Relapse Of Chronic Myelogenous Leukemia Research Articles

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

<h3></h3> Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that <i>MIR300</i> has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. <i>MIR300</i> is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates <i>MIR300</i> in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating <i>TUG1</i> long noncoding RNA that uncouples and limits <i>MIR300</i> function to cytostasis. Genetic and pharmacologic <i>MIR300</i> modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis <i>in vitro</i> and in patient-derived xenografts; hence, the importance of <i>MIR300</i> and PP2A activity for CML development and therapy. <h3>Significance:</h3> Tumor-naïve microenvironment–induced <i>MIR300</i> is the only tumor suppressor miRNA that induces CML LSC quiescence while inhibiting NK cell antitumor immune response, and CML LSC/progenitor cell apoptosis through its anti-proliferative and PP2A-activating functions, respectively. Thus, the importance of <i>MIR300</i> and PP2A-activating drugs for formation/survival and eradication of drug-resistant CML LSCs, respectively. <i>See related commentary by Broxmeyer, p. 13</i>. <i>This article is highlighted in the In This Issue feature, p. 5</i>

Basic knowledge on BCR-ABL1-positive extracellular vesicles.

Chronic myelogenous leukemia (CML) is a hematological malignancy characterized by the excessive proliferation of myeloid progenitors. In the case of CML, these extracellular vesicles (EVs) were shown to communicate with hematopoietic stem cells, mesenchymal stem cells, myeloid derived suppressor cells and endothelial cells determining a beneficial microenvironment for the CML clone. Moreover, as these EVs are marked through BCR-ABL1, they were shown to be useful in clinical research in determining the grade of molecular remission with further studies being needed to determine if they are better or worse at predicting CML relapse. More than this, we consider BCR-ABL1-positive EVs to represent only a stepping-stone for other malignancies that also present fusion genes that are loaded in EVs.

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.

The Interaction of Tumor Cells and Myeloid-Derived Suppressor Cells in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease characterized by the formation of the BCR-ABL fusion gene. At present, basic studies of the pathogenesis of relapse after stopping tyrosine kinase inhibitors (TKIs) treatment have mainly concentrated on two main aspects: the leukemia stem cells (LSCs) and the tumor microenvironment. However, whether relapse or non-relapse patients who discontinued TKIs therapy, LSCs are still exist. Among a variety of factors that compose the CML microenvironment, myeloid-derived suppressor cells (MDSC) are considered to be a strong contributor to the immunosuppressive tumor microenvironment. Here, we designed the study to investigate the potential relation between tumor cells and MDSC in CML and find risk factors for relapse after discontinuation. We detected the percentage of MDSC and the BCR-ABL (IS) transcript levels in bone marrow of 50 CML patients in chronic phase at our center. The data indicated that the frequency of MDSC had significant positive correlation with BCR-ABL (IS) transcript levels (Figure 1A). In addition, the counts of MDSC had significant difference at different response stages (Figure 1B), especially the M-MDSC, a subtype of MDSC. The percentage of M-MDSC was significantly higher in patients with newly diagnosed or complete hematological response (CHR) or major molecular response (MMR) compared with those of CML patients obtained complete molecular response (CMR) (Figure 1C). When K562 cells or CD34+ cells were cocultured with M-MDSC at a 1:10 ratio, K562 cells or CD34+ cells proliferated significantly at day 3 (Figure 1D and E). K562 subcutaneous tumor formation in BALB/c node mice confirmed that tumors weight and volume of the coculture group were higher than control. Then, we further investigated whether tumor cells have an impact on MDSC through microvesicles (MV). After adding K562-MV to peripheral blood mononuclear cells (PBMCs) from healthy donors, MDSC counts appeared significantly elevated in the different K562 cells counts group compared to the control group (Figure 1F).To analyze the roles of K562-MV collected before and after TKIs discontinuation on MDSC, we established a TKIs discontinuation model using the K562 cell, which emulates the cessation of TKIs treatment of CML patients in some extent. The results showed that regardless of the Imatinib or Dasatinib treatment, a significant increase was observed in the proportion of MDSC after TKIs treatment cessation compared with the TKIs treatment groups (Figure 1G). Experiments in vivo also proved K562-MV after different treatments promoted the proliferation of MDSC (Figure 1H and I). In conclusion, our study introduces the notion of the role of MDSC as mediators in the cross talk between tumor cells and the microenvironment. MDSC would provide a novel and useful model to predict the relapse of CML by establishing a type of new risk stratification system. MDSC could be also act as a promising target in the relapse of CML. In addition, we found a mutual promotion of proliferation of tumor cells and MDSC, this bidirectional interaction results in a vicious cycle by providing a protective niche against immune attacks. Therapeutic interventions modulating this interaction might accelerate the success of treatment-free remission. Figure 1 Disclosures No relevant conflicts of interest to declare.

Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia.

Imatinib-insensitive leukemia stem cells (LSCs) are believed to be responsible for resistance to BCR-ABL tyrosine kinase inhibitors and relapse of chronic myelogenous leukemia (CML). Identifying therapeutic targets to eradicate CML LSCs may be a strategy to cure CML. In the present study, we discovered a positive feedback loop between BCR-ABL and protein arginine methyltransferase 5 (PRMT5) in CML cells. Overexpression of PRMT5 was observed in human CML LSCs. Silencing PRMT5 with shRNA or blocking PRMT5 methyltransferase activity with the small-molecule inhibitor PJ-68 reduced survival, serial replating capacity, and long-term culture-initiating cells (LTC-ICs) in LSCs from CML patients. Further, PRMT5 knockdown or PJ-68 treatment dramatically prolonged survival in a murine model of retroviral BCR-ABL-driven CML and impaired the in vivo self-renewal capacity of transplanted CML LSCs. PJ-68 also inhibited long-term engraftment of human CML CD34+ cells in immunodeficient mice. Moreover, inhibition of PRMT5 abrogated the Wnt/β-catenin pathway in CML CD34+ cells by depleting dishevelled homolog 3 (DVL3). This study suggests that epigenetic methylation modification on histone protein arginine residues is a regulatory mechanism to control self-renewal of LSCs and indicates that PRMT5 may represent a potential therapeutic target against LSCs.

A clinical observation of Chinese chronic myelogenous leukemia patients after discontinuation of tyrosine kinase inhibitors.

Whether tyrosine kinase inhibitors (TKIs) can be safely discontinued is a key focus of chronic myelogenous leukemia (CML) at present. We report a clinical observation of TKIs cessation in Chinese CML patients and a probable connection between CML leukemia stem cells (LSCs) and relapse. In all, 22 of 1057 patients consented to participate in this observation. The average time of complete molecular response was 12.73 months after TKI withdrawal. LSCs could be flow cytometrically detected in most of the patients. However, the number of LSCs did not differ between the relapsers and non-relapsers. We evaluated the leukemogenetic ability of the LSCs by transplanting bone marrow into irradiated NOD/SCID mice. The results indicated that part of the bone marrow from the relapsers lead to leukemogensis in the mice. Besides, we found that LSCs-derived microvesicles might serve as a novel factor for the stratification of undetectable minimal residual disease and an early warning sign of relapse. In summary, post-TKI cessation relapse seems to show none association with the number of LSCs. A mouse xenograft model would provide a novel and useful method of analyzing LSCs function and predicting relapse. Microvesicles may provide important information about optimal molecular monitoring schedules in TKI discontinuation strategies.

Viral/Nonviral Chimeric Nanoparticles To Synergistically Suppress Leukemia Proliferation via Simultaneous Gene Transduction and Silencing.

Single modal cancer therapy that targets one pathological pathway often turns out to be inefficient. For example, relapse of chronic myelogenous leukemia (CML) after inhibiting BCR-ABL fusion protein using tyrosine kinase inhibitors (TKI) (e.g., Imatinib) is of significant clinical concern. This study developed a dual modal gene therapy that simultaneously tackles two key BCR-ABL-linked pathways using viral/nonviral chimeric nanoparticles (ChNPs). Consisting of an adeno-associated virus (AAV) core and an acid-degradable polymeric shell, the ChNPs were designed to simultaneously induce pro-apoptotic BIM expression by the AAV core and silence pro-survival MCL-1 by the small interfering RNA (siRNA) encapsulated in the shell. The resulting BIM/MCL-1 ChNPs were able to efficiently suppress the proliferation of BCR-ABL+ K562 and FL5.12/p190 cells in vitro and in vivo via simultaneously expressing BIM and silencing MCL-1. Interestingly, the synergistic antileukemic effects generated by BIM/MCL-1 ChNPs were specific to BCR-ABL+ cells and independent of a proliferative cytokine, IL-3. The AAV core of ChNPs was efficiently shielded from inactivation by anti-AAV serum and avoided the generation of anti-AAV serum, without acute toxicity. This study demonstrates the development of a synergistically efficient, specific, and safe therapy for leukemia using gene carriers that simultaneously manipulate multiple and interlinked pathological pathways.

