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Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation
Biology of Blood and Marrow Transplantation 10:178-185 (2004) 䊚 2004 American Society for Blood and Marrow Transplantation 1083-8791/04/1003-0004$30.00/0 doi:10.1016/j.bbmt.2003.10.006
Acute and Chronic Graft-versus-Host Disease after Ablative and Nonmyeloablative Conditioning for Allogeneic Hematopoietic Transplantation Daniel R. Couriel, Rima M. Saliba, Sergio Giralt, Issa Khouri, Borje Andersson, Marcos de Lima, Chitra Hosing, Paolo Anderlini, Michelle Donato, Karen Cleary, James Gajewski, Joyce Neumann, Cindy Ippoliti, Gabriela Rondon, Agueda Cohen, Richard Champlin Department of Blood and Marrow Transplantation, Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas Correspondence and reprint requests: Daniel R. Couriel, MD, Department of Blood and Marrow Transplantation, Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030-4095 (e-mail: [email protected]). Received July 2, 2003; accepted October 23, 2003
ABSTRACT In this study, we evaluated the influence of nonmyeloablative and ablative conditioning regimens on the occurrence of acute and chronic graft-versus-host disease (GVHD). One hundred thirty-seven patients undergoing matched-related sibling transplantations received the same GVHD prophylaxis. Myeloablative regimens included intravenous busulfan/cyclophosphamide (n ⴝ 45) and fludarabine/melphalan (n ⴝ 29). Patients in the nonmyeloablative group (n ⴝ 63) received fludarabine/idarubicin/cytarabine, cisplatin/fludarabine/idarubicin, and fludarabine/cyclophosphamide. The actuarial rate of grade II to IV acute GVHD was significantly higher (hazard ratio, 3.6; 95% confidence interval, 1.5-8.8) in patients receiving ablative regimens (36%) compared with the nonmyeloablative group (12%). The cumulative incidence of chronic GVHD was higher in the ablative group (40%) compared with the nonmyeloablative group (14%). The rates were comparable within the first 200 days and were significantly higher in the ablative group beyond day 200 (hazard ratio, 5.2; 95% confidence interval, 1.2-23.2). Nonrelapse and GVHD-related mortality were relatively low in both groups. The use of the described nonmyeloablative preparative regimens was associated with a reduced incidence of grade II to IV acute GVHD and chronic GVHD compared with the busulfan/cyclophosphamide and fludarabine/melphalan transplant regimens. It is interesting to note that nonrelapse mortality with nonmyeloablative regimens in older and more debilitated patients was low (14%) and comparable to that achieved with standard high-dose regimens in younger patients. © 2004 American Society for Blood and Marrow Transplantation
KEY WORDS Acute GVHD
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Preparative regimens
INTRODUCTION Myeloablative (MA) doses of chemotherapy and total body irradiation, typically used as preparative regimens in hematopoietic stem cell transplantation (HSCT), are associated with a high rate of morbidity and regimen-related mortality. In this respect, graftversus-host disease (GVHD) continues to be one of the main limitations of allogeneic transplantation [1-3]. This has largely limited the use of MA HSCT to life-threatening clinical indications in younger individuals without serious comorbidities. Hematologic 178
malignancies predominantly occur in older patients and often in patients with comorbidities who are not considered eligible for this treatment. Nonmyeloablative (NMA) preparative regimens have recently been developed as a means to reduce the morbidity related to hematopoietic transplantation [4-7]. These reduced-intensity regimens provide sufficient immunosuppression to achieve engraftment of an allogeneic bone marrow or blood stem cell graft and allow the immune-mediated graft-versus-malignancy effect to occur [8]. The syndrome of acute GVHD (aGVHD) is in
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part related to cytokines produced in response to the toxicity of the preparative regimen [9,10]. Intensive regimens can result in gastrointestinal (GI) mucosal injury with increased permeability and endotoxemia [11,12], which stimulates and amplifies a cytokine cascade that is central in the pathophysiology of aGVHD [12,13]. One theoretical advantage of the use of less toxic NMA preparative regimens may be a lower incidence of aGVHD. Because aGVHD is a risk factor for the development of chronic GVHD (cGVHD), its incidence may indirectly be reduced as well. The effect of preparative regimen intensity on the incidence of aGVHD and cGVHD has not yet been systematically addressed. In this study, we evaluated the influence of NMA and MA conditioning on the occurrence of aGVHD and cGVHD and nonrelapse mortality (NRM) in patients undergoing matchedrelated sibling transplantations and who received the same GVHD prophylaxis. We tested the hypothesis that use of a less toxic NMA preparative regimen would result in lower incidence of aGVHD and cGVHD.
METHODS We evaluated 137 consecutive patients who received HSCT as part of prospective clinical trials between June 1, 1996, and September 30, 2000. All patients received allogeneic hematopoietic transplants from an HLA-identical sibling donor. These studies were reviewed and approved by the University of Texas M.D. Anderson Cancer Institutional Review Board, and all patients provided written informed consent to participate. Information was obtained from the Department of Blood and Marrow Transplantation database, where all clinical data is systematically collected for every patient undergoing stem cell transplantation. Preparative regimens were considered MA if they produced profound pancytopenia for ⬎28 days without transplantation and if, after transplantation, hematopoietic recovery was completely donor derived (complete chimerism in ⬎80% of patients). NMA regimens were defined as those in which hematopoietic recovery would reliably occur within 28 days without transplantation and if, after transplantation, mixed chimerism could be documented in most patients [8]. MA preparative regimens included (1) busulfan 0.8 mg/kg intravenously (IV) every 6 hours on days ⫺7 to ⫺4 and cyclophosphamide 60 mg/kg on days ⫺3 and ⫺2 (IV BuCy) and (2) fludarabine 25 mg/m2 on days ⫺6 to ⫺2 and melphalan 90 mg/m2 on days ⫺3 and ⫺2 (FM), which is MA with this dose of melphalan. Hematopoietic transplantation occurred on day 0.
