Long-Term Disease-Free Survival After Nonmyeloablative

ORIGINAL ARTICLE Long-term disease-free survival after nonmyeloablative cyclophosphamide/ ﬂudarabine conditioning and related/unrelated allotransplantation for acute myeloid leukemia/myelodysplasia

RP Nelson Jr1,4,MYu2, JE Schwartz1, MJ Robertson1, R Hromas3, CA Fausel1, GH Vance4,5, SR Dlouhy5, JA Baute1, EA Cox1, LL Wood1, S Srivastava1, KA Robertson4, PR Haut4, SS Farag1,5,7, R Abonour6, K Cornetta1,5,7 and LD Cripe1

1Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 2Division of Biostatistics, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 3Division of Hematology/Oncology, University of New Mexico, Albuquerque, NM, USA; 4Division of Pediatric Hematology and Oncology, Department of Pediatrics, IU Melvin and Bren Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; 5Department of Medical and Molecular Genetics, IU Melvin and Bren Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; 6Department of Pathology and Laboratory Medicine, IU Melvin and Bren Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA and 7Department of Microbiology and Immunology, IU Melvin and Bren Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA

A total of 50 consecutive patients (median age, 57.5 years) Bone Marrow Transplantation (2010) 45, 1300–1308; with AML (n ¼ 30) or myelodysplasia (MDS, n ¼ 20) doi:10.1038/bmt.2009.348; published online 11 January 2010 underwent HLA matched related donor (MRD, n ¼ 27) or Keywords: nonmyeloablative; minimal intensity; trans- unrelated donor (MUD, n ¼ 23) peripheral blood hema- plantation; leukemia; myelodysplasia topoietic cell transplantation after nonmyeloablative CY/fludarabine (Flu) conditioning. GVHD prophylaxis included CsA (n ¼ 19)±mycophenolate mofetil (n ¼ 31). At a median follow-up of 59 months, 21 patients (42%) were alive without evidence of disease. By Kaplan–Meier Introduction analysis, year 1–4 disease-free survival (DFS) and OS estimates were 0.50/0.58, 0.40/0.46, 0.37/0.43 and Allogeneic hematopoietic cell transplantation (HCT) is an 0.37/0.41. MUD recipients were engrafted quickly established therapy for AML and myelodysplasia (MDS).1–5 (13.5 days) compared to MRD recipients (16 days) and Myeloablative conditioning regimens have traditionally relapsed/progressed less frequently (P ¼ 0.005). Overall been used for treatment; however, recognition of alloim- grade 3/4 acute GVHD (aGVHD) occurred in 26% in mune anticancer effects has redirected clinical HCT efforts the absence of antecedent mucositis and was associated toward methods that use less toxic conditioning for patients with chronic GVHD (cGVHD) and poor OS. Extensive with HLA matched donors.6–8 Phase I trials using reduced- cGVHD developed in 51.2% of 100 day survivors. Rates intensity conditioning (RIC) followed by allogeneic HCT of aGVHD, cGVHD and survival were similar between suggest that older patients with hematological malignancies MRD and MUD recipients. Of 14 survivors with experience decreased early TRM and morbidity, evidence cGVHD, 5 (35.7%) experienced resolution off immuno- for antitumor effects and variable rates of donor engraft- suppression, suggesting that tolerance with HLA matched ment and GVHD.9–13 grafts is possible at an advanced age by this method. This Early reports of RIC in AML/MDS showed high rates of study provides further evidence for prolonged DFS after engraftment and low TRM; results of studies with lengthy CY/Flu MRD allotransplantation for AML/MDS, and follow-up are mixed regarding the durability of disease extends the findings to older patients and those with responses.14–18 It is also not known to what extent a state of unrelated donors. tolerance and immunological reconstitution is possible in older patients.19–21 These questions are important, given that TRM and morbidity are significantly reduced when less intense conditioning is administered to older patients in whom these disorders are prevalent.22,23 Correspondence: Professor RP Nelson Jr, Division of Hematology and Although reduced-intensity regimens are associated with Oncology, Department of Internal Medicine, Indiana University School decreased toxicity to recipient tissues compared with high- of Medicine, 535 Barnhill Dr 473, Indianapolis, IN 46202-5289, USA. E-mail: [email protected] dose ‘conventional’ conditioning, most RIC regimens are Received 16 March 2009; revised 2 October 2009; accepted 6 October sufficiently intense to ablate recipient marrow function, and 2009; published online 11 January 2010 cells obtained from the recipient in the early post transplant Long-term DFS after NMAT for AML/MDS RP Nelson et al 1301 period are usually completely donor derived (full chimer- were given to recipients for prophylaxis against viral, ism). Khouri et al.7 Childs et al.9 and Storb et al.12 initially fungal and Gram-negative bacteria. CMV prophylaxis showed that the purine analogue, fludarabine (Flu), given consisted of acyclovir followed by peripheral blood with the alkylating agent CY or low-dose TBI permits monitoring beginning day þ 7 until day þ 100. At day engraftment without myeloablation. Such regimens are þ 100, monitoring was scheduled every 2 weeks until day associated with very low toxicity rates (that is, mucositis, þ 180. Patients with GVHD on active systemic immuno- hepatic injury), are characterized by an initial period of suppression that included glucocorticosteroids and/or mixed chimeric lymphohematopoietic engraftment and monoclonal antibodies were monitored