Optic Neuropathy due to Chronic Lymphocytic Leukemia Proven With Optic Nerve Sheath Biopsy and Chronic Myelogenous Leukemia Relapse Presenting With Central Nervous System Blast Crisis and Bilateral Optic Nerve Infiltration: Comment.

Optic Neuropathy due to Chronic Lymphocytic Leukemia Proven With Optic Nerve Sheath Biopsy and Chronic Myelogenous Leukemia Relapse Presenting With Central Nervous System Blast Crisis and Bilateral Optic Nerve Infiltration: Comment.

Isolated Ocular Manifestation of Relapsed Chronic Myelogenous Leukemia Presenting as Myeloid Blast Crisis in a Patient on Imatinib Therapy: A Case Report and Review of the Literature.

Blast phase in chronic myelogenous leukemia (CML) has rarely been reported to involve extramedullary sites like skin, lymph nodes, and central nervous system. Clinical history, characteristic hematologic findings (elevated leukocyte counts, myelocytic predominance, and basophilia), and Philadelphia chromosome are of high diagnostic significance especially in isolated extramedullary presentations. We describe a unique case of CML relapse with blast phase involving the eye. A 66-year-old man with a known diagnosis of CML on imatinib and in molecular remission for 3 years presented with a painful blind eye. Histologic examination revealed diffuse involvement of choroid, iris, vitreous humor, and the optic nerve by blast cells. The blasts expressed CD34, aberrant TdT, and a myeloid phenotype (CD13, CD33, and CD117). Fluorescence in situ hybridization (FISH) of vitreous fluid detected BCR-ABL1 gene rearrangement. Additionally, trisomy 8 and gains of 9 and 22 were seen which were not present in the initial diagnostic marrow study 3 years ago. At relapse, the bone marrow, peripheral blood, and the cerebrospinal fluid were not involved by CML. Patient received induction chemotherapy and single dose prophylactic intrathecal methotrexate and was maintained on antityrosine kinase therapy and eventually underwent allogenic stem cell transplantation.

Did splenectomy influence relapse of chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation?

Did splenectomy influence relapse of chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation?

Guest editorial: leukemia stem cell

Leukemia is a clonal, malignant hematological disorder. Recent studies have shown that at least in acute myelogenous leukemia (AML), the clonogenic and leukemogenic activities reside in a minor, primitive fraction of the leukemia cell population. In 1996, Dick et al. showed that the CD34CD38, but not other fractions of bone marrow cells from AML patients can reconstitute human AML in immunodeficient mice, demonstrating direct evidence for the presence of leukemia stem cells (LSCs). After this finding, the concept of the LSC becomes critical to understand the pathogenesis of leukemia. In this issue, four excellent review articles [1–4] focus on developmental mechanisms of LSCs to discuss how to eradicate them to obtain absolute cure of leukemia. Cancer should result from the accumulation of multiple genetic abnormalities. In hematopoiesis, the cells accumulating such mutations need to survive for a long time in the human body, and therefore, the main target for mutations should be hematopoietic stem cells (HSCs) that are capable of self-renewal. HSCs harboring multiple mutations can produce a large number of proliferating progenitor populations with identical mutations, which further increases the chance to get another critical hit to transform into LSCs. This outline of leukemia development reasonably fits the concept of blastic crisis from chronic myelogenous leukemia (CML). In CML, a HSC acquires BCR-ABL, resulting in clonal expansion of BCR-ABLexpressing hematopoietic progenitors and mature cells. These HSCs accumulate additional mutations, and their expanding myeloid or lymphoid progenitors finally transform into LSCs of myeloid or lymphoid blastic clones, respectively. In this context, BCR-ABL is an early mutation, and HSCs in CML patients could be regarded as ‘‘preLSCs’’. Imatinib mesylate, an inhibitor for kinase activity of BCR-ABL, induces hematological and cytogenetic remissions in nearly every patient in chronic phase of CML, preventing the transformation into blastic crisis, but the cessation of this drug results in relapse of CML in the majority of patients. Therefore, it is important to understand the molecular mechanisms to maintain BCR-ABLexpressing residual HSCs (pre-LSCs) in CML. Ito [1] summarizes key players mediating maintenance of such CML stem cells. Recent technological advances in genomics have revealed recurrent driver mutations in AML, demonstrating existence of the similar evolutionary processes for LSC development, in which genetic diversity caused by multiple mutations and clonal selection of a population with growth advantage should be critical. In this selection process, it is now clear that pre-LSCs possessing multiple mutations but still retaining ability to differentiate into all mature blood cells progressively become dominant. Chan and Majeti [2] especially focus on mutations related to epigenetic regulation, one of important groups of driver mutations, and summarize the current understanding of their roles in leukemogenesis. Targeting the LSC should be the ultimate therapeutic strategy to cure leukemias. Xenogeneic transplantation of human cells into immunodeficient mice has enabled to identify cell fractions enriched for LSCs. Ishikawa [3] summarizes the history of xenotransplantation models, and describes his NSG mouse model that is a powerful tool to discover functional properties of LSCs including their cell cycle status and location in the bone marrow. Using the K. Akashi (&) Department of Medicine and Biosystemic Sciences, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan e-mail: akashi@med.kyushu-u.ac.jp

