In an attempt to decrease toxicity in high-risk patients undergoing unrelated donor hematopoietic stem cell transplantation (URD HSCT), we tested a combination of cyclosporine (CSP) and mycophenolate mofetil (MMF) as graft-versus-host disease (GVHD) prophylaxis with the reduced-intensity conditioning regimen fludarabine/melphalan (Flu/Mel). A total of 22 adult patients with advanced myeloid (n=15) and lymphoid (n=7) malignancies were treated. All patients received Flu 25 mg/m2 for 5 days and Mel 140 mg/m2, with CSP 3 mg/kg daily and MMF 15 mg/kg three times a day. The median age was 49 years (range 18–66). Durable engraftment was seen in all but one patient with myelofibrosis. The 1-year nonrelapse mortality was 32%, 27% from GVHD. The cumulative incidence of acute GVHD grade 2–4 and 3–4 was 63 and 41%, respectively. With a median follow-up of 18 months, the disease-free survival (DFS) and overall survival (OS) are 55 and 59%, respectively. For patients with AML and MDS (n=14), the DFS and OS is 71%. For patients undergoing a second transplant (n=14), the DFS and OS is 57%. In conclusion, this regimen is associated with acceptable toxicity but high rates of GVHD in high-risk patients undergoing URD HSCT. Encouraging disease control for patients with advanced myeloid malignancies was observed.
The applicability of unrelated hematopoietic stem cell transplantation (URD HSCT) for patients with hematological malignancies has been greatly hampered by its early toxicity, with 100-day mortality ranging from 15 to 50%.1 These statistics have been generated from younger patients (<55 years) with normal organ function, with age and disease status at transplantation largely accounting for the variability in outcome. Older patients and those who have comorbidities or who have failed previous autologous transplants experience even greater toxicity, thus making them extremely high risk or ineligible for this procedure.2
Reduced-intensity conditioning (RIC) regimens have allowed high-risk patients to undergo allogeneic HSCT with decreased transplant-related toxicity. The RIC regimen fludarabine/melphalan (Flu/Mel), for example, has been used in conjunction with tacrolimus (FK-506) and mini-methotrexate (MTX) as graft-versus-host-disease (GVHD) prophylaxis, for high-risk patients with a variety of hematological malignancies, resulting in a relatively low 100-day nonrelapse mortality (NRM) of 37%.3 Excellent engraftment was also seen with this regimen (median donor chimerism of 100% at day 30), with an encouraging disease-free survival (DFS) at 1 year of 57 and 49%, for patients with early and advanced disease, respectively. In this series, the probability of grade 2–4 and 3–4 acute GVHD was 0.49 and 0.29, respectively.
The combination of cyclosporine (CSP) and mycophenolate mofetil (MMF) has been successfully developed as GVHD prophylaxis, with powerful synergism in animal models,4 and clinical efficacy in the nonmyeloablative transplant setting.5,6 In an attempt to further reduce regimen-related toxicity and time to engraftment in a high-risk URD HSCT population, we tested this regimen for GVHD prophylaxis, in combination with the Flu/Mel regimen. We herein report our results in 22 adult patients.
Patients and methods
Between December 2000 and April 2002, 22 consecutive patients were treated on this protocol. Patients had a suitable unrelated donor and were considered poor candidates for standard dose transplantation, based on risk factors outlined in Table 1. In all, 14 patients had failed a previous autologous transplant, with a median interval between transplants of 14.5 months (range 7–115 months). Exclusion criteria were severe organ dysfunction, including diffusion capacity (DCO) <40%, left ventricular ejection fraction <45%, and creatinine clearance <50 ml/min. Unrelated donors were consented and procured through the National Marrow Donor Program (NMDP) according to their standards and procedures. Patient characteristics are summarized in Table 2. The protocol was approved by the Institutional Review Board at City of Hope.
Diagnosis and status at transplantation
There were 15 patients with myeloid and seven with lymphoid malignancies. Most patients with myeloid malignancies had advanced disease at transplant, with only one of 10 AML patients in confirmed remission and three of four MDS patients with RAEB-t or worse. Most patients had poor risk cytogenetics before transplant. Details of cytogenetics, percent blasts at transplantation, and salvage therapy before transplant for these patients are described in Table 3. Patients with lymphoid malignancies had relapsed/refractory disease or concomitant therapy-related MDS (t-MDS). Most were heavily pre-treated (Table 4).
