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Relapse after allogeneic blood or marrow transplantation carries a very poor prognosis. Current strategies for management that include donor lymphocyte infusions (DLIs) and salvage chemotherapies are usually toxic and ineffective. Here we report the outcome of 10 patients with myeloid malignancies that received 5-azacytidine after a failed allogeneic bone marrow transplant. Of the 10 patients, 6 achieved a complete remission, 1 had stable disease, and 3 progressed after a median of 6 cycles administered. Only 1 patient has died (of disease progression), and no flares of graft-versus-host disease (GVHD) were observed with 5-azacytidine. As of latest follow-up, the median overall survival (OS) for the group was 422.5 days (127–1411). These results further suggest that 5-azacytidine is an active agent after failing an allogeneic bone marrow transplant, and prospective studies are warranted.
Patients who relapse after allogeneic blood and marrow transplant (BMT) for acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) have very poor prognoses . Survival is generally short with traditional salvage therapies such as further chemotherapy or donor lymphocyte infusions (DLI) [1,2]. Thus, there is a great need for new therapeutic approaches in this setting.
The DNA methyltransferase inhibitor 5-azacytidine is effective treatment for MDS, with 20% to 30% complete and partial responses as well as improved survival compared to best supportive care [3,4]. Preliminary data suggest that 5-azacytidine has similar activity in some subsets of AML [5,6]. Hypomethylating agents may play a role in effectively stabilizing MDS prior to allogeneic BMT [7,8]. Moreover, it appears that the drug may have immunolomodulatory properties that could potentially enhance the graft-versus-leukemia effect associated with allogeneic BMT [9,10]. Recent reports also suggest that 5-azacytidine may have activity in patients with relapse after allogeneic BMT [11–13]. On the basis of this information, 5-azacytidine has been used at our institution to treat patients with progressive AML or MDS after BMT.
We retrospectively identified all patients with myeloid malignancies who were treated with 5-azacytidine for relapse after allogeneic BMT between 2007 and 2009 at Johns Hopkins Hospital. There was a programmatic decision to consider treatment of relapses of myeloid malignancies after allogeneic BMT with 5-azacytidine in 2007, but the final decision on treatment was determined by the primary physician caring for the patient (whether to enroll on a clinical trial, give 5-azacytidine, or refer to hospice care, for instance). Evidence of recurrent disease was defined as decreasing or loss of donor chimerism usually with morphologic or cytogenetic persistence of the primary disease. Patients received supportive care including blood product support as needed. Patients were followed for morphological response, chimerism, and survival. This analysis was approved as being exempt from human subjects research by the Johns Hopkins Hospital institutional review board and written and electronic medical records were retrospectively analyzed. The survival analyses were completed as of May 1, 2010.
Chimerism was measured as follows. Unsorted and CD3+ cells are separated from peripheral blood, or unsorted bone marrow cells, using the RoboSep automated instrument (StemCell Technologies, Vancouver, Canada). This assay consists of PCR amplification of (15) microsatellite markers and the amelogenin locus using AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). The resulting PCR products are analyzed by capillary electrophoresis and the peak heights of the informative alleles are compared to calculate a percentage engraftment. In general, engraftment is calculated using 2 different microsatellite loci from a single PCR reaction. The true limit of detection for an individual reaction is both locus and PCR dependent. The formal limit of detection is 5% [14,15].
Between 2007 and 2009, 154 allogeneic transplants for 149 patients with myeloid malignancices were performed at Johns Hopkins Hospital. A total of 37 relapses were identified during that time frame, and 10 of these patients were identified as having received 5-azacytidine for disease progression after BMT (Table 1). One was previously reported . Their median age was 55 years (range: 25–67). Three were males, and 7 females. None of the patients were known to be positive for a FLT3 ITD, patients 1, 7, 9, and 10 were FLT3 ITD negative, and the rest were not tested. None of the patients had active graft-versus-host disease (GVHD) or were on immunosuppression at the time of starting therapy. None of the patients had blasts in peripheral blood at the time of starting therapy.
Patients received 5-azacytidine following different schedules as per the treating physician (Table 2), but most patients received 75 mg/m2/day for either 5 or 7 days. One patient received 5-azacytidine in combination with the histone deacetylase inhibitor entinostat as previously reported . The median number of cycles administered was 6 (range: 2–27).