Nilotinib -Induced Retroperitoneal Fibrosis In a Patient with Chronic Myelogenous Leukemia.

AbstractAbstract 449532 year old man was diagnosed with chronic myelogenous leukemia(CML) in accelerated phase. Karyotype analysis and fluorescent in situ hybridization of the leukemic clone revealed a 9; 22 translocation, loss of Y chromosome, and an interstitial deletion of chromosome 16. Despite treatment with imatinib and hydroxyurea, he progressed to myeloid blast crisis with the bone marrow showing three leukemic clones, including a second Ph' and trisomy 8, consistent with karyotype evolution. He underwent myeloablative stem cell transplant from a matched sibling donor, following which he was maintained on dasatinib which was discontinued 6 months later due to anasarca. At the time of discontinuation, he was in cytogenetic and molecular remission (CMR). He was then started on nilotinib 400mg twice daily. A year later he developed grade 3 hepatotoxicity with hyperbilirubinemia. Further workup revealed gallstones for which he underwent cholecystectomy. Despite surgery, his liver enzymes remained elevated and liver biopsy revealed chronic inflammation and fibrosis. Computed tomography scan of the abdomen showed a 2.6 × 5.5 cm periaortic mass in the retroperitoneum. Biopsy revealed fibrosis and chronic inflammation consistent with retroperitoneal fibrosis (RPF). Peripheral blood reverse transcriptase polymerase chain reaction for BCR-ABL showed patient to be in ongoing CMR.Therapy with imatinib mesylate (IM) is a standard of care for most patients with Ph+ CML. Resistance to imatinib occurs annually in 3% to 4% of patients with CML. Nilotinib is rationally designed in inhibiting the growth of 32 of 33 cell lines bearing mutations causing imatinib resistance, the exception being the T315I mutant. Reported adverse effects with nilotinib include elevated lipase levels, rash, indirect hyperbilirubinemia, myelosuppression, pancreatitis, QTc prolongation and sudden death. To our knowledge, RPF has not been previously reported with nilotinib use. We faced different diagnostic possibilities mainly (1) relapse of CML, (2) new onset of another postchemotherapy nonepithelial neoplasia and (3) idiopathic RPF (Ormond disease). Idiopathic RPF is a rare subtype of chronic periaortitis characterized by the presence of a fibroinflammatory mass surrounding the abdominal aorta and iliac arteries. It occurs predominantly in men in the fifth to sixth decade of life. Therefore, we considered this disease as statistically unlikely, although not impossible in this patient. In most cases secondary to malignant disease, RPF results from an exuberant desmoplastic response to retroperitoneal metastases—e.g. carcinoma of the prostate, breast, colon—or to the retroperitoneal primary tumour—e.g. Hodgkin's and non-Hodgkin lymphomas, various types of sarcomas. There have been no reported cases of RPF with CML. Among the drugs causing RPF most common ones are derivatives of ergot alkaloids, β-blockers, hydralazine, and analgesics.The estimation of the probability that a drug caused an adverse drug reaction (ADR) is usually based on clinical judgment. Naranjo et al developed a simple probability scale to assess the causality of ADR. The ADR was assigned to a probability category from the total score as follows: definite ≥ 9, probable 5 to 8, possible 1 to 4, doubtful ≤0 based on a simple questionnaire. We got a total score of 7 which falls in the probable category for nilotinib causing RPF. Mechanism of RPF from nilotinib use is unknown. In vitro and in vivo genetic toxicology studies conducted in animals found that nilotinib exhibited fibrotic changes in dog heart. Hematological findings, common in rats and monkeys, included elevated white counts attributed to drug-effects in the lymphoid tissues such as lymphoid hyperplasia and fibrosis. Therapy for patients with CML has grown in complexity. Nilotinib is a relatively new TKI and optimal management of patients on this drug requires intimate knowledge not only of response criteria and of timing but also of potential toxicities and how they may affect response to therapy and patient outcome. Disclosures:No relevant conflicts of interest to declare.