weekly. Initial permit autologous marrow recovery in the event of primary monitoring was by rapid peripheral blood culture. Later, allograft failure.9,12,13,24,25 These particular regimens may we incorporated quantitative PCR, an assay with a lower be designated as ‘minimally intense’ or ‘nonmyeloabla- limit of detection of 100 copies per ml (Viracor, Lee’s tive’.23,26–28 The Indiana University (IU) Stem Cell Trans- Summit, MO, USA).31 plantation Program adopted a CY/Flu regimen as a CY, 60 mg/kg, was administered i.v. on days –6 and À7 nonmyeloablative approach for treatment of hematological (total dose 120 mg/kg). Uroprotection consisted of forced malignancies in older patients or those whose risk for diuresis and sodium 2-mercaptoethane-sulfonate (Mesna). complications of myeloablative transplantation seemed too Fludarabine, 25 mg/m2, was given i.v. as a single daily high.9 The purpose of this study was to assess engraftment infusion for 5 consecutive days (days À5toÀ1; total dose, rates, toxicities and outcomes of patients transplanted for 125 mg/m2). Dosages of chemotherapy were adjusted for AML/MDS. obese patients (4120% ideal body weight) by using the following formula: dosing weight ¼ ideal body weight þ (actualÀideal weight)/2. Patients, donors and methods Patients 1–19 received CsA (3 mg/kg, adjusted for weight/24 h) every 12 h beginning day –1. Dose was Institutional review board-approved companion nonmye- adjusted according to fasting levels to achieve therapeutic loablative protocols were designed and implemented at the but nontoxic levels of 150–250 mg per 100 ml. Patients IU Cancer Center in May 2000, using the Childs et al.9 20–50 received CsA and mycophenolate mofetil (MMF); conditioning regimen for patients with a matched related 1000 mg b.i.d. i.v. or p.o. beginning day þ 1. Oral CsA was (MRD) or unrelated (MUD) donor. Inclusion criteria administered through day þ 100; in the absence of GVHD, considered the hematological malignancy, stage of disease it was tapered until discontinuation by approximately and subject age, and included the following: (1) high-risk day þ 180. MMF was administered through day þ 60 and MDS or AML, (2) relapsed advanced hematological discontinued if there was no GVHD. Although several malignancies after autograft or (3) refractory disease. The patients required CsA/MMF adjustments outside these following patients were considered eligible: (1) age over 50 parameters, the majority received CsA for at least 100 days with an indication for allogeneic transplantation or (2) a and for those who received, MMF, at least 45 days. comorbid condition or poor Eastern Cooperative Oncology Patients with no GVHD, an ANC 4500 per mm3 and Group (ECOG) Study performance status. Patient pre- peripheral blood chimerism 45% donor after day þ 30, or ference for lower-intensity conditioning was considered in those with evidence for disease progression, were eligible to selected cases following comprehensive counseling regard- have immunosuppression reduced or discontinued. Patients ing the differences between nonmyeloablative and myelo- who met the following criteria were eligible for a donor ablative SCT. Exclusion criteria included the following: (1) lymphocyte infusion (DLI) of 1 Â 106 CD3 þ cells per kg active central nervous system disease, (2) pregnancy, (3) (1 Â 105 for MUD) recipient body weight, to be given after SGOT more than three times the upper limit of normal, (4) day þ 45: (1) no GVHD, (2) ANC 4500 per mm3 and creatinine clearance o50 ml/h or glomerular filtration rate peripheral blood chimerism 45% donor and (3) evidence o50 ml/min per 1.73/m2, (5) earlier allogeneic transplant, of disease progression. If GVHD of any grade developed (6) abnormal fractional shortening by echocardiogram and between days þ 30 and þ 45, the patient was not eligible (7) lung diffusion capacity of o50% of predicted normal, for donor lymphocyte infusion. corrected for anemia. Unfractionated whole PBMC chimerism assessment was Donors were required to be HIV seronegative, hepatitis performed on DNA isolated from 2–3 ml whole blood B antigen-negative and HLA 6/6 or 5/6 MRD (HLA-A, (Gentra Kit Method; Gentra Systems, Minneapolis, MN, HLA-B and HLA-DR loci; serological class I, high- USA) analyzed by DNA RFLP or STR at the following resolution class II) or 6/6 matched unrelated.29 A total of time points: days þ 30, þ 60, þ 100, 6 months, 1 year and 49 patients underwent treatment with G-CSF followed by as clinically indicated.32,33 Evidence of donor engraftment apheresis that started on the fifth day as described.30 was defined as an ANC 4500 per mm3 at day þ 30 and Apheresis was performed on 1 additional day with the goal 45% donor chimerism. Time to resolution of cytopenias of obtaining 5 Â 106 CD34 þ cells per kg recipient body was defined as the number of days post transplant required weight. One related donor could not undergo G-CSF to reach an ANC 4500 per mm3 for 3 consecutive days and mobilization and provided BM harvested under general platelet transfusion independence for 7 days followed by anesthesia as per standard procedure. platelet count 420 000 per mm3 for 3 consecutive days. Patients were admitted to a dedicated BMT unit with The National Cancer Institute Common Toxicity Criteria laminar airflow or HEPA-filtered rooms under conditions version 2.0 was used for the evaluation of regimen-related of reverse isolation. Acyclovir, fluconazole and quinolone toxicity and GVHD was evaluated by established severity

