High Blast Counts Research Articles

Decitabine (DAC) in Combination with Donor Lymphocyte Infusions (DLIs) Can Induce Remissions of Overt Aml Relapses after Allogeneic Transplantation

Background: Treatment with the DNA hypomethylating agents (HMAs) 5-azacytidine (AZA) or DAC has become a well-established option for elderly MDS and AML patients (pts). Regarding their mechanism of action, HMAs not only reactivate tumor suppressor genes, but also lead to an immune activation response, by expression of Cancer/testis antigens (Almstedt et al., Leuk. Res. 2010; Goodyear et al., Blood 2010) and endogenous retroviruses (Chiappinelli et al., Cell 2015; Roulois et al., Cell 2015). Hypothesizing that HMA-mediated immune activation may sensitize blasts to subsequent DLIs, we and others showed that AZA combined with DLIs can induce long-term remissions in pts with relapsed MDS/AML after allogeneic transplantation, particularly with a low leukemic burden, and after failure to respond to DLIs alone (Steinmann et al., BMT 2015, Schroeder et al., BBMT 2015). As DAC is considered useful also in pts with higher WBC and higher blast counts, we now treated 18 pts (most of them with more aggressive hematologic relapse) with DAC+DLIs.Patients and Methods: Between 10/06 and 03/16, 18 pts (median age 63 years, range 29-76, 10 males) with hematological relapse after allogeneic transplantation were treated with DAC and (where feasible) DLIs. Thirteen pts had AML (genetic risk by ELN: 3x int-1, 4x int-2, 6x adverse (4 MK+), 3 had MDS, 1 had polycythemia vera, 1 had atypical CML. Sixteen and 2 pts had received reduced-toxicity conditioning (mostly FBM) and myeloablative conditioning, respectively. Donors: matched unrelated in 8 pts, matched sibling in 4, mismatched in 6. DLIs at relapse after 1st/2nd transplant in 15/3 pts (1st/2nd/3rd/4th relapse in 11/5/1/1 pts). Median duration from transplant to hematologic relapse was 306 days (range, 56-4943). Median % blasts before DAC was 4% in peripheral blood (pb, range, 0-40%), 30 % in bone marrow (range, 7-60%), the median WBC was 4,770/µl (range, 430-30,800). DAC (20 mg/m2 per day) was administered either as a standard 5-day (15 pts) or a 10-day course (3 pts). DLIs were to be given 7 days after the last HMA dose. Prior AZA as relapse treatment was allowed.Results: Of 18 pts with myeloid neoplasias (mostly AML) with hematologic relapse after allografting, 15 received DAC+DLIs (for 8 of them after previous DLI-only, for 3 after previous AZA+DLI), 3 pts only received DAC (median number of DAC cycles: 2, range 1-8, median number of DLIs: 2, range 1-10). Two AML pts (aged 64 and 76 yr, no previous AZA, 7 and 4% pb blasts, respectively) attained a complete hematologic and molecular remission after 5-day DAC+DLI, with a duration of 250+ and 391 days, respectively. One of them is still in CR with no signs of GvHD, the other died in CR because of liver GvHD and hemosiderosis. A third AML pt attained complete donor chimerism following DAC+DLI but died at day 35 because of a fungal pneumonia (concurrent Sorafenib). Eleven additional pts achieved a temporary disease control (median duration 93 days, range 34-212), 5 of them were subsequently re-transplanted. Four pts showed no response to DAC+DLI, 3 developed GvHD. Seven pts died of PD, 6 pts of infections, 1 pt of intracranial hemorrhage, 1 pt of GvHD.Conclusions: DAC in combination with DLIs showed good feasibility in pts with relapsed AML/MDS after allogeneic transplantation. Despite overall higher median WBC and blast expansion than a large previous cohort treated with AZA+DLI, clinical activity was comparable, and in 2/18 pts (11%) a complete hematologic and molecular remission was achieved (despite the presence of peripheral blood blasts). This approach is investigated prospectively within the RELDAC substudy of the EORTC trial AML21 of older AML patients (inDACtion vs. induction, NCT02172872). DisclosuresClaus:Janssen-Cilag: Consultancy, Honoraria, Other: Travel Funding; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Marks:Pfizer: Honoraria. Lübbert:Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding.

Targeting MDS and AML Stem Cells with AZD-9150 Mediated Inhibition of STAT3

Myelodysplastic syndrome (MDS) & Acute Myeloid Leukemia (AML) arise from a population of aberrant hematopoietic stem cells (HSCs) that have been altered by multiple genetic & epigenetic alterations. A preliminary genomewide transcriptomic and epigenomic analysis of stem and progenitor cells from MDS and AML patients revealed STAT3 upregulation with corresponding loss of promoter methylation in diseased samples (Will et al, Blood, 2012). We now demonstrate that STAT3 is overexpressed in highly purified FACS sorted disease initiating populations in MDS/AML. STAT3 expression was determined to be significantly higher in phenotypic LT-HSC (CD34+/CD38-/CD90+/Lin -ve), ST-HSC (CD34+/CD38-/CD90-/Lin -ve) and GMP (CD34+/CD38+/CD123+/CD45Ra+/Lin -ve) from 10 diseased and 6 control sorted samples. Transcriptomic data from another cohort of CD34+ cells from 183 MDS patients found significantly increased expression of STAT3 in MDS samples when compared to healthy controls and importantly, found that increased STAT3 expression was predictive of significantly adverse prognosis (P value < 0.01, median survival of 2.6 years compared to 5.8 years for group with lower STAT3). Clinical correlations revealed that MDS patients with higher STAT3 expression in stem/progenitor cells were significantly more anemic (Mean Hgb 9.4gm/dl vs 10.2gm/dl, P=0.002) and had higher blast counts (Mean Blast Count 7.1% vs 5.3%, P=0.02), further demonstrating STAT3 to be an adverse prognostic marker in MDS.To functionally determine the role of STAT3 in MDS/AML pathogenesis, we used AZD9150, a Gen 2.5 Antisense Oligonucleotide (ASO) specific inhibitor of STAT3 (Hong et al, SciTM, 2015). This ASO has recently demonstrated safety and single-agent antitumor activity in patients with refractory lymphoma in a phase 1 dose-escalation study. We evaluated its efficacy in multiple leukemic cell lines and primary MDS/AML samples and used an inactive structural analogue oligonucleotide as control. AZD9150 treatment led to significantly decreased viability in numerous AML and MDS derived cell lines. (N=7, P < 0.01). Loss of viability was accompanied by dose dependent increase in apoptosis in leukemic cells. Specific inhibition of STAT3, but not STAT5, was seen in AZD9150 treated leukemic cells in luciferase reporter assays. AZD9150 treatment also led to decreased expression of important oncogenic downstream genes including not only STAT3 itself, but also IL8, IL6, CXCR2 and others. In vivo studies using a leukemia xenograft model in NSG mice demonstrated that treatment with AZD9150 at 50mg/kg/day led to significantly improved survival compared to control oligonucleotide (p-value: 0.004).It is generally challenging to show uptake of oligonucleotide-based therapeutics by primary cancer samples. Here, we treated healthy stem cells and AML primary patient samples with AZD 9150 and determined by intracellular flow cytometry that the ASO were readily taken up by primary patient- or cord blood-derived HSCs, myeloid progenitors and lymphocytes in a time and dose-dependent manner. We then treated a cohort of MDS and AML primary samples (N= 7) with AZD9150 or control and found qualitatively decreased leukemic colony growth, enhanced erythroid colony formation, and increased myeloid differentiation following STAT3 inhibition.In conclusion, we demonstrate that STAT3 is upregulated in highly purified stem and progenitor cells in MDS and AML and is an adverse prognostic marker. Importantly, a clinically applicable oligonucleotide inhibitor of STAT3 shows preclinical in vitro and in vivo efficacy in MDS and AML models, thereby providing a rationale for further development and clinical testing in MDS and AML.Supported by LLS DisclosuresNo relevant conflicts of interest to declare.