Peptides Derived From Mutated BCR-ABL Elicit T Cell Immunity In CML Patients

AbstractAbstract 887Over 20 BCR-ABL mutations have been identified that result in imatinib resistance and relapse of chronic myelogenous leukemia (CML). CML is highly responsive to immunological manipulations and we therefore hypothesized that mutated BCR-ABL-derived peptides could serve as immunogenic tumor-specific targets. Herein, we present a multi-step strategy for identifying tumor-specific T cell epitopes generated from gene mutation. We first investigated whether peptides derived from 24 frequent BCR-ABL mutations could potentially bind 8 common class I MHC molecules by applying the well-validated prediction servers IEDB and NetMHC to tiled 9- and 10-mers around each mutation. More than 60 peptides were predicted to bind to one or more of the following alleles with IC50<1000: A*0201, A*0301, A*1101, B*0702, B*0801, B*1501, A*0101 or A*2402. From NetMHC, 24 of 84 (29%) were predicted as high (IC50<50), 42 (50%) as intermediate (IC50=50-500), and 18 (21%) as weak binders (1000> IC50>500). From IEDB, 9 of 61 (15%) were predicted as high, 35 (57%) as intermediate and 17 (38%) as weak binders. 24 of 84 mutated peptides (29%) and 24 of 61 mutated peptides (39%) were predicted as binding with at least two-fold higher affinity compared to parental peptides, using NetMHC and IEDB, respectively. These predictions indicated that cells from 7 of 9 imatinib-resistant CML patients had the potential to present at least one mutated BCR-ABL derived peptide by binding autologous HLA alleles (with IC50<1000). CML cells from 3 of the 5 patients had an E255K mutation and expressed HLA-A3, and were predicted to generate two promising candidate peptides: E255K-A247-255 (KLGGGQYGK, IEDB IC50=113; NetMHC IC50=192) and E255K-B255-263 (KVYEGVWKK, IEDB IC50=29; NetMHC IC50=28). Both peptides were predicted to bind HLA-A*0301 at least ten-fold more tightly than parental peptides. Using a competitive MHC binding assay, we confirmed that E255K-A and –B were high binders with IC50 scores of 208nM and 17nM, respectively and that they both had at least ten-fold fold greater affinity than parental peptides. In addition, E255K-B also bound to the other HLA-A3 superfamily members HLA-A*1101, HLA-A*3001, HLA-A*3101, HLA-A*6801 (IC50: 39–603nM). We next successfully generated T cell lines against E255K-B but not E255 K-A from a normal HLA-A3+ donor that demonstrated greater specificity against the mutated peptide (2330±325 SFC/million cells, by IFNγ-ELISPOT) than the parental peptide (1270±42 SFC/million cells). E255K-B is endogenously processed and presented since E255K-B reactive T cells also responded to HLA-A3+ antigen-presenting cells (APCs) that were transfected with a minigene encompassing 227 base pairs surrounding the E255K mutation (1900±85 SFC/million cells). Finally, we assessed the potential for E255K-B to stimulate T cell responses in CML patients. E255K-B elicits T cell immunity in vivo in that we could detect antigen-specific CD8+ T cell reactivity from two HLA-A3+ CML patients bearing the E255K mutation by IFNγ-ELISPOT and by antigen-specific tetramer staining. T cell responses could be abrogated in the presence of class I blocking antibody w6/32. For both patients, reactive T cells were polyfunctional, expressing GM-CSF, IP10 and TNFα in response to APCs pulsed with mutated peptide or expressing the E255K minigene. For Patient 2, E255K reactivity developed only in the setting of donor-derived engraftment following curative allogeneic stem cell transplantation. Further analysis to explore the kinetics of mutated peptide-specific immunity in relationship to burden of mutation-expressing leukemia cells is in progress. Our studies have demonstrated that leukemia-driven genetic alterations can provide immunogenic tumor-specific antigen targets associated with clinical response in vivo. Importantly, even though BCR-ABL mutations generate resistance to imatinib, they also create novel epitopes that can be effectively recognized by cytotoxic CD8+ T cells. Our studies support the development of specific, nontoxic and personalized vaccination strategies for eradication of drug-resistant CML. Disclosures:No relevant conflicts of interest to declare.