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NMA regimens included fludarabine 30 mg/m2/d on days ⫺6 to ⫺3, idarubicin 12 mg/m2/d on days ⫺6 to ⫺4, and cytarabine 2 g/m2 on days ⫺7 to ⫺3 (FlagIda); cisplatin 100 mg/m2 IV continuous infusion on days ⫺6 to ⫺3, fludarabine 30 mg/m2 on days ⫺4 and ⫺3, cytarabine 500 to 1000 mg/m2 on days ⫺4 and ⫺3 (PFA); and fludarabine 30 mg/m2 on days ⫺5 to ⫺3 with cyclophosphamide 750 mg/m2 on days ⫺5 to ⫺3 (FC). Seven patients in the FC group received rituximab 375 mg/m2 on day ⫺13 and 1000 mg/m2 on days ⫺6, ⫹1, and ⫹8. All these regimens have been used for standard chemotherapy and produce only transient myelosuppression without hematopoietic transplantation. The BuCy regimen was intended for younger patients without serious comorbidities. The FM regimen was intended as a potentially better tolerated MA regimen in older or medically infirm patients. Although it was initially considered a reduced-intensity regimen, it is now clear that its overall toxicity is comparable to that of MA regimens; it necessitates hematopoietic stem cell infusion for hematologic recovery. In this study, aGVHD, cGVHD, and GVHDrelated mortality were similar in patients receiving FM and IV BuCy. Thus, they were analyzed as a group. The NMA FlagIda regimen was intended for older patients with myeloid leukemias, and the NMA FC and PFA regimens were used for patients of all ages with lymphoid malignancies. The diagnoses treated included myelodysplastic syndrome/acute myelogenous leukemia (n ⫽ 61), chronic myelogenous leukemia/myeloproliferative disorders (n ⫽ 36), chronic lymphocytic leukemia (n ⫽ 8), and non-Hodgkin lymphoma (n ⫽ 32). All patients received their grafts from HLA genotypically identical siblings. The cell source was peripheral blood stem cells (PBSC) in most patients (n ⫽ 113; 82%). All patients received a common regimen of posttransplantation immunosuppressive therapy as prophylaxis against GVHD. This included tacrolimus starting at day ⫺2, with dose adjustments to maintain blood levels of 5 to 15 ng/mL, and methotrexate 5 mg/m2 on days 1, 3, and 6. Bone marrow recipients received an additional dose on day ⫹11 [14]. Tacrolimus was continued for at least 90 days in the absence of disease progression. Patients at low risk for relapse continued tacrolimus until 6 to 9 months after transplantation. Those at high risk for relapse or minimal residual disease had immunosuppression withdrawal at 3 to 6 months after transplantation. If grade II to IV aGVHD developed, methylprednisolone 2 mg/kg was generally administered. Chronic GVHD was initially treated with corticosteroids and tacrolimus. Patients with steroid-resistant aGVHD or cGVHD received alternative immunosuppressive therapy. We defined low-risk disease as the first chronic or 179
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accelerated phase of chronic myelogenous leukemia, acute myelogenous leukemia, or lymphoid malignancies in first complete remission and lymphoid malignancies in first chemosensitive relapse or second complete remission. All other patients were considered to have high-risk disease. Conditions classified as serious comorbidities included 1 or more of the following: (1) cardiac: coronary artery disease, congestive heart failure, serious or life-threatening arrhythmias, deep venous thrombosis, pulmonary embolism, and uncontrolled severe hypertension; (2) pulmonary: emphysema, asthma, history of acute respiratory failure necessitating mechanical ventilation and sarcoidosis; (3) renal: acute or chronic renal failure and hydronephrosis; (4) infections: fungal (Aspergillus species, Candida species, Torulopsis glabrata, or Fusarium species), viral (cytomegalovirus, varicella-zoster virus, or herpes simplex virus), mycobacterial (tuberculosis or myobacterium avium intercellulare), Pneumocystis carinii, and serious bacterial infections (septic shock or osteomyelitis); and (5) other conditions: pancreatitis, history of GI bleeding, stroke, seizures, other cancers, autoimmune conditions (inflammatory bowel disease, autoimmune hemolytic anemia, or GVHD in a previous transplant). GVHD Assessment
GVHD was assessed in 2 ways. The first way, which is the focus of this article, was to assess the occurrence of GVHD before immune manipulation to better determine the effect of the preparative regimen on the occurrence of GVHD. In this case, aGVHD was evaluated within 100 days after HSCT. The staging and grading of aGVHD was performed according to modified Glucksberg consensus criteria [15]. Biopsy samples from involved tissues were obtained in all 29 patients with grade II to IV aGVHD. The diagnosis of aGVHD was confirmed histologically in 24 (83%) of 29 patients with grade II to IV aGVHD. Seventeen (55%) of 31 skin biopsy samples were positive and confirmed the diagnosis of skin GVHD in 17 of 18 patients. The remaining patient had a negative skin biopsy with a simultaneous positive GI biopsy. Fourteen (70%) of twenty upper and lower GI biopsies confirmed the diagnosis of GI GVHD in 8 of 12 patients with clinical manifestations of GI GVHD. Six (50%) liver biopsies confirmed the diagnosis of liver GVHD in all 5 patients with abnormal bilirubin and alkaline phosphatase. The remaining patient had concomitant positive skin and GI biopsies. The diagnosis of cGVHD was based on clinical manifestations, including sicca syndrome, lichenoid changes on mucosal surfaces, lichenoid skin GVHD, sclerodermal GVHD, and GI strictures. Diarrhea or liver function abnormalities were considered part of the spectrum of cGVHD if they occurred beyond 100 days after the 180
last stem cell infusion and in the presence of a positive biopsy of the GI tract or liver, respectively. Chronic GVHD was classified as de novo, progressive, or relapsing [3]. De novo cGVHD was defined as occurring without a history of aGVHD. For relapsing cGVHD, cGVHD developed after a history of aGVHD that was successfully treated into complete remission. Patients with progressive cGVHD had aGVHD that failed to achieve remission with treatment and progressed into cGVHD. Second, we determined the incidence of all GVHD (ie, acute or chronic) without censoring at the time of immune manipulation. Only mortality was considered as a competing risk. Thus, we included GVHD that occurred after immune manipulation in both the MA and NMA groups. Chimerism
Hematopoietic chimerism was evaluated on bone marrow cells by restriction fragment length polymorphisms at the AY-29 or YNH24 loci and by cytogenetic studies in sex-mismatched cases [16]. These assays are able to detect mixed chimerism if more than 5% recipient or donor cells are present. Lineagerelated chimerism studies were not available on all patients and were not considered in this analysis. Mortality
GVHD-related mortality included death secondary to aGVHD or cGVHD independently of whether GVHD occurred before or after immune manipulation. Deaths due to infection in the setting of GVHD or in patients who were receiving immunosuppressive treatment for GVHD were also included in this group. NRM included all deaths unrelated to the recurrence or progression of malignancy. Statistical Analysis
The primary objective of the analysis was to assess the incidence of GVHD according to the intensity of the conditioning regimen while the patient received standard GVHD prophylaxis. For this purpose, the primary end points of the study were the incidence of grade II to IV aGVHD and cGVHD before immune modulation (including withdrawal of immune suppression) or donor lymphocyte infusions (DLI). Secondary end points were GVHD-related mortality, NRM, and the overall incidence of GVHD irrespective of immune modulation. The incidences of GVHD, GVHD-related mortality, and NRM were estimated by the method of Prentice et al. [17] to account for the differences in immune modulation, relapse, or mortality rates in the different conditioning regimens. Competing risks for the occurrence of GVHD before manipulation were withdrawal of immunosuppression, DLI, disease progression, and
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Table 1. Patient Characteristics Variable Sex (M/F) Age (y) Median Range No. patients >40 y (%) Diagnosis, n (%) AML/MDS CML CLL NHL Cell source, n (%) PBSC BM Disease risk group, n (%) High risk Low risk Prior chemotherapy regimens <2 >2 Serious comorbidities, n (%)
Overall (n ⴝ 137)
Ablative (n ⴝ 74)
Nonmyeloablative (n ⴝ 63)
P Value
85/52
47/27
38/25
NS
53 15-75 105 (77)
45 15-68 47 (63)
59 21-75 58 (92)
<.001 <.001
61 (45) 36 (26) 8 (6) 32 (23)
42 (57) 30 (40) 0 2 (3)
19 (30) 6 (9) 8 (13) 30 (48)
<.001
113 (82) 24 (18)
53 (72) 21 (28)
60 (95) 3 (5)
<.001
65 (48) 72 (52)
30 (40) 44 (60)
35 (56) 28 (44)
NS
88 (64) 49 (36) 63 (46)
54 (73) 20 (27) 29 (39)
34 (54) 29 (46) 34 (54)
.02 NS
AML indicates acute myelogenous leukemia; MDS, myelodysplastic syndrome; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin lymphoma; BM, bone marrow; NS, not significant.
death without GVHD. In addition, the occurrence of aGVHD was considered a competing risk for the estimation of the cumulative incidence of de novo cGVHD. Follow-up time was censored at the earliest of these events. Only death without GVHD was a competing risk for the estimation of the overall incidence of GVHD irrespective of immune manipulation, and only progression of malignancy was considered a competing risk for NRM. Median follow-up among survivors was 20 months (range, 6-48 months) overall, 19 months (range, 8-48 months) for the ablative group, and 23 months (range, 6-42 months) for the reduced-intensity group (P ⫽ .4). To ensure comparable follow-up time for the 2 conditioning regimen groups, we report the incidence of cGVHD at 18 months. NRM was estimated at 2 years. The Cox proportional hazards model was used to compare the outcomes of interest among patients receiving MA and NMA regimens. It was not possible to adjust for confounding by using multivariate analysis because of the sample size. Adjustment was therefore limited to bivariate analysis, when possible, with preparative regimen as a constant covariate. In addition, the distribution of potential confounding factors (age, stem cell source, diagnosis, and chimerism at day 30) was skewed in the NMA group (described below). This further limited our ability to conduct multivariate analysis. Adjustment for confounding in this case was limited to restricting the comparison of the regimen groups to the categories of the covariates that represented most patients in the NMA group. The
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assumption of constant proportional hazards over time was tested graphically [18]. When indicated, the maximized partial likelihood method was used to find the appropriate time breakpoint within which the hazards were proportional. Patient characteristics were compared by the Fisher exact and 2 tests. Two-sided P values of ⬍.05 were considered significant. Analysis was performed with Stata version 7.0 (Stata Corp., College Station, TX).
