Biphenotypic Leukemia Research Articles

Outcomes in Patients With Mixed Phenotype Acute Leukemia in Morocco

Mixed phenotype acute leukemia (MPAL) includes biphenotypic and bilineal types of leukemia, which constitute rare subtypes that require individualized therapy. Outcomes in Moroccan patients with MPAL are unknown. Among 1264 patients with acute leukemia, 20 were classified as having MPAL, including 17 with biphenotypic acute leukemia (1.3%) and 3 with bilineal leukemia (0.2%). There were 8 adults and 12 children. In 12 cases (60%), leukemic blasts expressed myeloid and T-lymphoid antigens, and, in 5 cases (25%), leukemic blasts expressed B lymphoid antigens plus myeloid antigens. Patients were initially treated on protocols for acute myeloid leukemia (n=4), acute lymphoblastic leukemia (ALL, n=14), or with palliative care (n=2). The probability of survival at 2 years in MPAL cases was 52%± 14%. Six of the 12 patients younger than 15 years remain alive versus 1 of 8 adult patients. Patients treated with ALL-directed therapy had significantly higher overall survival than those treated with acute myeloid leukemia-directed therapy (P=0.003). There was no association between the phenotypic characteristics and the clinical outcome (P=0.83). In conclusion, MPAL represents 1.5% of acute leukemia in Morocco. The prognosis is poor, but initial treatment with therapy directed toward ALL, improved supportive care, and the prevention of abandonment of therapy may improve outcomes in this subgroup of patients.

Second haematopoietic SCT using HLA-haploidentical donors in patients with relapse of acute leukaemia after a first allogeneic transplantation.

Haploidentical haematopoietic SCT (HSCT) using T-cell-replete grafts and post-transplant high-dose CY has found increasing acceptance. Our purpose was to evaluate the feasibility and outcome of this strategy as second HSCT incorporating donor change for acute leukaemia relapse after a first allogeneic transplantation. The courses of 20 consecutive adults (median age 37 years, 12 male) with AML (n=14), ALL (n=5) and acute bi-phenotypic leukaemia (n=1) were analysed retrospectively. Conditioning consisted of fludarabine, CY and either melphalan or TBIor tresosulfan+/-etoposide. Engraftment was achieved in 17 (85%), and a second remission was induced in 15 patients (75%) on day +30. The rate of grade II-IV acute GvHD was 35%, while chronic GvHD occurred in fivepatients. Most commonly observed grade III-IV toxicities were mucositis (30%), hyperbilirubinemia (20%), elevation of transaminases (20%) and creatinine (20%), while invasive fungal infection affected 30%. One-year non-relapse mortality (NRM) was 36%. At a median follow-up of 17 months, estimated 1-year OS was 45%, and 1-year relapse-free survival was 33%. This strategy was feasible and allowed for successful engraftment with a moderate rate of toxicity. Early outcome and NRM are at least comparable with results after a second HSCT from HLA-matched donors without donor change at HSCT2.

Integrated genome-wide genotyping and gene expression profiling reveals BCL11B as a putative oncogene in acute myeloid leukemia with 14q32 aberrations

Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluorescence in situ hybridization on 40 cases of acute myeloid leukemia with high BCL11B mRNA expression [2.5-fold above median; 40 out of 530 cases (7.5%)] revealed 14q32 abnormalities in two additional cases. In the four BCL11B-rearranged cases the 14q32 locus was fused to different partner chromosomes. In fact, in two cases, we demonstrated that the focal 14q32 amplifications were integrated into transcriptionally active loci. The translocations involving BCL11B result in increased expression of full-length BCL11B protein. The BCL11B-rearranged acute myeloid leukemias expressed both myeloid and T-cell markers. These biphenotypic acute leukemias all carried FLT3 internal tandem duplications, a characteristic marker of acute myeloid leukemia. BCL11B mRNA expression in acute myeloid leukemia appeared to be strongly associated with expression of other T-cell-specific genes. Myeloid 32D(GCSF-R) cells ectopically expressing Bcl11b showed decreased proliferation rate and less maturation. In conclusion, by an integrated approach involving high-throughput genome-wide genotyping and gene expression profiling we identified BCL11B as a candidate oncogene in acute myeloid leukemia.

Biphenotypic Acute Leukemia with t(15;17) Lacking Promyelocytic-Retinoid Acid Receptor α Rearrangement

Biphenotypic acute leukemias (BAL) account for less than 4% of all cases of acute leukemia. Philadelphia chromosome and 11q23 rearrangement are the most frequently found cytogenetic abnormalities. Since t(15;17) is almost always associated with acute promyelocytic leukemia, t(15;17) in BAL cases is extremely uncommon. We report here a rare and instructive case of BAL with t(15;17) and the successful treatment approach adopted. A 55-year old woman was referred to our hospital for an examination of elevated white blood cell (WBC) counts with blasts (WBC 13.4×109/L; 76% blasts). The blasts with acute lymphoblastic leukemia (ALL-L2, FAB) morphology co-expressed B-lymphoid and myeloid lineages, and a cytogenetic study revealed 4q21 abnormalities and t(15;17). However, promyelocytic-retinoid acid receptor α rearrangement was not detected by fluorescence in situ hybridization on interphase nuclei. Our patient was treated with chemotherapy for ALL and gemtuzumab ozogamicin without all-trans-retinoic acid, and has remained in hematologic first complete remission for more than 3.7 years.

Lateral pharyngeal wall myeloid sarcoma as a relapse of acute biphenotypic leukemia: a case report and review of the literature

IntroductionMyeloid sarcoma is a rare extramedullary malignant tumor composed of immature myeloid cells. The tumor can affect any part of the body. Involvement of the oral cavity and nasopharynx has been reported in 50 cases. We report a case describing myeloid sarcoma affecting the lateral pharyngeal wall.Case presentationA 31-year-old Arabian man who had acute biphenotypic leukemia treated with chemoradiation and allogeneic stem cell transplant was referred to our department with sore throat and a mass lesion in his lateral pharyngeal wall after failed antibiotic therapy. Biopsy of his lesion revealed myeloid sarcoma. He was referred to the Department of Hematology-Oncology for further evaluation that showed no other lesions.The patient was diagnosed with isolated extramedullary myeloid sarcoma of his lateral pharyngeal wall as a relapse of acute biphenotypic leukemia and managed with chemoradiation.ConclusionsMyeloid sarcoma of the pharynx is a rarely encountered malignancy in the practice of otolaryngology-head and neck surgery. It can develop de novo, but may also represent relapse of leukemia. Thus, it should be considered in the differential diagnosis of any pharyngeal lesions in patients with leukemia.

