Refractory Myeloid Leukemia Research Articles

Guideline for treating relapsed or refractory myeloid leukemia in children with Down syndrome.

Treatment of relapsed and refractory myeloid leukemia in Down syndrome (r/r ML-DS) poses significant challenges, as prognosis is dire and there is no established standard treatment. This guideline provides treatment recommendations based on a literature review and collection of expert opinions, aiming to improve overall and event-free survival of patients. Treatment options include fludarabine and cytarabine (FLA)±gemtuzumab ozogamicin (GO), azacytidine (AZA)±panobinostat, and hematopoietic stem cell transplantation (HSCT). Preferred approaches are AZA±panobinostat for cases with low blast count or FLA±GO for cases with high blast count, followed by HSCT after remission. Further research is crucial for the investigation of targeted therapies (e.g., BH3 mimetics, LSD1, JAK inhibitors).

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5592.

Survival outcomes of children with relapsed or refractory myeloid leukemia associated with Down syndrome

AbstractChildren with Down syndrome (DS) are at a significantly higher risk of developing acute myeloid leukemia, also termed myeloid leukemia associated with DS (ML-DS). In contrast to the highly favorable prognosis of primary ML-DS, the limited data that are available for children who relapse or who have refractory ML-DS (r/r ML-DS) suggest a dismal prognosis. There are few clinical trials and no standardized treatment approach for this population. We conducted a retrospective analysis of international study groups and pediatric oncology centers and identified 62 patients who received treatment with curative intent for r/r ML-DS between year 2000 to 2021. Median time from diagnosis to relapse was 6.8 (range, 1.1-45.5) months. Three-year event-free survival (EFS) and overall survival (OS) were 20.9 ± 5.3% and 22.1 ± 5.4%, respectively. Survival was associated with receipt of hematopoietic stem cell transplantation (HSCT) (hazard ratio [HR], 0.28), duration of first complete remission (CR1) (HR, 0.31 for > 12 months) and attainment of remission after relapse (HR, 4.03). Patients who achieved complete remission (CR) before HSCT, had an improved OS and EFS of 56.0 ± 11.8% and 50.5 ± 11.9%, respectively compared to those who underwent HSCT without CR (3-year OS and EFS of 10.0 ± 9.5%). Treatment failure after HSCT was predominantly because of disease recurrence (52%) followed by treatment-related mortality (10%). The prognosis of r/r ML-DS remains dismal even in the current treatment period and serve as a reference point for current prognostication and future interventional studies. Clinical trials aimed at improving the survival of patients with r/r ML-DS are needed.

Preclinical Validation of EZH2 and HDAC I Dual Inhibition As a Potent Therapy for Refractory Myeloid Leukemia Associated with Down Syndrome

