Sequential Intensified Conditioning and Tapering of Prophylactic Immunosuppressants for Graft-versus-Host Disease in Allogeneic Hematopoietic Stem Cell Transplantation for Refractory Leukemia Academic research paper on "Clinical medicine"

ASBMI

American Society for Blood and Marrow Transplantation

Sequential Intensified Conditioning and Tapering of Prophylactic Immunosuppressants for Graft-versus-Host Disease in Allogeneic Hematopoietic Stem Cell Transplantation for Refractory Leukemia

Qi-Fa Liu,1 Zhi-Ping Fan,1 Yu Zhang,1 Zu-Jun Jiang,2 Chun-Yan Wang,3 Dan Xu,1

Jing Sun, Yang Xiao, Huo Tan

For patients with advanced leukemia undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), a major obstacle to success, especially in those with a high leukemia cell burden, is relapse of the underlying disease. To improve the outcome of allo-HSCT for refractory leukemia, we investigated the strategy of sequential intensified conditioning and early rapid tapering of prophylactic immunosupressants therapy for graft-versus-host disease (GVHD) during the early stage after transplantation. A total of 51 patients with refractory leukemia (median age, 30.0years; unfavorable karyotypes, 49%) received fludarabine (Flu) 30 mg/m2/day and cytarabine 2 g/m2/day (on days — 10 to —6), 4.5 Gy total body irradiation (TBI)/day (on days —5 and —4), and cyclophosphamide (Cy) 60 mg/kg/day and etoposide 600 mg/day (on days — 3 and —2) for conditioning. Cyclo-sporine A (CsA) was withdrawn rapidly in a stepwise fashion to avoid overwhelming GVHD reactions if acute GVHD (aGVHD) did not develop at day 130. All 51 patients developed regimen-related toxicities (13 with grade III-IV); 93.9% of them achieved complete remission by day 130. Median follow-up was 41 months (range, 6.6 to 92.2 months); 5-year overall survival (OS) and disease-free survival (DFS) were 44.6% 6 8.1% and 38.2% 6 7.7%, respectively. Thirteen patients relapsed; the 3-year cumulative incidence of leukemia relapse was 33.3%. On multivariate analysis, cytogenetic status was the only significant pretransplantation factor. Survival was better in patients with grade I or II aGVHD than in those without aGVHD. Our data indicate that the sequential strategy of cytoreductive chemotherapy followed immediately by intensified myeloablative (MA) conditioning for allo-HSCTand rapid tapering of prophylactic immunosuppressants for GVHD in the early stage after transplantation has an acceptable toxicity profile and may be a better approach to treating refractory leukemia. Biol Blood Marrow Transplant 15: 1376-1385 (2009) © 2009 American Society for Blood and Marrow Transplantation

KEY WORDS: Cytoreductive chemotherapy, Myeloablative conditioning, Refractory leukemia

INTRODUCTION

The management of refractory leukemia in adults is very difficult. Although several salvage regimens have been used successfully to induce remission in this group of patients, the duration of remission is often brief, and there are few long-term survivors [1-3].

From the 'Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2Department of Hematology, Guangzhou General Hospital of Guangzhou Command, Guangzhou 510010, China; and Centre of Oncology and Hematology, First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510230, China. Financial disclosure: See Acknowledgments on page 1384. Correspondence and reprint requests: Qi-Fa Liu, Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China (e-mail: liuqifa@fimmu.com). Received May 12, 2009; accepted June 26, 2009 © 2009 American Society for Blood and Marrow Transplantation 1083-8791/09/1511-0004$36.00/0 doi:10.1016/j.bbmt.2009.06.017

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is commonly perceived as the only curative option for refractory leukemia [4-7]. Unfortunately, however, the outcome of allo-HSCT in patients with refractory leukemia depends largely on disease status at the time of transplantation [6-8]. Patients with an advanced disease who proceed directly to HSCT, especially those with a high leukemia cell burden, are likely to do poorly [4,7,9]. The relapse rate exceeds 50% in these patients after transplantation with a standard myeloablative (MA) regimen consisting of total body irradiation (TBI) or busulfan (Bu) combined with cyclophosphamide (Cy) [10-12]. Modifications of the MA conditioning regimen using various combinations of chemotherapy and radiotherapy have failed to reduce the risk of relapse in refractory patients [13,14]. Reduced-intensity conditioning (RIC) regimens have been advocated to reduce transplantation-associated toxicity in elderly or medically unfit patients [15,16]; however, in advanced leukemia, RIC may not

sufficiently control the disease to allow a graft-versus-leukemia (GVL) effect to occur, and disappointing results have been reported with RIC transplantation in patients with advanced disease [17]. The intensity of the conditioning regimen has been shown to directly affect the relapse rate and disease-free survival (DFS) after allo-HSCT for refractory leukemia [18,19].