Risk Factors For Relapse In Children Undergoing Preemptive Immunomodulatory Therapy After Allogeneic Hematopoietic Stem Cell (HSCT) For Acute Leukemia

IntroductionRelapse of leukemia following allogeneic HSCT is associated with a low survival rate and remains a major cause of transplant failure. To reduce risk of relapse after transplant, our institution introduced preemptive immunomodulatory therapy (IT) consisting of fast withdrawal of immunosuppression (FWI) and escalating doses of donor lymphocyte infusions (DLI). We performed a retrospective analysis of risk factors for relapse in children treated with preemptive post-transplant IT. Patients and methodsSixty-nine children (33 AML, 33 ALL, and 3 biphenotypic leukemia) underwent myeloablative HSCT followed by IT between 2005 and 2012. 61% were male. Stem cell sources included unmodified bone marrow (BM) in 26 (38%) or peripheral blood stem cells (PBSC) in 43 (62%) patients. Conditioning regimens were TBI-based in 41 (59%) and chemotherapy based in 28 (41%) patients. Donors were matched related in 29 patients (42%), 10/10 HLA allele-matched unrelated in 22 (32%) patients, and mismatched unrelated in 18 (26%) patients. Thirteen patients (19%) had evidence of disease at the time of transplant.58% of patients were enrolled in one of 2 prospective studies of IT open at our institution, while 42% of patients received IT as standard of care.Chimerism analysis was done at day 30±7 on whole PB or BM and cell subsets (CD3, CD14/15, CD19), using a semi-quantitative PCR-based method with a sensitivity of 1%. Confirmatory testing was done 2 weeks later. If residual host cells (mixed chimerism – MC), or if positive disease was found on post-transplant testing, FWI over 2-4 weeks was done. If MC persisted following FWI, DLI was initiated and given every 2-3 months until full donor chimerism (FDC) was documented or graft versus host disease (GVHD) developed. Patients who showed FDC in all subsets, or who developed GVHD prior to IT, became the observation group. Results36 patients (52%) had either FDC or acute GVHD and did not undergo IT; 15 (22%) patients underwent FWI and 18 (26%) underwent FWI followed by DLI. In patients undergoing IT, mean±SD time to FWI was 50.2±11 days post transplant, and mean time to first DLI was 135±62 days. Mean follow-up of living patients was 42±26 months. Kaplan Meier (KM) estimated event-free survival for the entire group was 72±5% at 24 months post transplant and 49±9% at 60 months post transplant. Events included relapse (N=20, 29%), death due to multi-organ failure (N=2, 3%), or death due to GVHD (N=4, 6%, only one of which was in the IT group). Out of 20 relapses, 6 were in IT group and 14 were in patients not receiving IT. Acute GVHD developed in 5 (15%) patients undergoing IT and in 17 (47%) patients not receiving IT. Chronic GVHD developed in 8 (24%) patients in IT group and 13 (36%) patients not receiving IT.Univariate KM analysis of risk factors for relapse in the entire cohort indicated that use of post-transplant IT (log rank p=0.047), pre-transplant serotherapy (p=0.04), negative disease prior to transplant (p=0.04), and cGVHD (p=0.002) we related to reduced risk of relapse. In multivariate Cox Regression analysis, lack of cGVHD was the only risk factor for relapse (p=0.004). Within a group of 33 patients who underwent post-transplant IT therapy, only the presence of disease prior to transplant was related to the increased risk of relapse (p=0.005). ConclusionsIn a cohort of children receiving preemptive post-transplant IT, the relapse rate is low (18%) and toxic death due to GVHD is low (3%). These results suggest that post-transplant IT is a safe and effective approach to decreasing the risk of relapse in pediatric patients. Additional studies are needed to optimize the use of post-transplant immunomodulatory therapy in patients with high-risk disease. Disclosures:No relevant conflicts of interest to declare.

Dose Dependent Enhancement Of Neutrophil Recovery By Infusion Of Notch Ligand Ex Vivo Expanded Cord Blood Progenitors: Results Of a Multi-Center Phase I Trial

IntroductionThere is a strong clinical need to overcome the increased early non relapse mortality (NRM) associated with delayed neutrophil recovery following cord blood transplant (CBT). Therefore we established a methodology using Notch ligand (Delta1) as a strategy for increasing the absolute number of marrow repopulating CB hematopoietic stem/progenitor cells (HSPC). We previously reported preliminary results of the first 10 patients in 2010 demonstrating the ability of Notch-expanded CB HSPC to provide rapid myeloid recovery post-CBT.1 Herein we present the updated results on 23 patients accrued to this trial aimed at assessment of efficacy as well as the feasibility of overnight shipment of the expanded cell product to three outside institutions. MethodsBetween July 2006 and March 2013, 23 patients with hematologic malignancies were enrolled in this prospective multi-center Phase I trial coordinated by the Fred Hutchinson Cancer Research Center in which one CB unit was ex vivo expanded prior to infusion. Conditioning consisted of Fludarabine (75mg/m2), Cyclophosphamide (120mg/kg) and TBI (13.2 Gy) over 8 days. On day 0, the unmanipulated CB unit was infused first followed 4 hours later by infusion of the freshly harvested expanded CB cells. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. All CB grafts were 4-6/6 HLA-matched (A/B antigen level, DRB1 allele level) to the recipient. Engraftment, NRM, relapse and GVHD were calculated using cumulative incidence rates to accommodate competing risks. Overall survival was analyzed using Kaplan-Meier estimates. ResultsPatient diagnosis was AML (n=16), ALL (n=5) and biphenotypic leukemia (n=2). Nine patients (39%) were ≥CR2 and 5 were MRD+ at the time of transplant. Median age was 28 years (range, 4-43) and weight 70 kg (range, 16-91) with a median follow-up of 614 days (range, 271-2443). 22 patients received the expanded graft with one product not meeting release criteria. The cell doses infused were significantly higher in the expanded CB graft: 2.7 (1.5-6.3) vs 6.9 (0.4-27.6) x107 TNC/kg, p<0.0008; 0.15 (0.02-0.57) vs 7.7 (0.62-49.5) x106 CD34/kg, p<0.0001. HLA-matching and ABO incompatibility of the expanded and unmanipulated products were similar. The incidence of neutrophil recovery was 95% (95% CI, 71-100) at a median of 13 days (range, 6-41 days) among the 22 patients receiving expanded CB cells which is significantly faster than that observed in 40 recipients of two unmanipulated units otherwise treated identically at a median time of 25 days (range, 14 to 45; p<0.0001). The incidence of platelet recovery (>20 x 10^9/L) was 77% (CI 95%: 53- 89) by day 100 at a median of 38 days (range, 19 – 134). There was one case of primary graft failure. Importantly, rate of neutrophil recovery correlated with CD34+ cell dose/kg with 8 out of 11 patients receiving greater than 8x106 CD34+cells/kg achieved an ANC ≥ 500/µl within 10 days. 21 patients were evaluable for in vivo persistence of the expanded cells. Ten (48%) demonstrated in vivo persistence beyond one month post infusion. The expanded cell graft was persistent at day 180 in 7 patients, and in those that survived to one year, dominance of the expanded cell graft persisted in one patient. The incidences of grade II-IV and III-IV acute GVHD was 77% (95% CI, 53-89) and 18% (95% CI, 5-36%), respectively; mild chronic GVHD was observed in 4 patients and severe chronic GVHD in one. Probability of OS was 62% (95% CI, 37-79%) at 4 years. Notably, the cumulative incidence of NRM at day 100 was 8% (95% CI, 14-24%) and at 4 years was 32% (95% CI, 8-40%). Nine patients died at a median time of 216 days (range, 31-1578 days) with respiratory failure/infection the most common cause (n=6). There were two relapses at day 156 and 365 post-transplant, with one death due to relapse. Secondary malignancy and primary graft failure were the other 2 causes of death. ConclusionsInfusion of Notch-expanded CB progenitors is safe and effective, significantly reducing the time to neutrophil recovery and risks of NRM during the first 100 days. An advantage for infusion of higher numbers of CD34+ cells/kg further demonstrates the need to develop methods that reproducibly provide even greater expansion of repopulating cells than currently achieved to improve efficacy and potentially cost effectiveness.1. Delaney C, et al, Nat Med. 2010 Feb;16(2):232-6. Disclosures:Delaney:Novartis: DSMB, DSMB Other; Biolife: Membership on an entity's Board of Directors or advisory committees; medac: Research Funding. Wagner:Novartis: Research Funding; cord use: Membership on an entity's Board of Directors or advisory committees.