Bone Marrow Transplantation Long-term DFS after NMAT for AML/MDS RP Nelson et al 1302 scales.34,35 Cytogenetic risk factors were applied according Table 1 Patient characteristics to Greenberg et al.36 and Slovak et al.37 Characteristic N % The National Marrow Donor Program (NMDP) guidelines for classiﬁcation of causes of death were used (form Age, years 190). Acute GVHD (aGVHD) was listed for patients who Median (57.5 years; range, 16–66) had severe, progressive aGVHD. If the patient had no o55 18 32 455 32 64 aGVHD symptoms and died of an opportunistic infection Female 19 38 while receiving immunosuppression, the infection was Male 31 62 considered primary and aGVHD a contributing cause of death. We considered chronic GVHD (cGVHD) as the HCT-CI score (median ¼ 3) 3 or less 29 58 primary cause of death for patients with severe, sympto- 4 or more 21 42 matic cGVHD. Infection or other complications were Sex-matched pairs 22 44 considered in these cases as a contributing cause of death. CMV-donor/recipient pairs positive 47 94 cGVHD was considered a contributing cause of death if the ABO-matched pairs 36 72 patient died of an opportunistic infection with controlled Donor age (median, 45) cGVHD while receiving immunosuppression. Infections MRD 27 54 that were indwelling venous catheter related were consid- MUD 23 46 ered primary causes of death. The initial study of patients with a variety of hemato- Diagnosis AML 30 60 logical malignancies ﬁnished accrual in October 2004 and Cytogenetic risk outcomes were analyzed. Given the favorable results, the Good 3 10 method was adopted at IU as a treatment plan for transplant Intermediate 11 37 candidates deemed not eligible for conventional conditioning. Poor 16 53 Patients were followed until death or 412 months (median, De novo 11 37 Secondary 16 63 629 days; survivors, 1715 days or 59 months). This report First remission 13 43 concerns the outcomes of consecutively and uniformly treated Second remission 9 30 patientswithAML/MDSfrom4May2000to10January Not in documented remission 7 23 2008, with survivor follow-up through 1 July 2009. Relapse (peripheral blasts) 1 3 Myelodysplasia 20 40 Subtypes Statistical analyses Refractory anemia 4 20 Baseline patient characteristics and survival information Refractory anemia with ringed sideroblasts 1 5 were compared between MRD and MUD groups and Refractory anemia with CLL 1 5 between those who received CsA and CsA/MMF. Fisher’s RAEB I 4 20 exact test was used for categorical variables to account for RAEB II 6 30 CMML 5 25 small sample sizes; the t-test was used for continuous IPSS variables. In some cases, when the normality assumption Intermediate-1 9 47 was in question, the nonparametric Wilcoxon’s signed-rank Intermediate-2 7 37 test was used for comparing two groups. The log-rank test High 3 16 was used for survival outcomes. When constructing the Abbreviations: HCT-CI ¼ hematopoietic cell transplant comorbidity index; Kaplan–Meier curve for survival, patients alive at the end IPSS ¼ International Prognostic Scoring System; MRD ¼ matched related of the study were regarded as censored. donor; MUD ¼ matched unrelated donor.