Approaches to improving outcomes in patients with refractory leukemia include administering salvage chemotherapy to decrease the leukemia cell burden before transplantation and increasing the intensity of the conditioning regimen. Suitable salvage regimens and intensified conditioning regimens must have acceptable toxicity and mortality, however. Recently, Schmid and coworkers [7,20] reported that a sequential regimen of salvage chemotherapy and RIC was effective and had an acceptable toxicity profile in allo-HSCT to treat acute myelogenous leukemia (AML) and myelodysplas-tic syndrome (MDS). But, despite these encouraging overall results, the relapse rate remains significant [7,20].

In addition to the antileukemic effect of conditioning regimens, the therapeutic efficacy of allo-HSCT also relies on the GVL reaction. Prophylactic transfusion of donor lymphocytes is commonly used to further exploit the GVL reaction in patients with refractory leukemia [21,22]. Clinical observations show that cyclosporine A (CsA) withdrawal can induce complete remission (CR), and this is generally used as firstline treatment in patients with relapsed leukemia after allo-HSCT [23-25]. Recently, our group introduced a regimen of sequentially intensified conditioning and tapering of prophylactic immunosuppressants for graft-versus-host disease (GVHD) in allo-HSCT for patients with refractory leukemia. The regimen consists of cytoreductive chemotherapy, followed immediately by intensified MA conditioning and then rapid tapering of prophylactic immunosuppressants for GVHD in the early stage after transplantation. A preliminary subgroup analysis of this trial suggested that patients with refractory leukemia might especially benefit from this strategy [26]. The differences between our trial and the trial reported by Schmid and coworkers are in the heterogeneity of the patients included, the drugs used for and intensity of conditioning, the interval between cytoreductive chemotherapy to conditioning, and the prophylactic immun-osuppressive therapy for GVHD. The current study was initiated to assess the feasibility and efficacy of this approach in patients with refractory leukemia in a multicenter trial involving 3 institutions in China.

PATIENTS AND METHODS

Patients and Eligibility Criteria

The study cohort comprised 51 patients with adult refractory leukemia divided into 2 groups. The first

group included 18 consecutive patients with refractory leukemia treated at Nanfang Hospital between May 2001 and May 2004; their records were reviewed retrospectively. The second group included 33 consecutive patients with refractory leukemia who were involved in a prospective multicenter trial at Nanfang Hospital, Southern Medical University, Guangzhou General Hospital of Guangzhou Command, and First Affiliated Hospital, Guangzhou Medical College between June 2004 and June 2008. The patients' median age at transplantation was 30 years (range, 14 to 53 years). They were categorized as having AML, acute lympho-blastic leukemia (ALL), acute biphenotypic leukemia (ABL), or chronic myelogenous leukemia with blast crisis (CML-BC) according to the World Health Organization classification system [27]. All patients who met at least 1 of the following criteria for refractory leukemia were included: (1) primary induction failure (PIF) after 2 or more cycles of chemotherapy, (2) relapse refractory to salvage combination chemotherapy, and (3) CML-BC resistant to tyrosine kinase inhibitors and PIF after 2 or more cycles of chemotherapy. The final inclusion criterion was age between 14 and 55 years. Exclusion criteria included creatinine clearance < 50 mL/min, bilirubin or transaminase level > 2 times the upper limit of normal, cardiac shortening fraction < 30%, and pregnancy. The study was performed in accordance with the modified Helsinki Declaration, and the protocol was approved by the respective ethical review boards before study initiation. All patients provided written informed consent.

HLA Typing

Standard serologic typing was used for HLA-A and -B, and genomic high-resolution molecular typing was used for HLA-DRB1 in HLA-matched sibling donors. Genomic high-resolution molecular typing was used for HLA-A, -B, and -DRB1 for HLA-mismatched family donors and unrelated donors.

Sequential Intensified Conditioning Regimen

The sequential intensified conditioning regimen for allo-HSCT has been described in detail previously [26]. In brief, patients received fludarabine (Flu) 30 mg/m2 and moderate- dose cytarabine (Ara-C), 2 g/m2, from day —10 to day —6, 4.5 Gy of TBI on days —5 and —4, and Cy 60 mg/kg and etoposide 600 mg/day on days —3 and —2.

GVHD Prophylaxis and Treatment

In our preliminary single-center study from May 2001 to May 2004, CsA only was administered for GVHD prophylaxis in all 10 patients undergoing HLA-matched sibling donor transplantation. In our multiple-center study, CsA with methotrexate (MTX) (on days +1 and +3) was administered for GVHD

prophylaxis in all 12 patients undergoing HLA-matched sibling donor transplantation. Patients receiving an unrelated donor, parent donor, or more than 1 locus HLA-mismatched sibling donor transplant received CsA + MTX (on days +1, +3, and +6) and human antithymocyte globulin (ATG [Thymoglobu-lin; Genzyme, Cambridge, MA]; 2.5 mg/kg/day on days —3, —2, and —1) as GVHD prophylaxis. CsA + MTX (on days +1, +3, and + 6), and mycophenolate mofetil (MMF; 0.5 g twice a day on days 0 to +28) was used in patients receiving a 1 locus HLA-mismatched sibling donor transplant as GVHD prophylaxis. Methylprednisolone 2 mg/kg/day was given to treat acute GVHD (aGVHD). ATG or ATG combined with CD25 monoclonal antibody (mAb) and another immunosuppressant agent was initially given to patients with glucocorticosteroid-resistant aGVHD. Corticosteroids and CsA were used initially to treat chronic GVHD (cGVHD), and the combination with various immunosuppressant agents was used to treat cGVHD that was unresponsive to initial therapy.