Children with Down Syndrome who are less than 5 years old have approximately 400-fold increased risk of developing myeloid leukemia. Even though children with myeloid leukemia associated with Down syndrome (DS-ML) are more sensitive to chemotherapy than their non-DS-ML counterpart, relapse and treatment-related toxicity needs to be eliminated to achieve better outcome for DS-ML children. Epigenetic alterations including DNA methylation and histone modifications play a key role in the pathogenesis of DS-ML. Enhancer of zeste homolog 2 (EZH2) is one of the epigenetic regulators that harbors high frequency of mutations in DS-ML. EZH2 is a catalytic subunit of PRC2 complex which suppresses gene expression by di- and tri-methylation of lysine27 on histone 3 (H3K27me2/3). EZH2 recruits class I histone deacetylases (HDAC, which remove acetyl groups from histones), on target genes for further suppression of gene expression. Using patient-derived xenograft models of DS-ML, we showed previously that combination of DNA hypomethylating agent azacitidine with HDAC inhibitor panobinostat or BCL2 inhibitor venetoclax showed significant improvement in median survival. Here we aimed to show synergistic effect of EZH2 inhibitor, GSK126, and class I HDAC inhibitor, romidepsin. The viability of CMK, a DS-ML cell line, and NTPL-60 and NTPL-386, in house generated DS-ML PDX lines, was determined by exclusion of propidium iodide-stained cells by flow cytometry. IC50 values for GSK126 and romidepsin were calculated using GraphPad Prism (dose-response curve). The IC50 of GSK126 was 40.5, 18.0, and 17.2 mM, while that of romidepsin was 10.2, 9.2, and 3.0 nM respectively. To determine synergy, the viability of cells exposed to a variety of concentrations of GSK126 and romidepsin alone or in combination was measured. Synergy index was calculated using relative risk ratio (RRR) formula as described previously (Quagliano et al, 56:36, Leuk Res, 2017). All the 3 DS-ML lines showed RRR value of less than 1 indicating synergistic inhibition of cell death by GSK126 and romidepsin treatment in ex vivo culture (Figure 1A). NTPL-386, a refractory DS-ML PDX line, which showed minimal response to chemotherapy (11-day improvement in median survival, Barwe et al., 134:2683, Blood, 2019), was chosen to determine the in vivo efficacy of GSK126 and romidepsin combination. NTPL-386 engrafted mice (n=5 each) were treated with GSK126 (100 mg/Kg) and/or romidepsin (2 mg/Kg) twice weekly for three weeks. Thus, one treatment cycle consisted of six doses. A separate cohort of mice were treated with four cycles of the combination, with two weeks rest between cycles. Disease progression was monitored by percentage of human cells in peripheral blood by flow cytometry. Kaplan-Meier survival estimates were plotted based on the time when mice were euthanized because they met pre-determined experimental endpoints. Compared to untreated mice, GSK126 or romidepsin treatment showed an increase in median survival by 8 days and 11 days respectively (Figure 1B, P <0.005). The combination of these drugs showed a remarkable improvement in survival (41 days). The difference in the median survival of the mice treated with the combination was statistically significant when compared to GSK126 or romidepsin (P <0.005). Further treatment for three additional cycles showed sustained remission upto 150 days post cell injection, when the study was terminated. Prolonged treatment showed a statistically significant improvement in survival compared to one treatment cycle (P <0.05). Further studies to characterize the mechanism of synergy are in progress. In summary, our data show a synergistic effect of EZH2 and HDAC class I inhibition on DS-AML growth and pave the path for clinical evaluation of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adoptive Immunotherapy with Cord Blood (CB) Grafts Targeting Shared Inherited Paternal Antigens (IPA) and/or Non-Inherited Maternal Antigens (NIMA) during Induction Treatment for High Risk Myeloid Malignancies: Safety and Preliminary Results