Response to Tyrosine Kinase Inhibitors Therapy and Donor Lymphocyte Infusion In Patients with Chronic Myelogenous Leukemia Relapsing After Allogenic Hematopoietic Stem Cell Transplantation.

AbstractAbstract 4482Tyrosine kinase inhibitors (TKIs) and donor limfocyte infusion (DLI) are nowadays possible treatment options to treat relapse of chronic myelogenous leukemia (CML) after allogeneic stem cell transplantation (alloHSCT). This report aim was to analyze management and outome of CML relapse after alloHSCT based on single centre experience.We retrospectively reviewed 8 patients treated with TKIs and/or DLI for CML relapse after alloHSCT. Study group chracteristic before transplantation: 8 patients (4 women, 4 men); median age 31 years (25-53); disease duration before alloHSCT 10 months (4-33); prior transplantation treatment: imatinib (n=8), nilotinib (n=1); CML phase: chronic phase 1 (n=7), chronic phase 2 (n=1); remission status: hematological (n=8), cytogenetic (n=4), molecular (n=3); donor type (identical sibling – 4, matched unrelated –3, 1 HLA-antigen mismatched unrelated – 1); stem cell source (bone marrow – 7, peripheral blood – 1); conditioning regimen (treosulfan and fludarabine – 7; busulfan and cyclophosphamide – 1); EBMT transplant risk score 2.5 (1-5). All transplantations were performed in intensive care, sterile air units. Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporine A and short course of standard dose methotrexate. The median number of transplanted cells: nucleated cells 3.3 × 10^8 (2.1-8.9); CD34(+) cells 3.6 × 10^6 (0.8-12.9); CD3(+) cells 19.3 × 10^6 (17.6-237)/kg recipient body weight. All patients engrafted and achieved full donor chimerism before day 100 after transplantation. Hematopoietic recovery was as follows: leukocytes to 1,0 G/l – median 21 days (12-39); granulocytes to 0,5 G/l - 21 (12-42); platelets to 50 G/l –23 (18-38). Only 3 patients had signs of acute GvHD – grade I (1pt – skin 2 degree; 2pts – skin 1 degree).8 patients relapsed at median time 5 months after HSCT (4-24). Type of relapse: hematologic –0, cytogenetic-4, molecular – 8. At the time of relapse four patients were still treated with immunosuppressive agents. The median donor chimerism at the relapse was 90% (40-100%) and in 5 cases was lower than 95%. All patients who relapsed started treatment with TKIs (imatinib-7; nilotinib-1). The madian treatment time is 10 months (2-50). Four of them are still treated with TKIs. Seven patients recieved also DLI – median 1.5 times (1-6). 7 of 8 patients patients achieved molecular remission and 1 patient a complete cytogenetic response. All patients who achieved remission showed evidence of conversion to complete donor chimerism.DLI have become the treatment of choise for CML patients who relapsed after allogenic HSCT. An alternative to DLI are now TKIs: imatinib or second line TKIs. Is the DLI still the “gold standard”? Or better chose only TKIs to achieve remission without the risk of GvHD? Or chose the combination with lower doses of DLI to maximise responses while minimising the risk of GvHD? We are still looking for optimal and most effective treatment option for these patients. Disclosures:No relevant conflicts of interest to declare.

Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

A retrospective analysis was performed of 118 patients with hematologic malignancies who received donor lymphocyte infusions (DLIs) after allogeneic stem cell transplantation (ASCT). Treatment was either given because of hematologic relapse (n = 44), molecular/cytogenetic relapse (n = 52), or other causes (n = 22). Molecular relapse was in most cases based on leukemia lineage-specific chimerism analysis. Patients with acute leukemia and myelodysplastic syndrome showed a 3-year survival of 42% if DLI treatment was given because ofmolecular relapse, compared to 16% in hematologic relapse (P < .006). In multivariate analysis, there was a correlation between response to DLI and nonhematologic relapse (risk ratio [RR] 3.36, P = .001), chronic graft-versus-host disease (cGVHD) (RR = 1.51, P = .005), and late relapse (RR = 2.06, P = .017). The overall incidences of acute GVHD (aGVHD) grades I-II and grades III-IV aGVHD were 33% and 8.5%, respectively. Probability of cGVHD was 33%. The development of aGVHD or cGVHD did not significantly influence the response of DLI in patients with molecular/cytogenetic relapse. However, the development of cGVHD was significantly associated with a better response in patients with hematologic relapse because only 4 of 29 patients without cGVHD responded compared to 7 of 12 with cGVHD (P = .007). The development of cGVHD increased significantly if DLI was given >12 months after ASCT (46% versus 27%, P = .04). In contrast, time between ASCT and start of DLI treatment had no significant influence on the risk of developing aGVHD. To conclude, monitoring of leukemia lineage-specific chimerism is of utmost importance for DLI response after ASCT.