A Canadian Blood and Marrow Transplant Group (CBMTG) Randomised Trial Comparing G-CSF Mobilized Peripheral Blood Versus G-CSF Stimulated Bone Marrow In Recipients Of Sibling Allografts For Hematologic Malignancies

G-CSF mobilized peripheral blood (G-PB) allografts lead to faster hematologic recovery than marrow grafts, but with more chronic GvHD(cGvHD). We hypothesized that G-CSF stimulated marrow (G-BM) may result in faster hematological recovery with less cGvHD. MethodsWe conducted a Phase III randomised multicenter trial of matched sibling G-PB vs G-BM allografts in adults with hematologic malignancies. The primary endpoint was time to treatment failure defined as the first occurrence of extensive cGvHD, relapse or death from any cause. Thirteen centers in Canada, Saudi Arabia, U.S., Australia and New Zealand enrolled patients. Adaptive stratification (minimization) balanced treatment groups by center, disease, disease risk and conditioning. Donors received G-CSF 5 mg/kg/d sc for 4 or 5 days and underwent apheresis on Day 5 and, if necessary, on Day 6 or marrow harvest on Day 5. Eligible recipients between 16 and 65 years received myeloablative conditioning and cyclosporine/methotrexate as GvHD prophylaxis. The study began before widespread adoption of the NIH GvHD consensus guidelines so cGvHD was characterized as limited or extensive. Immunophenotyping and functional assays performed on donor cells were correlated with incidence of cGvHD. ResultsBetween 2007 and 2012, 230 sibling donor-recipients were randomized to receive either a G-PB or G-BM allograft; 3 randomized to G-PB and 4 to G-BM were inevaluable (4 relapsed before transplant, 1 donor withdrew before collection, 1 recipient withdrew consent and 1 was ineligible (non-myeloablative conditioning)). Indication for allograft was AML (109; 49%), ALL (57; 25%); MDS/MPD(31; 14%), CML(13; 6%), lymphoproliferative disorder(NHL/MCL/CLL)(12; 5%) and biphenotypic leukemia(1). 77(70%)(G-PB) and 71(63%)(G-BM) recipients had low-risk disease (AML or ALL in CR1, RA, RARS, CML in 1st CP). Donor and recipient ages, sex and CMV immune status were balanced between groups.Donors tolerated mobilization well although one donor (G-PB) died of a cardiac event while receiving G-CSF. Median nucleated cells per kg were 7.59x108 (2.73-18.64) and 5.92x108 (0.94-13.23) in the G-PB and G-BM arms respectively (P<0.0001). Median CD34 cells per kg were 5.50x106 (2.03-23.94) and 3.22x106 (0.29-8.71) respectively (P<0.0001). 94% of G-PB donors underwent 1 apheresis. Median volume of harvested G-BM was 1127mL (350-1731mL).With a median follow-up of 36 months (range 9.6-48), using Cox proportional hazards multivariable modelling adjusted for the 4 minimization factors, time to treatment failure did not differ significantly between arms (HR 0.91; 95% CI 0.68-1.22; P= 0.52). There was a significantly delayed time to onset of overall cGvHD in the G-BM arm (HR=0.66; 95% CI 0.46-0.95; P=0.03) but time to onset of extensive cGvHD did not differ significantly between arms (HR=0.76; 95% CI 0.52-1.10; P=0.15). Other results are shown below:Table 1OutcomeG-PBG-BMp-value3-year OS56.8% (SE 5.2%)60.4% (SE 4.9%)0.903-year RFS55.6% (SE 5.2%)51.3% (SE 5.0%)0.34Grade II-IV aGvHD19.7% (SE 3.9%)15.1% (SE 3.4%)0.43Grade III-IV aGvHD9.9% (SE 3%)3.8% (SE 1.9%)0.09Any cGvHD by 2 yrs80.9% (SE 4.5%)68.4% (SE 5.1%)0.03Extensive cGvHD by 2 yrs67.1% (SE 5.0%)59.9% (SE 5.0%)0.15Neutrophil recovery, median d (range)17 (10-32)20 (12-38)<0.004Neutrophil engraftment by day +2894.5%85%0.0005Platelet recovery, median d (range)18 (5-91)21 (9-64)<0.0001Evaluation of donor immune populations showed that IFNg+ CD4 T cells and CD56bright NK cells were protective against cGvHD in G-PB and G-BM recipients. In contrast, specific B cell populations consistent with memory or exhausted B cells were associated with cGvHD in G-PB recipients (CD19+CD27+, p=0.05, CD19+ CD21lo, p=0.009) but not in G-BM. ConclusionsIn this large prospective randomized trial, the primary endpoint of time to treatment failure did not differ between G-PB and G-BM. OS and RFS were also not significantly different between arms. Neutrophil and platelet recovery were slower by a median of 3 days with G-BM. However, time to overall cGvHD was significantly delayed in G-BM recipients with non-significant trends of less acute and less extensive cGvHD with G-BM. cGvHD correlated with different cell populations in G-PB and G-BM suggesting that donor source can influence the mechanism of cGvHD. Further analyses are underway to determine the clinical impact of these differences. Disclosures:No relevant conflicts of interest to declare.

