B. Douglas Smith

Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms

Optimal reexpression of most genes silenced through promoter methylation requires the sequential application of DNA methyltransferase inhibitors followed by histone deacetylase inhibitors in tumor cell cultures. Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Bisulfite sequencing of the p15 promoter in marrow DNA during the first cycle of treatment showed heterogeneous allelic demethylation in three responding patients, suggesting ongoing demethylation within the tumor clone, but no demethylation in two nonresponders. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Gene demethylation correlated with the area under the aza-CR plasma concentration-time curve. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials.

Clonogenic Multiple Myeloma Progenitors, Stem Cell Properties, and Drug Resistance

Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury.

Results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse

In a randomized trial of therapy for FMS-like tyrosine kinase-3 (FLT3) mutant acute myeloid leukemia in first relapse, 224 patients received chemotherapy alone or followed by 80 mg of the FLT3 inhibitor lestaurtinib twice daily. Endpoints included complete remission or complete remission with incomplete platelet recovery (CR/CRp), overall survival, safety, and tolerability. Correlative studies included pharmacokinetics and analysis of in vivo FLT3 inhibition. There were 29 patients with CR/CRp in the lestaurtinib arm and 23 in the control arm (26% vs 21%; P = .35), and no difference in overall survival between the 2 arms. There was evidence of toxicity in the lestaurtinib-treated patients, particularly those with plasma levels in excess of 20 μM. In the lestaurtinib arm, FLT3 inhibition was highly correlated with remission rate, but target inhibition on day 15 was achieved in only 58% of patients receiving lestaurtinib. Given that such a small proportion of patients on this trial achieved sustained FLT3 inhibition in vivo, any conclusions regarding the efficacy of combining FLT3 inhibition with chemotherapy are limited. Overall, lestaurtinib treatment after chemotherapy did not increase response rates or prolong survival of patients with FLT3 mutant acute myeloid leukemia in first relapse. This study is registered at www.clinicaltrials.gov as #NCT00079482.

Immunoablative High-Dose Cyclophosphamide without Stem-Cell Rescue for Refractory, Severe Autoimmune Disease