BackgroundSeveral groups have reported use of haploidentical peripheral blood donor cells as adjunct to chemotherapy in remission induction of myeloid leukemia (Colvin et al, BBMT 2009, Gue et al, Blood 2011). Moreover, CB grafts have been associated with greater disease control and enhanced graft-versus-leukemia responses, as a consequence of the fetal-maternal interactions during pregnancy (van Rood et al, PNAS 2009 and PNAS 2012).We evaluated prospectively the feasibility and tolerability of infusing partially HLA matched cord blood units (CBUs) with specific alloreactive properties after salvage chemotherapy to patients (pts) with refractory myeloid leukemia.Patients and MethodsPts with confirmed diagnosis of AML or MDS, according to WHO classification (excluding acute promyelocytic leukemia) who had received two or more previous induction/re-induction regimens were eligible. All pts were treated at NYPH and all CBUs were obtained from the NCBP. The study was performed under FDA IND and was approved by the IRBs of both institutions. Pts received induction chemotherapy followed by CBU infusion (1-3 days later) without any immunosuppression.CBUs eligible for the study contained a pre-cryopreservation Total Nucleated Cell (TNC) dose of 0.5-2.5x107/kg. They were HLA matched at 4-6/6, HLA-A, -B intermediate resolution, -DRB1 high resolution. CBUs had undergone HLA typing of the mother, thus allowing determination of the paternal and maternal antigens. CBUs with shared IPA targets and/or NIMA match were selected for cell therapy.ResultsA total of 34 patients underwent CBU search. Mean CBU number per search was 134 (range: 7-250) with 58% potentially eligible CBUs per pt (range: 5-159). A total of 143 CBUs underwent testing including HLA confirmatory typing. At least one eligible CBU was identified for each pt. Higher HLA allele level match was preferred in the final selection.Of the 34 pts, 23 did not receive treatment because of progressive disease and death (n=8); other clinical trials/treatments (n=6); hematopoietic stem cell transplants (HSCT) (n=9); while one is currently undergoing evaluation. Ten pts have been treated (Table); 8 had AML, 7 with primary induction failure (PIF); one had myeloid blast phase CML and one with high risk MDS. Nine pts had poor risk karyotype and 4 had prior allografts. Median age was 52 years (range: 34-67); median number of prior treatments was 11 (2-15) and median HSCT-CI was 3 (0-7).Of the CBUs infused, six were 5/6 and four were 4/6 HLA matched. The allele level compatibility and presence of shared IPA targets and/or NIMA matches are shown in the Table. CBU median TNC was 90x107 (range: 63-151) and they tended to be “older” with a median cryopreservation time of 10 years. CBUs provided a mean cell dose of 1.66 x 107 TNC/kg (range: 1.1-2.4). Mean post-thaw CD34+ and CD3+ cell doses were 52x103/kg (range: 20-90) and 0.34x107/kg (range: 0.24-0.52; SD: 0.08), respectively, with excellent post-thaw CD34+ viability (93.5%-98.3%).No CBU infusion reactions or cytokine release syndrome were observed. One pt, recipient of a prior allograft with no history of GVHD, experienced skin rash 1 week after CBU infusion. The skin biopsy was consistent with GVHD. No GVHD was observed in the other 9 pts. One pt died of disseminated adenovirus infection early after treatment; viremia was present at the initiation of chemotherapy.Transient CBU chimerism was detected in 5 of the 10 pts as early as one week and as late as 6 months after CBU infusion. Four pts with median pre-treatment BM blast counts of 56% (range 13-94) had objective hematologic responses (Cheson B JCO 2003) with median post-treatment blast counts of 0% (range 0-12), 3 with morphologic leukemia free state and one with a partial response. One patient had aplastic marrow but with flow cytometry evidence of persistent leukemia; while 4 pts recovered with morphologic persistent leukemia. Six pts went on to receive HSCT. Two subsequently relapsed and one died of disseminated adenovirus, while 3 pts remain in remission.ConclusionUse of CBUs as adoptive immunotherapy for pts with refractory myeloid leukemia represents a safe, well tolerated treatment, in which the potential for alloreactivity can be enhanced through the selection of CBUs by targeting shared IPA and NIMA present in the patients. Further, smaller CBUs, unlikely to be adequate grafts for adult transplantation, can be utilized for cell therapy. [Display omitted] DisclosuresRoboz:Cellectis: Research Funding; AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy. Ritchie:Celgene: Consultancy, Other: Travel, Speakers Bureau; Pfizer: Consultancy, Other: Research funding to my institution; Incyte: Consultancy, Speakers Bureau; Astellas Pharma: Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Bristol-Myers Squibb: Other: Research funding to my institution; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau. Lee:Amgen: Consultancy; Baxalta: Consultancy; Clinipace: Consultancy; Alexion Pharmaceuticals: Consultancy.

Recipient T Cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells

Natural killer (NK) cells mediate surveillance for malignancy. In some chemotherapy refractory myeloid leukemia patients, adoptive transfer of NK cells from haploidentical donors can induce remission. We have previously shown that remission induction is linked to NK cell persistence at day +14, but the factors influencing NK cell persistence are unknown. To address this question, patient samples from a phase I trial of National Cancer Institute (NCI) IL-15 in whom either did or did not show NK cell expansion were compared with healthy donor control subjects. Before lymphodepleting chemotherapy, high absolute CD3+ count was predictive of patients who failed to expand their haploidentical NK cell graft. Interestingly, both groups had elevated expression of inhibitory receptors and decreased cytokine production compared with control subjects, suggestive of T cell exhaustion among all patients before haploidentical NK cell infusion. At day +14, however, haploidentical NK cell expanders had persistence of recipient CD8+ T cells with the most exhausted inhibitory phenotype (either PD-1high or dual PD-1+Tim-3+) and elevated expression of T-bet and Eomes compared with NK cell nonexpanders and control subjects. This suggested that maintenance of an exhausted T cell state at day +14 permits haploidentical NK cell expansion and supports further efforts to selectively deplete recipient T cells or modulate their dysfunction.

Treatment of relapsed or refractory acute myeloid leukemia.