Results 1 had AML with myeloﬁbrosis and 1 patient was in frank leukemic relapse. Of 30, 19 (63.3%) patients had leukemia Patients and donors secondary to MDS or treatment for an antecedent second Thirty-one men (62%) and 19 women (38%) underwent non- malignancy. The International Prognostic Scoring System myeloablative allogeneic transplantation (NMAT) for AML/ (IPSS) risk scores for patients with MDS are given in MDS from May 2000 to 10 January 2008. The median age was Table 1. The overall median donor age was 45 years (MRD, 57.5 years (range, 17–66; median age of the recipients of MUD 54; MUD, 30.5; P ¼ 0.091). graftswas57vs58yearsforMRD recipients). Among them, 80% were more than 50 years. Of 50, 46 (92%) recipients and 19 (38%) of donors were CMV positive before transplant. Engraftment and hematopoietic recovery Fifty-six percent of recipients were not in remission at the CD34 þ cell dosages ranged from 0.6 to 15.3 Â 106 time of transplant. Among them, 22 (44.0%) had AML in CD34 þ cells per kg (median, 4.95 Â 106 cells per kg) and ﬁrst (n ¼ 13) or second (n ¼ 9) CR; 2 were cytopenic after CD3 þ cell dosages ranged from 0.75 to 6.2 Â 108 per kg induction chemotherapy without increased marrow blasts; (median, 1.8) recipient body weight. An ANC 4500 per 2 had CMML that transformed to AML successfully mm3 was reached at a median time of 15 days (range, 12–23 induced to baseline CMML (coded as AML per NMDP days), 16 days for those with an MRD and 13.5 days for guidelines); 1 had secondary AML and concomitant CLL; those with a MUD (univariate P ¼ 0.048). All patients

Bone Marrow Transplantation Long-term DFS after NMAT for AML/MDS RP Nelson et al 1303 achieved an ANC of 500 per mm3 or more by 28 days after Both donor and recipient were CMV seronegative in three SCT. No patients experienced a decrease in ANC to o500 (6%) transplants. During the initial year (or until death if per mm3 for 3 consecutive days after having achieved survival o1 year) 62 (12.2%) of 759 post transplant direct neutrophil engraftment (secondary graft failure) or failed tests (shell culture early antigen detection or PCR) were to have evidence of at least 50% or more donor cell positive. Seventeen (34%) patients experienced CMV engraftment. Rates of complete donor chimerism in living activation beginning a median of 59 days post transplant patients at various time points were the following: day and it was resolved in 16 (94%) at a median of 90 days post þ 30, 56%; day þ 100, 55.3%; 6 months, 88.9% and 1 transplant. One patient had CMV enteritis in the context of year, 90%. Seven patients who achieved full donor aGVHD as a cause of death; one patient had CMV chimerism subsequently lost it; in each case, this was esophagitis, also with GVHD that responded to treatment. associated with disease relapse. No patients required Eleven skin cancers were diagnosed in six patients; all were DLI for encouragement of engraftment; ﬁve received excised without dissemination.41 DLI for disease recurrence. Nine patients failed to achieve Figures 1 and 2 show 1- to 4-year disease-free survival p80% donor chimerism at day þ 100. Three experi- (DFS)/OS estimates to be 0.50/0.58, 0.40/0.46, 0.37/0.43 enced disease progression never having achieved predominant and 0.37/0.41 by Kaplan–Meier analysis. OS and DFS donor chimerism; two relapsed/progressed after achieving were not different between MRD and MUD recipients 100% donor chimerism; one died of infection before achieving (P ¼ 0.633 and 0.859, respectively). There was no difference 100% donor chimerism; two converted to 100% donor and achieved CR after stopping immunosuppression and one patient with MDS achieved a transfusion-free mixed chimeric state (approximately 60% donor) for 14 months and then 1.0 transformedtoAMLday þ 426. 3 Matched related Multivariate analysis for time to ANC 4500 per mm All patients for MRD/MUD, including CD34 and CD3 cell dosages 0.8 Matched unrelated and GVHD prophylaxis (CsA vs CsA/MMF) rendered a P-value of 0.059. An ANC 42000 per mm3 was reached at a median time of 18 days (range, 14–71 days), 18 and 0.6 19 days for MRD and MUD recipients, respectively (univariate P ¼ 0.363). 0.4