Purine Analogues Based Induction Regimen Followed By Allo-HSCT Is An Effective Treatment Modality Of Philadelphia Chromosome-Positive Acute Myeloid Leukemia-Retrospective Analysis Of Polish Adult Leukemia Group (PALG)

Abstract Background Philadelphia chromosome positive acute myeloid leukemia (AML Ph+) is a rare hematological malignancy with dismal prognosis. It comprises 1-2% of all cases with AML. Overall survival (OS) of AML Ph+ patients (pts) is around 6 months irrespective of the treatment modalities. The best treatment option has not been clearly determined. Material and methods We present a retrospective, multicenter analysis of AML Ph+ cases diagnosed in last 12 years in Poland. We searched our onsite registries for the pts diagnosed with AML without preceding history of chronic myeloproliferative neoplasm. All the pts had Ph chromosome present in the classic GTG cytogenetics performed at the time of diagnosis of AML. Patients with biphenotypic acute leukemias were excluded from the analysis. To our knowledge, this analysis covers the biggest group of pts with AML Ph+. Results During the 12 years period, 17 adult pts with de novo AML Ph+ were identified among 1221 newly diagnosed AML pts in 13 hematological departments cooperating in PALG clinical trials. The overall incidence with de novoAML Ph+ among all AML patients was 1,39%. Clinical characteristics The AML Ph+ group consisted of 11 men and 6 women with the median (Me) age of 36 years old (range 18-70 y). The median WBC count (25,3 G/L), peripheral blood basophiles percentage (0,5%) and Hgb level (9.2 g/dl) at diagnosis in AML Ph+ did not differ significantly compared to that reported for AML of all types (WBC 13,4 G/L; Baso 0,3%; Hgb 8,5 g/dlL respectively). However, the AML Ph+ group had a higher PLT count at diagnosis than AML Ph- patients (108 vs 43,6 G/L, p50 G/L p&lt;0.0001) and a higher leukemic infiltration of bone marrow (78% vs 58%; p&lt;0.02). Splenomegaly, hepatomegaly and lymphadenopathy as assessed by physical examination were similarly common in AML Ph+ and Ph−. The majority of AML Ph+ were diagnosed as M2 (47%) and M4 (29%) according to FAB classification. The M0 and M1 FAB subtypes consisted equally of 12%. In nearly all cases of AML Ph+ the blasts expressed CD13, CD33, CD34 and CD117. Bone marrow cytogenetic results at diagnosis were available for all 17 pts. The t(9;22) was the sole chromosome abnormality in 11 cases. Six AML Ph+ cases exhibited additional aberrations besides t(9;22) and 5 cases had at least 1 extra abnormal related clone. One variant t(9;14;22) was also observed occurring as the sole chromosomal abnormality. Treatment Response and Outcome: All but two AML Ph+ pts received standard induction chemotherapy according PALG AML protocols (DA-6 pts; DAC-8 pts and DAF-1 pt) (Holowiecki J; JCO 2012). One patient received FLAG-Ida induction regimen and one low-dose Ara-C. Five pts underwent allogeneic stem cell transplantation (allo-SCT). After first course of induction therapy 8/16 pts (50%) achieved the complete remission (CR) and one patient died early. The CR rate in pts who received more intensive first induction regimen with purine analogues (PNA) (DAC; n=8, DAF; n=1 or FLAG-IDA; n=1) was higher than in the standard DA group (70% vs 16,7% respectively; p=0.056). All 7 patients who did not respond to first induction course received second induction cycle (CLAG-M-6 pts, and DAC +imatinib (IM) − 1 patient). The overall CR rate was 75% and is the highest published in the literature. The only factors associated with higher probability of CR achievement after one course of induction were WBC &gt;50 G/L (p=0.032) and more intensive induction therapy with PNA (p=0.034) in univariate analysis. The median OS of all patients with AML Ph+ was 7,1 months (mo). Higher (&gt;100 G/L) PLT count, achievement of CR and allo-SCT were associated with longer OS in univariate analysis (p=0.037; p=0.045 p&lt;0.01 respectively). However, in multivariate analysis the only factors that influenced OS were CR achievement (HR 10,6; 95%CI: 1.18-86.33; p=0.034). Five out of 17 pts received off label IM at different points of treatment course. Surprisingly, IM had no significant impact on OS in our group. Conclusions AML Ph+ has an aggressive clinical course and short OS. More intensive induction regimens with purine analogs increase response to treatment and make CR rate comparable to AML Ph -. The CR achievement and allo-SCT play a pivotal role in long term survival of AML Ph+ patients. Disclosures: Off Label Use: Imatinib in Ph+AML.

DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs or DNA Hypomethylating Agents in MLL-Rearranged Leukemia Cells

EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L that is currently under clinical investigation as a potential therapy for acute leukemias bearing MLL-rearrangements. Gene knockout and small molecule inhibitor studies have demonstrated that DOT1L is required for MLL-fusion protein–mediated leukemogenesis in model systems. In preclinical studies EPZ-5676 promoted cell killing of acute leukemia lines bearing MLL translocations in vitro while sparing those without MLL gene translocations and also caused sustained tumor regressions in a rat xenograft model of MLL-rearranged leukemia [Daigle et al. Blood 2013]. To support potential future clinical scenarios, we evaluated the activity of EPZ-5676 in combination with current standard of care agents for acute leukemias as well as other chromatin modifying drugs in cell proliferation assays with three human acute leukemia cell lines; Molm-13 (MLL-AF9 expressing acute myeloid leukemia (AML)), MV4-11 (MLL-AF4 expressing acute biphenotypic leukemia cell line) and SKM-1 (non-MLL-rearranged AML). We established a high density combination platform suitable for testing the anti-proliferative activity of a complete titration matrix of two agents with multiple replicate points to enable generation of statistically meaningful results. This platform was used to evaluate the anti-proliferative effects of EPZ-5676 combinations tested in a co-treatment model in which the second agent was added along with EPZ-5676 at the beginning of the assay, or in a pre-treatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. The drug combination analysis was performed using the Chou-Talalay method [Chou TC Pharmacological Reviews 2006]. Graphs representing values of combination index (CI) versus Fractional effect (Fa) known as Fa-CI plots were generated and synergy was evaluated. Drug synergy was statistically defined by CI values less than 1, antagonism by CI >1 and additive effect by CI equal to 1.We found that EPZ-5676 acts synergistically with the AML standard of care agents cytarabine or daunorubicin in Molm-13 and MV4-11 MLL-rearranged cell lines. However, in the non-rearranged SKM-1 cell line EPZ-5676 had no effect alone and did not act synergistically with cytarabine or daunorubicin.Moreover, a persistent combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the standard of care agents (Figure 1), suggesting that EPZ-5676 sets up a durable altered chromatin state that enhances the effect of chemotherapeutic agents in MLL-rearranged cells. We are currently exploring the mechanism of action of this synergy in more detail. [Display omitted] Our evaluation of EPZ-5676 in conjunction with other chromatin modifying drugs also revealed a consistent combination benefit including synergy with DNA hypomethylating agents.In summary, our results indicate that EPZ-5676 is highly efficacious as a single agent and is synergistic with other anticancer agents including AML standard of care drugs and DNA hypomethylating agents in MLL-rearranged cells. Disclosures:Klaus:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Daigle:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Iwanowics:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Johnston:Epizyme, Inc: Employment, Equity Ownership, Stock Options Other. Therkelsen:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Smith:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Moyer:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Copeland:Epizyme Inc. : Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Mersana: Membership on an entity's Board of Directors or advisory committees. Olhava:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Porter Scott:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Raimondi:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other.

