Peripheral Blood Blast Count Research Articles

Efficacy Of Azacitidine Versus Low-Dose Cytarabine In Patients With Acute Myeloid Leukemia - A Retrospective Single Center Experience

IntroductionAzacitidine (AZA) treatment has been shown to be superior to conventional care regimens including low dose cytarabine (LD-Ara-C) in acute myeloid leukemia (AML) patients with low bone marrow (BM) blast counts (20-30%). In contrast, data on efficacy of AZA in patients with blast counts exceeding 30% are scarce. Here we present a retrospective, single center analysis, comparing the efficacy and toxicity of AZA versus LD-Ara-C in AML patients with high BM blast counts (≥30%) prior to treatment. Patients and MethodsTwenty-seven patients receiving AZA and 38 patients receiving LD-Ara-C met the eligibility criteria for the analysis (age ≥18 years, documented BM blast count ≥30% prior to start of treatment and administration of at least one complete therapy cycle). Patients who underwent allogeneic transplantation following AZA treatment or received stem cell support following LD-Ara-C therapy were excluded from this analysis. Overall survival (OS) was estimated using the method of Kaplan and Meier. Comparison of OS between the AZA and LD-Ara-C group was done using the logrank test and by Cox regression adjusting for known confounders. ResultsPatient (age, ECOG status) and diseases characteristics (type of AML, cytogenetics, pretreatment, number of treatment cycles) did not differ significantly between the treatment groups, except for BM blast count (median 44% vs. 60% in the AZA and LD-Ara-C group, respectively; p=0.03) and peripheral blood blast count (median 6% vs. 56% in the AZA and LD-Ara-C group, respectively; p<0.01). Response rates to AZA treatment according to international working group (IWG) criteria were low with two patients achieving a complete remission (CR) and one patient showing partial remission (PR) after AZA treatment. In the LD-Ara-C cohort no CR was observed and two patients experienced a PR. Hematologic improvement (HI) rates according to IWG criteria did not differ between both treatment groups (any type of HI 33% vs. 24% in the AZA and LD-Ara-C group, respectively; p=0.41). In both cohorts, most common non-hematologic toxicities (CTCAE grade≥3) included febrile neutropenia, pneumonia and bleedings without significant differences regarding frequencies. As expected, skin involvement was more commonly observed in the AZA group (19% vs. 3%, p=0.04). One year survival rates were only 15% (95% CI 8-22%) and 13% (95% CI 7-19%) in the AZA and LD-Ara-C group, respectively. There was no statistically significant difference between the treatment groups (HR 1.2, p=0.41). Furthermore, there was no difference in hospitalization time (total days spent in hospital during treatment per patient-year of follow-up 29.4 vs. 27.2 in the AZA and LD-Ara-C group, respectively; RR 1.07 95% CI 0.95-1.21, p=0.23). In a multivariate analysis with OS as endpoint adverse cytogenetics (HR 2.24 95% CI 1.17-4.70, p<0.02) were significantly associated with inferior survival, whereas the treatment had no impact (AZA vs. LD-Ara-C HR 1.27 95% CI 0.67-2.40, p=0.46). ConclusionIn our center, treatment with AZA showed limited efficacy and no superiority to LD-Ara-C treatment in AML patients with BM blasts ≥30%. Disclosures:Dreger:Riemser Pharma : Consultancy, Honoraria, Research Funding.

Pilot Study Of Erlotinib In Patients With Acute Myeloid Leukemia

BackgoundPreclinical data suggest “off-target” efficacy of EGFR tyrosine kinase inhibitor erlotinib in MDS and AML by inducing apoptosis and promoting differentiation in myeloblasts (Boehrer et al., Blood, 2008). The cytotoxic effect of erlotinib on leukemic myeloblasts ex vivo occurs at concentrations below the plasma levels achievable with regular dosing of this drug. Two case reports, in literature, of patients with concomitant AML and lung cancer, treated with erlotinib and demonstrated AML response, corroborate these findings. The exact targets of erlotinib in MDS/AML are unknown but inhibition of SRC family kinases, Syk, mTOR, and drug efflux system has been suggested. Since normal CD34+ bone marrow cells are not affected by this agent, and no myelosuppression is seen in patients taking erlotinib, clinical application of this drug for the treatment of MDS/AML seems attractive. Clinical studies testing efficacy of erlotinib in MDS are undergoing. We report a pilot study of administration of erlotinib in AML. MethodsThis was a single-institution pilot study. The study was approved by scientific review committee and IRB. All patients signed informed consent. Eligibility included a confirmed diagnosis of AML per WHO classification (>20% blasts), no pre-existing history of MDS, newly diagnosed disease in patients older than 70 or relapsed disease in younger patients unfit for chemotherapy or refractory disease at any age. All cytogenetics and molecular profile categories were eligible. An ECOG performance status of 0-3 was acceptable. WBC count had to be less than 20,000/cumm, and hydroxyurea or other therapies to be discontinued at least 14 days prior to treatment; therefore, patients with high proliferative rate disease were excluded. Smokers were excluded due to potential diminished erlotinib plasma concentration with cigarette smoking. Drugs with potential interaction with erlotinib metabolism were not allowed. Erlotinib was given orally at 150 mg per day continuously in 28-day cycles. Bone marrow at baseline was evaluated for morphology, cytogenetics, flow-cytometry, and molecular profile (FLT3, NPM1, CEBPA mutations). CD34+ cells were separated from bone marrow aspirates taken at baseline, day 3-4, and day 8+1 of treatment, and frozen/banked for future correlative studies. Response was evaluated by bone marrow examination after each cycle. A flow-cytometry was performed on the bone marrow aspirate after the first cycle to be compared with baseline, specifically for evidence of differentiation. Given possibility of “slow/delayed response” with erlotinib, patients were planned to continue at least 3 cycles of treatment in the absence of disease progression or toxicity. Assessment of overall response was the primary endpoint of the study. ResultsBetween August 2010 and August 2012, a total of 11 patients were treated on this study at Indiana University Simon Cancer Center. One patient had relapsed AML, and 1 had relapsed/refractory disease; 9 patients were older than 70 with newly diagnosed AML. Two patients with newly diagnosed AML demonstrated modest response with the first cycle of treatment, with reduction of bone marrow blasts from 89% and 88% to 71% and 73%, respectively, but both experienced disease progression subsequently. Nine other patients had progression of the disease without any response. Of these, 6 completed at least one cycle of treatment, and 3 were taken off study earlier due to disease progression (per peripheral blood blast count) and clinical deterioration. None of the patients with circulating blasts showed meaningful response by total WBC or absolute peripheral blood blast count. Flow-cytometry of bone marrow aspirate following the first cycle compared to pre-treatment sample did not show evidence of differentiation in any of the 8 patients who completed at least one cycle of therapy. None of the patients experienced drug-related toxicity. ConclusionThis pilot study, which enrolled mostly older AML patients with newly diagnosed disease, did not demonstrate clinical response to erlotinib monotherapy when administered continuously at 150 mg per day. No evidence of differentiation was observed in AML blasts after 4 weeks of therapy. The treatment was well tolerated without drug-related adverse events. Disclosures:No relevant conflicts of interest to declare.