Prognosis for refractory or relapsed acute myeloid leukemia (AML) patients is poor, and allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only salvage option with curative potential for these patients. As the outcome of allogeneic HSCT in patients without complete remission (CR) status is unsatisfactory, it is important to attain a second CR before HSCT. An aggressive re-induction therapy for relapsed or refractory AML patients is often accompanied with severe infectious complications due to profound myelosuppression, which makes subsequent HSCT challenging. Furthermore, in AML patients with high-risk clinical features who do not achieve CR after remission induction chemotherapy (CT), allogeneic HSCT, preemptively performed immediately after re-induction CT or without prior CT, may induce durable remission in some patients. Determining the timing of HSCT and selecting a salvage regimen as a bridging therapy to HSCT are essential for avoiding severe treatment-related complications.

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Abstract Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated &amp;gt;50% inhibition, in another 40% it was 20-50% and in 20% inhibition was &amp;lt;20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p&amp;lt;0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML. Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3338.

Phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with advanced myeloid malignancies

We conducted a phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with relapsed, refractory myeloid leukemias. Twenty-eight patients received the construct intravenously at four dose levels (12, 18, 28 and 40 mg/m(2) per course) in a "3+3" study design. The dose-limiting toxicity was infusion-related allergic reaction including hypoxia and hypotension. The 28 mg/m(2) total dose was considered the maximally tolerated dose. Four patients developed a reduction in peripheral blood blasts of at least 50%. Three patients treated with the 10, 12 and 28 mg/m(2) doses showed a 38-50% reduction in bone marrow blasts. There was normalization of platelets in one patient treated with 40 mg/m(2). Pharmacokinetic analysis demonstrated that the highest blood levels achieved were 200-300 ng/mL which cleared with a half-life of ∼20 hours. Antigenicity was low with one patient at the 12 mg/m(2) dose and one patient at the 18 mg/m(2) dose (2/23, <10%) developing antibodies to the recombinant gelonin component after 28 days. We concluded that HUM-195/rGel can be safely administered in a multi-dose cycle to patients with advanced myeloid malignancies and warrants further investigation.

A phase 1 dose‐escalation study of ARRY‐520, a kinesin spindle protein inhibitor, in patients with advanced myeloid leukemias

ARRY-520 selectively inhibits the mitotic kinesin spindle protein (KSP), which leads to abnormal monopolar spindle formation and apoptosis. A phase 1 trial was conducted to establish the safety and the maximum tolerated dose (MTD) of ARRY-520 given as a 1-hour infusion in either a single dose or on a day 1, 3, and 5 divided-dose schedule per cycle in patients with advanced or refractory myeloid leukemias. Additional objectives were to characterize pharmacokinetics, assess preliminary clinical activity, and explore biomarkers of KSP inhibition with ARRY-520. A total of 36 patients with acute myelogenous leukemia (n = 34) or myelodysplastic syndromes (n = 2) with a median age of 66 years (range, 21-88 years) were enrolled: 15 in the single-dose schedule (dose levels: 2.5, 3.75, 4.5, and 5.6 mg/m(2)) and 21 in the divided-dose schedule (dose levels: 0.8, 1.2, 1.5, and 1.8 mg/m(2)/day). The MTD was 4.5 mg/m(2) total dose per cycle for both dose schedules. Dose-limiting toxicities included mucositis, exfoliative rash, hand-foot syndrome, and hyperbilirubinemia. Grades 3 or 4 reversible drug-related myelosuppression were observed in 33 of 36 patients. Plasma pharmacokinetic analyses revealed low clearance of ARRY-520 (~3 L/hour), a volume of distribution of ~450 L, and a median terminal half-life of >90 hours. Monopolar spindles were observed in blood mononuclear cells, through use of 4',6-diamidino-2-phenylindole nucleic acid stain and antitubulin antibodies. On the basis of the relative lack of clinical activity, further development of ARRY-520 as an antileukemic agent was halted. (Clinicaltrials.gov identifier NCT00637052).