Intensification Of Treosulfan and Fludarabine-Based Conditioning With 4 Gy TBI For Allogeneic Stem Cell Transplantation In Patients With Hematological Malignancies

BackgroundHematopoietic stem cell transplantation (HSCT) is so far the only potentially curative option for patients with aggressive hematological malignancies. Since many patients cannot find a suitable HLA-identical (related or unrelated) donor, it is essential to analyze the safety of alternative graft sources, such as haploidentical donors. The use of reduced-intensity conditioning regimens in patients with an advanced disease did not improve the outcome because of a higher incidence of relapse. Here we present an interim analysis of this phase II prospective clinical trial that investigates a new conditioning regimen for allogeneic HSCT based on a well established one (Fludarabine and Treosulfan) intensified adding 4 Gy TBI with the aim of maintaining an acceptable toxicity profile while reducing the incidence of relapse in high-risk patients (TrRaMM4Gy study, Eudract 2011-001534-42). Patients and MethodsNinety-six patients underwent allogeneic HSCT for AML (n=46), ALL (n=13), acute biphenotypic leukaemia (n=2), HD (n=8), NHL (n=7), MDS (n=6), Myelofibrosis (n=5), MM (n=4), CML (n=2), CMML (n=2) or CLL (n=1). The median age was 45 years (range 17-67). At the time of the transplantation most patients (n=76) were in advanced disease phase, while the remaining 20 patients were in early phase. Twenty-eight patients were enrolled for relapse after a previous allogeneic HSCT. Median time from diagnosis to transplantation was 443 days (range 56-5249). Median comorbodity index score (according to Sorror criteria) was 2 (range: 0-8). Sixty-two patients received the graft from haploidentical donors, 17 from MUD, 14 from HLA-identical sibling and 3 patients from a single cord blood unit. Median number of CD34+ and CD3+ cells/kg were 6.46 millions and 241 millions respectively. The conditioning regimen included Treosulfan (14 g/m2 for 3 days), Fludarabine (30 mg/m2 for 5 days) and 4 Gy TBI split in 2 fractions. Patients receiving HSCT from haploidentical donor (arm A) were also treated with ATG-Fresenius (10 mg/kg for 3 days) and Rituximab (200 mg/m2 in single dose), patients receiving HSCT from HLA-identical sibling, MUD or single cord blood unit and in early disease status (arm B) were also treated with ATG-Fresenius (5 mg/kg for 3 days) and Rituximab (200 mg/m2 in single dose), while patients receiving HSCT from HLA-identical sibling, MUD or single cord blood unit and in advanced disease status (arm C) did not receive any ATG or Rituximab. GvHD prophylaxis consisted of Sirolimus (target concentration 8-15 ng/ml, till day +60) and Mycophenolate Mofetil (10 mg/kg tid till day +30). ResultsNeutrophils engraftment was reported at a median time of 17 days (range: 9-46) in all 91 evaluable patients; five patients died of transplant-related causes (n=4) or disease progression (n=1) before day +15. Platelets engraftment occurred after a median time of 17 days (range: 4-153) in 75 patients (82%). At the first marrow evaluation on day +30 all the evaluable patients showed full donor chimerism, 77 patients (90%) were in complete remission and 9 were in stable disease or progression. After a median follow-up of 425 days, cumulative incidence of grade 2-4 and 3-4 aGvHD were 40% and 26% respectively. Cumulative incidence of moderate or severe cGvHD (according to NIH criteria) was 57%. Cumulative incidence of NRM at day +100 and +365 were 23% and 36% respectively; cumulative incidence of relapse at day +365 was 33%. Overall survival and progression-free survival 1 year after HSCT were 51% and 43% respectively. EBV reactivation occurred in 7 patients, but no PTLD was observed. ConclusionsThe new conditioning regimen we investigated proved to be feasible in this high-risk population. We did not observe any major difference in terms of NRM and aGvHD compared to the data from our previous trial (TrRaMM, Eudract 2007-5477-54) with similar inclusion criteria and patient characteristics, but a reduced-intensity conditioning regimen based on Treosulfan and Fludarabine. Interestingly, we observed a trend of lower relapse incidence, resulting in a better OS and PFS. The low toxicity profile of Treosulfan and Fludarabine should be considered as a myeloablative platform for further intensification; more studies are warranted in order to find the association for a conditioning regimen endowed with a low toxicity profile but still a strong anti-tumor activity. Disclosures:Bonini:MolMed SpA: Consultancy.

Unmanipulated Graft Haploindentical Allogeneic Stem Cell Transplantation (Haplo-SCT) In Acute Leukemia: A Long-Term Follow-Up Survey On Behalf Of The Acute Leukemia Working Party (ALWP) Of The European Group For Blood and Marrow Transplantation (EBMT)