Survival Stratification In Acute Myeloid Leukemia By Single Cell Signal Profiling

IntroductionDysregulation and mutations in signaling genes of cancer cells characterize more than half of the acute myeloid leukemia (AML) patients, and contribute to chemoresistance through regulation of cellular processes including apoptosis and DNA repair. We investigated if determination of single cell basal phosphorylation of signaling proteins reflected mutation of FLT3 and NPM1, cytogenetics, response to first course of chemotherapy or overall survival. MethodsWe employed flow cytometric single cell analysis of phosphoproteins central to signal transduction pathways in myeloid cancer cells and analyzed peripheral blood leukocytes from 93 acute myeloid leukemia (AML) patients. Blood samples from consecutively diagnosed AML patients with high peripheral blood blast counts (>7x109/L, >70% blasts) were collected after informed consent was given and biobanked by cryo-preservation. AML samples was thawed and equilibrated for one hour in a defined serum-free medium (StemSpan SFEM medium, Stem Cell Technologies) which include insulin and transferrin. All samples were viability controlled and validated for growth factor response. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) Alexa Flour 700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647; p38(pT180/pY182) and ERK2(pT202/pY204), SRC(pY418) and Akt(pT308), PDK1(pS241) and Akt(pS473), STAT1(pY701) and ribosomal protein S6(pS235/36), STAT3(pY705) and STAT5(pY694), CREB(pS133) and STAT3(pS727), ribosomal protein S6(pS240) and NFkB(pS529), 4EBP(pT37/pT45) and STAT6(Y641), without p-antibody and JNK(pT183/pY185). The lowest median signal for each phosphoprotein in AML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. Hierarchical clustering with the use of complete linkage were created using TM4, and Principal Component Analysis was carried out using Unscrambler X (CAMO Software). ResultsUnsupervised clustering revealed two distinct signature clusters based on low or elevated phosphorylation level among AML cells. A similar cluster signature was absent in endogenous non-leukemic lymphocytes from the same patients. No correlations between basal phosphorylation and prognostic mutations (FLT3 or NPM1), cytogenetics or response to first course of chemotherapy were found. In AML patients treated with intensive chemotherapy (n=45) the cluster with low phosphorylation level (n=19) correlated with significant (p=0.007) shorter overall survival. Principal component analysis verified the cluster analysis and guided a reduction to only three phosphoproteins (STAT3, 4EBP1, ribosomal protein S6) with statistically significant (p=0.014) stratification of survival. ConclusionLeukemic cells demonstrated a phosphorylation profile that reflected survival of the intensively treated patients, but surprisingly not correlated with mutational status of FLT3, NPM1, cytogenetics or first course remission status. This suggests that phosphoprotein determination in leukemic cells provide prognostic information so far not available with current diagnostics. The robust and relatively simple method of single cell signal profiling should be tested in clinical trials to examine its feasibility in therapy response prediction. More extensive mutational and epigenetic analyses are needed in search for the molecular origin of the low/high signal profiles.(A) Unsupervised hierarchical cluster analysis based on three phospho protein analysis stratified in two distinct clusters of patients receiving standard intensive induction chemotherapy (n=45; daunorubicin + cytarabine or idarubicin + cytarabine (3+7) similar to HOVON AML protocols 103 and 102, respectively). (B) Kaplan-Meier plot was performed calculating P value with the use mantel-Cox log-rank test. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High-Throughput Mutational Profiling Of Post-Myeloproliferative Neoplasm Acute Myeloid Leukemia Reveals Frequent Mutations In NRAS In JAK2V617F-Negative Post-MPN AML

BackgroundA subset of patients with the Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) transform to AML, and the prognosis of post-MPN AML patients is very poor. As such, new genomic insights are needed to define the mechanisms that govern transformation from MPN to AML, and to identify new therapeutic targets for clinical intervention in this poor-risk myeloid neoplasm. Importantly, patients with JAK2V617F mutant chronic-phase disease transform to JAK2 wildtype AMLs in approximately half of cases, indicating that diverse genomic paths lead to transformation. AimsTo characterize the genomic alterations, including point mutations, short indels, translocations, and copy number alterations, in 33 post-MPN AML samples. MethodsGenomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. We achieved a mean coverage depth of 511x (range 405-645). ResultsThe most common genomic alterations identified in post-MPN AML samples were mutations in JAK2 (51.5%), ASXL1 (48.5%), and IDH2 (30.3%). Mutually exclusive mutations in the genes of spliceosome components SRSF2, U2AF1, and SF3B1 were identified in 39% of patients in this cohort, suggesting that somatic mutations in splicing factors are a common genomic event in transformation from MPN to AML. These data were in contrast to de-novo AML, in which mutations in FLT3, NPM1, and DNMT3A are the most common disease alleles (NEJM 2013; 368: 2059), suggesting that the spectrum of genomic alteration in post-MPN AML differs from that in de novo AML.Within our cohort of post–MPN AML samples, 52% of the patients had JAK2V617F positive AML and 48% of our cohort presented with JAK2 wildtype AML. Notably, the two subsets of post-MPN AML had distinct mutational patterns. In the JAK2V617F mutant subgroup, we identified frequent mutations in ASXL1 (41.2%), TP53 (41.2%), and IDH2 (41.2%). In JAK2-wildtype AML subgroup we identified frequent ASXL1 mutations (56.3%) and NRAS point mutations (37.5%). NRAS mutations were exclusive of JAK2 mutations in the entire cohort, consistent with alternate disease alleles which activate signaling in JAK2V617F positive AML and in JAK2 wildtype AML. SETBP1 mutations were found in 19% of patients with JAK2 wildtype post-MPN AML but not in any patients with JAK2V617F-positive post-MPN AML.Several alterations not previously described in post-MPN AML, to the best of our knowledge, were identified in this cohort including MLL-PTD, which was observed in both JAK2V617F and wildtype JAK2 patients. Copy number analysis of our high-depth sequencing data allowed us to identify homozygous deletions of TET2 and ETV6 and MYC amplifications in post-MPN AML. Univariate analysis demonstrated TP53 mutations were associated with significantly impaired overall survival (p<0.001). IDH2 mutations were associated with higher peripheral blood blast count (p=0.017). ConclusionThe spectrum of genomic alterations in post-MPN AML is strikingly distinct from de novo AML. Furthermore, capture-based sequencing allowed us to show there are important differences in the mutational profile of JAK2V617F and JAK2 wildtype post-MPN AML. NRAS mutations may represent driver mutations in JAK2V617F negative post-MPN AML and further biologic evaluation of these mutations is needed. Most importantly, the identification of NRAS, IDH2, and MLL-PTD mutations in post-MPN AML suggest specific therapies including MAPK inhibition, IDH2 inhibition and dot1L/BRD4 inhibition may be of value in genotypically defined subsets of post-MPN AML. These finding help to further define the biology of leukemic transformation, as well as offer new therapeutic avenues for the treatment of post-MPN AML. [Display omitted] Disclosures:Rampal:Foundation Medicine: Consultancy. Verstovsek:Incyte Corporation: Research Funding.