Therapy was well tolerated with no unexpected or severe toxicities. One of the patients (a nonresponder) was admitted to the hospital for neutropenic fevers and fungal infection. Of the 10 patients treated with 5-azacytidine, 6 achieved complete remissions and 4 did not respond (1 stable disease, and 3 disease progressions) (Table 2). Achievement of complete remission was associated with complete loss of host chimerism in 4 patients and near loss in the 2 others. Three patients received DLI after treatment (2 after achieving a complete remission); 2 remain in complete remission, 1 of whom developed extensive chronic GVHD (cGVHD), and 1 died of disease progression. The median survival (from the time of relapse before starting 5-azacytidine) for the group was 422.5 (127–1411) days. Of the 6 patients who achieved a complete remission, 5 remain disease-free at a median follow-up of 624 (162–771) days. Of the 27 other relapses in the myeloid malignancy patients, only 5 remain alive.
The management of disease relapse after allogeneic BMT is challenging. DLI has traditionally been standard practice. Porter et al.  identified recipients of unrelated DLI for the treatment of relapsed disease from the NMDP database. Unrelated DLI was administered for relapsed AML to 23 patients. Forty-two percent (8 out of 19) of assessable patients with AML achieved a complete response. The estimated probability of DFS at 1 year after a complete response was 23%. Acute and cGVHD were commonly seen in these patients. Huff et al.  retrospectively analyzed 83 consecutive recipients of DLI after BMT. In relapsed acute myeloid leukemia (13 patients), DLI led to durable complete responses in 31% of patients. Grade II or higher acute GVHD (aGVHD) or cGVHD occurred in 43% patients and contributed to death in 16%. Schmid et al.  analyzed the outcome of 399 patients in first hematologic relapse included in the European Group for Blood and Marrow Transplantation. Of these, 171 received DLI and 228 did not. At 2 years, 21% of those receiving DLI were alive versus 9% of those who did not.
The low likelihood of achieving prolonged survival without comorbid GVHD after DLI provides the rationale for novel investigations and approaches. There are emerging data suggesting activity for 5-azacytidine for relapse after allogeneic BMT. Jabbour et al.  reported on 5-azacytidine (doses ranging from 16–40 mg/m2) as salvage therapy and on 8 patients as maintenance after allogeneic. The treatment was well tolerated with no grade 3–4 toxicities, and 5 of 9 patients with recurrent disease responded without exacerbations of GVHD. After a median follow up of 16 months, 14 patients are alive including 7 in CR. Lübbert et al.  administered 5-azacytidine for 3 days (100 mg/day) followed by DLI on day 10 every 21 days to patients relapsed after allogeneic BMT. The median survival for the 26 patients (who received a total of 60 courses of treatment) from the start of 5-azacytidine was 136 days. Toxicities related to the 5-azacytidine are hard to evaluate because of the addition for DLI.
In our cohort, treatment with 5-azacytidine was well tolerated, with sustained responses in many of the patients without exacerbations of GVHD. The potential immunologic antitumor effects of 5-azacytidine are still unclear. Hypomethylating agents including 5-azacytidine have been reported to reverse the loss of expression of tumor-specific, tumor-associated, or histocompatibility antigens on tumor cells, making them more susceptible to immune attack [19–23]. Hypomethylating agents may also enhance presentation of tumor antigens [9,10,24], which may be expected to generate a graft-versus-tumor reaction. The drug has also been reported to inhibit T cell proliferation and activation, decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (INF-γ), which may prevent GVHD and possibly graft-versus-tumor effects as well . It also induces FOXP3 expression in CD4+CD25− T cells both in vitro and in vivo .
Conversely, 5-azacytidine may induce cytotoxic T lymphocytes with activity against AML . Regardless of the exact mechanism of action, there are now several small studies that support the development of larger, prospective clinical trials to better characterize the safety and efficacy of 5-azacytidine after allogeneic BMT.
This research was supported in part by the grant P01CA15396 from the National Cancer Institute (Richard J. Jones, MD). Dr. Bolaños-Meade is an Investigator-2 Sistema Nacional de Investigadores (CONACYT, Mexico).
Financial disclosures: Steven D. Gore, MD: Celgene: Consultant, research support, and stock ownership (<10,000 US dollars). B. Douglas Smith, MD: Celgene: Consultant and honoraria. Other authors have nothing to disclose.