GVHD Probability of survival Grades 2–4 and 3–4 aGVHD occurred in 38.0 and 26.0% 0.2 of patients at a median of 27 and 21 days after MRD Log-rank test (MRD vs. MUD) P-value = 0.633 and MUD infusion, respectively. ‘Late onset’ aGVHD’ was diagnosed in 1 patient and 11 (22.0%) ﬁt the deﬁnition of 0.0 21,38 ‘serious GVHD’ given by Flowers et al. There was no 0 500 1000 1500 2000 2500 3000 difference in rates of any (P ¼ 0.15) or grade 3–4 (P ¼ 0.48) Days post BMT aGVHD in patients who received CsA or CsA/MMF. cGVHD or extensive cGVHD occurred after a median Figure 1 Kaplan–Meier (KM) curve for overall survival. of 184 days in 29 of 41 (70.7%) and 21 of 41 (51.2%) of 100-day survivors, respectively. The incidence of cGVHD was not different between MRD and MUD recipients 1.0 (P ¼ 0.305). At a median of 4.7 years, 8 of 21 (38.1%) Matched related All patients survivors were living with no GVHD, 3 (14.3%) had Matched unrelated limited and 10 (47.6%) had extensive cGVHD, and ECOG 0.8 performance status scores of 0 (n ¼ 18), 1 (n ¼ 2) and 2 (n ¼ 1). Two survivors required pulmonary specialty care for symptomatic bronchiolitis obliterans and no one 0.6 experienced malabsorption or chronic liver disease.39

0.4 Transplant-related complications and survival No patient experienced mucositis, lymphoproliferative disease or sinusoidal obstructive syndrome (SOS). Throm- 0.2 botic microangiopathy with hemolytic anemia occurred in Log-rank test (MRD vs. MUD) P-value = 0.859 two patients; one contributed to a patient’s death and one 40 was resolved with discontinuation of CsA. Nonhemato- Disease free survival distribution function 0.0 logical grade 4 toxicities included clinical/radiological 0 500 1000 1500 2000 2500 3000 ﬁndings consistent with obliterative bronchiolitis (n ¼ 3), all of whom had cGVHD. Of 50, 46 (92%) recipients and Days post BMT 19 (38%) donors were CMV positive before transplant. Figure 2 Kaplan–Meier (KM) curve for disease-free survival.

Bone Marrow Transplantation Long-term DFS after NMAT for AML/MDS RP Nelson et al 1304 in the DFS or OS rate between those with or without active required for engraftment. The rapid achievement of an disease (0.661). This may be explained by the high percentage ANC 4500 per mm3 observed in MUD recipients is of patients with high-risk cytogenetics in both groups and presumably due to the action of donor lymphocytes of low number of patients in frank relapse at the time of greater histocompatibility disparity in an environment transplant. devoid of the highly potent immunosuppressives such as Causes of death were disease relapse/progression (n ¼ antithymocyte globulin or alemtuzumab.43,44 In this regard, 13), infection (n ¼ 6), GVHD (n ¼ 6) and other (n ¼ 4). the time to aGVHD was 21 days for MUD recipients MRD and MUD recipients were more likely to die from compared to 27 days for MRD recipients. disease relapse and GVHD, respectively. Eight (16.0%) CY and Flu are active pharmacological agents against died during the first 100 days (two within 30 days), six of myeloid neoplasms; therefore, observed disease responses transplant-related causes and two of disease progression. cannot be unequivocally attributed to the allograft.45 OS in the AML group was 40% and in the MDS group it Nonetheless, it is important to realize that 39 (78%) of was 45% (P ¼ 0.726). Twenty-one surviving patients were the entire cohort had either disease past first remission, in remission a median of 4.7 years post transplantation with poor risk cytogenetics (AML), secondary AML, RAEB, ECOG performance status scores of 0 (n ¼ 18), 1 (n ¼ 2) CMML or ‘int-2’-‘high’ MDS as per IPSS. This is a patient and 2 (n ¼ 1). population that would not be expected to be successfully treated with chemotherapy alone. The fact that a significant fraction of patients experienced durable remissions (some Disease responses of whom whose cGVHD resolved) argues in favor of Of 28, 26 patients with active disease at the time of GVL effects active in the early post transplant period; this transplant were evaluable for disease responses and 19 interpretation agrees with Kumar et al., who suggest that (73.1%) experienced a complete response, 5 (19.2%) a slow lymphocyte recovery after matched-sibling allogeneic partial response, 1 each had stable disease and disease BMT for AML predicts subsequent relapse.46–50 In fact, the progression as the best response. Of 22 patients trans- overall relapse rate of 30.0% at 4.7 years is comparable to planted in remission, 14 of 20 evaluable maintained a that observed with RIC regimens or conventional allo- CR (70%). A total of 15 of 50 (30.0%) patients relapsed/ transplantation.1,51–53 The particularly low relapse rate progressed (MRD, 13; MUD, 2; P ¼ 0.005). The rate of with MUD transplants (9%) suggests that given sufficient disease progression or relapse was 70% in those who failed immunosuppression to effect engraftment, the risk of to achieve 100% donor chimerism by day þ 100; 86% of progression/relapse after MUD transplantation may be relapses/progressions occurred within the first year. Two of more fundamentally susceptible to increases in GVHD eight (25%) patients who relapsed reestablished lasting preventive intensity than the relative strength of condition- remissions with immunosuppressive withdrawal and DLI, ing.18 The results that we have observed in patients with respectively; there have been no relapses after 2 years of AML/MDS, many of whom had received chemotherapy as transplantation. part of disease management before transplant, cannot be necessarily extrapolated to patients treated for other hematological malignancies; this pertains particularly to Discussion CML, as shown by Sloand et al.54 Results of reduced-intensity or minimal intensity, non- Grigg et al.42 recently reported results of a clinical myeloablative, AML/MDS disease-specific investigations investigation using nonmyeloablative CY/Flu to prepare with 2 and at least 3 years of follow-up that preceded 34 patients with de novo AML in first CR for matched- this report are summarized in Tables 2 and 3. There is sibling allotransplantation. Patients experienced relatively one published minimally intensive allotransplant study in low incidences of grades 2–4 aGVHD (21%) and cGVHD AML/MDS with 2 years median follow-up, and one other (24%). Thirteen (38%) patients relapsed and estimated with at least 3 years of follow-up suitable for comparison. DFS and OS at 2 years were 56 and 68%, respectively.42 Scott et al.16 treated 38 patients (median age, 62 years) with The current investigation extends these findings, with truly nonmyeloablative TBI or TBI/Flu and CsA/MMF longer follow-up, to patients of advancing age and GVHD prophylaxis (n ¼ 36), and compared results with recipients of unrelated grafts, 75% of whom were not in a 112-patient cohort who received myeloablative BU/CY first CR at the time of transplantation. Mucositis and SOS over the same period. In their study, 3-year OS was were avoided, predominantly donor chimerism occurred in 27%, progression-free survival, 28% and nonrelapse all patients and DLI was not needed to encourage donor mortality, 41%, findings that did not differ significantly engraftment. Full donor chimerism was achieved by 6 months between conventional and nonmyeloablative groups.16 in most patients, which appears more rapid compared to Hegenbart et al. reported findings of 122 patients treated the radiation-based nonmyeloablative method, in which at multiple centers using the same regimen with median mixed donor/recipient T cell chimeras remained in more follow-up of 44 months. Cumulative mortalities from patients 6 months post transplant.27 Recipients of MUD disease progression were 47 and 33% at 2 years for MRD allografts experienced low rates of disease progression and MUD recipients, respectively. Overall 2-year survival and statistically equivalent rates of aGVHD, cGVHD was 48% and DFS, 44%.58 and survival compared with MRD recipients. CY/Flu The utility of mixed chimerism in limiting GVHD appears to suppress recipient resistance sufficiently to reported in animal models was not realized in this permit donor cells to deliver the antihematopoiesis activity study.25,61,62 A transient period of mixed chimerism was