Efectividad y seguridad del régimen FLAG-IDA en leucemias agudas resistentes o recidivantes

ObjectiveTo evaluate the effectiveness and safety of the FLAG-IDA regimen in patients with acute refractory and/or recurrent leukaemia. MethodDescriptive, retrospective, observational study of the clinical histories of patients with the FLAG-IDA regimen during the period of 2005-2010. Effectiveness was measured using objective response, progression-free interval, and global survival. Safety was measured using the NCI classification system of common toxicity criteria for adverse events. ResultsWe registered 12 patients (52.17±8.26 years in women, and 54.83±7.22 years in men), 11 cases were acute myeloid leukaemia (5 refractory, 3 in recurrence, 1 secondary to chronic refractory myeloid leukaemia (CML) and 2 secondary to myelodysplastic syndrome (MDS), one of which was refractory and the other had not been previously treated) and one case was acute refractory lymphoblastic leukaemia (ALL). Six patients (50%) reached a complete response (CR). One patient reached a partial response (PR), which was followed by another protocol that produced a CR, two died due to disease progression, and three due to secondary complications from treatment.The progression-free interval for patients that reached a CR was 24.38 weeks (6 months). Median global survival was 8.4 weeks.Mean time needed for the recovery of neutropenia was 23 and 37 days in the first and second cycle, respectively. The mean time required for recuperation of thrombocytopenia was 24 and 35 days in each cycle. ConclusionsThe FLAG-IDA induction regimen for the treatment of high-risk leukaemia patients is an established protocol, with good tolerance and acceptable toxicity levels that offers an opportunity for facilitating the transplantation of haematopoietic progenitors.

IL-2 Stimulated Treg Inhibit in Vitro Expansion of Haploidentical Natural Killer (NK) Cells, Which Is Partially Overcome with An IL-2-Diphtheria Toxin Fusion Protein In Vivo,

Abstract 3611Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory myeloid leukemia (AML). However, NK cells do not expand and persist in all patients despite lymphodepleting chemotherapy. In trials of adoptive NK cell therapy in solid tumors or lymphoma, host regulatory T cells (Treg) often expand in response to IL-2 given to stimulate donor NK cell expansion. Although murine studies report that Tregs inhibit NK cells, the influence of human Treg on NK cell proliferation and function is not well characterized. We studied the effect of allogeneic Tregs that were derived from human umbilical cord blood (UCB) as described by our group. Resting CFSE labelled NK cells or Teff were purified from healthy donors, and mixed with UCB Treg at various ratios. Unstimulated NK cells did not proliferate and thus IL-2 or IL-15 were added to the media at concentrations of 0.1, 0.25 and 0.5 ng/ml. In the absence of Treg, both cytokines induced equal NK cell proliferation at 5 days as measured by CFSE dilution in a concentration dependent manner. CFSE dilution was inhibited by Treg at a 1:1 ratio, especially at low cytokine concentrations. There were marked differences between the two cytokine conditions. Following IL-15 induced stimulation, the reduction in NK cell proliferation by Treg ranged from 1–35% (at different concentrations tested), whereas the inhibition of IL-2 stimulated NK cell proliferation ranged from 65–85%. Treg inhibition of NK cell proliferation could be measured at ratios as low as 1:8 in the presence of IL-2, but not IL-15. This inhibitory effect was partially explained by competition from CD25+ Tregs for IL-2. We measured Treg utilization of IL-2 by incubating NK cells with or without Treg in 0.5 ng/ml IL-2 for 4 days. The level of IL-2 with NK cells alone was 40 pg/ml vs. 17 pg/ml with Treg (compared to 330 pg/ml in IL-2-supplemented media without cells). Based on this data, we have incorporated host Treg depletion to enhance NK expansion after adoptive transfer to treat patients with refractory AML. As murine data from Blazar’s group shows that CTL therapy is enhanced by Treg depletion, we added one dose of denileukin diftitox (ONTAK®, Eisai Inc) at 12 mg/kg to our lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days, cyclophosphamide 60 mg/kg × 2 days for 12 AML patients. Haploidentical NK cells (CD3- and CD19-depleted PBMCs and overnight activated with IL-2 1000 U/ml) were infused on Day 0, followed by 6 doses subcutaneous IL-2 (9 million units) given every other day to promote in vivo NK cell expansion. Eleven of 12 patients were evaluable, having received at least 4 of 6 planned doses of IL-2. Blood and marrow were collected 7 and 14 days after infusion to assess NK cell and Treg expansion, as well as leukemia clearance. Of the 10 patients with interpretable day 7 chimerism data, 9 had detectable donor DNA (median 68% donor DNA). At day 14, 4 of the 12 patients (33%) had successfully expanded NK cells in vivo, with absolute donor derived NK cell counts of 480, 530, 1470 and 12390 cells/μL blood, improving on our previous 10% rate of in vivo NK cell expansion which was observed with the same regimen, without Treg depletion. In the 4 patients who expanded NK cells in vivo, there were no detectable Treg (defined as a CD25+CD4+FoxP3+ lymphocyte population) at either day 7 or day 14. In contrast, the presence of a bona fide Treg population at either day 7 [range 9.5–53%] or day 14 [27–71%] correlated with a lack of in vivo NK cell expansion at day 14. Clinically, 8 of the 11 evaluable subjects cleared leukemia (72%), 7 of whom recovered neutrophils (63% CRp) and 6 of whom went on to best donor transplant (45%). In summary, we demonstrate in vitro and in vivo suppression of NK cell proliferation by IL-2 stimulated Treg. This effect is not seen in vitro with IL-15. We have shown that the absence of host Treg correlates with in vivo NK cells expansion. Although an increased rate of donor NK expansion was observed with a single dose of denileukin diftitox, it did not completely overcome the IL-2 induced host Treg expansion. Future trials testing additional doses of denileukin difitox or other methods of Treg depletion, as well as the use of IL-15 are planned. Disclosures:No relevant conflicts of interest to declare.