Defined Central Memory and Stem Memory T Cell Phenotype of CD4 and CD8 CAR T Cells for the Treatment of CD19+ Acute Lymphoblastic Leukemia in an Automated Closed System

Relapsed and refractory B-precursor acute lymphoblastic leukemia (B-ALL) remains a major therapeutic problem. Chimeric antigen receptor (CAR) modified T cells targeting CD19 are promising treatment options for these patients with the potential to induce hematological remission in adult and pediatric patients with refractory B-ALL. Despite the promising data, some patients do not respond to T-cell treatment. Until now it is not possible to fully understand and predict critical factors for response or non-response, but proliferation and persistence of CAR T cells in vivo is an essential precondition for treatment efficacy. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to be the best candidates for a sustained in vivo expansion after T-cell therapy with small cell doses. Therefore, we set up a protocol for the generation of anti-CD19 CAR T cells in a closed system that is compliant to current GMP regulations. Starting samples were mononuclear cells from pediatric ALL patients at diagnosis and under chemotherapy using up to 100cc peripheral blood. After separation for CD4+/CD8+ cells, T cells were activated with anti-CD3/CD28 beads. The lentiviral vector encoded the anti-CD19 single-chain variable fragment, 4-1BB (CD137) co-stimulation and T cell receptor (TCR) zeta chain. The whole process including separation of T cells, activation, transduction and cultivation was performed in a closed and fully automated system. Despite a broad variety in cellular composition including high blast counts, low cell numbers and a rather exhausted phenotype in the starting fraction, a robust T-cell composition was achieved at day five after activation with a mean of 63% CD4+ and 37% CD8+ T cells and a transduction rate of up to 38 %. The vast majority of CAR T cells were of a Tcm (47%) and Tscm (44%) phenotype leading to a strong proliferative potential of more than 100-fold expansion. In addition, a reduced sensitivity to inhibitory signals was documented (programmed cell death protein 1 (PD-1) expression ≤10%). CAR T cells showed effective cytotoxic functionality when co-cultured with CD19+ target cells with only little background of the un-transduced control. At an effector to target ratio of 5:1 up to 80 % of the CD19+ target cells were killed. In addition, a significant release of Interferon gamma (IFN-γ), Tumor necrosis factor (TNF-α) and Interleukin-2 (IL-2) was detected upon recognition of the target cell lines, confirming a strong and target-specific Th1 response. In conclusion, generation of CAR T cells from small pediatric blood samples was feasible in a closed GMP-compatible fully automated system. Despite variety of cell numbers, cellular composition and T cell phenotype in the starting sample, a uniform T cell product of Tcm and Tscm could be produced with a balanced CD4 / CD8 ratio leading to high expansion potential and functionality of the T cell graft. DisclosuresBlaeschke:Miltenyi Biotec GmbH: Other: Miltenyi Biotec GmbH provided reagents free of charge.. Kaiser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment.

Short telomere length and its correlation with gene mutations in myelodysplastic syndrome

BackgroundTelomere erosion can lead to genomic instability and cancer progression. It has been suggested that the shortest telomere, not the average telomere length (TL), is critical for cell viability. Some studies have shown shorter TL in myelodysplastic syndrome (MDS) patients but the critically short telomeres, the variability of TL within individual patient has not been evaluated. Thus, we aimed to investigate the TL of MDS patients and assessed the association of TL with recurrent genetic mutations in MDS.MethodsWe measured the TL of bone marrow nucleated cells for diagnostic samples at a single-cell level by quantitative fluorescence in situ hybridization (Q-FISH) for 58 MDS patients and analyzed the minimum, median, average, standard deviation, average of the 0th to 10th percentile TL within a patient, and the proportion of cells with TL that is shorter than the lowest 10th percentile of the normal control (NC). The correlations of TL to clinical parameters, cytogenetic results, and genetic mutations were assessed.ResultsMDS patients showed eroded telomeres and narrow distribution compared to the NC (P < 0.001, P = 0.018, respectively). Patients with mutation showed significantly lesser cells with short TL, below the lowest 10th percentile of the NC (P = 0.017), but no differences in TL were found according to mutations/cytogenetic abnormalities except for CSF3R mutation. However, those patients with a high percentage (≥80 %) of cells with short TL showed poorer overall survival (P = 0.021), and this was an independent prognostic factor, along with TP53, U2AF1 mutation, and high BM blast count (P = 0.044, 0.001, 0.004, 0.012, respectively).ConclusionsThe shortest TL, which determines the fate of the cell, was significantly shorter, and higher burden of cells with short TL were found in MDS, which correlated with poor survival, suggesting the need to measure TL in single cells by Q-FISH.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0287-9) contains supplementary material, which is available to authorized users.

Targeted positron emission tomography imaging of CXCR4 expression in patients with acute myeloid leukemia.

Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.