Bone Marrow Transplantation Long-term DFS after NMAT for AML/MDS RP Nelson et al 1305 Table 2 Clinical investigations of RIC or minimal intensity conditioning and allotransplantation for AML/MDS and 2-year follow-up

First author, Median Donors Diseases Conditioning GVHD Median GVHD Survival year N age (years) prophylaxis survivor acute PFS OS follow-up 2–4 Chronic (months) extensive

Claxton, 200557 59 Haplo ¼ 6 AML, n ¼ 23 CY1000 mg/m2 Â 2 Tacrolimus 16 43% 2-year ¼ 50% n ¼ 23 MRD ¼ 6 Flu 25 mg/m2 Â 5 Sirolimus 50% MUD ¼ 10 MTX+ATG 5/6 URD ¼ 1

Hegenbart, 57.5 MRD ¼ 58 AML, n ¼ 122 200 cGy TBI (n ¼ 2) CsA+MMF 44 40% 2 year ¼ 44% 200658 MUD ¼ 64 CR ¼ 73.8% or 200 cGy TBI 36% 2 year ¼ 48% n ¼ 122 Not in CR ¼ 26.2% (n ¼ 36) Flu, 90 mg/m2

Grigg, 200742 45 MRD only De novo AML in ﬁrst Flu 125/m2 CsA and MTX 34.8 2-year ¼ 68% 2-year ¼ 56% N ¼ 34 remission CY 120 mg/kg Cytogenetics Intermediate ¼ 85% Poor ¼ 15%

Oran, 200759 55 MRD ¼ 59 AML, n ¼ 82 Flu 25 mg/m2 or Tacrolimus 29.4 39% PFS not n ¼ 112 MUD ¼ 53 MDS, n ¼ 30 30 mg/m2 for and low-dose reported (4 different PBHCs, n ¼ 56 CR ¼ 27% 4–5 days and Mel MTX+ATG protocols) BM, n ¼ 56 Not in CR ¼ 73% 100/m2 (n ¼ 11) (n ¼ 31) Cytogenetics or Mel 140/ Good risk ¼ 2.7% m2±gemtuzimab Intermediate ¼ 51.8% (n ¼ 46) Poor ¼ 42.9% or Mel 180/m2 (n ¼ 53) 44% 2-year ¼ 44%