High-dose cytotoxic therapy followed by autologous stem-cell transplantation has been proposed as a novel treatment for severe autoimmune disease [1, 2]. This approach was prompted by autoimmune animal models that demonstrated marked improvement or complete eradication of autoimmune disease after syngeneic marrow transplantation [3, 4]. In addition, allogeneic marrow transplantation (performed chiefly for aplastic anemia) has been reported to eradicate concurrent autoimmune disease [5, 6]. Allogeneic marrow transplantation is not routinely used to treat autoimmune disease because of substantial associated morbidity and mortality. Although interest in the use of high-dose cytotoxic therapy followed by autologous stem-cell transplantation to treat autoimmune disease is increasing, disease progresses or relapses early in many patients [7, 8]. It is unclear whether reappearance of the disease after autologous transplantation results from failure of high-dose therapy to eradicate autoaggressive lymphocytes, reinfusion of autoaggressive lymphocytes with the autograft, or renewed challenge from the autoantigen [7, 8]. However, the success of syngeneic transplantation in animal models and allogeneic transplantation in patients with autoimmune diseases suggests that high-dose cytotoxic therapy may be sufficient to eradicate autoaggressive lymphocytes [8]. We previously found that the immunoablative doses of cyclophosphamide used for transplantation can induce durable, complete remission (median follow-up > 10 years) without stem-cell rescue in most patients with severe aplastic anemia [9]. Because most cases of aplastic anemia result from immune suppression of hematopoiesis [10], high-dose cyclophosphamide without the addition of other cytotoxic immunosuppressive agents seems to ablate the autoaggressive lymphocytes. We also reported that high-dose cyclophosphamide spared hematopoietic stem cells because full hematopoietic recovery occurred [9]. Hematopoietic stem cells express high levels of aldehyde dehydrogenase, an enzyme responsible for cellular resistance to cyclophosphamide, and are therefore resistant to the cytotoxic effects of cyclophosphamide [11, 12]. We investigated the efficacy of high-dose cyclophosphamide without stem-cell rescue in patients with various severe autoimmune diseases. Methods Treatment Schedule Our study was approved by the institutional review boards of Johns Hopkins University and Hahnemann University. After giving informed consent, eight patients (Table 1 and Table 3) with refractory autoimmune disorders received cyclophosphamide (50 mg/kg of body weight per day) intravenously for 4 consecutive days. Granulocyte colony-stimulating factor therapy (5 g/kg per day) was started 6 days after the last dose of cyclophosphamide and was continued until the absolute neutrophil count reached 109 cells/L. Inclusion in the study required failure of two previous therapies. Patients were excluded if their cardiac ejection fraction was less than 0.45, their serum creatinine level was greater than 176.8 mol/L, or they were older than 70 years of age. Red blood cell transfusions were administered to patients with a hematocrit less than 0.25, and platelet transfusions were given to patients with platelet counts less than 20 109 cells/L or clinically significant bleeding. Complete remission required the absence of any clinical or serologic evidence of disease. Complete remission from lupus nephritis was defined as fewer than 10 dysmorphic erythrocytes per high-powered field, absence of cellular casts, and excretion of less than 1 g of protein per day without doubling of the serum creatinine level [13]. For patients with systemic lupus erythematosus, daily activity indices [14] were measured at 3-month intervals. Table 1. Patient Characteristics and Response to High-Dose Cyclophosphamide Table 3. Table 1. Continued Selected Case Reports Patient 1 was a 64-year-old man with a 35-year history of rheumatoid arthritis treated with prednisone and gold. The Felty syndrome had been diagnosed 3 years earlier when the patient developed a perirectal abscess and profound neutropenia (neutrophil count < 0.2 109 cells/L). Examination of bone marrow showed hypercellularity with myeloid maturation arrest. The patient was treated with myeloid growth factors and steroids but showed no response. He required frequent hospitalizations for recurrent infections. Before the patient received high-dose cyclophosphamide, he was positive for antineutrophil antibodies, the neutrophil count was 0.1 109 cells/L, the rheumatoid factor level was elevated, complement levels were depressed, and the Karnofsky score [15] was 40%. The patient tolerated high-dose cyclophosphamide well and had few side effects other than alopecia; he achieved a neutrophil count greater than 0.5 109 cells/L by day 15, and infections (perirectal abscess, pneumonia, and sinusitis) that were present at the time of treatment resolved. Two units of red blood cells and five platelet transfusions were required. The patient is in complete remission 21 months after treatment and has normal peripheral blood counts, has normal complement levels, and is negative for antineutrophil antibodies. He has not been receiving any immunosuppressive agents for more than 15 months. Patient 6 was a 23-year-old woman in whom lupus was diagnosed at 12 years of age after she presented with the Raynaud phenomenon and stomatitis. She later developed severe proteinuria, hyperlipidemia, polyarthralgia, and an extensive skin rash. Renal biopsy performed 4 years before initiation of high-dose cyclophosphamide therapy showed membranous nephropathy. The patient required hospitalization for lupus flares three to four times per year despite treatment with methylprednisolone (4 mg/d), hydroxychloroquine (400 mg/d), azathioprine (150 mg/d), and pulse-dose cyclophosphamide. Before high-dose cyclophosphamide therapy began, the hematocrit was 0.27, the leukocyte count was 2.8 109 cells/L, the platelet count was 278 109 cells/L, and the erythrocyte sedimentation rate was 104 mm/h. Anti-DNA antibodies were present at a titer of 1:320, the C3 level was 0.41 g/L, and the 24-hour urine protein level was 2 g. The patient tolerated high-dose cyclophosphamide well; side effects were alopecia and febrile neutropenia. A neutrophil count greater than 0.5 109 cells/L was reached on day 18, and only six units of red blood cells and three platelet transfusions were needed. The patient is in continuous complete remission 12 months after treatment; the erythrocyte sedimentation rate is 20 mm/h, no anti-DNA antibodies are present, the C3 level is 1.49 g/L, and the 24-hour urine protein level is 86 mg. Immunosuppressive therapy has been tapered to 1 mg of prednisone daily. Results High-dose cyclophosphamide was well tolerated and was associated with rapid hematologic recovery in all eight patients despite their poor medical condition at time of treatment. Four patients were hospitalized for complications of their autoimmune disease, and four patients were being treated for active infections at the time of cyclophosphamide therapy; the median Karnofsky score was 40% (range, 20% to 70%). The median time to achievement of a neutrophil count greater than 0.5 109 cells/L was 17 days (range, 11 to 22 days), and the median time to the last platelet transfusion was 16 days (range, 12 to 33 days). All patients experienced complete alopecia, and six patients required antibiotics for febrile neutropenia. No patient developed hemorrhagic cystitis or mucositis. Patients 2 and 4 eventually died of complications of autoimmune disease. Patient 2, who was treated for autoimmune hemolytic anemia, died of complications of immune thrombocytopenic purpura, which was not present when she was treated with high-dose cyclophosphamide. Autoimmune hemolytic anemia remained in complete remission until the patients death, 16 months after cyclophosphamide therapy. Patient 4 achieved brief remission of immune thrombocytopenic purpura and died of her disease 8 months later. Six patients remain alive, and five (patients 1, 3, 6, 7, and 8) have no symptomatic manifestations of their disease. In addition, four patients have no laboratory or clinical evidence of disease (Table 2). Patient 3, who has the Evans syndrome, shows continued improvement in blood counts; prednisone therapy is being tapered to 10 mg every other day. Patient 3 has been independent of transfusion for more than 10 months and has a normal hemoglobin level and a platelet count of 66 109 cells/L. One of the patients with lupus achieved complete remission; the other still has clinical and serologic evidence of the disease but continues to improve 14 months after treatment. Patient 7, who has the Felty syndrome, is in complete remission 3 months after cyclophosphamide therapy. Patient 10, who has chronic inflammatory demyelinating polyneuropathy, had progressive upper- and lower-extremity paralysis and was unable to walk. Plasmapheresis, intravenous immunoglobulin, and pulse-dose cyclophosphamide therapy had proven ineffective. Three months after therapy with high-dose cyclophosphamide, he has no neurologic manifestations, is not receiving immunosuppressive therapy, and can walk normally. Table 2. Laboratory Results* Discussion Most immunoablative therapy for severe autoimmune disease uses autologous stem-cell rescue after high-dose therapy with cyclophosphamide in combination with other immunosuppressive agents [8]. Although our study was small and the follow-up was relatively short, the results indicate that high-dose cyclophosphamide alone can be effective therapy for some patients with severe autoimmune disease. In addition, our study confirms that high-dose cyclophosphamide (50 mg/kg per day for 4 days) spares hematopoietic stem cells; the kinetics of bone marrow recovery after high-dose cyclophosphamide therapy without stem-cell rescue are similar to those of engraftment after autol