Transient and Myeloid Leukemia in Children with Down Syndrome Mosaic

Abstract 2539 Introduction:Transient leukemia (TL) occurs in 5 to 10% of newborns with Down syndrome (DS). In almost all cases it resolves spontaneously within 3 months, but 20–25% of the children develop myeloid leukemia (ML-DS) until the age of 4 years. TL and ML-DS can occur also in children without any clinical signs of Down syndrome, but with constitutional trisomy 21 due to mosaicism. It can be difficult to diagnose TL or ML-DS in these children and the treatment strategies have not been defined. Patients/Material:Between 1/2002 and 7/2011, 15 newborns and infants were diagnosed with DS mosaic. Nine of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL (table 1). In children without any stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic. Diagnostic work-up was performed according to standard guidelines: morphology, immunophenotyping (IP), cytogenetics and FISH (trisomy 21), molecular genetics (GATA 1 mutation screening). Screening of GATA1 mutations was done with direct sequencing of PCR product (Exon1, Exon2, and Exon3). For monitoring of GATA1 mutant clone qPCR have been used with patient specific TaqMan probes and primers. Mosaic was detected by cytogenetics or FISH in bone marrow, blood and/or fibroblasts.Table 1patient's characteristics.Age months median/ rangeWBC/μl median/ rangeHB [g/dl] median/ rangePlatelets median/rangeBlasts %PB median/ rangeBlasts %BM median/ rangeTrisomy 21 mosaicGATA 1TL n=90 (0 to 2)64,300 (4,000 to 196,000)11.2 (6 to 13.8)554,000 (25,000 to 655,000)65 (22 to 81)45.5 (24 to 67)4 blood 3 BM 1 fibroblast 1 lymphon=9ML-DS n=817 (8 to 36)7,300 (3,000 to 32,500)8.6 (7.1 to 13.2)57,000 (3,000 to 103,000)23 (3 to 51)31 (16 to 70)4 BM 2 fibroblastsn=8PB: peripheral blood; BM: bone marrow Results:All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. One patient died due to cardiovascular failure. In all patients GATA 1 mutation was confirmed. Minimal residual disease by qPCR (mutation-specific probes) or immunophenotyping (IP) revealed negativity in 3 out of 3 children monitored (follow-up 2 to 10.1 yrs).Two children with (unknown) trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation (one child), GATA1 mutation was identified retrospectively. Both children are alive in CR. Six children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.5 to 6.7 years, median 2.4 yrs). This was confirmed by MRD-monitoring, which achieved negativity after two treatment elements (qPCR <10−4 n=3; IP <10−3 n=6). In one child a distinct refractory myeloid leukemia population (GATA1mut negative/trisomy 21 negative) arose after the 1st induction. Due to treatment refractory, allogenic stem cell transplantation was applied. Conclusions:GATA1 mutated leukemia has to be excluded in all young children with AMKL (<5years old) to prevent overtreatment. Treatment with reduced intensity protocol like ML-DS 2006 seems to be effective and sufficient in children with trisomy 21 mosaic and GATA1 mutated ML-DS. Disclosures:No relevant conflicts of interest to declare.