The combination of valproic acid, all-trans retinoic acid and low-dose cytarabine as disease-stabilizing treatment in acute myeloid leukemia

BackgroundA large proportion of patients with acute myeloid leukemia (AML) are not fit for intensive and potentially curative therapy due to advanced age or comorbidity. Previous studies have demonstrated that a subset of these patients can benefit from disease-stabilizing therapy based on all-trans retinoic acid (ATRA) and valproic acid. Even though complete hematological remission is only achieved for exceptional patients, a relatively large subset of patients respond to this treatment with stabilization of normal peripheral blood cell counts.MethodsIn this clinical study we investigated the efficiency and safety of combining (i) continuous administration of valproic acid with (ii) intermittent oral ATRA treatment (21.5 mg/m2 twice daily) for 14 days and low-dose cytarabine (10 mg/m2 daily) for 10 days administered subcutaneously. If cytarabine could not control hyperleukocytosis it was replaced by hydroxyurea or 6-mercaptopurin to keep the peripheral blood blast count below 50 × 109/L.ResultsThe study included 36 AML patients (median age 77 years, range 48 to 90 years) unfit for conventional intensive chemotherapy; 11 patients responded to the treatment according to the myelodysplastic syndrome (MDS) response criteria and two of these responders achieved complete hematological remission. The most common response to treatment was increased and stabilized platelet counts. The responder patients had a median survival of 171 days (range 102 to > 574 days) and they could spend most of this time outside hospital, whereas the nonresponders had a median survival of 33 days (range 8 to 149 days). The valproic acid serum levels did not differ between responder and nonresponder patients and the treatment was associated with a decrease in the level of circulating regulatory T cells.ConclusionTreatment with continuous valproic acid and intermittent ATRA plus low-dose cytarabine has a low frequency of side effects and complete hematological remission is seen for a small minority of patients. However, disease stabilization is seen for a subset of AML patients unfit for conventional intensive chemotherapy.

Differential diagnosis of myelofibrosis based on WHO 2008 criteria: Acute panmyelosis with myelofibrosis, acute megakaryoblastic leukemia with myelofibrosis, primary myelofibrosis and myelodysplastic syndrome with myelofibrosis

The aim of this study was to characterize clinicopathological features of acute panmyelosis with myelofibrosis (APMF), acute megakaryoblastic leukemia with myelofibrosis (AMKL-MF), primary myelofibrosis (PMF) and myelodysplastic syndrome with myelofibrosis (MDS-MF) in order to provide the keys to the differential diagnosis of bone marrow (BM) fibrosis. We compared age, gender, splenomegaly, serum lactate dehydrogenase level, blood cell counts, blast counts in peripheral blood (PB) and BM, megakaryocyte counts, BM cellularity, dysplasia, and the karyotypes of patients with APMF (n=6), AMKL-MF (n=7), PMF (n=44), and MDS-MF (n=44). APMF showed hyperplasia of all three lineages, increase in megakaryocyte count with dysplasia and frequent abnormal karyotypes. AMKL-MF was associated with elevated BM blast counts, decreased BM megakaryocyte count with rare megakaryocytic dysplasia and chromosome 21 abnormality. PMF patients displayed splenomegaly, rare blasts in PB/BM, and JAK2 V617F mutation. MDS-MF patients showed pancytopenia, dysplasia in all three lineages and recurrent chromosomal abnormalities involving chromosome 5,7,12, and 17. Although differential diagnosis among APMF, AMKL-MF, PMF, and MDS-MF is very challenging due to the overlapping clinical and morphological features, meticulous investigation of the patient with respect to splenomegaly, blood cell count, PB and BM findings, and karyotype will serve as a guide to correct diagnosis.

Clinical features and outcome of T-cell acute lymphoblastic leukemia in patients older than 9 years: A single center experience of 110 patients from AIIMS, New Delhi, India.

7081 Background: It is known that T cell acute lymphoblastic leukemias (ALL) have poorer outcomes than their B cell counterparts. Data on T-ALL in the age group of &gt;9 years from India is minimal. Methods: This is a single institutional analysis of patients of above 9 years who were treated from January 2000 to December 2010. All patients who completed at least 4 weeks of induction therapy were analysed for various outcomes. Results: T-ALL formed 30% of all ALL in this age group. Of the 110 newly registered patients of T-ALL, the median age was 17 years (Range 10-50 years) with an M:F ratio of 5.9:1. Of this 62%, 30% and 18% patients belonged to 10-18, 19-30 and &gt; 31 years age group respectively. Eighteen (19%) and 2 (2%) and 33 (30%) had CSF, testicular and other extramedullary sites involvement respectively. Twenty eight per cent had a total leucocyte count (TLC) of above 100x109/L. Patients available for survival analysis were 104(94.5%). Complete remission (CR)rate was 68.2% and induction mortality was 14.4%. At a median follow up of 56.4 months 5 year leukemia free survival was 52.3% (median not attained). Twenty seven (38%) patients relapsed (median relapse time of 15.2 months, range 0.7 to 47.3 months), 55% during maintenance phase. The 5 year overall survival (OS) was 46.9% (median OS of 35.4 months). The 5 year OS of 10-18, 19-30 and &gt; 31 years age groups were 42.8%, 71% and 16.6% respectively (p value not significant). Not attaining CR in 1st induction, spontaneous tumor lysis syndrome and peripheral blood blast count of &gt; 80% were significant poor prognostic factors for survival. Conclusions: This is one of the largest study of T-ALL outcomes in patients above 9 years from a single center from India. Attainment of CR in 1st induction was the most important risk factor for survival. 5 year OS was 47%.

Azacytidine for acute myeloid leukemia in elderly or frail patients: a phase II trial (SAKK 30/07)

This phase II trial treated elderly or frail patients with acute myeloid leukemia (AML) with single-agent subcutaneous azacytidine at 100 mg/m2, on 5 of 28 days for up to six cycles. Treatment was stopped for lack of response, or continued to progression in responders. The primary endpoint was response within 6 months. A response rate ≥ 34% was considered a positive trial outcome. From September 2008 to April 2010, 45 patients from 10 centers (median age 74 [55–86] years) were accrued. Patients received four (1–21) cycles. Best response was complete response/complete response with incomplete recovery of neutrophils and/or platelets (CR/CRi) in eight (18%; 95% confidence interval [CI]: 8–32%.), 0 (0%) partial response (PR), seven (16%) hematologic improvement, 17 (38%) stable disease. Three non-responding patients stopped treatment after six cycles, 31 patients stopped early and 11 patients continued treatment for 8–21 cycles. Adverse events (grade ≥ III) were infections (n = 13), febrile neutropenia (n = 8), thrombocytopenia (n = 7), dyspnea (p = 6), bleeding (n = 5) and anemia (n = 4). Median overall survival was 6 months. Peripheral blood blast counts, grouped at 30%, had a borderline significant association with response (p = 0.07). This modified azacytidine schedule is feasible for elderly or frail patients with AML in an outpatient setting with moderate, mainly hematologic, toxicity and response in a proportion of patients, although the primary objective was not reached.

Predictive factors for all-trans retinoic acid-related differentiation syndrome in patients with acute promyelocytic leukemia

All-trans retinoic acid (ATRA) used for the treatment of APL can lead to the development of differentiation syndrome (DS), a potentially life threatening complication. Since ATRA is metabolized by cytochrome P450 (CYP) enzymes, we sought to identify drug interactions that might be associated with a higher risk for the development of DS in addition to other predictive factors related to the incidence of DS. We identified 60 consecutive patients with APL treated at our institution with ATRA from May 2004 until January 2010. Of the 60 patients identified, 29 (48%) developed DS within a median of 5days (range 1–31) of ATRA initiation. We did not find any difference in overall incidence of DS whether patients were on concurrent CYP 2C8, 2C9 or 3A4 inhibitors, inducers or substrates. In multivariable analysis, higher peripheral blood blast counts on admission (p=0.04) as well as higher body mass index (p=0.003) were associated with developing DS. Out of the 29 patients with DS, there were 4 early deaths of which 2 were attributed to DS compared to no early deaths in the patients who did not develop DS (p=0.05). Regarding disease-related outcomes, only CR rate was different between patients developing DS versus those who did not develop DS.