High-dose cyclophosphamide as single-agent, short-course prophylaxis of graft-versus-host disease

Because of its potent immunosuppressive yet stem cell-sparing activity, high-dose cyclophosphamide was tested as sole prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation (alloBMT). We treated 117 patients (median age, 50 years; range, 21-66 years) with advanced hematologic malignancies; 78 had human leukocyte antigen (HLA)-matched related donors and 39 had HLA-matched unrelated donors. All patients received conventional myeloablation with busulfan/cyclophosphamide (BuCy) and T cell-replete bone marrow followed by 50 mg/kg/d of cyclophosphamide on days 3 and 4 after transplantation. The incidences of acute grades II through IV and grades III through IV GVHD for all patients were 43% and 10%, respectively. The nonrelapse mortality at day 100 and 2 years after transplantation were 9% and 17%, respectively. The actuarial overall survival and event-free survivals at 2 years after transplantation were 55% and 39%, respectively, for all patients and 63% and 54%, respectively, for patients who underwent transplantation while in remission. With a median follow-up of 26.3 months among surviving patients, the cumulative incidence of chronic GVHD is 10%. These results suggest that high-dose posttransplantation cyclophosphamide is an effective single-agent prophylaxis of acute and chronic GVHD after BuCy conditioning and HLA-matched BMT (clinicaltrials.gov no. NCT00134017).