RESULTS Patient Characteristics
Patient characteristics varied significantly according to the preparative regimen (Table 1). The MA group patients were generally younger (median age, 45 years; P ⬍ .001). Less than half of these patients had high-risk disease, compared with 56% in the NMA groups (P ⫽ not significant). Similarly, a lower proportion of patients had serious comorbidities in the MA (39%) compared with the NMA (54%) group at the time of transplantation (P ⫽ not significant). A significantly higher proportion of patients in the NMA group had received more than 2 chemotherapeutic regimens before transplantation (P ⫽ .02). The stem cell source was bone marrow in 28% of the MA group, compared with 5% of those receiving NMA regimens (P ⬍ .001). In the MA group, 97% were myeloid malignancies. However, most patients in the NMA group (61%) had lymphoid malignancies (P ⬍ .001). 181
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Table 2. Incidence of GVHD before Immune Manipulation Regimen Myeloablative Nonmyeloablative Total
n*
No. aGVHD II-IV
Incidence† (95% CI)
67 56 123
23 6 29
34% (24-48) 11% (5-23)
n‡
No. cGVHD at 18 mo
Incidence† (95% CI)
70 55 125
25 7 32
40% (28-58) 14% (7-27)
*Patients with evidence of donor chimerism or aGVHD. †Cumulative incidence considering immune manipulation, second infusion, relapse, or death without GVHD as competing risks. ‡Patients with evidence of donor chimerism or cGVHD.
Seven patients had graft failure and were not considered at risk for GVHD; they were included only for the estimation of NRM. Of the remaining 130 patients, those who had evidence of engraftment by evaluation of mixed or complete chimerism (at day 30 for aGVHD and at day 100 for cGVHD) or by diagnosis of GVHD were evaluable for the incidence of GVHD. These included 123 patients for aGVHD and 125 patients for cGVHD. Four patients had aGVHD and 5 had cGVHD without assessment of chimerism on day 30 and 100, respectively. Acute GVHD
Overall Incidence and Severity. The incidence of grade II to IV aGVHD was 36% (95% confidence interval [CI], 26%-49%) in the MA group compared with 12% (95% CI, 6%-25%) in the NMA group (hazard ratio [HR], 3.6; 95% CI, 1.5-8.8; Table 2; Figure 1). This effect was consistent when comparison was performed among patients who were older than 40 years and those who received PBSCs (Table 3), after adjusting for the number of prior chemotherapy regimens received or the disease risk category. Ten patients (8%) developed grade III to IV aGVHD. The number of patients with grade III to IV aGVHD was low with all preparative regimens. The
incidence was higher in the MA (12%; 95% CI, 6%23%) than in the NMA group (4%; 95% CI, 1%14%), but this was not statistically significant (P ⫽ .1). The PFA and the FlagIda regimens had no cases of grade III to IV aGVHD. Organ Involvement. Skin was the organ most frequently involved (n ⫽ 18; 15%), followed by GI GVHD (n ⫽ 12; 10%) and liver (n ⫽ 5; 4%; Table 4). When individual regimens were analyzed for organ involvement, there was a significantly lower proportion of skin involvement in the NMA group (P ⫽ .03) and a higher proportion of GI involvement in the FM group (P ⫽ .01) compared with the other regimens. Only 5 patients with grade II to IV aGVHD had liver involvement (4%). Liver was the only organ involved in 3 patients, all of whom were in the BuCy group. Chimerism and aGVHD. In the NMA group, 32 evaluable patients (57%) had complete donor chimerism, and 23 (41%) had mixed chimerism at day 30. Most patients (89%) in the MA group had complete donor chimerism at day 30. Among patients who had complete donor chimerism at day 30, the incidence of grade II to IV aGVHD was 36% in the MA group and 19% in the NMA group (HR, 2.3; 95% CI, 0.9-6.2). This effect is only marginally significant, but it is consistent with the overall higher incidence in the MA group compared with the NMA group. Among patients receiving NMA regimens, there was a trend for a reduced rate of grade II to IV aGVHD in patients with mixed chimerism. This ocTable 3. Incidence of aGVHD by Stem Cell Source, Age, and Diagnosis
Variable
Figure 1. Cumulative incidence of acute GVHD. Patients receiving nonmyeloablative regiments were compared with those receiving myeloablative regimens. Progression of malignancy, immunosuppression withdrawal, donor lymphocyte infusion, and death without GVHD were considered competing risks. 182
Peripheral blood stem cells Myeloablative Nonmyeloablative Age >40 y Myeloablative Nonmyeloablative Myeloid malignancy Myeloablative Nonmyeloablative
n*
No. aGVHD II-IV
48 53
19 6
43 51 65 23
HR
95% CI
3.9
1.5-9.7
14 6
3
1.1-7.7
22 2
4.3
1.0-18.5
*Patients with evidence of donor chimerism or aGVHD.
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Table 4. Acute GVHD Grade II to IV: Organ Involvement Regimen
n
Skin, n (%)
GI, n (%)
Liver, n (%)
BuCy FM Nonmyeloablative Total
41 26 56 123
8 (19) 6 (23) 4 (7)* 18 (15)
3 (7) 6 (23)* 3 (5) 12 (10)
3 (7) 1 (4) 1 (2) 5 (4)
*Indicates a statistically significant difference in organ involvement in comparison with the other preparative regimens.
curred in 1 patient with mixed chimerism (4%) and 5 patients with complete donor chimerism (16%), but it was not statistically significant (HR, 0.5; 95% CI, 0.1-2.4). The effect of mixed chimerism could not be assessed among the MA group because of the small number of patients. Chronic GVHD
The cumulative incidence of cGVHD at 18 months was 40% (95% CI, 28%-58%) for the MA group and 14% (95% CI, 7%-27%) for patients receiving NMA regimens. The increased rate among the MA group was not constant over time, and the Cox model assumption of constant proportional hazards was not met. Up to day 200 after transplantation, the rates of cGVHD were comparable in the MA and NMA groups (HR, 1.1; 95% CI, 0.4-3.4). Beyond day 200, the rate was significantly higher for the MA group (HR, 5.2; 95% CI, 1.2-23.2; Figure 2). This effect was consistent when comparison was done among patients who were older than 40 years and for those who received PBSC, after adjusting for the number of prior chemotherapy regimens received or for the disease risk category. The cumulative incidence of de novo cGVHD was
Figure 3. Cumulative incidence of all GVHD. Patients receiving nonmyeloablative (NMA) regimens were compared with those receiving myeloablative (MA) regimens. Only mortality was considered a competing risk. Patients undergoing immunosuppression withdrawal, DLI, or both were included in both the NMA (n ⫽ 44) and MA (n ⫽ 29) groups.
10% (95% CI, 5%-22%) and 6% (95% CI, 2%-19%) in the MA and NMA groups, respectively. The rates were not significantly different between these 2 groups, and the HR did not vary over time (HR, 2.4; 95% CI, 0.6-9.9). This suggests that the higher rate among the MA group could be attributed mostly to a higher rate of progressive and relapsing cGVHD, highlighting the importance of aGVHD as a risk factor. Chimerism and cGVHD. Among patients with complete donor chimerism at day 30, we still observed a trend for an increased rate of GVHD for the MA group (HR, 1.7; 95% CI, 0.7-4.2). Similarly to what was observed in the overall group, the HR varied over time; however, this was not statistically significant and did not warrant a separate analysis before and after day 200 after transplantation. In the NMA group, there was a trend for a lower rate of cGVHD for patients with mixed chimerism compared with those with complete chimerism; 1 of 23 patients with mixed chimerism developed cGVHD, compared with 6 of 30 of patients with complete chimerism (HR, 0.3; 95% CI, 0.03-2.4). Similarly, the corresponding proportions for day 100 were 1 of 24 and 6 of 16 (HR, 0.2; 95% CI, 0.02-1.5). GVHD after Early Immunosuppression Withdrawal and DLI
Figure 2. Cumulative incidence of chronic GVHD. Patients receiving nonmyeloablative regimens were compared with those receiving busulfan/cyclophosphamide and fludarabine/melphalan. Progression of malignancy, immunosuppression withdrawal, donor lymphocyte infusion, and death without GVHD were considered competing risks.