on behalf of the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). BackgroundThe use of an unmanipulated graft is increasingly adopted in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) from haploidentical family donors. MethodsThis survey aimed to assess the outcome of adult patients with de novo acute leukemia (AL) who were reported to the EBMT registry, and who received an unmanipulated peripheral blood (PB) and/or bone marrow (BM) graft from a haploidentical family donor between 2007 and 2011. 106 patients with consolidated follow-up and fully audited data were selected for the purpose of this analysis. ResultsThe median folllow-up was 36.2 months (range, 1.3-66.1). 74 pts received a haplo-SCT for acute myeloid leukemia (AML, 70%), 31 pts for acute lymphoblastic leukemia (ALL, 28%) and 1 pt for acute biphenotypic leukemia (1%). The haplo-SCT was the first allogeneic transplant for all the patients. However, 17 pts had previously received an autologous SCT (16%). 26 pts were transplanted in CR1 (24%), 27 pts in CR2 or higher CR (26%) and 53 pts in more advanced disease (50%). The number of HLA mismatches was 2 in 16 pts (15%), 3 in 14 pts (13%), 4 in 27 pts (26%) and 5 in 44 pts (46%). Median age at time of haplo-SCT was 46 years (range, 18-66). 81 pts out of 106 received a reduced-intensity conditioning regimen (RIC, 76%). 92 pts received anti-thymocyte globulins (87%) as part of the conditioning regimen and 5 pts received alemtuzumab (4.7%). Stem cell source was bone marrow (BM) for 37 pts (35%) and peripheral blood (PB) for 69 pts (65%). Median dose of infused CD34+ cells was 2x10e6/kg (range, 0.85-5.3) for BM and 8x10e6/kg (1.72-13,19) for PB. Donor median age was 38 years (range, 19-71). 26 male pts received a graft from a female donor (24%). CMV donor/host serostatus was as follow: neg/neg for 13 pts (12%), pos/neg for 9 pts (9%), neg/pos for 25 pts (24%) and pos/pos for 57 pts (55%), missing for 2 pts. Finally, the Karnofsky Performance Status (KPS) at time of haplo-SCT was >80% for 74 pts (71%), < or = 80% for 30 pts (29%) and missing for 2 pts. 98 out of 106 pts engrafted (96.1%), with a median time for reaching ANC >0.5x10e9/L of 17.5 days (range, 11-63). The cumulative incidence of grade II or higher acute GvHD was 30±4%. The 2-years incidence of chronic GvHD was 39±5% (n=41: 23 limited, 18 extensive). At 3 years, the estimates of leukemia-free survival (LFS) for pts transplanted in CR1, CR2 or in advanced disease were 54±10%, 37±9%, 12±5%, respectively. The estimates of overall survival (OS) were 67±10%, 48±10% and 11±5%, respectively. The cumulative incidences of relapse (RI) were 37±10%, 26±9% and 59±7%, respectively. Non-relapse mortality (NRM) incidences were 8±6%, 37±9% and 29±6%.In a multivariate analysis including all relevant factors for LFS, disease status at transplant was found to be significant (HR=2.89; 95%CI:1.73-4.83; p=0.0001) in conjunction with the number of HLA mismatches >3 (HR=1.69; 95%CI: 1-2.86; p=0.05). Disease status and number of HLA mismatches (>3) were also significant risk factors for OS: (HR=3.79; 95%CI:2.24-6.39; p<10-3) and (HR=1.87; 95%CI: 1.06-3.30; p=0.03,), respectively. For RI, 2 factors were identified: disease status (HR=3.94; 95%CI: 2.10-7.39; p<10-3) and patient age (≥35y) (HR=2.57; 95%CI: 1.13-5.85; p=0.02). Finally, the number of HLA mismatches (>3) was the only significant parameter associated with NRM (HR=3.06; 95%CI: 1.05-8.93; p=0.04). ConclusionThese data suggest that unmanipulated graft haplo-SCT is a valid treatment option in acute leukemia. We are currently completing this EBMT survey to include all patients reported to the registry (> 350 patients). The current LFS and NRM rates support the use of such transplant approach as part of the treatment algorithms for adult acute leukemia patients with an indication to allogeneic SCT but lacking an HLA-identical sibling or matched-unrelated donor. Disclosures:No relevant conflicts of interest to declare.

Human NUP98/Iqcg Fusion Gene Induced An Acute Myelomonocytic Leukemia In Mice

NUP98 gene encoding a nucleoporin located at 11p15 has been reported to be fused with about 30 different partner genes by chromosomal translocations in hematological malignances. NUP98/IQCG was cloned in a patient of Myeloid/ T-lymphoid bi-phenotypic leukemia with 47, XX, t(3;11)(q29q13;p15)der(3)(q29), +21 karyotype. However, NUP98/IQCG's ability for leukemogenesis has not been identified yet.In this study, we established a retrovirus-mediated murine bone marrow transduction and transplantation (BMT) model of NUP98/IQCG to investigate its oncogenicity. In our model, half of the NUP98/IQCG mice developed a penetrable and transplantable acute myelomonocytic leukemia ,which was similar to the phenotype of patient with t(3;11). It suggested that NUP98/IQCG could induce the disease development.To investigate how the fusion gene promoted leukemogenesis, we transduced NUP98/IQCG into primary bone marrow cells, and found that NUP98/IQCG-expressing cells retained the ability to generate colonies in serial replating, while seldom control cells did, which indicated the increasing self-renewal ability caused by NUP98/IQCG. Meanwhile, when induced by Macrophage-Colony Stimulating Factor, NUP98/IQCG -expression bone marrow cells showed enhanced proliferation in vitro. Further molecular mechanism studies revealed that NUP98/IQCG could be involved in both NFκB and CREB pathways during leukemia development.In summary, we showed that NUP98/IQCG promoted leukemogenesis in BMT mouse model through increasing the bone marrow cells' proliferation and self-renewal capacity, which explained the fusion gene's oncogenicity in patient with t(3;11). Our mouse model will be a powerful tool both to investigate the leukemogenic mechanism of NUP98-related fusion gene, and to find the drugs for treating the disease. Disclosures:No relevant conflicts of interest to declare.

Phase II Prospective Multicentre Study Testing The Efficacy and Safety Of a Clofarabine (Clo), I.v. Busulfan (Bu) and Antithymocyte Globulins (ATG)-Based Reduced-Intensity Conditioning Regimen (RIC) Before Allogeneic Stem Cell Transplantation (allo-SCT) For High-Risk Myelodysplastic Syndrome Or Acute Leukemia: The Cloric Trial