Knockdown of SOD1 sensitizes the CD34+ CML cells to imatinib therapy

Leukemia stem cell is thought to be one of the leading causes of imatinib resistance and the resultant relapse of chronic myelogenous leukemia (CML). Eradicating the leukemia stem cells holds the promise of CML treatment. In this study, we found that the CD34+ subpopulation in the CML cell line K562 had a higher expression of SOD1 than that in the CD34 negative cells. Knockdown of SOD1 in CD34+ cells had no significant effects on cell survival and growth, while it sensitized the CD34+ cells to imatinib therapy. N-acetyl-L cysteine (NAC) blocked the pro-apoptotic effects of SOD1 knockdown, suggesting the antioxidant effects of SOD1 was essential for the resistance of CD34+ cells to imatinib therapy. In summary, our results suggest that antagonizing the enhanced endogenous antioxidant activity in leukemia stem cells sheds lights on CML therapy.

Response to Tyrosine Kinase Inhibitor Therapy in Patients with Chronic Myelogenous Leukemia Relapsing in Chronic and Advanced Phase Following Allogeneic Hematopoietic Stem Cell Transplantation

Tyrosine kinase inhibitors (TKI) have been used to treat relapse of chronic myelogenous leukemia (CML) after allogeneic stem cell transplant (HSCT), with responses seen predominantly in chronic phase (CP) patients. This study aimed to analyze the response to TKI therapy and overall survival for patients relapsing predominantly in advanced phase. We retrospectively reviewed 22 patients treated with imatinib (n=20) and/or dasatinib (n=6) for relapsed CML after HSCT; 8 patients were in CP, and 14 patients had advanced disease. Seven patients also received donor lymphocyte infusions. Hematologic, cytogenetic, and molecular responses were analyzed. Nineteen patients (86%) achieved complete hematologic response (CHR), 17 patients (77%) achieved complete cytogenetic response (CCR), and 14 patients (64%) achieved complete molecular response (CMR). In advanced phase patients, 11 (79%) achieved CHR, 10 (71%) CCR, and 8 (57%) achieved CMR. Grade 3 or 4 cytopenias occurred in 10 cases. With median follow-up of 31.5 months from relapse, 14 (64%) patients remain alive, 13 in CMR. In multivariate analysis, the achievement of CMR was significantly correlated with OS with an odds ratio of 20.5 (95% confidence interval 2.3-182) P=.007. TKI therapy is capable of inducing durable molecular responses for CML relapsing after HSCT, both in chronic and advanced phases. The achievement of CMR appears to be crucial in providing long-term disease control for these patients.

Donor Lymphocyte Infusions (DLI) After Peripheral Blood Stem Cell Transplantation. A Retrospective Analysis of 357 Patients by the Chronic Leukemias Working Party EBMT.

