Martin PJ, Hansen JA. Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRB1 first domain. Immunogenetics 1991; 33: 267–275. 24 Saral R, Burns WH, Laskin OL, Santos GW, Lietman PS. Acyclovir prophylaxis of herpes-simplex-virus infections. N Engl J Med 1981; 305: 63–67. 25 Slavin MA, Osborne B, Adams R, Levenstein MJ, Schoch HG, Feldman AR et al. Efficacy and safety of fluconazole for fungal infections after marrow transplant – a prospective, randomized, double-blind study. J Infect Dis 1995; 171: 1545–1552. 26 Boeckh M, Bowden RA, Goodrich JM, Pettinger M, Meyers JD. Cytomegalovirus antigen detection in peripheral blood leukocytes after allogeneic marrow transplantation. Blood 1992; 80: 1358–1364. 27 Durnam DM, Anders KR, Fisher L, O’Quigley JO, Bryant EM, Thomas ED. Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridization assay. Blood 1989; 74: 2220–2226. 28 Ugozzoli L, Yam P, Petz LD, Ferrara GB, Champlin RE, Forman SJ et al. Amplification by the polymerase chain reaction of hypervariable regions of the human genome for evaluation of chimerism after bone marrow transplantation. Blood 1991; 77: 1607–1615. 29 Sullivan KM, Weiden PL, Storb R, Witherspoon RP, Fefer A, Fisher L et al. Influence of acute and chronic graft-versus-host disease on relapse and survival after bone marrow transplantation from HLAidentical siblings as treatment of acute and chronic leukemia. Blood 1989; 73: 1720–1728. 30 Gratwohl A, Hermans J, Goldman JM, Arcese W, Carreras E, Devergie A et al. Risk assessment for patients with chronic myeloid leukaemia before allogeneic blood or marrow transplantation. Lancet 1998; 352: 1087–1092. 31 Passweg JR, Walker I, Sobocinski KA, Klein JP, Horowitz MM, Giralt SA, Chronic Leukemia Study Writing Committee of the International Bone Marrow Transplant Registry. Validation and extension of the EBMT Risk Score for patients with chronic myeloid leukaemia (CML) receiving allogeneic haematopoietic stem cell transplants. Br J Haematol 2004; 125: 613–620. 32 Radich JP, Gehly G, Gooley T, Bryant E, Clift RA, Collins S et al. Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients. Blood 1995; 85: 2632–2638. 33 Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the presence of competing risks: new representations of old estimators. Stat Med 1999; 18: 695–706. 34 Feinstein LC, Sandmaier BM, Hegenbart U, McSweeney PA, Maloney DG, Gooley TA et al. Non-myeloablative allografting from human leucocyte antigen-identical sibling donors for treatment of acute myeloid leukaemia in first complete remission. Br J Haematol 2003; 120: 281–288. 35 Gregoire V, Hittelman WN, Rosier JF, Milas L. Chemo-radiotherapy: radiosensitizing nucleoside analogues (Review). Oncol Rep 1999; 6: 949–957.


997 36 Mattsson J, Uzunel M, Brune M, Hentschke P, Barkholt L, Stierner U et al. Mixed chimaerism is common at the time of acute graftversus-host disease and disease response in patients receiving nonmyeloablative conditioning and allogeneic stem cell transplantation. Br J Haematol 2001; 115: 935–944. 37 Uzunel M, Mattsson J, Brune M, Johansson JE, Aschan J, Ringden O. Kinetics of minimal residual disease and chimerism in patients with chronic myeloid leukemia after nonmyeloablative conditioning and allogeneic stem cell transplantation. Blood 2003; 101: 469–472. 38 Sloand E, Childs RW, Solomon S, Greene A, Young NS, Barrett AJ. The graft-versus-leukemia effect of nonmyeloablative stem cell allografts may not be sufficient to cure chronic myelogenous leukemia. Bone Marrow Transplant 2003; 32: 897–901.

39 Lange T, Deininger M, Brand R, Hegenbart U, Al-Ali H, Krahl R et al. BCR-ABL transcripts are early predictors for hematological relapse in chronic myeloid leukemia after hematopoietic cell transplantation with reduced intensity conditioning. Leukemia 2004; 18: 1468–1475. 40 Or R, Shapira MY, Resnick I, Amar A, Ackerstein A, Samuel S et al. Nonmyeloablative allogeneic stem cell transplantation for the treatment of chronic myeloid leukemia in first chronic phase (comment in: Blood 2003 Jun 15; 101(12): 5084; author reply 5084–5; PMID: 12788790). Blood 2003; 101: 441–445. 41 Das M, Saikia TK, Advani SH, Parikh PM, Tawde S. Use of a reduced-intensity conditioning regimen for allogeneic transplantation in patients with chronic myeloid leukemia. Bone Marrow Transplant 2003; 32: 125–129.

Leukemia