IntroductionClofarabine (Clo) is a purine analogue which was developed to overcome limitations and to incorporate the favorable pharmakokinetic properties of fludarabine and cladribine. This agent has also a significant antileukemic activity. Thus, can one exploit both the antileukemic and immunosuppressive effects of Clo for further improving outcome after RIC allo-SCT for patients with high-risk myelodysplastic syndrome (MDS) or acute leukemia. Here we report the results of a prospective multicentre trial testing the use of Clo in replacement of fludarabine in combination with i.v. Busulfan (Bu) and ATG in 30 patients with high-risk MDS/acute leukemia (clinicaltrials no. NCT00863148). Patients and MethodsThirty patients (male n=18, female n=12) from 6 centres were included in this study between October 2009 and August 2012. Sixteen patients were diagnosed with high-risk MDS (n=5) or acute myeloid leukemia (AML, n=11), while 13 patients had high-risk acute lymphoblastic leukemia (ALL, Ph+ n= 2, Ph- n=11) and 1 patient a biphenotypic leukemia. All patients were in first (AML/MDS, n= 10; ALL/biphenotypic n=10) or second (AML n= 3; ALL n= 4) complete remission, or in stable disease (MDS n=3) at time of transplant. Median age at transplant was 58.8 years (range: 20.5-64.5). The median interval between diagnosis and transplant was 6 months (range: 3.8-124). Karnofsky's performans status at transplant was: 100% (n=19); 90% (n=6); and 80% (n=4).The RIC regimen consisted of: i.v. Clo 30 mg/m²/day for 4 days (day-8 to day-5), i.v. Bu 3.2 mg/Kg/day for 2 days (day-4 and day-3) and ATG (Thymoglobuline) 2.5 mg/kg/day for 2 days (day -2 and day-1). All patients received G-CSF-mobilized PBSCs and cyclosporine alone for GVHD prophylaxis, irrespective of the type of donor (sibling donors n=14; 10/10 MUD, n=16). For the purpose of this study, the single case of binephotypic leukemia was considered as ALL for comparison between AML/MDS and ALL patients. The primary endpoint of the trial was the assessment of leukemia-free survival (LFS) at one year after allo-SCT. ResultsEngraftment was observed in all patients (100%). Median time for neutrophils (>500 /µL) and platelets (>50.000/µL) recovery was 18 (range: 14-26) and 12 (range: 0-23) days, respectively. With a median follow-up of 23 months (range: 12-37), the 1-year and 2-year overall survival (OS), leukemia-free survivals (LFS), relapse incidence (RI) and non relapse mortality (NRM) rates were 63±9% and 58+-10%, 57±9% and 53+-9%, 40±9% and 44+-9%, and 3.3±3% and 3.3±3%, respectively. Thirteen patients relapsed (43%) at a median time of 3.5 months (range: 2.3-13.1) after allo-SCT. Overall, 13 patients died, with the cause of death being relapse in 11 and GVHD in 2 (including one after donor lymphocyte injection).1-year and 2-year OS were significantly higher for AML/MDS patients compared to ALL patients (75±10% vs 50±13%, and 75±10% vs 37±14%, p=0.04). There were trends for higher 1-year and 2-year LFS (69±12% vs 43±13%, and 69±12% vs 34±13%, p=0.08) and lower RI (57±14% vs 25±11%, and 66±14% vs 25±11%, p=0.05) for AML/MDS patients compared to ALL patients. Finally, 1-year and 2-year NRM were similar between both groups (AML/MDS: 6±6% vs 0%, p=0.36). ConclusionThis phase 2 prospective multicentre trial shows that a Clo-i.v. Bu-ATG RIC regimen prior to allo-SCT in high-risk MDS/leukemia is feasible allowing for full engraftment and very low toxicity. Disease control appears to be satisfactorily, especially in AML/MDS, warranting a prospective comparison with other widely used fludarabine-based RIC regimens (e.g. Fludarabine, i.v. Bu, ATG versus Clo, i.v. Bu, ATG).Genzyme/Sanofi provided clofarabine and financial support for the study. Disclosures:No relevant conflicts of interest to declare.

Different Approaches Of Allogeneic Stem Cell Transplantation For Acute Leukemias In Suzhou (China) Lead To Similar Outcome Compared With European Group For Blood and Marrow Transplantation (EBMT) Results: A Pair-Matched Analysis

The first affiliated hospital of Soochow University initiated a program of stem cell transplantation for hematological malignancies in 2001 and has done until December 2011 a total of 460 transplants. The EBMT presently handles a registry with information on more than 450 000 transplants including over 100 000 transplants for Acute Leukemias. In order to evaluate the outcome of patients transplanted in Suzhou and to compare it with results from the EBMT, we collected the necessary information on all patients transplanted in Suzhou and we did a pair matched analysis.In Suzhou, the patient population studied consisted of 382 first allografts (322 adults and 60 children less than 18 years old).There were 223 Acute Myelocytic Leukemias (AML), 148 Acute Lymphoblastic Leukemias (ALL) and 11 biphenotypic leukemias. The median age of the patients was 32 years (4-63). By cytogenetics, 9% of the patients were good risk, 57% intermediate risk and 34% poor risk. Molecular biology was not available. 286 patients were transplanted in first remission (CR1), 63 in CR2 and 30 in more advanced disease. Donors were identical siblings in 57%, unrelated in 30%, mismatched relatives in 13%. The source of stem cells was bone marrow (BM) (harvested after stimulation of the donor by GCSF) in 40%, Peripheral Blood (PB) in 36%, BM + PB in 17% , Cord Blood in 4% and other in 3%. 97% of the patients received myeloablative conditioning (MAC) with no Total Body Irradiation (TBI) in 76%, and 3% reduced intensity conditioning (RIC). 98% of the patients engrafted. Acute Graft versus Host Disease (aGVH) grade >=2 occurred in 43%. The cumulative incidence of chronic GVHD at 2 years was 26±2%. With a median follow up of 21 months (1-143), 2 year overall survival (OS), Leukemia free survival (LFS), Relapse Incidence (RI) and non Relapse Mortality (NRM) in patients allografted in CR1 were 76± 3, 65± 3, 18± 2 and 17 ± 2% and in CR2 52± 7, 36± 7, 37± 7 and 28 ± 6%. TBI in MAC was associated with poorer results (p< 0.01 for OS,LFS,RI). By multivariate analyses, The NRM was lower with PB (p=0.04) and PB+BM (p= 0.01) compared to BM and the OS higher ( p=0.05 and 0.01 respectively). In a pair matched analysis 246 adult patients from Suzhou transplanted in CR with BM and/or PB were matched with 484 patients from the ALWP EBMT registry. Matching factors were age, diagnosis (AML and ALL), disease status (CR1 or CR2), Cytogenetics and donor origin (identical siblings, unrelated, mismatched relatives). Patients from Suzhou were transplanted more recently ; the interval from diagnosis to transplant was shorter (138 days vs 154 days, p<0.0001). TBI was less frequently used for MAC both for AML and ALL (p< 0.0001 for each) and PB + BM was used in 17% of the cases versus 0% for EBMT (p < 0.0001). By univariate analyses the results were: Suzhou versus EBMT: OS 72 ± 3 vs 61 ±2 % (p= 0.05), LFS 62 ± 3 vs 55 ± 2% (p= 0.17), RI 19 ± 3 vs 22 ± 2% (p= 0.25), NRM 20 ± 3 vs 22 ± 2% (p = 0.67), incidence of chronic GVH 30± 3 vs 48 ± 2% (p< 0.0001).By multivariate analyses, there was no significant difference for OS (HR: 0.9 range 0.59-1.39), LFS (HR: 0.93, range 0.63-1.38), RI ( HR: 0.72, range 0.41-1.28) and NRM (HR: 1.21, range 0.70-2.10). [Display omitted] We conclude that the results from Suzhou are identical to those obtained using the EBMT database, even though some approaches (GCSF stimulated BM, BM+PB, conditioning regimen) were different. Disclosures:No relevant conflicts of interest to declare.