CsA Withdrawal

CsA was withdrawn rapidly in a stepwise fashion (ie, total dose reduced by 20%/week) to avoid an overwhelming GVHD reaction in patients who did not develop aGVHD by day +30 posttransplantation. For those who developed aGVHD, methylpredniso-lone was added to the regimen.

Infection Prophylaxis

Oral sulfamethoxazole and norfloxacin were given to all patients. Acyclovir was given daily from the beginning of conditioning therapy to engraftment, and then daily for 7 days every 2 weeks for up to 1 year after transplantation. Ganciclovir was given for 2 weeks before transplantation for prophylaxis of cyto-megalovirus (CMV) infection, and then again during periods of CMV viremia within 1 year after transplantation. Antifungal agents were administered 5 days before transplantation. Fluconazole (0.3 g$day—1 for up to +60 days after transplantation) was used in patients with no history of invasive fungal infection (IFI); those with a history of IFI received i.v. itraconazole (0.4 g$day— !), voriconazole (0.4 g$day— !), or Am-Bisome (2 mg$kg— 1 day—!). Oral itraconazole and voriconazole were started when the peripheral white blood cell (WBC) count exceeded 2.0 x 109/L and was discontinued after 90 days posttransplantation.

Evaluation Points and Definitions

Our data were analyzed on December 31, 2008. Evaluation points included hematopoietic engraft-ment, regimen-related toxicities (RRTs), infections, disease response, relapse, mortality, and overall survival (OS) and DFS from transplantation, as well as

the incidence and severity of aGVHD and cGVHD. Hematopoietic engraftment was defined as the first of 2 consecutive days with an absolute neutrophil count (ANC) in the peripheral blood (PB) exceeding 0.5 x 109/L and the first of 3 days with an absolute platelet count exceeding 20 x 109/L without transfasion support. RRTs were graded according to Bear-man's criteria [28]; aGVHD and cGVHD were graded as described previously [29]. Cytogenetic subgroups were classified according to criteria described previously [30]; CML-BC accompanied by complex aberrations was considered unfavorable, and the other subgroups were considered intermediate. On days 0, + 15 (neutrophil engraftment), and +30, disease response was assessed by bone marrow (BM) aspiration. Complete remission (CR) was defined as < 5% blasts with no evidence of dysplasia in the BM and no manifestations of leukemia outside the hematopoi-etic system. Partial remission (PR) was defined as < 30% blasts with or without extramedullary leukemia. No response was defined as a failure to meet the criteria for CR or PR. Donor chimerism in the PB and BM was analyzed on days +15, +30, +90, and + 180 using fluorescein in situ hybridization (FISH) in sex-mismatched transplantation and short tandem repeat analysis in sex-matched transplantation. Complete chimerism (CC) was defined as > 95% donor cells detected; mixed chimerism (MC), as 5% to 95% donor cells detected. Molecular and cryptogenic relapse was assessed based on chimerism status and presence of the tumor target gene marker; relapse was defined as a > 5% decrease in donor chimerism status or the reappearance of the tumor target gene marker. Hematologic relapse was defined by the reappearance of blasts in the PB, by any manifestation of leukemia outside the hematopoietic system, or by > 5% blasts in the BM smear.

Follow-Up

All patients were followed up weekly for the first 6 months, monthly between 6 to 24 months, every 3 months between 25 to 36 months, and every 6 months beyond 36 months after transplantation.

Statistical Analysis

Numerical variables were analyzed as categories based on their values being below or above the median of the entire cohort, as indicated in the Results section. For comparison of group characteristics (eg, RRTs), the c2 test, Fisher's exact test, and Student's t-test were used for univariate analysis. aGVHD and cGVHD were analyzed as time-dependent variables. The relationships between GVHD and infection, relapse, and OS were analyzed using the Spearman rank test. OS and DFS were estimated using the Kaplan-Meier method. The log-rank test and a Cox

proportional hazards regression model were used for analysis of risk factors for time-to-event variables. Probabilities of nonrelapse mortality (NRM) and relapse mortality were calculated using reciprocal cumulative incidence estimates to account for competing risks. The SPSS software package (SPSS, Chicago, IL) was used for all data analysis.