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Fifty-nine patients underwent early withdrawal of immunosuppression for recurrence or progression of malignancy. Nineteen of these patients additionally received a DLI for disease control. Five patients received a DLI without immunosuppression withdrawal. The proportion of patients receiving immune manipulation was significantly higher in the NMA group (70%) than in the MA group (39%; P ⬍ .01). We 183
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Table 5. Cumulative Incidence of GVHD Regimen
n*
No. GVH
Myeloablative Nonmyeloablative Total
72 58 130
38 23 61
Incidence at 18 mo†
95% CI
55% 41%
48-63 29-57
*Excluded were patients with primary graft failure: 2 in the myeloablative group and 5 in the nonmyeloablative group. †Cumulative incidence of GVHD considering mortality as a competing risk.
evaluated all GVHD (both acute and chronic) in both groups, including patients who underwent immune manipulation. Overall, the MA group had a significantly higher incidence of GVHD (55%) compared with the NMA (41%; HR, 1.8; 95% CI, 1.1-3.0; P ⫽ .03; Table 5; Figure 3). NRM and GVHD-Related Mortality
NRM at 2 years was similar for the MA (19%; 95% CI, 12%-31%) and NMA (15%; 95% CI, 8%29%; HR, 1.4; 95% CI, 0.6-3.3; Figure 3) groups. The causes of NRM at 2 years are listed in Table 6. The GVHD-related mortality rate was low for both groups: 8% (95% CI, 3%-18%) in the MA group and 9% (95% CI, 3%-22%) in the NMA group (HR, 1.1; 95% CI, 0.3-4.1; Table 6). DISCUSSION T-cell alloreactivity remains central in the pathophysiology of both aGVHD and cGVHD. However, research over the last decade supports additional mechanisms that involve other cell types and multiple cytokines in the pathophysiology of aGVHD. In this model, the conditioning regimen causes tissue damage and the release of proinflammatory cytokines, including tumor necrosis factor-␣, interleukin (IL)-1, and IL-6 [13]. Donor T cells respond to host alloantigens with activation and release of the T-helper 1 cytokines IL-2 and interferon-␥. Interferon-␥, in conjunction with endotoxins from GI bacteria that circulate systemically after mucosal damage, activates macrophages and natural killer cells [9,12,13]. This activation results in further release of inflammatory cytokines and amplifies the tissue damage caused by the immune-mediated graft-versus-host reaction [13]. In this study, we tested the hypothesis that a less intensive and toxic NMA regimen would be associated with less aGVHD and, consequently, cGVHD in patients receiving the same GVHD prophylaxis. Comparison of GVHD that occurs after NMA and MA regimens is difficult because of differences in patient characteristics and risk factors for GVHD. NMA transplantations were developed to extend the 184
use of HSCT to older patients and those with comorbidities who were not eligible for MA regimens. Thus, the NMA transplant recipients were at higher risk for aGVHD and cGVHD. Many centers have used unique posttransplantation immunosuppressive therapy with NMA transplants, and this precludes direct comparison with MA regimens. Others have intentionally discontinued immunosuppressive therapy early after NMA transplantations to enhance graftversus-leukemia effects. Early termination of immunosuppression may be associated with a higher incidence of GVHD, lower overall survival, and an increase in transplant-related mortality [19]. We have chosen to use the same posttransplantation immunosuppressive therapy for NMA regimens as with MA transplantations and to continue this therapy for at least 3 months in the absence of disease progression. This facilitates comparison of the effect of the preparative regimen on the incidence and severity of GVHD. An additional limitation to the comparison of regimens is the heterogeneity of the patient and transplant characteristics, which is inherent to the current indications for NMA regimens and favors the MA group. Most patients who received NMA regimens in our population were older than 40 years and had received PBSCs; they were therefore at an increased risk of GVHD. To adjust for this heterogeneity, we compared outcomes within these categories of patients because the skewed distribution precluded the use of multivariate analysis. Differences in the number of prior chemotherapy regimens and the disease risk group may be additional confounders in the comparison of the effects of preparative regimens. We could conduct only bivariate analysis to adjust for these factors because of the small sample size. The incidence of grade II to IV aGVHD was significantly lower in NMA regimens compared with the MA group. This effect was consistent among patients ⱖ40 years old, those who received PBSCs, and those with complete donor chimerism. It was also independent of the number of prior chemotherapy regimens or the disease risk category. As previously reported [20], we also found that there was a trend to a lower incidence of aGVHD in
Table 6. Causes of Nonrelapse Mortality Variable
Myeloablative (n ⴝ 74)
Nonmyeloablative (n ⴝ 63)
GVHD Graft rejection Infection Multiorgan failure Hemorrhage Other Total
6* 0 1 3 1 4 15
5* 1 2 0 0 1 9
*One death occurred beyond 18 mo after transplantation.
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patients with mixed chimerism in the NMA group. The incidence of severe (grade III to IV) aGVHD was low in all regimens, and no statistically significant differences were found between groups. These conclusions were maintained when patients undergoing early withdrawal of immunosuppression, DLI, or both were included in the analysis. Similarly to aGVHD, we observed a lower incidence of cGVHD among patients receiving NMA regimens. This became significant beyond 200 days after transplantation. Most (76%) of the cases in the MA group were progressive or relapsing cGVHD, and this highlights the importance of aGVHD as a risk factor. This increased rate was independent of the number of prior chemotherapy regimens or the disease risk category, and it persisted among older patients and those receiving PBSC. Differences in the incidence of GVHD were still present when patients who had undergone immunosuppression withdrawal or DLI were included in the comparison of both groups. NRM and GVHD-related mortality were relatively low with all regimens. It is interesting to note that NRM with NMA regimens in older and more debilitated patients was low and comparable to that achieved with standard highdose regimens in younger patients. In conclusion, the use of the described NMA preparative regimens was associated with a reduced incidence of grade II to IV aGVHD and cGVHD compared with MA transplantation regimens. Our data suggest that the reduction in the incidence of aGVHD and cGVHD is independent of factors such as older age, use of PBSCs, and more prior chemotherapy treatments or advanced disease.
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15.
16.
REFERENCES 1. Martin PJ, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute graft-versus-host disease: initial treatment. Blood. 1990;76:1464-1472. 2. Martin PJ, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute graft-versus-host disease: secondary treatment. Blood. 1991;77:1821-1828. 3. Vogelsang GB, Wagner JE. Graft-versus-host disease. Hematol Oncol Clin North Am. 1990;4:625-639. 4. Giralt S, Estey E, Albitar M, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy. Blood. 1997;89:4531-4536. 5. Giralt S, Thall PF, Khouri I, et al. Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies under-
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17.
18.