Phase II Trial Of Medium-Dose VP-16, Cyclophosphamide and Total-Body Irradiation Conditioning Regimen Before Allogeneic Stem Cell Transplantation For Acute Lymphoblastic Leukemia In Adults

BackgroundThe prognosis of adult patients (pts) with acute lymphoblastic leukemia (ALL) is dismal, and allogeneic stem cell transplantation (allo-SCT) is performed in most pts. However, even for pts treated with allo-SCT using the standard regimen of cyclophosphamide with total body irradiation (CY/TBI), the prognosis is not satisfactory due to a high rate of relapse. We previously reported excellent outcomes in adult pts with ALL undergoing allo-SCT conditioned with medium-dose VP-16, CY and TBI (medium-dose VP/CY/TBI). We therefore conducted a prospective nationwide multicenter phase II trial (UMIN trial number 000001672) to evaluate the efficacy of this conditioning regimen. MethodsThe eligibility criteria of this study were as follows: (1) diagnosis of ALL or acute biphenotypic leukemia (ABL), (2) aged 15-49 years, (3) in CR, (4) first SCT, (5) PS (ECOG) 0-2, (6) intact organ function, and (7) HLA-serologically 6/6 matched donor. Pts with Burkitt leukemia were ineligible for this study. Conditioning regimen consisted of medium-dose VP (15 mg/kg once daily i.v. for two days) and CY (60 mg/kg once daily i.v. for two days) combined with fractionated TBI (total dose: 12 Gy). Stem cell source was limited to bone marrow (BM) or peripheral blood stem cell (PBSC). The primary endpoint of this study was event-free survival (EFS) at one year after SCT, and the events were defined as death or relapse. The expected 1-year EFS was estimated to be 75%, and the threshold 1-year EFS was estimated to be 55%, on the basis of our previous observations. ResultsBetween February 2009 and August 2011, 52 pts were enrolled, and 50 pts met the criteria. Among 50 eligible pts, the median age was 33.5 years (range, 17-49 years), and 15 pts (30%) were over 40. Twenty-three (46%) pts were female. Forty-eight (96%) pts had ALL and 2 (4%) had ABL. Nineteen (38%) pts were Philadelphia chromosome (Ph)-positive. Forty-seven (94%) pts were in first CR at SCT, and 3 (6%) in second CR. Among pts with ALL in first CR (n=45), 38 (84%) had high-risk disease. Twenty-six (52%) pts underwent SCT from a related donor and 24 (48%) from an unrelated donor. Forty (80%) pts received BM and 10 (20%) received PBSC. All pts achieved neutrophil engraftment. The incidence of grade II-IV and III-IV acute graft-versus-host disease (GVHD) was 38% and 8%, respectively, and that of chronic GVHD was 54%. After 1-year follow-up of the final enrollment (range, 379-1218 days), the 1-year EFS was 76% (95% confidence interval (CI): 62-86%, Figure). One-year overall survival was 80% (95% CI: 66-89%). No pts died within 100 days after SCT and 1-year non-relapse mortality (NRM) was 14% (95% CI: 6-25%). No secondary malignancies were observed during the-follow-up period. The 100-day and 1-year relapse rate was 2% (95% CI: 0-9%) and 10% (95% CI: 4-20%), respectively. The 1-year EFS for pts with high-risk and standard-risk disease was 76% (95% CI: 59-87%) and 71% (95% CI: 26-92%), Ph-positive and negative pts was 79% (95% CI: 53-92%) and 76% (95% CI: 56-88%), and pts over 40 years and under 40 years was 73% (95% CI: 44-89%) and 77% (95% CI: 59-88%), respectively. In univariate analysis, high-risk disease, Ph-positivity and higher age were not significant risk factors for EFS. ConclusionsThe results demonstrate that the conditioning regimen of medium dose VP/CY/TBI for allo-HCT in adult pts with ALL enable good disease control without increase in NRM, even for relatively high age pts and pts with high-risk disease. A phase III trial comparing this regimen with standard CY/TBI regimen for adult pts with ALL is warranted. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

Phase II trial of clofarabine with topotecan, vinorelbine, and thiotepa in pediatric patients with relapsed or refractory acute leukemia

Outcomes for children with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) are dismal. In an effort to improve outcomes, we performed a phase I/II study of a novel clofarabine based combination regimen called TVTC. Herein, we report the response rates of patients in the phase II portion of the study. Seventeen patients with R/R ALL, AML, or biphenotypic leukemia were enrolled. Sixteen patients were evaluable for response. Patients were treated at the maximum tolerated dose (MTD) from the phase I portion of the study (clofarabine 40 mg/m(2) /day IV × 5 days, topotecan 1 mg/m(2) /day IV continuous infusion × 5 days, vinorelbine 20 mg/m(2) /week IV × 3 weeks, thiotepa 15 mg/m(2)/day IV × 1 day). The primary endpoint was overall response rate (ORR), defined as CR or CR without platelet recovery (CRp). The ORR was 69% (10 CR, 1 CRp). Among the 11 responders, 9 (82%) proceeded to hematopoietic stem cell transplantation. The most common grade 3+ non-hematologic toxicities were febrile neutropenia (82%) and transient transaminase elevation (47%). TVTC demonstrates significant activity in patients with R/R acute leukemia. The activity in R/R AML patients was very encouraging, with 8 of 12 (67%) patients achieving a CR/CRp. Patients with high risk de novo AML may benefit from incorporation of TVTC therapy into frontline treatment regimens. This regimen warrants further exploration in a larger cohort of patients with R/R leukemia.

MicroRNA-126–mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

Lineage Classification of Childhood Acute Lymphoblastic Leukemia according to the EGIL Recommendations: Results of the ALL-BFM 2000 Trial

Flow cytometry immunophenotyping (FCM) is an undispensable tool for the diagnosis and for the treatment stratification of childhood acute lymphoblastic leukemia. The correlation of the EGIL-classification with prognostically relevant parameters like age, prednisone response and risk group is analyzed. Between March 2000 and June 2009 12 patients less than 1 year of age, 1 836 patients with 1 to less than 6 years, 620 patients with 6 to less than 10 years, 615 patients with 10 to less than 15 years and 275 patients with 15 to less than 19 years were analyzed with a comprehensive 4-color antibody panel and classified according to the EGIL recommendations. Bone marrow or peripheral blood mononuclear cells were isolated by ficoll gradient centrifugation, washed and stained with fluorochrome-conjugated antigen-specific monoclonal antibodies. Cell preparations were acquired and analyzed on a flow cytometer. Centralized FCM was performed for 2 775 patients (82.6%) with B-cell precursor acute lymphoblastic leukemia, 493 patients (14.7%) with T-cell acute lymphoblastic leukemia and 90 patients (2,7%) with biphenotypic acute leukemia. There was a slight overall predominance of male (56.1%) over female (43.9%) patients. Patients with B-cell precursor ALL had a slightly more favourable outcome with a 10 y pEFS of 78 ± 1.0%, compared to patients with a T-ALL or BAL (biphenotypic acute leukemia) phenotype with a 10 y pEFS of 74 ± 1.8% (n.s.) or 69 ± 9.0% (p<0.009), respectively. FCM according to the EGIL recommendations not only provides diagnostic lineage determination and subclassification but also enables an initial prognostic orientation before MRD (minimal residual disease)-based risk stratification becomes available.