Clinical Observation of Fludarabine、 Intermediate-dose Ara-C and G-CSF in Treatment for Refractory or Relapsed Acute Myeloid Leukemia(26 Cases)

Objective To observe the therapeutic effects and side effects of FLAG regimen(fludarabine, Ara-C, and granulocyte-colony stimulating factor( C-CSF) for refractory or relapsed acute myeloid leukemia( AML). Methods Twenty-six AML were treated with fludarabine plus intermediate-dose Ara-C and G-CSF,of whom 15 cases belonged to refractory and 11 cases belonged to relapsed. Results After two courses of treatment, 14 cases were completely relieved (53. 8% ) and 5 cases were partially relieved (19.2% ). The overall effective rates was 73.1%. The main side effects were severe myelosuppression and non-hematological toxicity was mild. Conclusion FLAG regimen was very effective for refractory or relapsed acute myeloid leukemia and was well tolerated. The treatment-related mortality rate was low,so it provided a treatment choice for these patients. Key words: Leukemia, myeloid; Fludarabine; Cytarabine; Granulocyte colony-stimulating factor; Treatment outcome

Phase 2 study of FLAGM (Fludarabine+high-dose ara-C+G-CSF+Mitoxantrone) for refractory or relapse acute myeloid leukemia (AML): a report from the Japan Adult Leukemia Study Group (JALSG)

Phase 2 study of FLAGM (Fludarabine+high-dose ara-C+G-CSF+Mitoxantrone) for refractory or relapse acute myeloid leukemia (AML): a report from the Japan Adult Leukemia Study Group (JALSG)

PR1 vaccination in myeloid malignancies

The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation is testament to the effectiveness of the immune system in recognizing and eliminating leukemia cells. The successful identification of a range of leukemia-associated antigens (LAAs) that drive the GVL response in recent years has stimulated research in the development of vaccines to treat hematological malignancies. Here, we review the current experience with the PR1 vaccine. PR1 is a nine amino acid, HLA-A*0201-restricted peptide, shared by two myeloid LAAs, proteinase (PR)3 and neutrophil elastase (NE). PR3 and NE are found in the primary (azurophil) granule proteins of normal granulocytes and are overexpressed in myeloid leukemia cells. PR1 induces powerful HLA-A*0201-restricted CD8+ T-cell responses that selectively kill myeloid leukemia cells in vitro. The detection of low frequencies of PR1-specific CD8+ T cells in patients with chronic myeloid leukemia and at higher frequencies in patients entering molecular remission after allogeneic stem cell transplantation supports the concept that there is natural immunity to PR1, which can be boosted further by vaccination to enhance immunity to leukemia. Preliminary reports indicate that PR1 peptide vaccination induces significant increases in PR1-specific CD8+ T cells, with rapid and durable remissions in some patients with myeloid leukemia. These promising early results point the way to optimizing the administration of peptide vaccines to improve the treatment of otherwise refractory myeloid leukemias.

Tissue chimerism analysis after donor lymphocyte infusion: a case of deceptive evidence

A 31-year-old female with refractory extramedullary myeloid leukemia relapse underwent peripheral blood stem-cell transplantation from her HLA-identical brother. Hematopoietic recovery followed disappearance of the lesions. Studies three-months post-transplant showed complete chimerism (CC). Fourteen months after transplantation, the patient presented with an increasing number of extramedullary sites of biopsy-proven disease relapse (as nodular skin lesions). Bone marrow was in remission with maintenance of CC. However, tissue chimerism analysis showed full recipient-cell population. After one course of conventional dose chemotherapy followed by mobilized donor-lymphocyte infusions (DLI), a complete response was achieved. DLI continued monthly but she developed new skin lesions accompanied by multiple cervical masses. Bone marrow and tissue chimerism revealed both recipient and donor cell population. We conclude that tissue chimerism analysis after DLI may not accurately document the cell origin.