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going allogeneic progenitor cell transplantation. Blood. 2001;97: 631-637. Khouri IF, Saliba RM, Giralt SA, et al. Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: low incidence of toxicity, acute graft-versus-host disease, and treatment-related mortality. Blood. 2001; 98:3595-3599. McSweeney PA, Niederwieser D, Shizuru JA, et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graftversus-tumor effects. Blood. 2001;97:3390-3400. Champlin R, Khouri I, Shimoni A, et al. Harnessing graftversus-malignancy: non-myeloablative preparative regimens for allogeneic haematopoietic transplantation, an evolving strategy for adoptive immunotherapy. Br J Haematol. 2000;111:18-29. Hill GR, Crawford JM, Cooke KR, et al. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood. 1997;90:3204-3213. Couriel DR, Hicks K, Giralt S, et al. Role of tumor necrosis factor-alpha inhibition with inflixiMAB in cancer therapy and hematopoietic stem cell transplantation. Curr Opin Oncol. 2000; 12:582-587. Fegan C, Poynton CH, Whittaker JA. The gut mucosal barrier in bone marrow transplantation. Bone Marrow Transplant. 1990; 5:373-377. Hill GR, Ferrara JL. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood. 2000;95:2754-2759. Ferrara JL. The cytokine modulation of acute graft-versus-host disease. Bone Marrow Transplant. 1998;21(suppl 3):S13-S15. Przepiorka D, Khouri I, Ippoliti C, et al. Tacrolimus and minidose methotrexate for prevention of acute graft-versushost disease after HLA-mismatched marrow or blood stem cell transplantation. Bone Marrow Transplant. 1999;24:763-768. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825-828. Yam PY, Petz LD, Knowlton RG, et al. Use of DNA restriction fragment length polymorphisms to document marrow engraftment and mixed hematopoietic chimerism following bone marrow transplantation. Transplantation. 1987;43:399-407. Prentice RL, Kalbfleisch JD, Peterson AV, et al. The analysis of failure times in the presence of competing risks. Biometrics. 1978;34:541-554. Hess KR. Graphical methods for assessing violations of the proportional hazards assumption in Cox regression. Stat Med. 1995;14:1707-1723. Michallet M, Bilger K, Garban F, et al. Allogeneic hematopoietic stem-cell transplantation after nonmyeloablative preparative regimens: impact of pretransplantation and posttransplantation factors on outcome. J Clin Oncol. 2001;19:3340-3349. Mattsson J, Uzunel M, Brune M, et al. Mixed chimaerism is common at the time of acute graft-versus-host disease and disease response in patients receiving non-myeloablative conditioning and allogeneic stem cell transplantation. Br J Haematol. 2001;115:935-944.
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Acute and Chronic Graft-versus-Host Disease after Ablative and Nonmyeloablative Conditioning for Allogeneic Hematopoietic Transplantation Daniel R. Couriel, Rima M. Saliba, Sergio Giralt, Issa Khouri, Borje Andersson, Marcos de Lima, Chitra Hosing, Paolo Anderlini, Michelle Donato, Karen Cleary, James Gajewski, Joyce Neumann, Cindy Ippoliti, Gabriela Rondon, Agueda Cohen, Richard Champlin Department of Blood and Marrow Transplantation, Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas Correspondence and reprint requests: Daniel R. Couriel, MD, Department of Blood and Marrow Transplantation, Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030-4095 (e-mail: [email protected]). Received July 2, 2003; accepted October 23, 2003
ABSTRACT In this study, we evaluated the influence of nonmyeloablative and ablative conditioning regimens on the occurrence of acute and chronic graft-versus-host disease (GVHD). One hundred thirty-seven patients undergoing matched-related sibling transplantations received the same GVHD prophylaxis. Myeloablative regimens included intravenous busulfan/cyclophosphamide (n ⴝ 45) and fludarabine/melphalan (n ⴝ 29). Patients in the nonmyeloablative group (n ⴝ 63) received fludarabine/idarubicin/cytarabine, cisplatin/fludarabine/idarubicin, and fludarabine/cyclophosphamide. The actuarial rate of grade II to IV acute GVHD was significantly higher (hazard ratio, 3.6; 95% confidence interval, 1.5-8.8) in patients receiving ablative regimens (36%) compared with the nonmyeloablative group (12%). The cumulative incidence of chronic GVHD was higher in the ablative group (40%) compared with the nonmyeloablative group (14%). The rates were comparable within the first 200 days and were significantly higher in the ablative group beyond day 200 (hazard ratio, 5.2; 95% confidence interval, 1.2-23.2). Nonrelapse and GVHD-related mortality were relatively low in both groups. The use of the described nonmyeloablative preparative regimens was associated with a reduced incidence of grade II to IV acute GVHD and chronic GVHD compared with the busulfan/cyclophosphamide and fludarabine/melphalan transplant regimens. It is interesting to note that nonrelapse mortality with nonmyeloablative regimens in older and more debilitated patients was low (14%) and comparable to that achieved with standard high-dose regimens in younger patients. © 2004 American Society for Blood and Marrow Transplantation
KEY WORDS Acute GVHD
●
Preparative regimens
INTRODUCTION Myeloablative (MA) doses of chemotherapy and total body irradiation, typically used as preparative regimens in hematopoietic stem cell transplantation (HSCT), are associated with a high rate of morbidity and regimen-related mortality. In this respect, graftversus-host disease (GVHD) continues to be one of the main limitations of allogeneic transplantation [1-3]. This has largely limited the use of MA HSCT to life-threatening clinical indications in younger individuals without serious comorbidities. Hematologic 178
malignancies predominantly occur in older patients and often in patients with comorbidities who are not considered eligible for this treatment. Nonmyeloablative (NMA) preparative regimens have recently been developed as a means to reduce the morbidity related to hematopoietic transplantation [4-7]. These reduced-intensity regimens provide sufficient immunosuppression to achieve engraftment of an allogeneic bone marrow or blood stem cell graft and allow the immune-mediated graft-versus-malignancy effect to occur [8]. The syndrome of acute GVHD (aGVHD) is in
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part related to cytokines produced in response to the toxicity of the preparative regimen [9,10]. Intensive regimens can result in gastrointestinal (GI) mucosal injury with increased permeability and endotoxemia [11,12], which stimulates and amplifies a cytokine cascade that is central in the pathophysiology of aGVHD [12,13]. One theoretical advantage of the use of less toxic NMA preparative regimens may be a lower incidence of aGVHD. Because aGVHD is a risk factor for the development of chronic GVHD (cGVHD), its incidence may indirectly be reduced as well. The effect of preparative regimen intensity on the incidence of aGVHD and cGVHD has not yet been systematically addressed. In this study, we evaluated the influence of NMA and MA conditioning on the occurrence of aGVHD and cGVHD and nonrelapse mortality (NRM) in patients undergoing matchedrelated sibling transplantations and who received the same GVHD prophylaxis. We tested the hypothesis that use of a less toxic NMA preparative regimen would result in lower incidence of aGVHD and cGVHD.
METHODS We evaluated 137 consecutive patients who received HSCT as part of prospective clinical trials between June 1, 1996, and September 30, 2000. All patients received allogeneic hematopoietic transplants from an HLA-identical sibling donor. These studies were reviewed and approved by the University of Texas M.D. Anderson Cancer Institutional Review Board, and all patients provided written informed consent to participate. Information was obtained from the Department of Blood and Marrow Transplantation database, where all clinical data is systematically collected for every patient undergoing stem cell transplantation. Preparative regimens were considered MA if they produced profound pancytopenia for ⬎28 days without transplantation and if, after transplantation, hematopoietic recovery was completely donor derived (complete chimerism in ⬎80% of patients). NMA regimens were defined as those in which hematopoietic recovery would reliably occur within 28 days without transplantation and if, after transplantation, mixed chimerism could be documented in most patients [8]. MA preparative regimens included (1) busulfan 0.8 mg/kg intravenously (IV) every 6 hours on days ⫺7 to ⫺4 and cyclophosphamide 60 mg/kg on days ⫺3 and ⫺2 (IV BuCy) and (2) fludarabine 25 mg/m2 on days ⫺6 to ⫺2 and melphalan 90 mg/m2 on days ⫺3 and ⫺2 (FM), which is MA with this dose of melphalan. Hematopoietic transplantation occurred on day 0.