Reduced PLCG1 expression Is Associated with Inferior Survival in Myelodysplastic Syndromes

The PLCG1 gene encodes phospholipase C g isoform, which is involved in diverse physiological and pathological cellular processes through catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerole. Until now, roles of phospholipase C g 1 in hematopoiesis have been shown in animal models. Plcg1 deficient mouse lacks erythropoiesis and die in early embryonic stage. In zebrafish model, it was demonstrated that phospholipase C g 1 is required in granulocyte maturation. Physiological and/or pathological role of phospholipase C g 1 in human hematopoiesis has not been elucidated. In human, the PLCG1 gene is located on long arm of chromosome 20. Deletion of long arm of chromosome 20 (del(20q)) is commonly observed in myelodysplastic syndromes (MDS). Previously, we determined the common deleted region (CDR) of del(20q) in MDS, and the PLCG1 gene is located within the CDR. Reduced expression of genes located in the CDR due to haploinsufficiency may play role in molecular pathogenesis of MDS. Therefore, we analyzed PLCG1 expression in bone marrow mononuclear cells in MDS patients with or without del(20q), and investigated its clinical significance in the present study.Mononuclear cells separated from bone marrow samples taken at the time of diagnosis with written informed consent from patients were used. To analyze PLCG1 expression, quantitative RT-PCR was performed. Total RNA was extracted from mononuclear cells and subjected to cDNA synthesis. Real-time RT-PCR was carried out using cDNA as template by the TaqMan probe method (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems) were performed. The human PLCG1 primer-probe set was from Applied Biosystems. A total of 109 MDS patients, 65 males and 44 females with median age of 69 years (range: 22-91 years), with (n=20) or without (n=89) del(20q), were included in the present study. They were classified as RCUD (n=11), RCMD (n=55), RARS (n=11), RAEB-1 (n=16), and RAEB-2 (n=16) according to WHO classification. They were categorized in four IPSS risk groups, low risk (n=21), intermediate-1 risk (n=52), intermediate-2 risk (n=20), and high risk (n=6).Relative PLCG1 expression level was significantly reduced in MDS patients with del(20q) compared to control subjects (n=20) (P=0.011). Median values of relative PLCG1 expression level in MDS patients with del(20q) and control subjects were 0.94 and 1.96. In addition, relative expression level of PLCG1 in whole MDS cohort significantly lower than that in control subjects (P=0.046). Expression patterns of PLCG1 among, were not different. Median values of relative PLCG1 expression level in five WHO-subtypes, RCUD, RCMD, RARS, RAEB-1, and RAEB-2 were 1.50, 1.55, 1.29, 1.13, and 1.12, respectively, but no statistically difference was observed. WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) showed trend in association with reduced PLCG1 expression compared with those with low blast counts (RCUD, RCMD, and RARS) (median value: 1.01 vs. 1.54, P =0.11). To investigate prognostic implication of PLCG1 expression in MDS, we analyzed impact of PLCG1 expression on overall survival (OS). Based on PLCG1 expression level, 109 patients were divided into four groups, high (Q1), intermediate (Q2, Q3), and low (Q4) quartiles. Kaplan-Meier analysis demonstrated that the lowest quartile (Q4) showed significantly worse survival compared with remaining quartiles (Q1-Q3) (P =0.0015). The estimated 5-year OS rates in Q1-3 group and Q4 group were 63.2% and 29.7%, respectively. Percentage of patients with WHO-subtypes with high blast count (RAEB-1 and RAEB-2) was significantly higher in Q4 than other quartiles (46.9% vs 22.1%, P =0.019 by Fisher's exact test).The present study demonstrated that reduced PLCG1 expression is associated with inferior clinical outcome, indicating that PLCG1 expression could be a useful prognostic marker in MDS. Association between reduced PLCG1 expression and WHO-subtypes with high blasts counts, suggesting that PLCG1 dysfunction play a role in disease progression. DisclosuresNo relevant conflicts of interest to declare.

Association of Vascular Endothelial Growth Factor A (VEGFA) and its Receptor (VEGFR2) Gene Polymorphisms with Risk of Chronic Myeloid Leukemia and Influence on Clinical Outcome.

Vascular endothelial growth factor A (VEGFA) and its kinase insert domain receptor (VEGFR2/KDR) were reported to be upregulated in chronic myeloid leukemia (CML); however, the influence of polymorphisms in VEGFA and VEGFR2 in CML pathogenesis and therapeutic response, have not yet been elucidated. We aimed to analyze these polymorphisms in 212 CML patients and 212 healthy controls by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. The VEGFA+936C>T polymorphism did not differ significantly between the CML patients and controls. The frequency of CT genotype was higher in CML patients than in controls (25 vs. 18%), higher in males than in females (29 vs. 18%), was more prevalent in the patients with splenomegaly (p = 0.03), and was negatively associated with lactate dehydrogenase (LDH) levels (p = 0.01). The frequency of VEGFR2 mutant T-allele was higher in CML patients than controls (p < 0.0001). In the dominant model, patients having the combined AT and TT genotypes were associated with 2.6-fold higher risk of CML [odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.71–3.97, p < 0.0001]. VEGFR2 AT genotype was significantly associated with high blast count (p = 0.006), minor hematological response (p = 0.03) and poor cytogenetic response (p = 0.003), indicating its role in therapeutic resistance. In contrast, poor molecular response was observed in patients with TT genotype (p = 0.02). VEGFA+936C>T polymorphism was found to have synergistic interaction with VEGFR2+1416A>T in inflating the risk for CML further (P(interaction) = 0.0002). Our results indicate that VEGFR2+1416A>T polymorphism may be a useful marker in assessing the disease progression in CML patients. In addition, VEGFA+936C>T was observed to have additive effect in inflating the risk further.

Potential roles of microRNA-29a in the molecular pathophysiology of T-cell acute lymphoblastic leukemia.

Recent evidence has shown that deregulated expression of members of the microRNA-29 (miR-29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR-29 in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T-ALL) has not been investigated. Here, we show that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T-ALL patients. Furthermore, miR-29a levels are extremely reduced in T-ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR-29a mimics into Jurkat cells revealed the downregulation of several predicted targets (CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR-29a levels in Jurkat and Molt-4 T-ALL cells led to the demethylation of many genes commonly methylated in T-ALL. Overall, our results suggest that reduced miR-29a levels may contribute to the altered epigenetic status of T-ALL, highlighting its relevance in the physiopathology of this disease.