Phase I study of the ribonucleotide reductase inhibitor 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) in combination with high dose cytarabine in patients with advanced myeloid leukemia

This Phase I dose escalation study was based on the hypothesis that the addition of 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) to cytarabine would enhance cytarabine cytotoxicity. The primary objective of the study was to establish the maximum tolerated dose of 3-AP when given in combination with a fixed dose of cytarabine. Twenty-five patients with relapsed or refractory myeloid leukemia were enrolled to three dose levels of 3-AP. Cytarabine was administered as a 2 h infusion at a fixed dose of 1,000 mg/m2/day for 5 consecutive days. Escalating doses of 3-AP as a 2 h infusion were administered on days 2 through 5. The 3-AP infusion preceded the start of the cytarabine infusion by 4 h. In general, the toxicities observed with the combination were similar to the expected toxicity profile for cytarabine when utilized as a single agent at this dose and schedule. However, two of three patients developed dose-limiting methemoglobinemia at the highest 3-AP dose studied (100 mg/m2). Transient reversible methemoglobinemia was documented in 11 of 15 patients enrolled at the 75 mg/ m2 dose level. Objective evidence of clinical activity was observed in four patients. The combination of 3-AP and cytarabine given on this schedule is feasible in advanced myeloid leukemia. The recommended Phase II dose is 75 mg/m2/day of 3-AP on days 2-5 given prior to cytarabine administered at a dose of 1,000 mg/m2/day over 5 consecutive days. Methemoglobinemia is a common toxicity of this combination and requires close monitoring.

Neutrophil granule proteins as targets of leukemia-specific immune responses

Recent progress in the identification of leukemia antigens has stimulated the development of vaccines to treat hematological malignancies. Here we review the identification and characterization of the myeloid leukemia-specific antigens proteinase 3 and neutrophil elastase found in the primary (azurophil) granule proteins of granulocytes and their precursors. A peptide 'PR1' derived from these proteins induces powerful HLA-A0201-restricted CD8 T-cell proliferation. PR1-specific T cells are cytotoxic to leukemia and myelodysplastic syndrome progenitors, and occur at low frequencies in normal individuals. Frequencies are higher in patients with myeloid leukemias, and highest in patients with chronic myeloid leukemia entering molecular remission after allogeneic stem cell transplantation. These observations, together with the known association of autoimmunity to proteinase 3 and neutrophil elastase in Wegener's granulomatosis, support the concept that there is a natural immunity to primary granule proteins which can be boosted to enhance immunity to leukemia. Preliminary reports indicate that PR1 peptide vaccination induces significant increases in PR1-specific cytotoxic T cells with rapid and durable remissions in some patients with advanced myeloid leukemias. These promising developments in antileukemia vaccines have stimulated research to optimize vaccine delivery and modify regulation of natural T-cell immunity to primary granule proteins to improve treatment of otherwise refractory myeloid leukemias and myelodysplastic syndrome.

Prognostic factors for outcomes of patients with refractory or relapsed acute myelogenous leukemia or myelodysplastic syndromes undergoing allogeneic progenitor cell transplantation

Allogeneic progenitor cell transplantation is the only curative therapy for patients with refractory acute myelogenous leukemia or myelodysplastic syndromes. To identify prognostic factors in these patients, we performed a retrospective analysis of transplantation outcomes. Patients were selected if they had undergone an allogeneic transplantation between January 1988 and January 2002 and were not in remission or first untreated relapse at the time of transplantation. A total of 135 patients were identified. The median age was 49.5 years (range, 19-75 years). At the time of transplantation, 39.3% of patients had not responded to induction therapy, 37% had not responded to first salvage therapy, and 23.7% were beyond first salvage. Forty-one patients (30%) received unrelated donor progenitor cells. Eighty patients (59%) received either a reduced-intensity or a nonmyeloablative regimen. A total of 104 (77%) of 135 patients died, with a median survival time of 4.9 months (95% confidence interval, 3.9-6.6 months). The median progression-free survival was 2.9 months (95% confidence interval, 2.5-4.2 months). A Cox regression analysis showed that Karnofsky performance status, peripheral blood blasts, and tacrolimus exposure during the first 11 days after transplantation were predictive of survival. These data support the use of allogeneic transplantation for patients with relapsed or refractory acute myelogenous leukemia/myelodysplastic syndromes and suggest that optimal immune suppression early after transplantation is essential for long-term survival even in patients with refractory myeloid leukemias.