Abbreviations: ATG ¼ antithymocyte globulin; Flu ¼ ﬂudarabine; haplo ¼ haploidentical; Mel ¼ melphalan; Mel ¼ melphalan; MMF ¼ mycophenolate mofetil; MRD ¼ matched sibling; MUD ¼ matched unrelated; PBHCs ¼ peripheral blood hematopoietic cells; URD ¼ unrelated donor.

Table 3 Clinical investigations of RIC or minimal intensity (nonmyeloablative) conditioning allotransplantation for AML/MDS: 3- or 4-year follow-up

First author, year n, median Donors Conditioning GVHD prophylaxis Median survivor OS Progression-free age (year) follow-up (months) (year, %) survival

Tauro, 200560 76, MRD ¼ 35 Flu 150/m2 Mel 140/m2 Alemtuzumab 36 3, 41 3 years: 37% 52 MUD ¼ 41 MRD ¼ 45% MUD ¼ 31% Hallemeier, 200655 51, MRD ¼ 21 TBI, 550 cGy CsA+steroids or MTX 44 3, 37 3 years: 37% 44 MUD ¼ 30 CY 120 mg/kg MRD ¼ 47% MUD ¼ 27% Scott, 200616,a 38, MRD ¼ 26 200 cGy TBI (n ¼ 2) CsA+MMF 20.4 3, 28 3 years ¼ 27% 62 MUD ¼ 12 or 200 cGy TBI (n ¼ 36) Flu, 90 mg/m2 Valcarcel, 200718 93, MRD only Flu 150 mg/m2 CsA and MTX or MMF 43 4, 43 4 years ¼ 43% 53 BU, 8–10 mg/kg Nelson, 2009 50, MRD ¼ 27 Flu 125/m2 CsA±MMF 58 4, 41 4 years ¼ 37% (current submission) 57.5 MUD ¼ 23 CY 120 mg/kg MRD ¼ 44% MUD ¼ 39%

Abbreviations: Flu ¼ ﬂudarabine; Mel ¼ melphalan; MMF ¼ mycophenolate mofetil; MRD ¼ matched related donor; MUD ¼ matched unrelated donor. aOne arm of nonmyeloablative versus myeloablative retrospective comparison.

followed by the achievement of full donor chimerism tional conditioning and associated with the development of relatively soon compared with a radiation-based minimally extensive cGVHD and poor survival in multivariate intensive nonmyeloablative regimen.27 Clinically detectable analysis. This ﬁnding provides evidence that injury to host mucositis was avoided and engraftment was initially tissues is not a requirement for aGVHD with this method; characterized as mixed; nonetheless, the incidence of grade rather, the GVH reaction is likely activated by direct 3–4 aGVHD was similar to that observed after conven- or indirect antigen presentation to donor lymphocytes