Donor/recipient matching and stem cell source
High-resolution polymerase chain reaction-sequence-specific-primers (PCR-SSP) for HLA class I and II was performed.7 Donor/recipients were fully matched at HLA A, B, C, and DRB1 in 17 pairs, while five had HLA class I allele mismatches. Stem cell source, which was determined by the NMDP donor center, was bone marrow (n=11) and peripheral blood (n=11). The median mononuclear and CD-34 cell dose was 0.9 × 108/kg and 5.6 × 106/kg, respectively. There was no significant difference in the number of CD-34 cells between marrow and blood products (4.3 × 106 vs 5.9 × 106, P=0.33), but mononuclear cell dose was lower from bone marrow products (0.7 × 108 vs 4.8 × 108, P=0.013).
Patients were conditioned with Flu 25 mg/m2 intravenously daily for 5 days (day −7 to day −3), and Mel 140 mg/m2 intravenously on day −2. Donor-derived bone marrow or filgrastim-stimulated peripheral blood stem cells were infused on day 0. Mel was dosed based on actual body weight.
GVHD prophylaxis consisted of CSP 3.0 mg/kg intravenously daily, starting on day −1, switched to an oral dose of approximately three times the intravenous dose when oral intake resumed; and MMF 15 mg/kg intravenously three times a day beginning on day 0, switched to the same dose when oral intake resumed. CSP was adjusted in the first month according to renal function and to maintain a therapeutic level (100–300 ng/ml). Monitoring of whole blood CSP levels was performed twice a week. Tapering of MMF was started on day +28, and discontinued over 1–2 months, followed by tapering of CSP over 3–6 months, if GVHD was not present.
Antimicrobial prophylaxis included levofloxacin, low-dose amphotericin (0.15 mg/kg/day), trimethroprim/sulfametoxasole, and acyclovir according to our hospital guidelines. Monitoring for CMV viremia was performed twice a week from day +21 to day +100, using the shell vial method. Patients with a positive culture were started pre-emptively on gancyclovir or valgancyclovir for 6 weeks.
Acute GVHD was graded by established criteria.8 Chronic GVHD was evaluated in patients surviving beyond 100 days, and classified as limited or extensive9 Patients developing grade 2 or greater acute GVHD were treated with methylprednisolone, 1 mg/kg twice a day. Chronic GVHD was treated with CSP and prednisone. Steroid refractory GVHD was treated with alternative immunosuppressive agents, at the discretion of the investigator.
Toxicity was graded according to Bearman criteria.10 Bone marrow examinations were performed around days +30, +100, and +365, and were evaluated for morphology, cytogenetics, FISH, and chimerism using short tandem repeats (STRs).
Overall survival (OS) was measured from the date of transplant until death or last contact. DFS was defined for all patients as the transplant date to the date of confirmed progression, relapse, or death. Time-to-event curves were estimated using the Kaplan–Meier method and we calculated P-values based on the log-rank test. We applied the Cox proportional hazards regression to estimate the association between survival and continuous prognostic variables. Univariate analysis was performed to determine the effects of various prognostic factors on OS, DFS, and NRM. The Fisher's exact test was used for comparing categorical variables against other categorical variables. All P-values are two-sided unless otherwise noted. The data were analyzed as of March 2003.
Engraftment and chimerism
All patients showed evidence of hematopoietic engraftment. The median time to ANC greater than 500/μl was 13 days (range 10–71 days). By stem cell source, the median time to ANC greater than 500/μl was 17 days (range 10–71 days) and 13 days (range 11–18) for bone marrow and PBSC, respectively. All patients became platelet transfusion independent at a median time of 19 days. One of five patients with a major ABO mismatch developed transient pure red cell aplasia. One patient with myelofibrosis had secondary graft failure in the setting of severe GVHD, but durable engraftment was seen in all other cases. Day 30 chimerism by STR analysis from bone marrow mononuclear cells was >90% donor in 20 tested patients.