RESULTS

Patient, Donor, and Transplant Characteristics

Patients' characteristics are summarized in Table 1. Within the study population, 3 patients had comorbid-ities; 2 had hypertension, and 1 had hypertension and diabetes mellitus. Two patients had a history of pulmonary tuberculosis, and 11 had a history of IFI before transplantation. Active Aspergillus pneumonia was diagnosed in 3 patients at the time of transplantation. Before conditioning, the median percentage of leuke-mic blasts in the BM was 26% (range, 8% to 92%; mean, 33.6% 6 23.0%). Six patients had extramedul-lary disease before conditioning, including 2 with central nervous system leukemia (CNSL), 1 with soft tissue infiltration, and 3 with lymph node infiltration.

Table 1. Patient Characteristics before Transplantation

Male/female 36 (70.6%)/15 (29.4%)

Median age, years (range) 30 (14-53)

Diagnosis

AML 20 (39.2%)

ALL 16 (31.4%)

ABL 9 (17.6%)

CML-BC-AML 6 (11.8%)

Cytogenetic subgroup

Favorable 4 (7.8%)

Intermediate 18(35.3%)

Unfavorable 20 (39.2%)

Unknown 9 (17.6%)

Stage of treatment before transplantation

Primary induction failure 29 (58.9%)

Refractory relapse after CRI < 6 months 9 (17.6%)

Refractory relapse after CRI > 6 months 7(13.7%)

Refractory relapse after CR2 3 (5.9%)

Previous autologous transplantation 3 (5.9%)

Median marrow blasts before conditioning 26% (8.0-92.0%)

(range)

Extramedullary disease at time of 6 (11.6%)

transplantation

CNSL 2

Soft tissue and lymph node involvement 4

Number of chemotherapy cycles before

transplantation

Two 2 (3.9%)

Three 11 (21.6%)

Four 9 (17.6%)

Five or six 12(23.5%)

More than six 17(33.3%)

Median (range) 5(2-13)

Median time from diagnosis to transplantation, 215 (85-1253)

days (range)*

AML indicates acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; ABL, acute biphenotypic leukemia; CML-BC, chronic myelogenous leukemia with blast crisis; CNSL, central nervous system leukemia.

*Median time in CML-BC was defined as blast crisis to transplantation.

The cytogenetic subgroups according to the criteria [30] were favorable in 4 cases, intermediate in 18 cases, and unfavorable in 20 cases. The karyotype was unknown in 9 cases. All but 1 patient (who died of bacterial septicemia during the conditioning) received a stem cell transfusion. Donor and transplant characteristics are summarized in Table 2.

Hematopoietic Engraftment and Chimerism

All patients achieved hematopoietic engraftment, except for 1 patient who died of bacterial septicemia during conditioning and 1 patient who died of an RRT on day 13 after transplantation. The median time to neutrophil engraftment was 12 days (range, 8 to 19 days), and the median time to platelet engraft-ment was 15 days (range, 10 to 28 days). Donor chimerism was analyzed in the BM at the time of neutrophil engraftment and on day 130 after transplantation. All 49 evaluable patients achieved donor chimerism, including 17 (33.3%) with CC and 32 (62.7%) with MC (with donor chimerism ranging from 58% to 93%) at the time of neutrophil engraftment, and 48 of these 49 patients (98.0%) had CC and 1 had MC by day 1 30 after transplantation.

RRT and Infection within 100 Days Posttransplantation

The conditioning was tolerated by all patients except 1 patient who died of an RRT (ie, heart toxicity). The incidence of RRT was 100% according to Bearman's criteria [28]. Toxicity was most common in the gastrointestinal tract (39/51). Overall, 33 patients developed grade I toxicity and 13 (25.5%) had grade III-IV toxicity in 1 or more organs (1 organ, n =19; 2 organs, n =22; 3 or more organs, n = 10). Significant central nervous system (CNS) toxicity was not observed. Three patients developed seizures related to high CsA levels within 1 month after transplantation. Organ toxicity is summarized in Table 3.

Table 2. Donor and Transplant Characteristics

Donor type (n = 50)

HLA-matched sibling donor 22 (44.0%)

HLA-mismatched sibling donor 4 (8.0%)

One allele mismatched 2 (4.0%)

Two alleles mismatched 2 (4.0%)

HLA-mismatched family donor 5 (10.0%)

One allele mismatched 2 (4.0%)

Two alleles mismatched 3 (6.0%)

HLA-matched unrelated donor 12(24.0%)

HLA-mismatched unrelated donor 7(14.0%)

One allele mismatched 3 (6.0%)

Two alleles mismatched 4 (8.0%)

Stem cell source (n = 50)

Mobilized PBSCs 35 (70.0%)

BM 9 (18.0%)

Mobilized PBSCs + mobilized BM 6 (12.0%)

Median CD34+ cells per graft, x l06/kg (range) 8.7 (4.0-21.4)

PBSCs indicates peripheral blood stem cells; BM, bone marrow.