Decitabine Improves Outcomes in Older Patients with AML and Higher Blast Counts

S192 salvage 31% (9/29), (c) intense first-salvage and less-intense secondsalvage 4% (2/45), (d) less-intense firstand less intense secondsalvage 4% (1/23) (Table 1). Conclusions: Pending evaluation of other covariates (CR1 duration, age, transplant in first CR, cytogenetics etc.), our data suggest that intensity of first-salvage had no significant effect on CR rate with second-salvage therapy. This suggests that the policy of regarding various salvage regimens as identical when limiting number of prior treatments for purposes of study eligibility is reasonable. 225 Decitabine Improves Outcomes in Older Patients with AML and Higher Blast Counts Table 1 Patient Characteristics for Both Arms of DACO-016 Trial Characteristic DAC TC N(%) 127 (47) 144 (53) Median age [years] 73 73 Male sex [%] 56 65 Secondary AML [%] 24 30 t-AML [%] 5 1 BM Blast count Median Blast Percent 55% 55% Pts. with > 30% BM blasts [%] 100 100 Pts. with > 50% BM blasts [%] 57 55 Adverse Cytogenetics [%] 37 33 WBC Median (range) 2.3 (0.3-13.9) 3.2 (0.5-14.8) Pts. with WBC > 15 0 0 ECOG PS (%] 0-1 76 74 2 24 26 1-yr OS 37% 27% Median Relapse-Free Survival (m) 9.5 7.7 30-day mortality [%] 10 6 Grade 3-4 Heme toxicity (%) anemia 33 23 neutropenia 23 20 neutropenic fever 42 25 Thrombocytopenia 34 35 Tapan Kadia, Xavier Thomas, Anna Dmoszynska, Agnieszka Wierzbowska, Mark Minden, Christopher Arthur, Peter De Porre, Liang Xiu, Margaret Doyle, Jacqueline Bussolari, Hagop Kantarjian Leukemia, MD Anderson Cancer Center; Hopital Edouard Herriot; Haematooncology and Bone Marrow Transplantation, Medical University of Lublin; Medical University of Lodz; Princess Margaret Cancer Centre; Royal North Shore Hospital; Janssen; Janssen Research & Development, USA; Janssen-Cilag Ltd Introduction: Hypomethylating agents such as decitabine (DAC) and 5-azacytidine (AZA) have been shown to improve outcomes in older patients with high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with 20-30% bone marrow (BM) blasts. The AML-001 trial compared AZA vs. conventional care ([CC]; which included supportive care [SC], intensive chemotherapy [IC], or low-dose cytarabine [LDAC] ) in older patients with AML and BM blasts > 30%. AZA was associated with a trend towards improvement in OS (10.4 vs. 6.5 months, HR1⁄40.84; p1⁄40.083) compared to CC (Dombret, et. al. EHA 2014). Methods: DACO-016 was a multicenter, randomized, phase III trial of DAC vs. treatment choice ([TC]; SC or LDAC) in older patients with AML (Kantarjian, et. al. JCO 2012). In order to have a cohort comparable to the AZA-001 trial, we selected a subset of patients from DACO-016 with BM blast counts >30% and WBC count < 15. We then analyzed the clinical characteristics and outcomes of these patients. The OS data is based on the mature data set of DACO-016 with the clinical cutoff in October 2010, having a median follow-up of 30.7 months. Results: Of the 485 pts on the trial, 271 (56%) met the criteria to be analyzed, 127 (47%) who received DAC and 144 (53%) who had TC. Patient characteristics for both arms are summarized in Table 1. Patients on the DAC arm received a median of 4 cycles (range, 1-33) compared to a median of 2 (1-34) cycles on the TC arm. The CR/CRi rate was 27% for DAC vs. 11% for TC. This was associated with a significantly improved OS for patients receiving DAC (8.6 vs. 4.7 months, HR1⁄40.67; p1⁄40.0033)(Figure 1). This represented an absolute improvement of 3.9 months or a relative improvement of 83% over TC. The relapse-free survival was 9.5 vs. 7.7 months for DAC vs. TC. When censored at the time of subsequent therapy, DAC maintained a Clinical Lymphoma, Myeloma & Leukemia June 2015 significant OS advantage compared to TC (9.3 vs. 5 months, HR 0.69; p1⁄40.011). The 30-day mortality was similar for DAC vs. TC (10% vs. 6%), but DAC was associated with a higher rate of grade 3-4 hematologic toxicity (Table 1). Conclusion: DAC is an effective treatment in older patients with AML and may also improve outcomes in patients with higher BM blast counts.

Incidence of and risk factors for acute myeloid leukemia involvement of the central nervous system

![Graphic][1] It is thought that unlike acute lymphoblastic leukemia, adult acute myeloid leukemia (AML) rarely disseminates to the central nervous system (CNS). Therefore, routine lumbar puncture (LP) is rarely performed in patients with newly diagnosed AML, and the incidence of CNS leukemic infiltrate in AML patients is unknown. Because induction chemotherapy for AML includes high-dose cytarabine that penetrates the blood-brain barrier, cytarabine may eliminate any CNS disease present at diagnosis. However, high-dose cytarabine has not been included in recent induction chemotherapy protocols designed primarily for elderly patients with AML. To determine the incidence of CNS leukemia in these patients and to identify risk factors for CNS disease, we reviewed the medical records of 1,412 patients who received induction therapy at The University of Texas MD Anderson Cancer Center between January 2000 and December 2012 (144 months). Of these patients, 779 (55.2%) were male, and the median age at diagnosis was 62 years (range, 14–89 years). In the majority of the patients (1370/1412; 97.0%), LP was performed only if clinically indicated. Of these patients, 45 (3.4%) had CNS disease (12 at diagnosis and 33 during the course of the disease). The incidence of CNS leukemia among 42 AML patients (median age, 41 years; range, 14–49 years) who underwent LP at diagnosis as part of an investigational treatment regimen (N=8; 19%) was significantly higher (P<0.0001). The median time from diagnosis to CNS disease detection was 8 months (range, 0–120 months). Compared with that of Caucasian patients, CNS leukemia rates were higher among African American patients (odds ratio [OR], 4.1; 95% confidence interval [CI], 1.9–8.9) and patients of Asian (OR, 4.8; CI, 1.3–15.8) or Hispanic (OR, 2.5; CI, 1.0–5.9) descent. Induction chemotherapy included high- or medium-dose cytarabine in 826 patients (52.8%), low-dose cytarabine in 187 patients (11.9%), and no cytarabine in 552 patients (35.3%). The CNS disease incidence among patients who received cytarabine (3.2%) was similar to that of patients who did not receive high dose cytarabine (3.3%). Compared with patients who had no CNS disease, patients who had CNS dissemination had more bone marrow blasts at diagnosis (P = 0.003) and higher lactate dehydrogenase levels (P = 0.001). The CNS disease incidence among 132 AML patients with acute monoblastic leukemia (French-American-British classification M5; 7.6%) was significantly higher than that of patients with other AML subtypes (2.8%; P = 0.004). Conversely, the incidence of CNS disease among patients with prior myelodysplastic syndrome was only 0.6% (P = 0.05). Age; sex; cytogenetic abnormalities including complex karyotype, t(8:21), and inv16; and molecular aberrations including NPM1 , FLT3, and RAS mutations were not associated with an increased incidence of CNS dissemination. In a multivariate logarithmic model, only ethnicity (African American or Asian descent), high bone marrow blast count, and high LDH levels predicted CNS involvement. Patients with or without CNS disease had similar overall survival rates. However, the median disease-free survival (DFS) duration of patients without CNS disease (29 months; 95% CI, 19 - 39), was significantly longer than that of patients with CNS disease (7 months; 95% CI, 2 - 12; P<0.0001). Taken together, our data suggest that, contrary to the current dogma, AML spreads into the CNS in nearly 20% of newly diagnosed patients. Patients who received high-, low-, or no cytarabine had similar incidences of post-treatment CNS infiltration. Risk factors for post-treatment CNS infiltration were a high number of bone marrow blasts, acute monoblastic leukemia, and African American or Asian descent. The DFS duration of patients with CNS dissemination was significantly shorter than that of patients with no CNS dissemination, suggesting that the CSF is a sanctuary for residual AML. Therefore, further studies that determine whether, in whom, and at what treatment stage AML patients should undergo LP or receive intrathecal chemotherapy are warranted. Disclosures: No relevant conflicts of interest to declare. [1]: /embed/inline-graphic-2.gif