Toxicity was graded according to the Bearman criteria.10 There were no grade 4 toxicities. Four patients developed transient pulmonary infiltrates around the time of engraftment, which responded rapidly to short courses of corticosteroids. Two patients had mild reversible elevation of liver enzymes, including one of seven patients (14%) who received Mylotarg before URD HSCT. Grade 2 stomatitis was seen in nine patients (45%). Four patients died before day 100, all from complications of GVHD. The 100-day NRM was 18%. Three additional patients died between days +100 and +365 from nonrelapse causes, two from GVHD, and one from herpes simplex (HSV) encephalitis, for a 1-year NRM of 32%. By univariate analysis, we did not detect any pre-transplant factor that predicted for NRM, but the small number of patients in this study results in insufficient power to draw any firm conclusions.
CMV viremia was detected in six patients (18 patients at risk based on positive donor/recipient serology pre-transplant) during the first 4 months post transplant. Four of them had CMV disease at a median of 33 days post transplant. Two of the latter patients had severe GVHD and died as a result of it.
Other opportunistic infections
Three additional opportunistic infections occurred, including one case each of Aspergillus pneumonia, HSV encephalitis, and RSV pneumonia.
A total of 14 patients developed grade 2–4 acute GVHD for a cumulative incidence of 63% (95% CI, 43–84%). Nine of these had grades 3–4, for a cumulative incidence of 41% (95% CI, 20–61%). Chronic GVHD was seen in 14 of 19 (73%) (95% CI, 48–98%) evaluable patients, 10 extensive and four limited according to Seattle criteria. The current mean dose of prednisone for these patients is 8.7 mg/day (range, 0–40 mg/day). Six of nine (27%) deaths on this protocol were related to GVHD.
Survival and DFS
Currently, 13 patients are alive for a projected 1 year OS of 59% (95% CI 39–79%), with a median follow-up of 18 months (range 11–27 months). The 1-year DFS is 55% (95% CI 34–75%), with 12 patients in continuing complete remission (CR) (Figure 1).
Disease-specific outcome and second transplant outcome
Myeloid disease (Table 3)
In all, 13 of 14 patients with AML or MDS had no evidence of disease around day +30, by bone marrow morphology and cytogenetics. One patient was not evaluable due to early, lethal GVHD. One patient relapsed at 2 months post transplant with malignant ascites and subsequently died. Two patients died from nonrelapse causes (one HSV encephalitis, one GVHD). For this group of patients (AML or MDS), the 1-year OS and DFS is 71% (95% CI 46–95%) (Figure 2). One patient with myelofibrosis died early post transplant from severe GVHD.
Lymphoid disease (Table 4)
Two patients with follicular non-Hodgkin's lymphoma (NHL) and one with large cell transformation of follicular NHL died of severe GVHD within 4 months post transplant, and were not evaluated for response. The remaining patient with follicular lymphoma achieved a PR but developed progressive disease at 10 months post transplant. One patient with refractory Hodgkin's disease (HD) did not respond to treatment, and died of progressive disease at 9 months post transplant. One patient with cutaneous T-cell lymphoma (CTCL) and one with diffuse large cell NHL are alive without evidence of disease, at 16 and 13 months post transplant, respectively.
Eight of 14 patients (7/9 myeloid, 1/5 lymphoid) undergoing an URD HSCT following a failed autologous transplant are alive without disease. Two patients (14%) died before day 100 from GVHD, two additional patients died after day 100 from nonrelapse causes (one GVHD, one HSV encephalitis), and two patients died from relapsed disease (one AML, one HD). At a median follow up of 17 months, the OS and DFS for this group is 57% (95% CI 31–83%) (Figure 3).
The benefit of URD HSCT could be greatly expanded to patients with contraindications to conventional myeloablative transplant by reducing early toxicity related to the preparative regimen. As such, the Flu/Mel regimen has been shown to be effective in high-risk patients with hematological malignancies, providing sufficient immunosuppression and cytoreduction, with limited early mortality.3 This regimen relies to a large extent on a graft-versus-malignancy effect,11 which may be more pronounced in the URD HSCT setting, by virtue of a greater minor histoincompatibility.12,13
By substituting CSP/MMF as GVHD prophylaxis instead of FK-506/MTX, we attempted to further decrease early toxicity and time to engraftment for high-risk adult patients undergoing URD HSCT, conditioned with Flu/Mel. We demonstrated that this regimen provides adequate immunosuppression to allow engraftment in all patients. In addition, we found an encouraging 100-day NRM of 18%, related to GVHD rather than organ dysfunction. This compares favorably to the 37% NRM reported with FK506/MTX,3 although a definitive conclusion in this regards cannot be made due to the small number of patients in our study and the indirect, nonrandomized comparison between studies. However, our result may be related in part to the absence of MTX in the regimen. Indirect evidence for this hypothesis includes a low incidence of serious liver toxicity. We did not observe any cases of veno-occlusive disease of the liver (VOD) in the seven patients who received Mylotarg before URD HSCT, in contrast to the experience recently reported with this drug, describing a 17% incidence of VOD after HSCT, 11% fatal.14 Mucositis was observed in 45% of our patients (Bearman grade 2); again, indirect comparison with MTX-based regimens in this respect is difficult due to intrinsic problems with mucositis grading systems, but one study using Flu/Mel and FK-506/MTX reported Bearman grade 2 stomatitis in nine of 11 patients (82%),15 suggesting that the avoidance of MTX is important in minimizing regimen-related toxicity.