Table 3. Organ Toxicity According to Bearman's Criteria

Grade 0, n (%) Grade I or II, n (%) Grade III or IV, n (%)

Single Multiple Total Single Multiple Total Single Multiple Total

Heart 11 (61.1) 21 (63.6) 32 (62.7) 6 (33.3)

Bladder 15 (83.3) 25 (75.8) 40 (78.4) 2(11.1)

Kidneys 16 (88.9) 29 (87.9) 45 (88.2) 2(11.1)

Lungs 18(100) 32 (97.0) 50 (98.0) 0 (0.0)

Liver 14(77.8) 27 (81.8) 41 (80.4) 3 (16.6)

CNS 17 (94.4) 31 (90.9) 37 (93.9) 1 (5.6)

Mucosa 10(55.6) 19 (60.6) 29 (56.7) 6 (33.3)

Gut 3(16.7) 9 (27.3) 12 (23.5) 13 (72.2)

CNS indicates central nervous system.

The incidence and degree of RRTs did not differ significantly between the 18 patients in our single-center retrospective group and the 33 patients in our multicenter group (Table 3).

Within the first 100 days posttransplantation, 26 patients developed 37 episodes of infection. Fifteen patients had bacterial infections, including 1 patient with tuberculosis who had a history of pulmonary tuberculosis; 4 patients had IFI; 6 patients had viral infections; 8 patients had mixed infections (6 bacterial and fungal; 2 bacterial and viral); and 4 patients had infections of unknown etiology. In addition, 19 patients had CMV viremia, not considered an infection. Infection was not related to aGVHD (gs = 0.737; P = .072). The site of infection was identified in 35 of the 37 episodes of infection, including 4 bacteremia, 2 septicemia + lung, 3 septicemia + perianal, 2 septicemia + intestinal, 12 lung, 4 mouth + esophagus, 5 stomach + intestine, 2 soft tissue, and 1 bladder. Bacteremia was detected in 11 patients; 9 patients had symptoms of sepsis or septic shock, and 1 patient developed CMV pneumonia. In the 3 patients with active fungal infection before transplantation, infection progressed in 1 patient, and infection was controlled after transplantation in 2 patients. Four patients died of infection within 100 days posttransplantation.

Disease Response

To identify patients who might not benefit from our sequential intensified conditioning approach, we examined the BM on day 0 in all 50 evaluable patients. The median percentage of blasts in the BM was 7.5% (range, 0% to 38%; mean, 9.1% 6 7.8%). The percentage of blasts in the BM was significantly lower on day 0 than at the start of conditioning (34.5% 6 23.6%; P < .001). At the time of neutrophil engraft-ment, 43 patients (87.8%) had < 5% blasts in the BM, and all 49 evaluable patients had achieved donor chimerism. On day +30, 46 patients (93.9%) achieved CR, 2 patients achieved PR, and 1 patient achieved NR. Two patients with PR had ALL and AML, accompanied by submaxillary lymph node enlargement and mediastinal enlargement, respectively, be-

9 (27.3) 15 (29.4) 1 (5.5) 3(9.1) 4 (7.8)

4(12.1) 6 (11.8) 1 (5.5) 4(12.1) 5 (9.8)

3(9.1) 5 (9.8) 0 (0.0) 1 (3.0) 1 (2.0)

1 (3.0) 1 (2.0) 0 (0.0) 0 (0.0) 0 (0.0)

5(15.2) 8(15.7) 1 (5.6) 2(6.7) 3 (5.9)

2(6.1) 3 (5.9) 0 (0.0) 0 (0.0) 0 (0.0)

14(42.4) 20 (39.2) 2(11.1) 5(15.2) 7(13.7)

21 (63.6) 34 (66.7) 2(11.1) 3(9.1) 5 (9.8)

fore transplantation. Both of them had < 5% blasts in the BM, but, because they had extramedullary leukemia, they achieved only PR. Both developed aGVHD and achieved CR after withdrawal of CsA. Although the patient with NR achieved donor chimerism after transplantation, the rate of donor chimerism was 48% at the time of neutrophil engraftment and 32% by day +30. This patient did not achieve remission of leukemia after withdrawal of CsA and chemotherapy and died of leukemia progression.

Grade I-IV aGVHD occurred in 23 of 49 evaluable patients (46%) by day +30 (grade I, n =7; grade II, n = 8; grade III, n = 4; grade IV, n = 4). The median day of onset was day +21 (range, day +7 to day +28). Skin, liver, and gut involvement was seen in 21, 15, and 14 patients, respectively. Of the 26 patients who did not develop GVHD by day +30, 15 developed aGVHD (grade I, n = 4; grade II, n = 8; grade III, n = 3) after gradual reduction or withdrawal of immunosuppres-sant agents. In the 7 patients with leukemia recurrence, aGVHD occurred in 4 of the 6 patients who received treatment including donor lymphocyte infusion (DLI) and in 1 patient after withdrawal of CsA. cGVHD developed in 21 of 42 (50.0%) patients who survived for more than 100 days, including 2 patients who developed cGVHD after DLI. The median interval from transplantation to onset of de novo cGVHD was 141 days (range, 91 to 266 days). Twelve patients had limited disease, and 9 patients had extensive disease.