Decitabine improves outcomes in older patients with acute myeloid leukemia and higher blast counts

The median age at diagnosis of patients with acute myeloid leukemia (AML) is 66 years, and most patients are > 60 years of age.[1] Due to comorbidities and adverse-risk disease, many older patients may not benefit from intensive chemotherapy.[2, 3] Hypomethylating agents such 5-azacytidine (AZA) and decitabine (DAC) have been used as “lower-intensity” therapy in older patients with AML and may be associated with outcomes similar to intensive therapy.[4, 5] In a post-hoc analysis of a randomized phase III trial of AZA vs. conventional care regimens (CCR) in patients with higher-risk myelodysplastic syndrome (MDS) and 20–30% blasts, AZA produced a response rate of 18% and was associated with an improvement in overall survival (OS) compared to CCR (24.5 vs. 16 months, p=0.001).[6] Subsequent reports have confirmed the activity of AZA in older patients with AML and in those unfit for intensive chemotherapy.[4, 7–9]

Allo- HSCT for Advanced Myelodysplastic Syndrome:MSKCC Vs MDACC

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is the only curative treatment for myelodysplastic syndrome (MDS). Relapse, infections and graft-versus-host disease (GvHD) are the main causes of treatment failure. We compared the outcomes of patients receiving T cell depleted (TCD) grafts at Memorial Sloan-Kettering Cancer Center (MSKCC) with patients receiving unmodified grafts at MD Anderson Cancer Center (MDACC) for advanced MDS (RAEB-1 and 2).Adult patients transplanted between 2001 -2012 were included in this retrospective analysis. All recipients of TCD grafts (N=60) received myeloablative conditioning (MAC) and antithymocyte globulin (ATG) to prevent graft rejection. None of them received post-transplant GvHD prophylaxis. Of the 129 recipients of unmodified grafts, 87 received MAC and 42 reduced intensity conditioning (RIC); GvHD prophylaxis consisted of tacrolimus and mini-dose methotrexate in the majority of patients (N=113). ATG was given to all matched unrelated donor (MUD) recipients. Patients in the unmodified group had more therapy-related MDS (MDS-t), very poor risk cytogenetics by IPSS-R at diagnosis and bone marrow (BM) blast count >5% at transplant. Only the TCD group had mismatched donors (Table 1).Univariate analysis identified a lower incidence of grade II-IV acute GvHD in the TCD group with 100-day cumulative incidence (CI) of 13.3% vs. 34.1% in the unmodified group (p=0.031). There was no difference in grade III-IV acute GvHD with a 10% CI in both groups at day-100 (p=0.546). The incidence of chronic GvHD was lower in the TCD group with a CI at 3-yrs of 3.4% vs. 44.3% in the unmodified group (p < 0.001). The non-relapse mortality (NRM) in both groups was similar. CIs at day 100, 1yr, and 3 yrs in the TCD group were 8.3%, 20.2% and 32.7% vs. 12.4%, 22.5% and 28.1% in the unmodified group (p=0.628). Relapse was lower in the TCD group, with CI at 1 and 3 yrs of 8.5% and 15.5%, vs. 31.0% and 39.4% in the unmodified group (p=0.002). Since the unmodified recipients had worse disease characteristics, further analyses in patients with good/intermediate risk cytogenetic showed that the relapse incidence was similar between these subgroups, with 3-yr CIs of 7.9% in TCD vs. 18% in unmodified group (p=0.185). The most common causes of death in the TCD group were infections (32%) and relapse (28%), while in the unmodified group it was relapse (55%), GVHD (20%) and infections (13%). Considering the differences in disease characteristics between the groups, multivariate regression models were performed for relapse-free survival (RFS) and overall survival (OS) adjusting for MDS-t, high-risk cytogenetics at diagnosis and high blast count at HSCT. No significant differences were observed between the groups for RFS (HR=1.44, p=0.128) and OS (HR= 1.35, p=0.236) (Table 2). High-risk cytogenetics at diagnosis (very poor risk) was the only significant prognostic factor for RFS (HR=5.32, p<0.001) and OS (HR=4.81, p<0.001).Allo-HSCT is an effective treatment for patients with MDS with similar long term survival with either unmodified or TCD allografts. TCD is associated with a lower incidence of acute and chronic GVHD and without an increased risk of relapse.Table 1CharacteristicsTCD (N=60)Unmodified (N= 129)p-valueMedian follow-up, months (range)43.4 (3.8-119.5)49.4 (12.2-136.3)Age, years (range)57.1 (21.9-72.0)57.0 (19.0-72.0)0.769Female gender34 (56.7%)45 (34.9%)0.008MDS-t7 (11.7%)43 (33.3%)0.003Cytogenetic risk at diagnosis ( IPSS-R)0.009Good25 (42.4%)48 (37.2%)Intermediate15 (25.4%)19 (14.7%)Poor11 (18.6%)15 (11.6%)Very poor8 (13.6%)47 (36.4%)Missing1Blasts at transplant< 0.001< 5%48 (81.4%)57 (46.3%)5-9%11 (18.6%)35 (28.5%)10-19%031 (25.2%)Missing16Donor type< 0.001MRD21 (35.0%)65 (50.4%)MUD25 (41.7%)64 (49.6%)MMD14 (23.3%)0Stem cell source0.002BM5 (8.3%)46 (35.7%)PB55 (91.7%)83 (64.3%)Table 2:Multivariate analysisVariablesOS HR (95% CI)RFS HR (95% CI)Disease etiology0.5180.511De novo11MDS-t0.87 (0.56-1.34)0.87 (0.56-1.33)Cytogenetic risk at diagnosis IPSS-R)< 0.001 < 0.001 Good11Intermediate1.41 (0.74-2.68)1.39 (0.73-2.64)Poor2.06 (1.13-3.73)2.49 (1.40-4.44)Very poor4.81 (2.88-8.02)5.32 (3.22-8.80)Blasts at transplant0.2860.502< 5%115-9%1.11 (0.70-1.77)1.03 (0.65-1.62)10-19%1.57 (0.91-2.71)1.37 (0.80-2.34)Type of transplant0.2360.128TCD11Unmodified1.35 (0.82-2.19)1.44 (0.90-2.31) DisclosuresNo relevant conflicts of interest to declare.