Abstract 3309Poster Board III-197Donor Lymphocyte Infusions (DLI) constitute a potent therapeutic option for treating relapse of chronic myelogenous leukemia (CML) after hematopoietic stem cell transplantation (SCT) inducing durable remissions in the majority of patients. A number of factors is known to influence the efficiency of DLI. A preliminary analysis of EBMT data had suggested that DLI efficiency might be inferior after peripheral blood stem cell transplantation (PBSCT) as compared to DLI following bone marrow transplantation (BMT) (Schmid et al. ASH, 2005). To control for a number of other factors that were not known at the time of the previous analysis, we repeated this analysis based on the results of 357 patients treated with DLI following PBSCT (N=108) and BMT (N=249). We limited the analysis to patients who relapsed after standard intensity conditioning SCT from HLA-identical family donors in first chronic phase of disease. The median age of patients was 39 years (range 18-60) with predominance of males (59%). 53% of patients with known data were CMV positive and in 44% of the patients there was a sex-mismatch with the stem cell donor. SCTs have been performed between 1994 and 2007 (median year: 1998) and the conditioning treatment included total body irradiation (TBI) in 68% and T cell depletion in 44% of patients. 92% of patients with known data achieved complete remission after SCT while grade II-IV acute GvHD occurred in 18% of patients and extensive chronic GvHD in 17% of patients. Median time to relapse was 17 months (range 0.6-129) and median time from SCT to first DLI infusion was 23 months (range 0.6-142). Looking at the patients with known data at the time of first DLI infusion, the relapse could be classified as molecular/cytogenetic in 63%, hematologic in 27% and transformed in 10% of patients. The median year of first DLI was 2000 ranging from 1995 to 2007. As the initial DLI infusion, 9% of patients received <1×10e6, 62% 1.1-10×10e6 and 29% received >10×10e6 CD3+ cells/kg. However, the comparative analysis of groups based on the stem cell source revealed that the group of patients transplanted with PBSCs included significantly more males (68 vs. 56%), were older (median age 42 vs. 39) and underwent more frequently T cell depletion at SCT (72 vs. 34%,). PBSCTs have been performed more recently (median year 1999 vs. 1997) and both duration of remission and time from SCT to first DLI were shorter after PBSCT (median duration: 12 vs. 21 months and 14 vs. 26 months respectively). The initial cell dose in patients from PBSCT arm was significantly lower than in BMT group (≤10×10e6 CD3+/kg in 89% vs. 65% of patients). Similarly to the previous study, we also observed a trend towards superior overall survival after DLI in BMT group compared to PBSCT group, especially in the early post-transplant period. The actuarial probability of survival at five years from DLI was 77% in PBSCT group and 79% in BMT group. However the differences were not statistically significant (p=0.77). The source of stem cells did not influence the occurrence of molecular/cytogenetic remissions after DLI (80% vs. 77%) grade II-IV acute GVHD (16% vs. 16%), chronic GVHD (23% vs. 30%) and myelosuppression (10% vs. 16%). In order to search for factors having impact on survival of analyzed patients, we performed both univariate and multivariate survival analyses. The univariate analysis revealed that interval from SCT to DLI longer than 2 years (p=0.001), date of DLI after 2000 (p=0.026) and molecular/cytogenetic stage of relapse at DLI (p<0.001) were associated with favorable survival. Similarly, the multivariate Cox analysis identified interval between SCT and DLI (HR= 0.50, CI: 0.3-0.8; p=0.01 for after 2 years), date of DLI (HR=0.63, CI:0.4-1.0; p=0.07 for after 1999), and stage of relapse (HR=2.8, CI:1.2-6.5; p=0.02 for HemCR and HR=3.6, CI:1.8-7.0; p<0.001 for missing data group), but not for stem cell source (HR=0.95, CI 0.56-1.6 ;p=0.86) as independent factors affecting survival. Based on our retrospective data from EBMT registry covering a period of 14 years of SCT and DLI, it seems that the PBSCT does not affect the efficiency of DLI compared to BMT. Therefore, keeping all limitations of a retrospective analysis in mind, it seems that differences in efficacy of DLI do not influence the decision whether PBSC or BM should be used as stem cell source for allogeneic SCT in CML in first chronic phase. DisclosuresNo relevant conflicts of interest to declare.

Polymyositis complicating donor lymphocyte infusion after stem cell transplantation for relapsed chronic myeloid leukemia: report of a case and review of literature

Polymyositis may occur along with other manifestations of chronic graft vs host disease after allogeneic bone marrow transplantation (BMT). Donor lymphocyte infusion (DLI) could produce durable remissions in relapsed patients with chronic myelogenous leukemia (CML) but it may contribute to the development of polymyositis. We report in this study a 25-year-old man who suffered from a relapse of CML 4 years after a sibling human leukocyte antigen-matched allogenic BMT. The patient developed polymyositis 18 months after DLI. Mini-pulse therapy with methylprednisolone was effective for his proximal weakness and elevated creatine phosphokinase. There was no relapse of symptoms of polymyositis on tapering of the medication.

1107. Gene Therapy Clinical Trials for Relapsed Leukemia with Infusions of the Suicide-Gene Transduced Donor Lymphocytes in Japan

Donor lymphocytes function as a double-edged sword in allogeneic stem cell transplantation (allo-SCT), that is, they attack leukemic cells (graft versus leukemia-GVL) as well as patient’s normal tissues such as liver, intestines, and skin, (graft versus host disease-GVHD). The strong GVL reaction has made an infusion of donor lymphocytes (DLI) an effective treatment for relapsed leukemia after allo-SCT, especially for relapse of chronic myelogenous leukemia. However, the use of DLI is severely limited by the risk of a potential life- threatening complication, GVHD.