Relapse and Outcome

Thirteen patients (26.5%) relapsed, at a median time of 4.5 months (range, 3.0 to 8.0 months) after transplantation, as a result of hematologic disease in 8, a cytogenetic disorder in 2, and extramedullary disease in 3. The cumulative rate of relapse at 3 years posttransplantation was 33.3%. Nine patients (69.2%) relapsed during the first 6 months posttransplantation; these patients had some poor prognostic characteristics in common, including high-risk

cytogenetics (n = 5), ALL (n = 5), and PIF (n = 7). Ten patients received treatment, and 3 patients abandoned treatment. Six patients received treatment including DLI, and 4 patients received only chemotherapy and/or immunosuppressant withdrawal because of the lack of a suitable donor. Five patients achieved CR, 1 patient achieved PR, and 4 patients achieved NR. All patients who achieved CR received DLI; 1 patient experienced hematologic relapse, 2 patients had a cytogenetic disorder, and 2 patients sustained extramedullary relapse.

The Spearman rank test demonstrated a negative correlation between disease relapse and aGVHD (rs = —0.418; P = .004), as well as a negative correlation between relapse and cGVHD (rs = —0.355; P = .023). However, OS was not related to aGVHD (rs = — 0.070; P = .631) or cGVHD (rs = 0.120; P = .457). The Pearson c2 analysis indicated that the rate of disease relapse did not differ significantly among the various types of disease (c2 = 2.443; P = .486) (Table 4).

OS and DFS

Twenty-six patients were alive at a median follow-up of 41 months (range, 6.6 to 92.4 months). Causes of death included leukemia progression (n = 1), infection (n = 6), RRT (n = 1), leukemia relapse (n = 6), GVHD (n = 9), and second tumor (n = 2, including lung cancer in 1 patient and posttransplantation lym-phoproliferative disease in 1 patient). The 100-day posttransplantation mortality was 21.6% (n = 11, including RRT in 1, infection in 3, aGVHD in 5, disease progression in 1, and posttransplantation lympho-proliferative disease in 1). Overall, the 1—, 3 — , 5—, and 7-year OS and DFS were 57.7% 6 7.1% and 48.0% 6 7.1%, 49.0% 6 7.6% and 42.4% 6 7.3%, 44.6% 6 8.1% and 38.2% 6 7.7%, and 44.6% 6 8.1% and 38.2% 6 7.7%, respectively (Fig. 1). The 3-year OS and DFS did not differ significantly in our single-center and multicenter groups (P = .642 and .522, respectively). OS and DFS among all disease types (AML, ALL, ABL, and CML-BC-AML) also did not differ significantly according to Kaplan-Meier analysis (P = .803 and .839, respectively).

The development of aGVHD had a significant effect on outcome. In the posttransplantation period, when aGVHD developed, OS and DFS were better

Table 4. Diagnosis and Relapse

Diagnosis Relapse, n (%) Nonrelapse, n (%) С P

AML 5 (25.0) 15 (75.0) 2.443 .486

ALL 6 (37.5) 10(62.5)

ABL 1 (11.1) 8 (88.9)

CML-BC-AML 1 (16.7) 5 (83.3)

AML indicates acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; ABL, acute biphenotypic leukemia; CML-BC, chronic myelogenous leukemia with blast crisis.

Figure 1. OS and DFS.

in patients with grade I or II aGVHD compared with patients without aGVHD. However, grade III-IV aGVHD was associated with high mortality and poor outcome (P = .010 for OS and P = .006 for DFS; log-rank, multigroup comparison) (Figure 2). In contrast, cGVHD had no significant effect on survival.

Survival Analysis

Risk factors for survival are presented in Table 5. Four pretransplantation variables were associated with better OS on univariate analysis: stage at transplantation (others vs PIF), lower circulating blasts at transplantation (none vs present), BM infiltration by < 30% blasts, and favorable cytogenetic status. Four pretransplantation variables also were associated with better DFS on univariate analysis: younger age at transplantation, lower circulating blasts at transplantation (none vs present), BM infiltration by < 30% blasts, and favorable cytogenetic status. Using a Cox regression model for multivariate analysis, cytogenetic status (high riskvs other: P = .001, hazard ratio [HR] = 0.224 for OS; P = .045, HR = 0.382 for DFS) was the only significant pretransplantation factor. In contrast, age, sex, diagnosis, circulating blasts at transplantation, BM infiltration by blasts at transplantation, number of chemotherapy cycles before transplantation, time from diagnosis to transplantation, CD341 cell count in the graft, and HLA typing were not predictive of outcome.