We decided to use MMF in a t.i.d. schedule based on preliminary clinical and pharmacological data available at the time we designed our regimen, indicating that the twice a day regimen was associated with a high incidence of acute GVHD in a fully ablative URD setting (Richard Nash and Hans-Peter Kiem, personal communication). In spite of this, acute GVHD remained a significant problem for our patients, accounting for six of nine deaths. The risk of grade 3–4 acute GVHD, in this older patient population, was 41%. Although this is similar to the 39% reported with FK-506/MTX,3 lower toxicity from our regimen has not translated into less GVHD. Based on the limited data from this study, the combination of CSP/MMF did not clearly reduce the risk of acute GVHD, but the relatively small number of patients prevents drawing any firm conclusions. The risk of chronic GVHD of 73% in our series is similar to other reports using Flu/Mel3 or fully ablative conditioning regimens for URD HSCT.16
A critical question regarding RIC regimens is their ability to control advanced hematological malignancies. It has become apparent from the initial experience with nonmyeloablative regimes that some cytoreduction is important for these patients.5,17 In this regards, encouraging results were seen for our patients with AML and MDS, who had advanced disease at transplantation in most cases. Only one patient from this group of 14 has relapsed, and 10 are alive in remission. Patients with as high as 60% blasts in the bone marrow, and with poor-risk cytogenetics, remain in CR beyond 1 year after HSCT. Most of these patients, however, had few circulating blasts, (Table 3) and their marrows were generally hypocellular (data not shown), indicating a lower leukemia burden. These features may be important in selecting candidates with AML and MDS for this type of HSCT.
Patients with lymphoid malignancies had a poor outcome in this study, with three of seven dying from GVHD, and two additional patients relapsing post transplant. The small number of evaluable patients precludes any meaningful conclusion regarding the benefit of this approach for this disease category. Nevertheless, we have observed a very gratifying response in the patient with CTCL, consistent with our previous impression of a significant GVL effect for this disease.18 Other reports suggest a possible role for RIC regimens and nonmyeloablative regimens in the treatment of relapsed indolent lymphomas, HD, and multiple myeloma.19,20,21
Previous autologous transplant has emerged as a strong contraindication for standard allogeneic transplant, given its dismal prognosis from extremely high toxicity.2,22,23 In contrast, eight of 14 patients in our study are alive without disease beyond 1 year after second HSCT, underscoring the lower toxicity of RIC regimens in this setting. Recent reports confirm this observation.3,15
It is expected that RIC regimens will allow a growing number of older and sicker patients with hematological malignancies to benefit from the life-saving potential of URD HSCT. Our encouraging 1-year DFS of 55% for the entire group, and 71% for patients with advanced AML or MDS seems comparable or better than standard dose regimens for younger patients. Whether RIC regimens are superior to standard dose regimens for lower risk patients, based on their lower toxicity, will likely depend on the cytoreductive and immunosuppressive properties of the particular regimen in a given clinical scenario. This question should be addressed in large comparative trials.
In conclusion, Flu/Mel with CSP/MMF as GVHD prophylaxis was associated with excellent engraftment and an acceptable NRM in high-risk patients undergoing URD HSCT. Results were particularly encouraging for patients with advanced AML and MDS, including those failing a previous autologous transplant. However, GVHD remains a major cause of death with this regimen and new strategies in this direction are urgently needed.
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We acknowledge Diana Garcia for her assistance in manuscript preparation. We also thank other people who contributed to this study including Thomas Morrison, Allison Sano, Kathy Patane, and Popsie Gaytan.
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