Multiple Mechanisms Leading to ARID2 defects in Myeloid Neoplasms

Next generation sequencing (NGS) and single nucleotide polymorphism arrays (SNP-A) contribute to more precise identification of the spectrum of somatic mutations and karyotypic abnormalities in myeloid neoplasms. The diversity of individual defects and their combinations corresponds to the tremendous clinical heterogeneity. Identification of key driver genes remains a fundamental component to understanding the immense data generated from this technology and the contributions to leukemogenesis.In this project, we evaluated 1200 cases of MDS and AML. Somatic mutations of AT rich interactive domain 2 (ARID2) were found in myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), primary acute myeloid leukemia (pAML) or secondary AML (sAML). All ARID2 mutations occurred in either frameshift (at p.S1489 and p.T1645) or nonsense (at p.E65, p.S154 and p.Q1637) configurations, consistent with loss of function. We have identified a total of 5 mutant cases, all of somatic origin. Study of clonal architecture elucidated that ARID2 mutations were ancestral events in 50% of mutant cases as defined by variant allelic frequencies. By SNP-A, a commonly deleted region on chr.12q was defined by mapping minimally affected lesions in 9 patients with MDS, MPN, sAML or pAML. Haploinsufficient expression of ARID2 was confirmed by expression array analysis in the cases with del(12q), which is compatible to the frequent incidence of heterozygous ARID2 loss-of-function mutations. Del(12q) was more frequent in high-risk phenotype with higher blast counts. In addition, significantly lower expression of ARID2 was also observed in 22 out of 183 patients with diploid 12q. Interestingly, amplification of locus was not found in any of the cases studied by SNP-A. Altogether, such lesions of defective ARID2 were pathogenic in more than 10% of cases with myeloid neoplasms.ARID2 is encoding one of the components of SWI/SNF complex and involved in chromatin remodeling. Therefore, we stipulate that other genes which function together with ARID2 might be affected with somatic mutations or deletions. Further analyses demonstrated the presence of other somatic mutations and deletions affecting SWI/SNF complex, including ACTL6B (N=53) and SMARCD3 (N=66). While SWI/SNF complex lesions were mutually exclusive, concomitant subclonal mutations in such affected cases were commonly observed in RAS pathway genes, including K/NRAS, NF1 and PTPN11. To the contrary, ARID1B, which negatively regulates chromatin remodeling, is predominantly activated in the cohort with similar phenotype. While germline mutations of multiple genes in SWI/SNF complex are reported to be associated with Coffin-Siris syndrome, no family or past history characteristic of this congenital disorder was observed in our cohort.Further clues into the function of ARID2 in myeloid neoplasms came from studying splicing dysfunction in U2AF1 mutant cases. Deep RNA sequencing in the cases with U2AF1 mutations (p.S43F and p.Q157P), showed a consistent loss of ARID2 exon 8 (predominantly noted in sAML). Two types (whole and partial) of exon skipping led to frameshift in the transcript creating a stop codon. Targeted RT-PCR confirmed the transcriptome sequencing results in primary bone marrow samples of the cases with U2AF1 but not in the corresponding wild-type cases. These results are compatible with the genetic finding that spliceosomal mutations were not concomitantly observed in the cases with SWI/SNF complex defects, suggesting misspliced transcript with nonsense decay consequences is enough pathogenic to preclude additional spliceosomal mutations.To validate functional consequences of ARID2 loss, knockdown experiment using ARID2-shRNA transduction in K562 and HL60 cell lines were performed. Knockdown of ARID2 generally demonstrated cell cycle arrest in G2 phase prior to entry into the S-phase, which was partly caused by decreased expression of CDKL3 and CCND3. Hb staining with Benzidine showed impairment of erythroid differentiation in ARID2 knockdown K562, which was confirmed by cytological examination.In sum, multiple mechanisms of defective ARID2 including somatic mutations, haploinsufficiency and phenocopy due to spliceosomal mutations can be involved in ARID2-mediated leukemogenesis. Together with the other components, novel lesions of SWI/SNF complex constitute a group of tumor suppressor genes in myeloid neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Factors for Outcome in Patients (pts) with Refractory and Relapsed Acute Lymphocytic Leukemia (ALL) Treated with Inotuzumab Ozogamicin (IO), a CD22 Monoclonal Antibody