DISCUSSION

For patients with advanced leukemia undergoing allo-HSCT, a major obstacle to success is relapse of

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Figure 2. Influence of acute GVHD on OS (P = .010) and DFS (P = .006).

the underlying disease. In these patients, outcome depends largely on the leukemia burden at the time of transplantation [8]. With standard MA regimens consisting of TBI or Bu combined with Cy, the relapse rate (RR) is > 50% for patients who undergo HSCT with advanced-stage disease [10-12]. Intensified conditioning can reduce the leukemia burden before transplantation and improve the CR rate and long-term survival in patients with advanced leukemia after allo-HSCT [7,18-20]. The Flu/Ara-C regimen for salvage chemotherapy in patients with refractory and relapsed leukemia has a reported 40% to 50% overall probability of achieving CR [1-3,31]. Based on these results, we used the Flu/Ara-C regimen followed by a myeloablative regimen, in our patients with refractory leukemia who underwent allo-HSCT. To identify those patients who might benefit from our intensified conditioning regimen, we performed a BM examination on day 0, and found a markedly lower percentage of blasts in the BM on day 0 than before the start of

conditioning. On day +30 posttransplantation, 48 of 49 patients achieved CR of the BM with no evidence of dysplasia in the BM. These results confirm the suitability of using Flu/Ara-C with the MA regimen in refractory leukemia to decrease the leukemic burden before allo-HSCT and increase the CR rate after allo-HSCT.

RRTs remain a major obstacle when intensified conditioning regimens are used in allo-HSCT [32,33]. Flu is widely used for HSCT conditioning in both MA and nonmyeloablative (NMA) regimens. Replacing Cy with Flu in MA conditioning can decrease the rate of RRTs and treatment-related mortality (TRM) [34-36]. The combination of Ara-C and TBI/Cy has been investigated at some transplantation centers [18,37-41]. Some studies have reported that adding high-dose Ara-C to MA conditioning can increase pulmonary, neurologic, and cardiac toxicity [37,38,41]; however, other studies have found that this addition reduced leukemia relapse with no

Table 5. Risk Factors for Outcomes

OS DFS

Univariate Multivariate (HR) Univariate Multivariate (HR)

Sex, male versus female NS NS NS NS

Age, less than versus greater than or equal to NS NS .025 (2.541) NS

median

Diagnosis, AML versus other NS NS NS NS

Stage at HSCT, PIF versus other .048 (0.387) NS NS NS

Circulating blasts at HSCT, none versus present .007 (7.340) NS .025 (3.767) NS

BM infiltrated by blasts at HSCT, < 30% versus > .007 (3.27l) NS .036 (2.318) NS

Number of chemotherapy cycles before HSCT, NS NS NS NS

less than versus more than or equal to median

Time from diagnosis to HSCT, less than versus NS NS NS NS

greater than or equal to median

Cytogenetic status, high risk versus other .001 (0.224) .017(0.280) .003 (0.310) .045 (0.382)

CD34+ counts in the graft, less than versus NS NS NS NS

greater than or equal to median

HLA type, matched versus mismatched NS NS NS NS

OS indicates overall survival; DFS, disease free survival; AML, acute myelogenous leukemia; HSCT, hematopoietic stem cell transplantation; PIF, primary induction failure; BM, bone marrow; NS, not significant.

increase in toxicity [18,39,40]. Most of the studies that reported increased RRTs used 3 g/m2 x 6 to 12 doses of Ara-C [37,38,41]. Recently, Schmid and coworkers [7,20] reported that a sequential regimen of Flu/Ara-C/amsacrine for salvage chemotherapy and RIC was effective and had an acceptable toxicity profile in allo-HSCT for refractory AML and MDS. In our single-center phase I study, we observed no significant increase in RRTs in the intensified conditioning regimen consisting of Flu/Ara-C and TBI/Cy compared with the standard TBI/Cy conditioning for patients under the age of 50 years undergoing allo-HSCT for refractory leukemia [26]. In this multicenter study, for patients under age 55 years, the incidence of RRTs was similar to that in the single-center study. Regarding the association of RRTs with Ara-C, most studies have demonstrated an association between RRTs and high-dose Ara-C; the use of moderate doses of Ara-C in the present study might explain the lower incidence of RRTs that we found. Infection is a common complication after allo-HSCT, especially in the early period after transplantation. Some studies have indicated that intensified conditioning is complicated by an increased infection rate [42-44]. We did not perform a direct, randomized comparison of intensified conditioning with standard TBI-based regimens; however, the incidence of infections in our cohort appeared to be similar to that in patients with CR status who underwent allo-HSCT with a standard conditioning regimen in a corresponding time period within 100 days posttransplantation (data not shown).