TdT expression in acute myeloid leukemia with minimal differentiation is associated with distinctive clinicopathological features and better overall survival following stem cell transplantation

The diagnostic criteria for acute myeloid leukemia (AML), not otherwise specified, with minimal differentiation (AML-M0, French–American–British classification), have been refined in the 2008 World Health Organization (WHO) classification. Terminal deoxynucleotidyl transferase (TdT) expression in AML-M0 has been proposed by others as a surrogate for RUNX1 (runt-related transcription factor 1) mutations, a mutation associated with distinct gene expression profiles in AML-M0. In this study, we investigated the significance of TdT expression in AML-M0 cases defined using the 2008 WHO classification criteria. Demographic, laboratory and clinical information were obtained from the hospital medical records. Statistical analysis was performed using Student's t-test, log-rank test and Fisher's exact test. The study group included 30 AML-M0 patients (male:female=19:11; median age: 60 years). In all, 10 cases of AML-M0 were positive for TdT(+) and 20 cases were negative for TdT(−). Patients with TdT+ AML-M0 had higher peripheral blood and bone marrow blast counts compared to patients with TdT− AML-M0 (P=0.01). TdT expression in AML-M0 was not associated with a distinct immunophenotype. Monoclonal IgH and TCR gene rearrangements were frequent, but independent of TdT expression in AML-M0. TdT expression in AML-M0 correlated with trisomy 13 and inversely correlated with aberrations of chromosomes 5 and 17. Among six patients with AML-M0 who received a stem cell transplant, overall survival was significantly longer for the three TdT+ patients compared with the three TdT− patients (P=0.03). In the TdT+AML-M0 subgroup, the three patients with stem cell transplant had better overall survival compared with five patients who did not receive stem cell transplant (P=0.01). We conclude that AML-M0, as currently defined in the 2008 WHO classification, can be divided into two groups based on TdT expression. Although there is a need to assess a greater number of patients, our results suggest that TdT positivity in AML-M0 identifies a subset of patients with a better prognosis after stem cell transplant.

Many questions raised by a question on JAK1/2 inhibitors in primary myelofibrosis

TO THE EDITOR: Firstly, I congratulate Chul Won Jung for the perspectives on the role of JAK1/2 inhibitors in the management of primary myelofibrosis (PMF) [1]. I would like to share some of my thoughts on the same topic. In comparison with conventional chemotherapeutic drugs, the expectations of targeted therapies are high. Imatinib, which targets BCR-ABL, and all-trans retinoic acid (ATRA) that targets PML-RARa, have made major impacts on medicine. It was expected that JAK1/2 inhibitors would have similar, almost magical, effects in JAK-positive myeloproliferative neoplasms. However, the data to date have shown that ruxolitinib, the first of the JAK1/2 inhibitors, has failed to live up to these expectations. Ruxolitinib reduces constitutional symptoms and reduces spleen size in PMF [2]. However, the observations of reduction in constitutional symptoms and the decrease in spleen size after treatment with a JAK1/2 inhibitor in patients with wild-type JAK2 create doubts about the targeted nature of this drug and the role of JAK2 in the pathogenesis of PMF. In addition, it is hard to imagine how ruxolitinib reduces mortality without having a significant impact on the peripheral blood blast count, marrow fibrosis, cytogenetic remission, or reduction of JAK2 V617F allele burden. How does a targeted drug reduce mortality without having a significant effect on the disease biology? It would have been enlightening had this question been addressed; previous articles have not touched upon this topic. Severe splenomegaly has been associated with poor outcomes after hematopoietic stem cell transplant (HSCT). Given this, the study author predicts that ruxolitinib administered prior to HSCT may improve the outcomes of HSCT in PMF. However, as mentioned in the penultimate section of the article, it is difficult to understand how this drug would have any impact on the outcome of HSCT by decreasing the proinflammatory cytokine levels when the mechanism of action for this effect is still unknown. The outcomes of trials of ruxolitinib in PMF have raised many questions and provided a few answers. What is the specificity of ruxolitinib as a targeted drug? Are there any other undiscovered pathways in the pathogenesis of PMF that hinder the action of ruxolitinib? These are some of the many questions that further studies need to answer. In my opinion, then and only then will the question Will JAK1/2 inhibitors change the standard of care for myelofibrosis? be answered appropriately.

Rapid Rate of Peripheral Blood Blast Clearance Accurately Predicts Complete Remission in Acute Myeloid Leukemia

AbstractAbstract 3551 Background:With intensive chemotherapy, many acute myeloid leukemia (AML) patients will enter complete remission (CR). Empirically, the bone marrow is typically examined 14 days after initiating induction therapy, and re-treatment is commonly considered if significant residual blasts remain. However, many patients receiving single induction will enter CR without additional therapy despite substantial amounts or residual marrow blasts on day 14, leaving considerable uncertainty about the value of early marrow assessments. An emerging alternative approach to predict the efficacy of induction therapy includes the assessment of peripheral blood blast (PBB) dynamics. Rapid clearance of PBB, determined either by review of manual differential counts or flow cytometry is predictive of CR (likelihood of 76–90%) and overall survival. We investigated whether mathematical modeling of early PBB dynamics using automated complete blood cell (CBC) counts and the manual differential could further refine our ability to predict CR. Patients and Methods:We identified 111 adult patients with circulating PBB who underwent curative-intent, single-cycle induction chemotherapy for newly diagnosed AML between April 1999 and December 2011. Therapy regimens included “7+3”-like (56.8%), and regimens of similar/higher (29.7%) or lower (13.5%) intensity. PBBs were quantified as WBC count from the automated CBC times percentage of blasts by manual differential (100 cells). Cytogenetic abnormalities, NPM1/Flt3 status, day 14 and recovery bone marrow data were extracted from patient records. In the 62 patients with >3 measurable PBB counts, the rate of PBB clearance was calculated by fitting an exponential decay curve to the data points of absolute PBB counts, starting on day 1 of chemotherapy. This subgroup of patients had similar baseline parameters as the whole group, including age, WBC count at diagnosis, cytogenetic risk, percent of secondary AML, therapy regimens were similar, and CR rates were identical (69 vs. 68%). Results:An exponential decay curve [N(t) = N0×e-xt, with x=decay constant] resulted in an excellent goodness of fit of early PBB dynamics (mean r2=0.93). Rapid PBB clearance was highly predictive of CR achievement, with an optimal cut-off of x=1.4 (corresponding to a 4.2-log reduction in tumor burden if maintained over the course of a weeklong chemotherapy) based on the receiver operating characteristic (ROC) curve. All but 1 of the 27 patients with x>1.4 achieved CR (positive predictive value [PPV]=96%) the only non-responder with x>1.4 had a combination of negative prognostic factors including secondary AML, unfavorable cytogenetics, older age, and lower intensity treatment. PPV of PBB clearance rate of 96% for predicting CR compared favorably to alternative previously published approaches such as day of PBB clearance or percentage of day 14 bone marrow blasts (84 and 85% respectively in our study). Day 14 marrow assessments did not add prognostic information in 26/27 patients who had fast PBB clearance rate (x>1.4). In univariate analyses, CR achievement was significantly correlated with a higher PBB clearance rate, younger age, primary AML, more favorable cytogenetics, and treatment intensity. In multivariate analyses including age, primary vs. secondary AML, cytogenetics, type of therapy, and PBB clearance rate (57 patients), only the PBB clearance rate remained statistically significantly associated with CR achievement. Importantly, information from CBC differentials is routinely available in most institutions and associated costs are low. Unlike determination of the exact day of PBB clearance that coincides with profound cytopenia, the PBB clearance rate is measured while PBB are still abundant, rendering the latter method less prone to sampling and observer errors. Conclusion:our findings suggest that early marrow assessment may not be necessary in AML patients who experience rapid PBB clearance upon induction treatment initiation as they have an almost 100% chance of achieving CR. Disclosures:Vainstein:Neumedicines Inc: Employment.