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NMA regimens included fludarabine 30 mg/m2/d on days ⫺6 to ⫺3, idarubicin 12 mg/m2/d on days ⫺6 to ⫺4, and cytarabine 2 g/m2 on days ⫺7 to ⫺3 (FlagIda); cisplatin 100 mg/m2 IV continuous infusion on days ⫺6 to ⫺3, fludarabine 30 mg/m2 on days ⫺4 and ⫺3, cytarabine 500 to 1000 mg/m2 on days ⫺4 and ⫺3 (PFA); and fludarabine 30 mg/m2 on days ⫺5 to ⫺3 with cyclophosphamide 750 mg/m2 on days ⫺5 to ⫺3 (FC). Seven patients in the FC group received rituximab 375 mg/m2 on day ⫺13 and 1000 mg/m2 on days ⫺6, ⫹1, and ⫹8. All these regimens have been used for standard chemotherapy and produce only transient myelosuppression without hematopoietic transplantation. The BuCy regimen was intended for younger patients without serious comorbidities. The FM regimen was intended as a potentially better tolerated MA regimen in older or medically infirm patients. Although it was initially considered a reduced-intensity regimen, it is now clear that its overall toxicity is comparable to that of MA regimens; it necessitates hematopoietic stem cell infusion for hematologic recovery. In this study, aGVHD, cGVHD, and GVHDrelated mortality were similar in patients receiving FM and IV BuCy. Thus, they were analyzed as a group. The NMA FlagIda regimen was intended for older patients with myeloid leukemias, and the NMA FC and PFA regimens were used for patients of all ages with lymphoid malignancies. The diagnoses treated included myelodysplastic syndrome/acute myelogenous leukemia (n ⫽ 61), chronic myelogenous leukemia/myeloproliferative disorders (n ⫽ 36), chronic lymphocytic leukemia (n ⫽ 8), and non-Hodgkin lymphoma (n ⫽ 32). All patients received their grafts from HLA genotypically identical siblings. The cell source was peripheral blood stem cells (PBSC) in most patients (n ⫽ 113; 82%). All patients received a common regimen of posttransplantation immunosuppressive therapy as prophylaxis against GVHD. This included tacrolimus starting at day ⫺2, with dose adjustments to maintain blood levels of 5 to 15 ng/mL, and methotrexate 5 mg/m2 on days 1, 3, and 6. Bone marrow recipients received an additional dose on day ⫹11 [14]. Tacrolimus was continued for at least 90 days in the absence of disease progression. Patients at low risk for relapse continued tacrolimus until 6 to 9 months after transplantation. Those at high risk for relapse or minimal residual disease had immunosuppression withdrawal at 3 to 6 months after transplantation. If grade II to IV aGVHD developed, methylprednisolone 2 mg/kg was generally administered. Chronic GVHD was initially treated with corticosteroids and tacrolimus. Patients with steroid-resistant aGVHD or cGVHD received alternative immunosuppressive therapy. We defined low-risk disease as the first chronic or 179
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accelerated phase of chronic myelogenous leukemia, acute myelogenous leukemia, or lymphoid malignancies in first complete remission and lymphoid malignancies in first chemosensitive relapse or second complete remission. All other patients were considered to have high-risk disease. Conditions classified as serious comorbidities included 1 or more of the following: (1) cardiac: coronary artery disease, congestive heart failure, serious or life-threatening arrhythmias, deep venous thrombosis, pulmonary embolism, and uncontrolled severe hypertension; (2) pulmonary: emphysema, asthma, history of acute respiratory failure necessitating mechanical ventilation and sarcoidosis; (3) renal: acute or chronic renal failure and hydronephrosis; (4) infections: fungal (Aspergillus species, Candida species, Torulopsis glabrata, or Fusarium species), viral (cytomegalovirus, varicella-zoster virus, or herpes simplex virus), mycobacterial (tuberculosis or myobacterium avium intercellulare), Pneumocystis carinii, and serious bacterial infections (septic shock or osteomyelitis); and (5) other conditions: pancreatitis, history of GI bleeding, stroke, seizures, other cancers, autoimmune conditions (inflammatory bowel disease, autoimmune hemolytic anemia, or GVHD in a previous transplant). GVHD Assessment
GVHD was assessed in 2 ways. The first way, which is the focus of this article, was to assess the occurrence of GVHD before immune manipulation to better determine the effect of the preparative regimen on the occurrence of GVHD. In this case, aGVHD was evaluated within 100 days after HSCT. The staging and grading of aGVHD was performed according to modified Glucksberg consensus criteria [15]. Biopsy samples from involved tissues were obtained in all 29 patients with grade II to IV aGVHD. The diagnosis of aGVHD was confirmed histologically in 24 (83%) of 29 patients with grade II to IV aGVHD. Seventeen (55%) of 31 skin biopsy samples were positive and confirmed the diagnosis of skin GVHD in 17 of 18 patients. The remaining patient had a negative skin biopsy with a simultaneous positive GI biopsy. Fourteen (70%) of twenty upper and lower GI biopsies confirmed the diagnosis of GI GVHD in 8 of 12 patients with clinical manifestations of GI GVHD. Six (50%) liver biopsies confirmed the diagnosis of liver GVHD in all 5 patients with abnormal bilirubin and alkaline phosphatase. The remaining patient had concomitant positive skin and GI biopsies. The diagnosis of cGVHD was based on clinical manifestations, including sicca syndrome, lichenoid changes on mucosal surfaces, lichenoid skin GVHD, sclerodermal GVHD, and GI strictures. Diarrhea or liver function abnormalities were considered part of the spectrum of cGVHD if they occurred beyond 100 days after the 180
last stem cell infusion and in the presence of a positive biopsy of the GI tract or liver, respectively. Chronic GVHD was classified as de novo, progressive, or relapsing [3]. De novo cGVHD was defined as occurring without a history of aGVHD. For relapsing cGVHD, cGVHD developed after a history of aGVHD that was successfully treated into complete remission. Patients with progressive cGVHD had aGVHD that failed to achieve remission with treatment and progressed into cGVHD. Second, we determined the incidence of all GVHD (ie, acute or chronic) without censoring at the time of immune manipulation. Only mortality was considered as a competing risk. Thus, we included GVHD that occurred after immune manipulation in both the MA and NMA groups. Chimerism
Hematopoietic chimerism was evaluated on bone marrow cells by restriction fragment length polymorphisms at the AY-29 or YNH24 loci and by cytogenetic studies in sex-mismatched cases [16]. These assays are able to detect mixed chimerism if more than 5% recipient or donor cells are present. Lineagerelated chimerism studies were not available on all patients and were not considered in this analysis. Mortality
GVHD-related mortality included death secondary to aGVHD or cGVHD independently of whether GVHD occurred before or after immune manipulation. Deaths due to infection in the setting of GVHD or in patients who were receiving immunosuppressive treatment for GVHD were also included in this group. NRM included all deaths unrelated to the recurrence or progression of malignancy. Statistical Analysis
The primary objective of the analysis was to assess the incidence of GVHD according to the intensity of the conditioning regimen while the patient received standard GVHD prophylaxis. For this purpose, the primary end points of the study were the incidence of grade II to IV aGVHD and cGVHD before immune modulation (including withdrawal of immune suppression) or donor lymphocyte infusions (DLI). Secondary end points were GVHD-related mortality, NRM, and the overall incidence of GVHD irrespective of immune modulation. The incidences of GVHD, GVHD-related mortality, and NRM were estimated by the method of Prentice et al. [17] to account for the differences in immune modulation, relapse, or mortality rates in the different conditioning regimens. Competing risks for the occurrence of GVHD before manipulation were withdrawal of immunosuppression, DLI, disease progression, and
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Table 1. Patient Characteristics Variable Sex (M/F) Age (y) Median Range No. patients >40 y (%) Diagnosis, n (%) AML/MDS CML CLL NHL Cell source, n (%) PBSC BM Disease risk group, n (%) High risk Low risk Prior chemotherapy regimens <2 >2 Serious comorbidities, n (%)
Overall (n ⴝ 137)
Ablative (n ⴝ 74)
Nonmyeloablative (n ⴝ 63)
P Value
85/52
47/27
38/25
NS
53 15-75 105 (77)
45 15-68 47 (63)
59 21-75 58 (92)
<.001 <.001
61 (45) 36 (26) 8 (6) 32 (23)
42 (57) 30 (40) 0 2 (3)
19 (30) 6 (9) 8 (13) 30 (48)
<.001
113 (82) 24 (18)
53 (72) 21 (28)
60 (95) 3 (5)
<.001
65 (48) 72 (52)
30 (40) 44 (60)
35 (56) 28 (44)
NS
88 (64) 49 (36) 63 (46)
54 (73) 20 (27) 29 (39)
34 (54) 29 (46) 34 (54)
.02 NS
AML indicates acute myelogenous leukemia; MDS, myelodysplastic syndrome; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin lymphoma; BM, bone marrow; NS, not significant.