A clinically relevant population of leukemic CD34+CD38- cells in acute myeloid leukemia

Relapse of acute myeloid leukemia (AML) is thought to reflect the failure of current therapies to adequately target leukemia stem cells (LSCs), the rare, resistant cells presumed responsible for maintenance of the leukemia and typically enriched in the CD34(+)CD38(-) cell population. Despite the considerable research on LSCs over the past 2 decades, the clinical significance of these cells remains uncertain. However, if clinically relevant, it is expected that LSCs would be enriched in minimal residual disease and predictive of relapse. CD34(+) subpopulations from AML patients were analyzed by flow cytometry throughout treatment. Sorted cell populations were analyzed by fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities (when present) and by transplantation into immunodeficient mice to determine self-renewal capacity. Intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity reliably distinguished leukemic CD34(+)CD38(-) cells capable of engrafting immunodeficient mice from residual normal hematopoietic stem cells that exhibited relatively higher ALDH activity. Minimal residual disease detected during complete remission was enriched for the CD34(+)CD38(-)ALDH(int) leukemic cells, and the presence of these cells after therapy highly correlated with subsequent clinical relapse. ALDH activity appears to distinguish normal from leukemic CD34(+)CD38(-) cells and identifies those AML cells associated with relapse.

Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay

FLT3 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells and plays an important role in stem cell survival and differentiation. Two different types of functionally important FLT3 mutations have been identified. Internal tandem duplication mutations arise from duplications of the juxtamembrane portion of the gene and result in constitutive activation of the FLT3 protein. This alteration has been identified in approximately 20% to 30% of patients with acute myelogenous leukemia and appears to be associated with a worse prognosis. The second type of FLT3 mutation, missense mutations at aspartic acid residue 835, occurs in approximately 7.0% of acute myelogenous leukemia cases. These mutations also appear to be activating and to portend a worse prognosis. Identification of FLT3 mutations is important because it provides prognostic information and may play a pivotal role in determining appropriate treatment options. We have developed an assay to identify both internal tandem duplication and D835 FLT3 mutations in a single multiplex polymerase chain reaction. After amplification, the polymerase chain reaction products are analyzed by capillary electrophoresis for length mutations and resistance to EcoRV digestion. Here we describe the performance characteristics of the assay, assay validation, and our clinical experience using this assay to analyze 147 clinical specimens.

FLT3 ligand impedes the efficacy of FLT3 inhibitors in vitro and in vivo

We examined in vivo FLT3 inhibition in acute myeloid leukemia patients treated with chemotherapy followed by the FLT3 inhibitor lestaurtinib, comparing newly diagnosed acute myeloid leukemia patients with relapsed patients. Because we noted that in vivo FLT3 inhibition by lestaurtinib was less effective in the relapsed patients compared with the newly diagnosed patients, we investigated whether plasma FLT3 ligand (FL) levels could influence the efficacy of FLT3 inhibition in these patients. After intensive chemotherapy, FL levels rose to a mean of 488 pg/mL on day 15 of induction therapy for newly diagnosed patients, whereas they rose to a mean of 1148 pg/mL in the relapsed patients. FL levels rose even higher with successive courses of chemotherapy, to a mean of 3251 pg/mL after the fourth course. In vitro, exogenous FL at concentrations similar to those observed in patients mitigated FLT3 inhibition and cytotoxicity for each of 5 different FLT3 inhibitors (lestaurtinib, midostaurin, sorafenib, KW-2449, and AC220). The dramatic increase in FL level after chemotherapy represents a possible obstacle to inhibiting FLT3 in this clinical setting. These findings could have important implications regarding the design and outcome of trials of FLT3 inhibitors and furthermore suggest a rationale for targeting FL as a therapeutic strategy.