Proteomic Profiling of AML Bone Marrow-Derived Mesenchymal Stem Cells (MSC) Reveals Marked Differences From Normal MSC.

Abstract 2355The interaction of bone marrow (BM) mesenchymal stem cells (MSC) and acute myeloid leukemia (AML) cells creates a microenvironment (McrEnv) that supports and regulates the survival and proliferation of leukemic cells. These same BM McrEnv interactions can also create a sanctuary that protects subpopulations of AML blasts from chemotherapy. The mechanisms by which the BM-MSC McrEnv effects these changes remain unclear and the heterogeneity of these effects across different subtypes of AML (FAB, WHO or cytogenetics) is unknown. Furthermore, the functional differences between normal BM-MSC and AML BM-MSC biology are undefined. To address this we set out to perform proteomic profiling comparing normal and AML BM derived-MSC and to ascertain how AML BM-MSC protein expression patterns correlated with AML blast protein expression. We cultured BM samples for 4 to 8 weeks in MEM-alpha media with 20% fetal calf serum to isolate AML-MSC (n=106), and NL-MSC (n=70). Cells defined as MSC were positive for CD90 and CD105 and negative for CD45 by flow cytometry. Whole cell protein lysates were prepared. Matched AML protein samples prepared from mononuclear cell fractions from the same BM collection were available for most cases (n=96). We generated a custom Reverse Phase Protein Array (RPPA) from these samples and probed the array with 151 validated antibodies. Statistical analysis was performed by two-way ANOVA using Tukey's test to identify differential expression of individual proteins between the samples and Ingenuity pathway analysis was performed to elucidate differential pathway utilization.Comparison of AML-BM-MSC to NL-BM-MSC demonstrated similar levels of expression for 66 proteins (43.7%) but 85, 67 and 28 were different at the p-value of < 0.05, < 0.01 and <0.0001 with a false discovery rate (FDR) of 0.05, 0.01, 0.0001 respectively. By function, the 28 proteins with greatest differential expression (Italic = lower in AML-MSC) included cell cycle regulators (P21, Cyclin D1, CDK4), adhesion/integrins (CD49b, CD31, and galectin 3, signaling pathway members and their targets (Smad1, Smad4, Stat1, Stat5, pPDK1, GSK3), apoptosis regulators (Bak, BCLXL, Smac,) growth and proliferation regulators (pCREB, EIF2α, FOXO1α, Sirt1 Strathmin) and pIRS, cleaved Notch1, HSP90, TP53 CK2 and PP2A. Using mixed linear effectors protein expression levels in AML-MSC did not show correlation with patient’s age (< 50 >), gender, blast count in BM or peripheral blood, or the percent of CD34 positivity. Protein expression in AML-BM-MSC from cases with favorable cytogenetics had significantly lower levels of GAB2, P27 P70S6K, SMAC and 14.3.3e and cases with unfavorable cytogenetics had significantly lower levels of antiapoptosis proteins Bax, Bad and BCL-XL and higher levels of Smac as well as lower levels of phosphor-FOXO3a and pELK. Levels of ARC were higher in cases with intermediate cytogenetics. Ingenuity pathway analysis also demonstrated differential utilization of several families of proteins regulating signal transduction, apoptosis and transcription and connected to surface growth factor receptors and adhesion molecules. As anticipated for cells of different origin, the expression patterns were completely different between AML BM-MSC and AML blasts for 131 of 151 proteins (86.1%) (Tukey’s p-value <0.0001 and FDR 0.0001). These results suggest that protein expression in AML MSC is markedly different from that of NL-MSC. Differential expression was observed in multiple functional groups suggesting that AML-MSC are functionally distinct from NL-MSC. Since MSC influence adjacent and nearby AML blasts it is likely that these variances impact AML blast biology. Additional analysis is underway to determine if recurrent patterns of protein expression exist in AML-BM-MSC, how these differ from protein expression patterns in NL-MSC, and whether AML-BM-MSC protein expression patterns correlate with AML-blast protein expression patterns. Correlation between MSC patterns and AML-blast patterns would provide therapeutically targetable sites in MSC that could be exploited to influence AML blast biology. Disclosures:No relevant conflicts of interest to declare.

Predicting Duration of Neutropenia After Successful Intensive Induction Chemotherapy for Acute Myeloid Leukemia or High-Grade Myelodysplastic Syndromes

Abstract 4336 Background:Chemotherapy-induced neutropenia (CIN) is a major cause of morbidity and mortality in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) due to the risk of overwhelming infection. Although the risk for infections increases with longer duration of neutropenia, no attempts have been undertaken to predict time of neutrophil recovery in AML/MDS patients following intensive chemotherapy. In unpublished work, we have successfully used mathematical modeling of peripheral blood blast kinetics following induction chemotherapy to accurately predict the likelihood of subsequent complete remission (CR). In this retrospective study, we investigated whether data derived from mathematical modeling of early peripheral blood neutrophil or blast dynamics could predict the duration of CIN in AML/MDS patients undergoing intensive induction chemotherapy. Patients and Methods:We retrospectively analyzed a cohort of 59 consecutive patients with newly diagnosed AML or MDS who achieved CR after induction chemotherapy after 1 course of chemotherapy with a 7 + 3-like regimen. Daily peripheral blood neutrophil and blast counts were recorded from the initiation of chemotherapy until the day of neutrophil recovery (defined as an absolute neutrophil count [ANC] ≥ 500 cells/μL). In patients with ≥ 3 measurable neutrophil or blast counts within the first five days of data collection, the rate of peripheral blood neutrophil and blast clearance was calculated by fitting an exponential decay curve to the data points starting on day 1 of chemotherapy. Results:The average age of the study cohort was 54 years (range 25 to 78). Seventy-six percent had primary AML. Cytogenetic risk was favorable in 32%, intermediate in 37%, and adverse in 27% of patients. The 23 patients with initial ANC < 500 cells/μL had a significantly longer duration of neutropenia than the 36 patients with initial ANC ≥ 500 cells/μL (mean: 29 versus 26 days, range: 21 – 39 versus 21 – 34, p=0.045). Neither age nor percentage of blasts in the bone marrow at diagnosis predicted neutropenia duration. In patients for whom decay rates could be modeled, there was no association between duration of neutropenia and decay rate for neutrophils (n = 36) or blasts (n = 20). There was also no association with the day that neutrophils first started to decline (defined as the first drop < 70% of the initial neutrophil count) or the first day of blast clearance from peripheral blood. Seven patients received granulocyte colony-stimulating factor (G-CSF; 1 prior to chemotherapy for antecedent myelodysplastic syndrome and 6 between days 15 and 43 for prolonged neutropenia and/or febrile neutropenia); the duration of neutropenia in this group did not differ significantly from that of the cohort as a whole. Conclusion:While kinetics of normal blood counts following initiation of intensive chemotherapy do not appear helpful to predict the duration of subsequent neutropenia, pre-treatment ANC levels provide limited information, with patients who are severely neutropenic prior to induction chemotherapy for AML having a longer average duration of CIN. This observation may provide some clinical guidance on the risk-adapted use of G-CSF in patients with AML/MDS undergoing induction chemotherapy. Disclosures:Vainstein:Neumedicines Inc: Employment.