death without GVHD. In addition, the occurrence of aGVHD was considered a competing risk for the estimation of the cumulative incidence of de novo cGVHD. Follow-up time was censored at the earliest of these events. Only death without GVHD was a competing risk for the estimation of the overall incidence of GVHD irrespective of immune manipulation, and only progression of malignancy was considered a competing risk for NRM. Median follow-up among survivors was 20 months (range, 6-48 months) overall, 19 months (range, 8-48 months) for the ablative group, and 23 months (range, 6-42 months) for the reduced-intensity group (P ⫽ .4). To ensure comparable follow-up time for the 2 conditioning regimen groups, we report the incidence of cGVHD at 18 months. NRM was estimated at 2 years. The Cox proportional hazards model was used to compare the outcomes of interest among patients receiving MA and NMA regimens. It was not possible to adjust for confounding by using multivariate analysis because of the sample size. Adjustment was therefore limited to bivariate analysis, when possible, with preparative regimen as a constant covariate. In addition, the distribution of potential confounding factors (age, stem cell source, diagnosis, and chimerism at day 30) was skewed in the NMA group (described below). This further limited our ability to conduct multivariate analysis. Adjustment for confounding in this case was limited to restricting the comparison of the regimen groups to the categories of the covariates that represented most patients in the NMA group. The
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assumption of constant proportional hazards over time was tested graphically [18]. When indicated, the maximized partial likelihood method was used to find the appropriate time breakpoint within which the hazards were proportional. Patient characteristics were compared by the Fisher exact and 2 tests. Two-sided P values of ⬍.05 were considered significant. Analysis was performed with Stata version 7.0 (Stata Corp., College Station, TX).
RESULTS Patient Characteristics
Patient characteristics varied significantly according to the preparative regimen (Table 1). The MA group patients were generally younger (median age, 45 years; P ⬍ .001). Less than half of these patients had high-risk disease, compared with 56% in the NMA groups (P ⫽ not significant). Similarly, a lower proportion of patients had serious comorbidities in the MA (39%) compared with the NMA (54%) group at the time of transplantation (P ⫽ not significant). A significantly higher proportion of patients in the NMA group had received more than 2 chemotherapeutic regimens before transplantation (P ⫽ .02). The stem cell source was bone marrow in 28% of the MA group, compared with 5% of those receiving NMA regimens (P ⬍ .001). In the MA group, 97% were myeloid malignancies. However, most patients in the NMA group (61%) had lymphoid malignancies (P ⬍ .001). 181
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Table 2. Incidence of GVHD before Immune Manipulation Regimen Myeloablative Nonmyeloablative Total
n*
No. aGVHD II-IV
Incidence† (95% CI)
67 56 123
23 6 29
34% (24-48) 11% (5-23)
n‡
No. cGVHD at 18 mo
Incidence† (95% CI)
70 55 125
25 7 32
40% (28-58) 14% (7-27)
*Patients with evidence of donor chimerism or aGVHD. †Cumulative incidence considering immune manipulation, second infusion, relapse, or death without GVHD as competing risks. ‡Patients with evidence of donor chimerism or cGVHD.
Seven patients had graft failure and were not considered at risk for GVHD; they were included only for the estimation of NRM. Of the remaining 130 patients, those who had evidence of engraftment by evaluation of mixed or complete chimerism (at day 30 for aGVHD and at day 100 for cGVHD) or by diagnosis of GVHD were evaluable for the incidence of GVHD. These included 123 patients for aGVHD and 125 patients for cGVHD. Four patients had aGVHD and 5 had cGVHD without assessment of chimerism on day 30 and 100, respectively. Acute GVHD
Overall Incidence and Severity. The incidence of grade II to IV aGVHD was 36% (95% confidence interval [CI], 26%-49%) in the MA group compared with 12% (95% CI, 6%-25%) in the NMA group (hazard ratio [HR], 3.6; 95% CI, 1.5-8.8; Table 2; Figure 1). This effect was consistent when comparison was performed among patients who were older than 40 years and those who received PBSCs (Table 3), after adjusting for the number of prior chemotherapy regimens received or the disease risk category. Ten patients (8%) developed grade III to IV aGVHD. The number of patients with grade III to IV aGVHD was low with all preparative regimens. The
incidence was higher in the MA (12%; 95% CI, 6%23%) than in the NMA group (4%; 95% CI, 1%14%), but this was not statistically significant (P ⫽ .1). The PFA and the FlagIda regimens had no cases of grade III to IV aGVHD. Organ Involvement. Skin was the organ most frequently involved (n ⫽ 18; 15%), followed by GI GVHD (n ⫽ 12; 10%) and liver (n ⫽ 5; 4%; Table 4). When individual regimens were analyzed for organ involvement, there was a significantly lower proportion of skin involvement in the NMA group (P ⫽ .03) and a higher proportion of GI involvement in the FM group (P ⫽ .01) compared with the other regimens. Only 5 patients with grade II to IV aGVHD had liver involvement (4%). Liver was the only organ involved in 3 patients, all of whom were in the BuCy group. Chimerism and aGVHD. In the NMA group, 32 evaluable patients (57%) had complete donor chimerism, and 23 (41%) had mixed chimerism at day 30. Most patients (89%) in the MA group had complete donor chimerism at day 30. Among patients who had complete donor chimerism at day 30, the incidence of grade II to IV aGVHD was 36% in the MA group and 19% in the NMA group (HR, 2.3; 95% CI, 0.9-6.2). This effect is only marginally significant, but it is consistent with the overall higher incidence in the MA group compared with the NMA group. Among patients receiving NMA regimens, there was a trend for a reduced rate of grade II to IV aGVHD in patients with mixed chimerism. This ocTable 3. Incidence of aGVHD by Stem Cell Source, Age, and Diagnosis
Variable
Figure 1. Cumulative incidence of acute GVHD. Patients receiving nonmyeloablative regiments were compared with those receiving myeloablative regimens. Progression of malignancy, immunosuppression withdrawal, donor lymphocyte infusion, and death without GVHD were considered competing risks. 182
Peripheral blood stem cells Myeloablative Nonmyeloablative Age >40 y Myeloablative Nonmyeloablative Myeloid malignancy Myeloablative Nonmyeloablative
n*
No. aGVHD II-IV
48 53
19 6
43 51 65 23
HR
95% CI
3.9
1.5-9.7
14 6
3
1.1-7.7
22 2
4.3
1.0-18.5
*Patients with evidence of donor chimerism or aGVHD.
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Table 4. Acute GVHD Grade II to IV: Organ Involvement Regimen
n
Skin, n (%)
GI, n (%)
Liver, n (%)
BuCy FM Nonmyeloablative Total
41 26 56 123
8 (19) 6 (23) 4 (7)* 18 (15)
3 (7) 6 (23)* 3 (5) 12 (10)
3 (7) 1 (4) 1 (2) 5 (4)
*Indicates a statistically significant difference in organ involvement in comparison with the other preparative regimens.
curred in 1 patient with mixed chimerism (4%) and 5 patients with complete donor chimerism (16%), but it was not statistically significant (HR, 0.5; 95% CI, 0.1-2.4). The effect of mixed chimerism could not be assessed among the MA group because of the small number of patients. Chronic GVHD
The cumulative incidence of cGVHD at 18 months was 40% (95% CI, 28%-58%) for the MA group and 14% (95% CI, 7%-27%) for patients receiving NMA regimens. The increased rate among the MA group was not constant over time, and the Cox model assumption of constant proportional hazards was not met. Up to day 200 after transplantation, the rates of cGVHD were comparable in the MA and NMA groups (HR, 1.1; 95% CI, 0.4-3.4). Beyond day 200, the rate was significantly higher for the MA group (HR, 5.2; 95% CI, 1.2-23.2; Figure 2). This effect was consistent when comparison was done among patients who were older than 40 years and for those who received PBSC, after adjusting for the number of prior chemotherapy regimens received or for the disease risk category. The cumulative incidence of de novo cGVHD was
Figure 3. Cumulative incidence of all GVHD. Patients receiving nonmyeloablative (NMA) regimens were compared with those receiving myeloablative (MA) regimens. Only mortality was considered a competing risk. Patients undergoing immunosuppression withdrawal, DLI, or both were included in both the NMA (n ⫽ 44) and MA (n ⫽ 29) groups.