Bone Marrow Transplantation Long-term DFS after NMAT for AML/MDS RP Nelson et al 1306 through specific receptors propagated by selectively active a busulfan-Cytoxan versus Cytoxan-total body irradiation as cytokines.63–68 The onset appeared 1–2 weeks later than preparative regimen: a report from the Group d’Etudes de la that usually observed after standard conditioning/prophy- Greffe de Moelle Osseuse. Blood 1992; 79: 2578–2582. laxis and MMF appeared to add little, if any, anti-GVHD 2 Chopra R, Goldstone AH. Modern trends in bone marrow activity to CsA alone in this context, as predicted by Mohty transplantation for acute myeloid and acute lymphoblastic leukemia. Curr Opin Oncol 1992; 4: 247–258. et al.69 Thirty percent of patients experienced no cGVHD, 3 Morgenstern GR, Powles RL. Allogeneic bone marrow 47% with cGVHD have tolerated reductions in immuno- transplantation for acute myeloid leukemia in first remission. suppression and 33% with extensive cGVHD have Haematol Blood Transfus 1981; 26: 139–142. experienced resolution and are receiving no GVH therapy. 4 Speck B. [Indications for bone marrow transplantation in Given the mean current survivor age of 63 years, tolerance aplastic anemia and acute myeloid leukemia]. Schweiz Med induction appears possible in older patients conditioned in Wochenschr 1973; 103: 508–511. this manner without disease recurrence over extended 5 Thomas ED, Buckner CD, Banaji M, Clift RA, Fefer A, periods. Flournoy N et al. One hundred patients with acute leukemia We are currently studying the potential for eliminating in treated by chemotherapy, total body irradiation, and allo- vivo alloreactive T cells, a form of negative selection in the geneic marrow transplantation. Blood 1977; 49: 511–533. post transplant period, with an inhibitor of activated T cells 6 Giralt S, Estey E, Albitar M, van Besien K, Rondon G, Anderlini P et al. Engraftment of allogeneic hematopoietic that is approved for solid organ rejection prevention, for progenitor cells with purine analog-containing chemotherapy: 70–72 a number of reasons. First, over the years in the HLA harnessing graft-versus-leukemia without myeloablative ther- matched setting, the addition of broadly active immuno- apy. Blood 1997; 89: 4531–4536. suppressants or lymphocyte depletion has yet to limit the 7 Khouri IF, Keating M, Korbling M, Przepiorka D, Anderlini GVHD problem compared with standard calcineurin- P, O’Brien S et al. Transplant-lite: induction of graft-versus- inhibitor/MTX prophylaxis, without the consequence of malignancy using fludarabine-based nonablative chemother- higher relapse/progression rates. Second, patients referred apy and allogeneic blood progenitor-cell transplantation as to our institution for NMAT were of advanced age or with treatment for lymphoid malignancies. J Clin Oncol 1998; 16: comorbid conditions and intermediate to poor cytoge- 2817–2824. netics, but not in florid relapse; therefore, a potent, well- 8 Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G et al. Nonmyeloablative stem cell transplantation tolerated therapy for older recipients with high-risk features and cell therapy as an alternative to conventional bone marrow and low tumor burden is needed. Third, we saw no transplantation with lethal cytoreduction for the treatment of apparent additional anti-GVHD prophylactic activity with malignant and nonmalignant hematologic diseases. Blood adjunctive MMF.69,73 Finally, the rapid engraftment 1998; 91: 756–763. dynamic, relatively early achievement of full donor 9 Childs R, Clave E, Contentin N, Jayasekera D, Hensel N, chimerism and potent GVL effects presumably delivered Leitman S et al. Engraftment kinetics after nonmyeloablative soon after transplant, appears to support such a concept.44 allogeneic peripheral blood stem cell transplantation: full A minimal intensity HCT allotransplant approach might donor T-cell chimerism precedes alloimmune responses. Blood then be considered an attractive therapeutic option for 1999; 94: 3234–3241. those of advancing age with AML/MDS and either an 10 Grigg A, Bardy P, Byron K, Seymour JF, Szer J. Fludarabine- based non-myeloablative chemotherapy followed by infusion HLA MRD or MUD. of HLA-identical stem cells for relapsed leukaemia and lymphoma. Bone Marrow Transplant 1999; 23: 107–110. 11 Sandmaier BM, McSweeney P, Yu C, Storb R. Nonmyelo- Conflict of interest ablative transplants: preclinical and clinical results. Semin Oncol 2000; 27:78–81. The authors declare no conflict of interest. 12 Storb R, Yu C, Sandmaier B, McSweeney P, Georges G, Nash R et al. Mixed hematopoietic chimerism after hematopoietic stem cell allografts. Transplant Proc 1999; 31: 677–678. Acknowledgements 13 Sykes M, Preffer F, McAfee S, Saidman SL, Weymouth D, Andrews DM et al. Mixed lymphohaemopoietic chimerism We acknowledge the work of Judy Amlin, Kelly Ward, and and graft-versus-lymphoma effects after non-myeloablative Elizabeth Taylor, who took excellent care of the participants therapy and HLA-mismatched bone-marrow transplantation. in this study, and Patty Fredenburgh for care coordination. Lancet 1999; 353: 1755–1759. We also thank the community physicians for their kind referral 14 Aoudjhane M, Labopin M, Gorin NC, Shimoni A, Ruutu T, of patients, for the dedicated nursing staff in the BMT Units Kolb HJ et al. Comparative outcome of reduced intensity at Indiana University and the Clarian Health System, and the and myeloablative conditioning regimen in HLA identical supportive administration at Indiana University School of sibling allogeneic haematopoietic stem cell transplantation Medicine and the IU and Melvin and Bren Simon Cancer for patients older than 50 years of age with acute myeloblastic Center. leukaemia: a retrospective survey from the Acute Leukemia Working Party (ALWP) of the European group for Blood and Marrow Transplantation (EBMT). Leukemia 2005; 19: References 2304–2312. 15 de Lima M, Anagnostopoulos A, Munsell M, Shahjahan M, 1 Blaise D, Maraninchi D, Archimbaud E, Reiffers J, Devergie Ueno N, Ippoliti C et al. Nonablative versus reduced-intensity A, Jouet JP et al. 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