Spliceosome Mutations Involving SRSF2, SF3B1 and U2AF35 in World Health Organization Defined Chronic Myelomonocytic Leukemia; Prevalence, Clinical Correlates and Prognosis

AbstractAbstract 1711 Background:Mutations in genes of the splicing machinery, such as SF3B1, SRSF2 and U2AF35 are common in patients with myelodysplastic syndromes [MDS] (Nature 2011;478:64) and chronic myelomonocytic leukemia [CMML] (Haematologica 2012;Epub). In MDS, SRSF2 gene mutations are an independent risk factor for shortened over-all (OS) and leukemia-free survival (LFS) (Blood 2012;119:3578). In MDS with ring sideroblasts (RS), SF3B1 mutations have a high prevalence (∼50%), but do not influence either, the OS or the LFS (Blood 2012;119:569). We carried out this study to evaluate the prevalence, clinical correlates and prognosis of the aforementioned spliceosome mutations in CMML. Methods:The study included 227 patients with WHO defined CMML who were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. DNA was interrogated in the three most frequent spliceosome genes with somatic mutations; SRSF2, SF3B1 and U2AF35. Results I:Prevalence and clinical correlatesAmong the 227 study patients, 153 (67%) were male, median age was 71 years (range, 17–90 years) and 192 (85%) met the WHO criteria for CMML-1. Ninety (40%) patients had SRSF2 mutations (86% CMML-1), 13 (6%) had SF3B1 mutations (75% CMML-1) and 20 (9%) had U2AF35 mutations (95% CMML-1). One-hundred and twenty three (54%) patients had at least one of three spliceosome mutations (86% CMML-1). Mutational hot spots were P95 for SRSF2 (P95L-n=36/H-n=32/R-n=13/A-n=1), K700E (n=7) and H662Q (n=2) for SF3B1, and Q157 (Q157R-n=5/P-n=5/G-n=1) and S34F (n=7) for U2AF35. Seven patients (54%) with SF3B1 mutations had ≥1% RS, with 5 (38%) showing ≥15% RS. Mutations involving all three spliceosome genes were mutually exclusive. The cytogenetic distribution based on the Spanish risk stratification system (Haematologica 2011;96:375) was; SRSF2 mutations: 69 (77%) low risk, 11 (12%) intermediate risk, and 10 (11%) high risk (+8-n=3, del/monosomy 7-n=2, monosomal karyotype-n=5); SF3B1 mutations: 8 (62%) low risk and 5 (38%) intermediate risk; U2AF35 mutations: 15 (75%) low risk, 3 (15%) intermediate risk and 2 (10%) high risk (p=0.89). The distribution of mutations according to the MD Anderson prognostic scoring system [MDAPS] (Blood 2002;99:840) was; SRSF2 - low-n=41, intermediate-1-n=26, intermediate-2-n=18, high-n=5, SF3B1- low-n=7, intermediate-1-n=3, intermediate-2-n=2, high-n=1, and U2AF35- low-n=11, intermediate-1-n=5, intermediate-2-n=3, high-n=1 (p=0.73). There was no statistically significant difference, among the three mutation groups, in prognostically relevant parameters, including gender distribution, median age, hemoglobin values, platelet counts, peripheral blood (PB) and BM blast counts, absolute neutrophil counts (ANC) and absolute monocyte counts (AMC). The only notable difference was that patients with the SF3B1 mutation had a lower median white blood cell count (p=0.04) and a lower absolute lymphocyte count (p=0.045). Results II:Prognostic impact of spliceosome mutationsAt a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. Median survivals for patients with mutations involving SRSF2, SF3B1 and U2AF35 were 24, 17 and 12 months, respectively. In univariate analysis, the presence of SRSF2 (p=0.67), SF3B1 (p=0.96) or U2AF35 (p=0.49) mutations had no prognostic impact on OS. Similarly, none of the three spliceosome mutations affected LFS; corresponding p values were 0.55 for SRSF2, 0.9 for SF3B1 and 0.38 for U2AF35 mutations respectively. We then examined possible prognostic value of having none of these mutations (n=104) vs otherwise (n=123) and the results were once again negative (p=0.87). Conclusions:SRSF2 is the most frequently mutated spliceosome gene in CMML, but neither it nor SF3B1 or U2AF35 mutations affect overall or leukemia-free survival in CMML. Furthermore, the current study suggests limited genotype-phenotype association, save for the already established association between SF3B1 mutations and RS. Disclosures:No relevant conflicts of interest to declare.

Prognostic Interactions Between SRSF2, ASXL1, and IDH Mutations in Primary Myelofibrosis and Determination of Added Value to Cytogenetic Risk Stratification and DIPSS-Plus