In addition to the antileukemic effect of conditioning regimens, the therapeutic efficacy of allo-HSCT also relies on the GVL reaction, which is based on the interaction of the host's antigen-presenting cells and the donor's immunocompetent cells. Generally, the occurrence of GVHD is considered to be associated with the GVL reaction. It is known that leukemia relapse after allo-HSCT can be successfully treated by inducinga GVL reaction [7,20,45,46]. DLI, CsAwithdrawal, and cytokines (eg, interferon-alpha and interleukin-2) are generally used to induce a GVL reaction in relapsed patients after allo-HSCT. CsA withdrawal accompanied by GVHD and the GVL reaction has been observed in some patients undergoing allo-HSCT and has been used as first-line treatment in patients with leukemic relapse after allo-HSCT [23-25]. In our cohort of patients, 2 of 3 patients who did not achieve CR by day 130 achieved CR after the occurrence of aGVHD grade II and III, respectively, and another patient without GVHD did not achieve CR from a rapid tapering of prophylactic immunosuppressants for GVHD in the early stages posttransplantation. In our cohort, Kaplan-Meier analysis also indicated better OS and DFS in patients with grade I or II aGVHD compared with patients without aGVHD. The induction of GVL through rapid tapering of prophylactic

immunosuppressants during the early stages posttransplantation is also supported by the low relapse rate and excellent survival in our entire cohort. Our strategy of GVHD prophylaxis and CsA withdrawal resulted in a higher incidence of aGVHD, but the aGVHD-related lethality was acceptable, and the incidence of cGVHD was only 45.2% (19/42; 2 patients developed cGVHD after DLI).

The combination of Flu/Ara-C and the TBI/Cy conditioning regimen was intended to increase anti-leukemic efficacy and decrease the leukemia burden before transplantation. Flu/Ara-C chemotherapy is aimed mainly at rapidly proliferating malignant cells, and TBI may have an antileukemic effect on less rapidly proliferating or even quiescent leukemic cells. This approach has proven highly effective, as demonstrated by the number of leukemic cells in the BM on day 0 and the rate of CR on day 130 posttransplantation in our cohort. The success of the GVL reaction in decreasing the rate of leukemia relapse depends on the leukemia burden after HSCT. Rapid tapering of prophylactic immunosuppressants for GVHD in the early stages of the transplantation protocol was intended to accelerate the GVL reaction in patients with a lower leukemia burden after allo-HSCT. This concept also proved highly effective, with 7-year OS and DFS of 44.6% 6 8.1% and 38.2% 6 7.7%, respectively, and a cumulative relapse rate of 33.3% at 3 years posttransplantation in our cohort. These results compare favorably to those of previous studies on treating refractory leukemia using standard or intensified conditioning regimens and allo-HSCT [4,5,14,47,48]. A formal comparison of our results with those from other intensified conditioning regimens is difficult because of the heterogeneity of the patient populations, differences in the intensity of conditioning, and transplantation approaches. Recently, Schmid and coworkers [7,20] reported a very promising study that improved OS and DFS for refractory AML and MDS through a sequential regimen of Flu/Ara-C/amsacrine chemotherapy and RIC for allo-HSCT, along with prophylactic DLI. But, despite these encouraging overall results, the relapse rate in this study was still significant. Our strategy for patients with refractory leukemia undergoing allo-HSCT is similar to that described by Schmid and coworkers, but our results are superior to theirs with respect to OS and DFS. Along with the heterogeneity of the patients included in our cohort, our protocol differed from theirs in terms of the intensity of, and drugs used in, conditioning, the interval between cytoreductive chemotherapy and conditioning, the prophylactic immunosuppres-sive therapy for GVHD, and other aspects. Notably, Schmid and coworkers used amsacrine in their protocol, whereas we did not. In contrast to the results reported by Schmid and coworkers [7,20], our univariate analysis data indicate that PIF at transplantation

was an unfavorable prognostic factor. However, multi-variate analysis of potential risk factors for outcome identified favorable cytogenetic status as the only pre-transplantation variable with a strong association with better OS and DFS (P = .001 and .045, respectively). A reasonable interpretation of our findings is that the patients with PIF received the same number of chemotherapy cycles as other patients. Although our protocol appears to prolong the survival of patients with refractory leukemia, it cannot overcome the obstacle of cytogenetic status; patients with high-risk cytogenetic status have lower OS and DFS. In previous studies, patients with refractory ALL had a higher relapse rate and shorter OS than those with refractory AML after allo-HSCT [7,20,49]; however, to our surprise, in our study, relapse rate and OS were similar in patients with ALL and those with AML. This finding might be related to the beneficial profile of our approach, or possibly to the small size of our cohort, which did not allow differentiation among all types of leukemia. In the present study, although the mortality of RRTs was only 2% (1/50), the day 1100 TRM was 21.6% (11/51), with a 17.6% mortality (9/51) for GVHD. Decreasing TRM, especially the lethality of GVHD, is a worthy goal that merits further study.

In conclusion, our data suggest that sequential cytoreductive chemotherapy and MA conditioning in allo-HSCT for adult refractory leukemia is well tolerated. Although this approach cannot overcome the obstacle of cytogenetic status, it appears to improve OS and DFS. The sequential strategy of cytoreductive chemotherapy followed immediately by intensified MA conditioning for allo-HSCT and rapid tapering of prophylactic immunosuppresants for GVHD during the early stages posttransplantation may represent a step forward in the treatment of refractory leukemia.

ACKNOWLEDGMENTS

Financial disclosure: The authors have nothing to disclose.

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