Background:IO was found to be highly active in pts with refractory-relapsed ALL, with an overall response rate of 58% and a median survival of 6.3 months (mos). Identifying factors associated with different outcomes on IO therapy may help select patients for this treatment and advice of prognosis.Methods:A total of 89 pts treated with IO on previous studies were analyzed. IO was given at 1.3-1.8 mg/m2 IV x 1 every 3-4 weeks or weekly (0.8 mg/m2 Day 1, 0.5 mg/m2 Days 8 and 15) every 3-4 weeks. Pretreatment factors associated with achieving marrow complete response (CR) and with survival were analyzed using standard statistical methods.Results:Baseline characteristics of the 89 pts are summarized in Table 1. Overall, 17 pts (19%) achieved CR, 29 (33%) had CRp, and 6 pts (7%) had bone marrow CR with incomplete recovery (Cri). 5 pts (6%) died within 4 weeks of starting therapy. The ORR was 58%. The rate of cytogenetic CR by morphologic responses was as follows: 8 cytogenetic CR/10 morphologic CR (80%); 16 cytogenetic CR/22 morphologic CRp (73%); and 4 cytogenetic CR/4 morphologic CRi (100%). The median survival of patients with at least marrow CR was 9 mos versus 3.4 mos for those without marrow CR (p<0.001). By multivariate analysis, a high peripheral blood absolute blast count (ABC) and low platelet count were independently associated with a lower likelihood of achieving at least marrow CR. With a median follow-up of 23 mos, (5-44), the median overall survival of pts who received IO was 6 mos; the median survival was 5 mos with the single-dose schedule and 9.5 mos with the weekly schedule. The median remission duration was 12 mos (1-year rate, 48%) (Figure 1A). Baseline characteristics independently associated with worse survival included adverse cytogenetics [complex karyotype, translocation (4;11), translocation (9;22), abnormal chromosome 17], disease beyond first salvage, and high peripheral blood ABC. Pts with 0, 1-2 or 3 adverse factors had a median survival of 42+, 7.5, and 2.4 mos, respectively (Figure 1B). To assess the benefit of achieving a marrow CR, we repeated the multivariate survival analysis using a 6-week landmark that excluded 5 pts who died within 6 weeks. The median survival was 9.2 mos and 3.4 mos for patients with and without marrow CR (p<0.001). The multivariate analysis selected the achievement of response as independently associated with survival improvement [HR=0.5 (95% CI=0.28-0.89); p=0.02]. Disease with complex karyotype, translocation (4;11), translocation (9;22), and abnormal chromosome 17 [HR=2.9 (95% CI=1.29-6.62); p=0.009], and disease status beyond first Salvage regimen [HR=1.2 (95% CI=2.16-3.91); p=0.01] were independently associated with significant worse survival. Pts with 0, 1- 2 or 3 adverse factors had a median survival of 42+, 8, and 3 mos, respectively (Figure 1C).Conclusion:Our current analyses identified a subset of adult pts with ALL in whom outcome of therapy with IO can be differentially predicted.Table 1Patients’ characteristicsN (%)ParameterSingle-Dose, n=49Weekly, n=40Overall, n=89Age, year≤183 (6)3 (8)6 (7)≥6012 (24)13 (33)25 (28)ECOG, PS0-144 (90)36 (90)80 (90)≥25 (10)4 (10)9 (10)Salvage StatusS113 (27)16 (40)29 (33)>S136 (73)24 (60)60 (67)PB ABC, x 109/L<1.033 (67)25 (63)58 (65)≥1.016 (33)15 (38)31 (35)WBC, x 109/L<4.028 (57)17 (43)45 (51)4-1111 (22)16 (40)27 (30)>1110 (20)7 (18)17 (19)BM blasts, %<203 (6)8 (20)11 (12)20-4910 (20)8 (20)18 (20)50-698 (16)6 (15)14 (16)≥7028 (57)18 (45)46 (52)Platelets, x 109/L<10043 (88)28 (70)71 (80)≥1006 (12)12 (30)18 (20)KaryotypeDiploid8 (16)9 (23)17 (19)Ph-positive9 (18)8 (20)17 (19)Translocation (4;11)6 (12)3 (8)9 (10)Complex13 (27)11 (28)24 (27)Abnormal chromosome 176 (12)5 (13)11 (12)Other6 (12)4 (10)10 (11)Prior ASCT7 (14)2 (5)9 (10)Prior HCVAD regimen35 (71)32 (80)67 (75)CD22-positive, %≥9028 (57)31 (78)59 (66)70-8914 (29)7 (18)21 (24)50-697 (14)2 (5)9 (10) [Display omitted] [Display omitted] [Display omitted] DisclosuresKadia:GSK: Research Funding; ARIAD: Honoraria. Hagop:ARIAD: Research Funding; Pfizer: Research Funding; Amgen: Research Funding.

The prognostic significance of an inv(3)(q21q26.2) in addition to a t(9;22)(q34;q11.2) in patients treated with tyrosine kinase inhibitors

In chronic myelogenous leukemia, BCR-ABL1 positive detection of cytogenetic abnormalities in addition to the t(9;22) is thought to portend a poor prognosis; however, not all abnormalities associated with the t(9;22) have the same impact. Inv(3) defines a group of aggressive neoplasms with poor response to conventional treatment options. In this study, four cases with the t(9;22) and inv(3) treated with tyrosine kinase inhibitors (TKI) were investigated. In three cases, the inv(3) was not detected at the initial diagnosis and the patients initially responded to TKI therapy; the inv(3) was detected at blast crisis in all three cases, and one case had both abnormalities at the initial presentation, but this case presented as acute myeloid leukemia. In all cases, detection of an inv(3) was associated with a high blast count and a lack of response to treatment regimens including TKI. All patients died within months from the detection of inv(3). This indicates that cases with the t(9;22) and inv(3) have a clinical course similar to that of cases with an inv(3) and no other therapeutically targetable abnormality.

Azacitidine and low‐dose cytarabine in palliative patients with acute myeloid leukemia and high bone marrow blast counts – a retrospective single‐center experience

We retrospectively analyzed and compared the efficacy and toxicity of azacitidine (AZA) and low-dose cytarabine (LD-Ara-C) in 65 palliative patients with acute myeloid leukemia (AML) showing high bone marrow blast counts (≥30%) before start of treatment. Twenty-seven and 38 patients received AZA and LD-Ara-C, respectively. The median patient age was 71 yr. Patient and disease characteristics did not differ between the treatment groups, except for BM blast counts, and peripheral leukocyte and blast counts which were significantly higher in the LD-Ara-C group. AZA and LD-Ara-C were first-line treatment in 12 (44%) and 17 patients (45%), respectively. Response and hematologic improvement rates were low and similar in both treatment groups. In both treatment groups, most common non-hematologic toxicities included febrile neutropenia, pneumonia, and bleedings without significant differences regarding frequencies. Estimated 1-yr survival rates were 15% (95% CI 8-22) and 13% (95% CI 7-19) in the AZA and LD-Ara-C groups, respectively, without statistically significant difference. In multivariate analysis (n = 65), previous treatment (HR 2.27, 95% CI 1.00-5.22, P = 0.05) and adverse cytogenetics (HR 2.50, 95% CI 1.20-5.22, P = 0.02) were independent predictors of poor survival. In our center and within the limitations of a retrospective study, both treatment regimens showed similar but limited efficacy in palliative patients with AML and high BM blast counts.

Multicenter study evaluating the impact of hypomethylating agents as bridging therapy to hematopoietic stem cell transplantation in myelodysplastic syndromes

Allogeneic hematopoietic stem cell transplantation (alloSCT) is currently the only curative treatment modality for myelodysplastic syndromes (MDS). The treatment paradigm for MDS has changed in recent years with the introduction of hypomethylating agents (HMAs). The present retrospective multicenter study was designed to assess the effects of pre-transplant HMA on transplant outcome and determine which patients would benefit most from this therapy. A total of 109 patients who received alloSCT at one of five institutions between 2007 and 2010 were enrolled in this study regardless of pre-transplant HMA therapy. 81 of the 109 patients enrolled were treated with HMA prior to alloSCT. 28 patients received alloSCT without HMA bridging. The distributions of WHO classification groups and IPSS scores were similar between the two groups (P = 0.752 and P = 0.265, respectively). Pre-transplant HMA did not affect OS (P = 0.244), and there were no differences in response to HMA therapy within the HMA-treated group. The cumulative incidence of NRM was not significantly different between the two groups (P = 0.500). However, for patients with a high blast count (>5 % of bone marrow at the time of diagnosis), pre-transplant HMA therapy had a NRM benefit (83.3 vs. 48.6 %, P = 0.014).