AbstractAbstract ▪430▪This icon denotes a clinically relevant abstract Background:The Dynamic International Prognostic Scoring System (DIPSS-plus) uses eight risk factors to predict overall survival (OS) in primary myelofibrosis (PMF): unfavorable karyotype, peripheral blood (PB) blast count ≥1%, platelet count <100 × 109/L, white blood cell count (WBC) >25 × 109/L, hemoglobin level <10 g/dL, red blood cell transfusion need, constitutional symptoms, and age >65 years (JCO 2011;29:392). Among these risk factors, karyotype holds a dominant role in predicting both overall (OS) and leukemia-free (LFS) survival (Blood 2011;118:4595). More recently, certain PMF-associated somatic mutations (IDH, EZH2, ASXL1, SRSF2) have been reported (in print or at recent meetings) to carry prognostic relevance for OS or LFS in PMF while other mutations (JAK2, MPL, TET2, SF3B1) did not. The purpose of the current study was to examine the individual and combined prognostic relevance of the former in the context of cytogenetic risk stratification and DIPSS-plus. Methods:An updated Mayo Clinic database of 1008 karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify study patients in whom DNA at time of referral was available for mutation analysis. OS and LFS were calculated from the time of referral, which was also the time of study sample collection. Results:In an ongoing project, a total of 397 PMF patients have so far been studied for IDH1/2 (n=367), SRSF2 (n=355), ASXL1 (n=260) and EZH2 (n=43) mutations. The corresponding mutational frequencies were 5.4% (20/367), 14.4% (51/355), 31.2% (81/260) and 7% (3/43). These mutations were not exclusive of either each other or JAK2 or MPL mutations and a significant clustering was noted between SRSF2 and IDH mutations; IDH mutational frequency was 21% (10/48) in SRSF2-mutated versus 3% (9/279) in unmutated cases (P<0.0001). Mutational hotspots were P95 in exon 1 for SRSF2 and exons 1, 2 and 5 for ASXL1.OS and LFS effect have so far been examined for ASXL1, SRSF2 and IDH 1/2 mutations; in univariate analysis, all three mutations significantly affected OS (ASXL1 RR 1.9, 95% CI 1.4–2.7; SRSF2 RR 1.7, 95% CI 1.2–2.5; IDH 1/2 RR 1.8, 95% CI 1.1–3.1) whereas only IDH 1/2 (RR 5.5, 95% CI 2.5–11.8) and SRSF2 (RR 3.5, 95% CI 1.7–7.0) but not ASXL1 (p=0.29) mutations affected LFS. The observed prognostic relevance for OS was sustained during multivariable analysis that included ASXL1 with either SRSF2 or IDH mutations, as co-variates, whereas both IDH and SRSF2 mutations remained significant for LFS.Based on the above observations, study patients were classified into two groups: i) unmutated for ASXL1, SRSF2 and IDH1/2 (all unmutated; n=146) and ii) mutated for at least one of the three mutations (one or more mutated; n=130); the latter group constituted 62%, 52%, 18% and 27% of DIPSS-plus high, intermediate-2, intermediate-1and low risk patients, respectively (p<0.0001). The corresponding rates for patients with favorable and unfavorable karyotype were 47% and 43% (p=0.7) and for normal vs. abnormal karyotype were 53% and 36% (p=0.008). The “all unmutated group”, compared to the “one or more mutated group” enjoyed a significantly superior OS (RR 0.5, 95% CI 0.4–0.7) and LFS (RR 0.3, 95% CI 0.2–0.7) that was DIPSS-plus-independent for OS (p=0.03) and karyotype-independent for LFS (p=0.004). The prognostic value of distinguishing “all unmutated” from “one or more mutated” patient groups was most evident for DIPSS-plus intermediate-2 or lower risk groups (p=0.002) and its added value for high-risk group was limited (p=0.23) (Figure). Conclusions:A concomitant analysis strategy reveals that SRSF2, ASXL1 and IDH mutations carry inter-independent prognostic relevance for overall or leukemia-free survival in PMF. Any one of these three mutations is more likely to occur in patients with normal karyotype and such an event predicts shortened survival that is not accounted for by DIPSS-plus and a higher risk of leukemic transformation that is independent of karyotype. The current study suggests that such information might be useful in treatment decisions involving intermediate-2 or lower risk patients. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Compassionate Use Program (CUP) with Ruxolitinib in Mexican Patients with Primary Myelofibrosis (PMF), Post – Polycythemia, Vera Myelofibrosis (PPV – MF), and Post–Essential Thrombocythemia Myelofibrosis (PET – MF)

AbstractAbstract 5067 IntroductionMyelofibrosis (MF) is a myeloproliferative neoplasm (MPN) that is characterized by ineffective hematopoiesis and symptomatic burden such as cytopenias, and splenomegaly. The prevalence of MF symptoms is relatively uniform across the 3 main subtypes: primary MF (PMF), post – polycythemia vera MF (PPV MF), and post – essential thrombocythemia MF (PET MF). Available therapies (AT) in Mexico, includes hydroxyurea (HU), interferon (IFN), for ET, besides phlebotomy for PV, as well as androgens, steroids, chemotherapy, IFN, thalidomide, EPO and radiotherapy for PMF. The only potentially curative therapy is allogeneic hematopoietic stem cell transplantation (HSCT) but treatment-related mortality remains high. Recently ruxolitinib an oral Janus kinase–2 inhibitor (JAK-2) has been approved to treat patients with MF. The safety and efficacy of Ruxolitinib has been evaluated 528 patients from COMFORT I and COMFORT II trials. ObjectiveTo report the first clinical experience in a CUP with an oral JAK–2 Inhibitor (ruxolitinib) in 40 MF patients refractory to AT in Mexico. Patients and MethodsPMF patients and PPV/PET MF eligible for the ruxolitinib CUP, were diagnosed according to the 2008 WHO criteria, irrespective of JAK2 mutation status; classified as high risk; intermediate risk level 2; or, intermediate risk level 1 with an enlarged spleen; with a peripheral blood blast count of < 10%; adequate renal and liver function, platelet count >100×109/L, all of them were treated with best available therapies in Mexico. Therapy with ruxolitinib was administered to all patients. Doses were adjusted according to the platelet counts.Clinical and demographic characteristics were assessed at baseline and during the follow-up. The analyzed characteristics were: Demographic: age, gender; Clinical: Risk group, Bone marrow fibrosis grade, spleen size, ECOG, MPN subtype, JAK mutation, CBC counts;Spleen size at baseline and at week 20 of treatment. ResultsThe median age was 64. 2 (Interval 41 – 75). Gender distribution was 46% female and 54% male. Patients according to the International Prognostic Scoring System (IPSS) were distributed in the following risk category: 10% low risk; 53. 3% intermediate – 1 risk; 26. 7 % intermediate – 2 risk; and 20% high risk. Bone marrow fibrosis grade distribution was: Grade I 46. 15%; Grade II 23. 07%; Grade III 30. 76%. Spleen length below costal margin: <10 cm, 80. 7%; 10 to <20 cm, 11. 5% and > 20 cm, 7. 7% of patients. Spleen Average size was 9. 58 cm below costal margin at the beginning of treatment. ECOG 0 was present in 15% of patients; ECOG 1, 73%; and ECOG 2, 12% of patients. The disease subtype distribution was: PMF 45%; PPV-MF 36% and PET-MF 46%. JAK mutation was positive for 19% of patients, 46% were negative, and 35% did not have mutation analysis.The findings from the baseline to week 20 were: Splenomegaly decreased from 9. 58 cm (avg) to 4. 5 cm (avg) (53. 02% of reduction); Median Hemoglobin level was 13. 2 gr/dL at baseline versus 10. 24 gr/dL (22. 42% of decrease). Median Platelet count was 437, 000/mm3 at baseline versus 341, 503/mm3 at week 20 of treatment (21. 85% of decrease).Median time of treatment is 20. 1 weeks.Hematologic adverse events presented: Hemoglobin level decrease events (CTCAE grade 2 and 3) resolved with transfusion support. Platelet count decreases (CTCAE grade 2) were transitory and resolved after two weeks of treatment interruption. ConclusionData showed demonstrate principal characteristics of this MF cohort. Until recently, most treatments provided only palliative care with no single treatment addressing all of the complications and symptoms of the MF. Ruxolitinib showed a primary therapeutic benefit as reduction in splenomegaly and significant improvement in MF-related symptoms in this cohort of Mexican patients with myelofibrosis. Disclosures:No relevant conflicts of interest to declare.