10% (95% CI, 5%-22%) and 6% (95% CI, 2%-19%) in the MA and NMA groups, respectively. The rates were not significantly different between these 2 groups, and the HR did not vary over time (HR, 2.4; 95% CI, 0.6-9.9). This suggests that the higher rate among the MA group could be attributed mostly to a higher rate of progressive and relapsing cGVHD, highlighting the importance of aGVHD as a risk factor. Chimerism and cGVHD. Among patients with complete donor chimerism at day 30, we still observed a trend for an increased rate of GVHD for the MA group (HR, 1.7; 95% CI, 0.7-4.2). Similarly to what was observed in the overall group, the HR varied over time; however, this was not statistically significant and did not warrant a separate analysis before and after day 200 after transplantation. In the NMA group, there was a trend for a lower rate of cGVHD for patients with mixed chimerism compared with those with complete chimerism; 1 of 23 patients with mixed chimerism developed cGVHD, compared with 6 of 30 of patients with complete chimerism (HR, 0.3; 95% CI, 0.03-2.4). Similarly, the corresponding proportions for day 100 were 1 of 24 and 6 of 16 (HR, 0.2; 95% CI, 0.02-1.5). GVHD after Early Immunosuppression Withdrawal and DLI
Figure 2. Cumulative incidence of chronic GVHD. Patients receiving nonmyeloablative regimens were compared with those receiving busulfan/cyclophosphamide and fludarabine/melphalan. Progression of malignancy, immunosuppression withdrawal, donor lymphocyte infusion, and death without GVHD were considered competing risks.
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Fifty-nine patients underwent early withdrawal of immunosuppression for recurrence or progression of malignancy. Nineteen of these patients additionally received a DLI for disease control. Five patients received a DLI without immunosuppression withdrawal. The proportion of patients receiving immune manipulation was significantly higher in the NMA group (70%) than in the MA group (39%; P ⬍ .01). We 183
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Table 5. Cumulative Incidence of GVHD Regimen
n*
No. GVH
Myeloablative Nonmyeloablative Total
72 58 130
38 23 61
Incidence at 18 mo†
95% CI
55% 41%
48-63 29-57
*Excluded were patients with primary graft failure: 2 in the myeloablative group and 5 in the nonmyeloablative group. †Cumulative incidence of GVHD considering mortality as a competing risk.
evaluated all GVHD (both acute and chronic) in both groups, including patients who underwent immune manipulation. Overall, the MA group had a significantly higher incidence of GVHD (55%) compared with the NMA (41%; HR, 1.8; 95% CI, 1.1-3.0; P ⫽ .03; Table 5; Figure 3). NRM and GVHD-Related Mortality
NRM at 2 years was similar for the MA (19%; 95% CI, 12%-31%) and NMA (15%; 95% CI, 8%29%; HR, 1.4; 95% CI, 0.6-3.3; Figure 3) groups. The causes of NRM at 2 years are listed in Table 6. The GVHD-related mortality rate was low for both groups: 8% (95% CI, 3%-18%) in the MA group and 9% (95% CI, 3%-22%) in the NMA group (HR, 1.1; 95% CI, 0.3-4.1; Table 6). DISCUSSION T-cell alloreactivity remains central in the pathophysiology of both aGVHD and cGVHD. However, research over the last decade supports additional mechanisms that involve other cell types and multiple cytokines in the pathophysiology of aGVHD. In this model, the conditioning regimen causes tissue damage and the release of proinflammatory cytokines, including tumor necrosis factor-␣, interleukin (IL)-1, and IL-6 [13]. Donor T cells respond to host alloantigens with activation and release of the T-helper 1 cytokines IL-2 and interferon-␥. Interferon-␥, in conjunction with endotoxins from GI bacteria that circulate systemically after mucosal damage, activates macrophages and natural killer cells [9,12,13]. This activation results in further release of inflammatory cytokines and amplifies the tissue damage caused by the immune-mediated graft-versus-host reaction [13]. In this study, we tested the hypothesis that a less intensive and toxic NMA regimen would be associated with less aGVHD and, consequently, cGVHD in patients receiving the same GVHD prophylaxis. Comparison of GVHD that occurs after NMA and MA regimens is difficult because of differences in patient characteristics and risk factors for GVHD. NMA transplantations were developed to extend the 184
use of HSCT to older patients and those with comorbidities who were not eligible for MA regimens. Thus, the NMA transplant recipients were at higher risk for aGVHD and cGVHD. Many centers have used unique posttransplantation immunosuppressive therapy with NMA transplants, and this precludes direct comparison with MA regimens. Others have intentionally discontinued immunosuppressive therapy early after NMA transplantations to enhance graftversus-leukemia effects. Early termination of immunosuppression may be associated with a higher incidence of GVHD, lower overall survival, and an increase in transplant-related mortality [19]. We have chosen to use the same posttransplantation immunosuppressive therapy for NMA regimens as with MA transplantations and to continue this therapy for at least 3 months in the absence of disease progression. This facilitates comparison of the effect of the preparative regimen on the incidence and severity of GVHD. An additional limitation to the comparison of regimens is the heterogeneity of the patient and transplant characteristics, which is inherent to the current indications for NMA regimens and favors the MA group. Most patients who received NMA regimens in our population were older than 40 years and had received PBSCs; they were therefore at an increased risk of GVHD. To adjust for this heterogeneity, we compared outcomes within these categories of patients because the skewed distribution precluded the use of multivariate analysis. Differences in the number of prior chemotherapy regimens and the disease risk group may be additional confounders in the comparison of the effects of preparative regimens. We could conduct only bivariate analysis to adjust for these factors because of the small sample size. The incidence of grade II to IV aGVHD was significantly lower in NMA regimens compared with the MA group. This effect was consistent among patients ⱖ40 years old, those who received PBSCs, and those with complete donor chimerism. It was also independent of the number of prior chemotherapy regimens or the disease risk category. As previously reported [20], we also found that there was a trend to a lower incidence of aGVHD in
Table 6. Causes of Nonrelapse Mortality Variable
Myeloablative (n ⴝ 74)
Nonmyeloablative (n ⴝ 63)
GVHD Graft rejection Infection Multiorgan failure Hemorrhage Other Total
6* 0 1 3 1 4 15
5* 1 2 0 0 1 9
*One death occurred beyond 18 mo after transplantation.
GVHD and Conditioning Regimens
patients with mixed chimerism in the NMA group. The incidence of severe (grade III to IV) aGVHD was low in all regimens, and no statistically significant differences were found between groups. These conclusions were maintained when patients undergoing early withdrawal of immunosuppression, DLI, or both were included in the analysis. Similarly to aGVHD, we observed a lower incidence of cGVHD among patients receiving NMA regimens. This became significant beyond 200 days after transplantation. Most (76%) of the cases in the MA group were progressive or relapsing cGVHD, and this highlights the importance of aGVHD as a risk factor. This increased rate was independent of the number of prior chemotherapy regimens or the disease risk category, and it persisted among older patients and those receiving PBSC. Differences in the incidence of GVHD were still present when patients who had undergone immunosuppression withdrawal or DLI were included in the comparison of both groups. NRM and GVHD-related mortality were relatively low with all regimens. It is interesting to note that NRM with NMA regimens in older and more debilitated patients was low and comparable to that achieved with standard highdose regimens in younger patients. In conclusion, the use of the described NMA preparative regimens was associated with a reduced incidence of grade II to IV aGVHD and cGVHD compared with MA transplantation regimens. Our data suggest that the reduction in the incidence of aGVHD and cGVHD is independent of factors such as older age, use of PBSCs, and more prior chemotherapy treatments or advanced disease.
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