Risk-stratified outcomes of nonmyeloablative HLA-haploidentical BMT with high-dose posttransplantation cyclophosphamide

Related HLA-haploidentical blood or marrow transplantation (BMT) with high-dose posttransplantation cyclophosphamide (PTCy) is being increasingly used because of its acceptable safety profile. To better define outcomes of nonmyeloablative (NMA) HLA-haploidentical BMT with PTCy, 372 consecutive adult hematologic malignancy patients who underwent this procedure were retrospectively studied. Risk-stratified outcomes were evaluated using the refined Disease Risk Index (DRI), developed to stratify disease risk across histologies and allogeneic BMT regimens. Patients received uniform conditioning, T-cell-replete allografting, then PTCy, mycophenolate mofetil, and tacrolimus. Six-month probabilities of nonrelapse mortality and severe acute graft-versus-host disease were 8% and 4%. With 4.1-year median follow-up, 3-year probabilities of relapse, progression-free survival (PFS), and overall survival (OS) were 46%, 40%, and 50%, respectively. By refined DRI group, low (n = 71), intermediate (n = 241), and high/very high (n = 60) risk groups had 3-year PFS estimates of 65%, 37%, and 22% (P < .0001), with corresponding 3-year OS estimates of 71%, 48%, and 35% (P = .0001). On multivariable analyses, the DRI was statistically significantly associated with relapse, PFS, and OS (each P < .001). This analysis demonstrates that the DRI effectively risk stratifies recipients of NMA HLA-haploidentical BMT with PTCy and also suggests that this transplantation platform yields similar survivals to those seen with HLA-matched BMT.

Outcomes of Nonmyeloablative HLA-Haploidentical Blood or Marrow Transplantation With High-Dose Post-Transplantation Cyclophosphamide in Older Adults

PURPOSE Recent advances in nonmyeloablative (NMA), related HLA-haploidentical blood or marrow transplantation (haplo-BMT) have expanded the donor pool. This study evaluated the effect of age on NMA haplo-BMT outcomes in patients age 50 to 75 years. PATIENTS AND METHODS A retrospective analysis was performed of 271 consecutive patients with hematologic malignancies, age 50 to 75 years, who received NMA, T-cell-replete haplo-BMT with high-dose post-transplantation cyclophosphamide. RESULTS The median age was 61 years, with 115 patients (42%) age 50 to 59, 129 (48%) age 60 to 69, and 27 (10%) age 70 to 75 years. Overall, 84% of patients had intermediate- or high-/very high-risk disease. The 6-month probabilities of grade 3 or 4 acute graft-versus-host disease (GVHD) and nonrelapse mortality (NRM) were 3% and 8%, respectively. Patients in their 50s, 60s, and 70s had 6-month NRM probabilities of 8%, 9%, and 7%, respectively (P=.20). With a median follow-up of 4 years, corresponding 3-year progression-free survival probabilities were 39%, 35%, and 33% (P=.65), and corresponding 3-year overall survival probabilities were 48%, 45%, and 44% (P=.66). Three-year progression-free survival probabilities were 40% in acute myeloid leukemia (n=65), 39% in aggressive non-Hodgkin lymphoma (n=83), and 37% in indolent or mantle-cell lymphoma (n=65). Older patient age was associated with a significantly higher risk of grade 2 to 4 acute GVHD but not grade 3 to 4 acute or chronic GVHD. No statistically significant associations were found between older age (relative to age 50 to 59 years or as a continuous variable) and NRM, relapse, or survival. CONCLUSION NMA haplo-BMT with post-transplantation cyclophosphamide has encouraging safety and survival outcomes in patients age 50 to 75 years. In patients otherwise fit for BMT, the results support consideration of this approach despite advanced age.