Frequency and Prognostic Relevance ofFLT3Mutations in Saudi Acute Myeloid Leukemia Patients

The Fms-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that plays a key role in cell survival, proliferation, and differentiation of hematopoietic stem cells. Mutations of FLT3 were first described in 1997 and account for the most frequent molecular mutations in acute myeloid leukemia (AML). AML patients with FLT3 internal tandem duplication (ITD) mutations have poor cure rates the prognostic significance of point mutations; tyrosine kinase domain (TKD) is still unclear. We analyzed the frequency of FLT3 mutations (ITD and D835) in patients with AML at diagnosis; no sufficient data currently exist regarding FLT3 mutations in Saudi AML patients. This study was aimed at evaluating the frequency of FLT3 mutations in patients with AML and its significance for prognosis. The frequency of FLT3 mutations in our study (18.56%) was lower than many of the reported studies, FLT3-ITD mutations were observed in 14.4%, and FLT3-TKD in 4.1%, of 97 newly diagnosed AML patients (82 adult and 15 pediatric). Our data show significant increase of FLT3 mutations in male more than female (13 male, 5 female). Our results support the view that FLT3-ITD mutation has strong prognostic factor in AML patients and is associated with high rate of relapse, and high leucocytes and blast count at diagnosis and relapse.

Overexpression of Bcl2 protein predicts chemoresistance in acute myeloid leukemia: Its correlation with FLT3

Potential prognostic biomarkers in acute myeloid leukemia (AML) can be identified by understanding the cellular pathway and molecular changes underlying leukemogenesis. Deregulation of apoptosis is one of the important features of AML and to understand the molecular mechanism underlying apoptosis and its contribution to tumor progression, this study aimed to evaluate anti-apoptotic Bcl2 protein expression in AML and correlate with FLT3 parameters for their role in prognosis of disease.Bcl2 and FLT3 protein expression was quantified by flow cytometry on leukemic blasts in total 174 de novo AML, myelodysplastic syndrome (MDS) and aplastic anemia patients. FLT3 internal tandem duplication (ITD), Tyrosine kinase domain (TKD) point mutations and quantification of mRNA level was carried out using PCR and RT-PCR methods. The incidence of Bcl2 positivity was 71% in AML patients. Bcl2 positivity was significantly associated with CD34+ and CD117+ AML. Bcl2 positivity tended to be associated with reduced DFS while Bcl2 positivity with FLT3 protein positivity was significantly associated with reduced DFS. In multivariate analysis, Bcl2+ and combined Bcl2+/FLT3 protein+ along with high WBC count emerged as poor prognostic factors for reduced DFS and high blast count for predicting reduced OS. In MDS patients, the incidence of Bcl2 expression was high while in aplastic anemia patients, incidence of Bcl2 expression was low.Patients with Bcl2 and FLT3 protein positivity showed significantly reduced DFS suggesting parallel role of these proteins in imparting chemoresistance to the leukemic cells.

Prognostic Implications Of PRAME Expression Levels In Myelodysplastic Syndromes

The preferentially expressed antigen of the melanoma (PRAME) gene was first identified in melanoma tissue as a tumour-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. While expression level of PRAME is quite low in most of normal tissues of human, including bone marrow normal CD34+ cells, except for testis, PRAME overexpression is observed in various human cancers, including hematological malignant disorders. Although clinical and biological significance of PRAME expression in human cancers has not been fully elucidated, it has been demonstrated that high PRAME expression is associated poor clinical outcomes in a number of solid cancers. Association between PRAME expression and clinical outcomes in hematological malignancies has not been clarified. In acute myeloid leukemia, high expression of PRAME was shown to be associated with worse progression-free survival or overall survival by some research groups but not by other groups. Our previous study demonstrated that inhibition of PRAME expression in leukemic cells results in cell cycle arrest and induction of apoptosis, suggesting oncogenic function of PRAME expression in leukemogenesis. Clinical significance of PRAME expression in myelodysplastic syndromes (MDS) also has not been fully elucidated. In the present study, we examined PRAME expression of bone marrow cells in MDS patients to clarify clinical significance of PRAME expression.Bone marrow samples of MDS patients were used for analysis. Samples were taken at the time of diagnosis with written informed consent from patients. Total RNA extraction, cDNA synthesis and quantitative real-time RT-PCR by the TaqMan probe method using an ABI 7500 real-time PCR system (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems), were performed. Expression levels were obtained using the standard curve method in each experiment, after normalization with the GAPDH gene for each sample in duplicate wells. The human PRAME primer-probe sets were from Applied Biosystems (assay ID: Hs00196132_m1). Data including patients' demographic, disease status, medical history, clinical and laboratory findings, and outcome, were collected from medical records and laboratory data base.A total of 111 MDS patients, 68 males and 43 females with median age of 69 years (range: 20-91 years) were included in the present study. They were classified as RCUD (n=15), RCMD (n=61), RARS (n=8), RAEB-1 (n=15), and RAEB-2 (n=12) according to WHO classification. Based on the IPSS, they were categorized in four risk groups, low risk (n=29), intermediate-1 risk (n=55), intermediate-2 risk (n=23), and high risk(n=4). Expression level of PRAME was varied among the MDS patients analyzed. Median value of relative PRAME expression level of 111 MDS patients and that of 19 control subjects were 0.073 and 0.07, and there was no significant difference in distribution of expression level of PRAME. However, when we compared expression patterns of PRAME among five WHO-subtypes, statistical difference was observed (P=0.0116). Relative PRAME expression level in WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) was significantly higher than that in WHO-subtypes with less blast counts (RCUD, RCMD, RARS) (median value: 0.44 vs. 0.05, P=0.0012). To investigate prognostic implication of PRAME expression in MDS, we analyzed impact of PRAME expression on overall survival (OS). Based on PRAME expression level, 111 patients were divided into two categories, ‘high expression group' (above median value) and ‘low expression group' (below median value). Kaplan-Meier analysis demonstrated that high expression group showed significantly poorer overall survival than low expression group (P=0.0165). The estimated 5-year OS rates in high expression group and low expression group were 63.5% and 78.4%, respectively.The present study demonstrated that high PRAME expression is associated with poorer clinical outcome, indicating that PRAME expression could be a useful prognostic marker in MDS. Biological significance of PRAME expression in MDS is unclear. Expression level of PRAME was higher in WHO-subtypes with high blasts counts, suggesting that PRAME may play role in disease progression in MDS. Further studies should be necessary to clarify clinicopathological and biological significance of PRAME expression in MDS. Disclosures:No relevant conflicts of interest to declare.