Predictors of Greater Than 80% Two-Year Mortality in Primary Myelofibrosis: A Mayo Clinic Study of 884 Karyotypically-Annotated Cases

Abstract▪2806▪This icon denotes a clinically relevant abstract Background:A high degree of prognostic certainty is required to recommend or discourage high risk treatment procedures in primary myelofibrosis (PMF). The Dynamic International Prognostic Scoring System (DIPSS-plus) uses eight risk factors to predict overall survival (OS) in PMF: unfavorable karyotype, peripheral blood (PB) blast count ≥1%, platelet count <100 × 109/L, white blood cell count (WBC) >25 × 109/L, hemoglobin level <10 g/dL, red blood cell transfusion need, constitutional symptoms, and age >65 years (Gangat et al. JCO 2011;29 :392). The presence of four or more of these risk factors defines high-risk disease. purpose:The purpose of the current study was to enhance the prognostic weight of some of the DIPSS-plus risk factors with the intent to identify one or two parameters that can reliably predict death in the first two years of disease. Methods:An updated Mayo Clinic database of 884 karyotypically-annotated patients with PMF was used. Calculations of 2-year mortality rates and variables considered for prognostic value were from time of referral to the Mayo Clinic. Cytogenetic risk categorization per DIPSS-plus was further refined to capture additional prognostic information from monosomal karyotype (MK) (Vaidya et al. Blood 2011;117 :5612) and inv(3)/i(17q) abnormalities (Caramazza et al. Leukemia 2011;25 :82). Receiver operating characteristic (ROC) analysis was employed to define best discriminant levels. Results:To date, 564 (64%) deaths have been documented. Each one of the aforementioned DIPSS-plus risk factors was associated with a 2-year mortality rate that ranged from 42% (PB blast count ≥1%) to 60% (unfavorable karyotype). High-risk disease per DIPSS-plus was associated with 57% two-year mortality. The only risk factors that were associated with >80% two-year mortality were MK (n =19) and inv(3)/i(17q) abnormalities (n =8) and both were associated with significantly worse survival, compared to other unfavorable karyotype (n =102): HR (95% CI) of 5.1 (3.1−8.4) and 3.9 (1.7−8.8), respectively. ROC analysis identified PB blast counts of 2% and 9% (AUC 0.62) and WBC of 43 × 109/L (AUC 0.66) as best discriminant levels for predicting 2-year mortality; OS was significantly worse in the presence of PB blast >9% (HR 4.1, 95% CI 2.8–6.1) vs. 2% to 9% (HR 1.8, 95% CI 1.5–2.2) vs. <2%; the corresponding 2-year mortality rates were 73%, 46% and 25%. OS was also significantly worse in the presence of WBC ≥40 × 109/L (HR 2.8, 95% CI 2.2–3.6) vs. 26–39 × 109/L (HR 1.6, 95% CI, 1.2–2.1) vs. <26 × 109/L; the corresponding 2-year mortality rates were 63%, 42% and 28%. Two-year mortality rates exceeded 80% in the presence of any two of the following: PB blast count >9%, WBC ≥40 × 109/L or unfavorable karyotype other than MK or inv(3)/i(17q). Conclusions:A greater than 80% 2-year mortality in PMF is predicted by the presence of MK, inv(3)/i(17q) abnormalities, or any two of the following: PB blast >9%, WBC ≥40 × 109/L, other unfavorable karyotype. Such patients and those with high DIPSS-plus risk should be referred to allogenic stem cell transplantation earlier than later. Disclosures:No relevant conflicts of interest to declare.

IKZF1 Deletion Is Strongly Associated with Risk of Relapse in Intermediate Risk Group in JACLS ALL02 Cohort

Abstract 1455▪▪This icon denotes a clinically relevant abstract Introduction:Despite the progress of the current therapy, approximately 20 % of pediatric patients with B-cell-presursor (BCP)-ALL experience relapse who had no conventional adverse prognostic factor. Recently, alteration of the IKZF1 gene has been reported to be associated with a poor outcome in pediatric BCP-ALL without BCR-ABL. In addition, it is also reported that a significant proportion of these cases with the alteration of IKZF1 shared the point mutation of JAK2 and over-expression of cytokine receptor-like factor 2 (CRLF2). Herein, in order to assess the prognostic value of these genetic abnormalities in Japanese cohort, we conducted genetic analysis of IKZF1, JAK2 and CRLF2 in pediatric BCP-ALL. Materials and Methods:Diagnostic bone marrow or peripheral blood samples of 215 pediatric BCP-ALL patients treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol from April, 2002 to May, 2008 were examined in this study. All patients were categorized into high risk (HR) group defined as follows;(1) initial white blood cell counts more than 10,000 /μl, (2) age at diagnosis is > 10 years and (3) good response to initial predonisolone (PSL) treatment ( blast counts in peripheral blood is less than 1,000 /μl after one week PSL treatment). Ph+ALL and infantile ALL patients were excluded in this protocol. The patients with Down syndrome were also excluded in this genetic analysis. The deletion of IKZF1 was determined using multiplex ligation-dependent probe amplification (MLPA) in 212 patients whose diagnostic DNA samples were available. The expression of isoform of IKZF1 was determined by RT-PCR in 113 patients whose diagnostic RNA samples were available. The expression level of CRLF2 was also determined by real time RT-PCR in 112 patients and over-expression was defined as over ten times more than median expression value. The presence of P2RY8-CRLF2 fusion was examined by RT-PCR or MLPA in the patients with CRLF2 over-expression or IKZF1 deletion. JAK2 mutations were also determined by direct sequencing of the exon 16, 20 and 21 in the patients with IKZF1 deletion. Results:The deletion of IKZF1 gene was present in 19 of 212 (9.0 %) patients. In detail, the mono-allelic deletion of entire IKZF1 gene was present in 10 of 19 patients. On the other hand, the expression of dominant-negative IK6 isoform was present in 9 of 112 (8.0%) patients including 12 patients with IKZF1 deletion. The expression of IK6 was present in 4 of 12 (33.3%) IKZF1 deleted patients. Interestingly, 5 of 9 patients with IK6 expression had no alteration of IKZF1 gene. In terms of CRLF2, over-expression was detected in 16 of 112 patients (14.3 %). However, P2RY8-CRLF2 fusion was not detected in these 16 patients with altered CRLF2 expression. Strikingly, none of the patients with IKZF1 deletion (n=19) had either P2RY8-CRLF2 fusion or JAK2 exon 16, 20 and 21 mutation. Patients with IKZF1 gene deletion had significantly worse relapse rate than those without IKZF1 deletion (7/19 vs 22/193, p<0.01). On the contrary, none of the patients with IK6 expression experienced relapse. Discussions:This study confirmed that the presence of IKZF1 deletion was strongly correlated with risk of relapse in intermediate risk group in JACLS ALL02 cohort. Thus, we expect that IKZF1 deletion is an independent predictor of treatment outcome and represents a candidate of prognostic marker to be integrated in future algorithms for early risk stratification in pediatric BCP-ALL. Strikingly, point mutation of JAK2 exon 16, 20 and 21 or P2RY8 -CRLF2 fusion was rarely present even in BCP-ALL patients with IKZF1 deletion. There might be unrevealed class I mutation cooperating with IKZF1 alteration in Japanese BCP-ALL cohort. Disclosures:No relevant conflicts of interest to declare.

Targeting mTOR and JAK2 in Xenograft Models of CRLF2-Overexpressing Acute Lymphoblastic Leukemia (ALL)

Abstract 249CRLF2 genomic rearrangements that lead to overexpression have been identified in a subset of children with clinically high-risk B-precursor ALL and are highly associated with activating JAK2 mutations (Harvey et al., Blood 2010; Mullighan et al., PNAS 2009 and Nat Genet 2009). These children frequently respond poorly to current intensive cytotoxic chemotherapy regimens and suffer high rates of relapse and mortality. New therapies for these patients are urgently needed. These leukemias exhibit gene expression profiles similar to those of BCR-ABL1 positive ALL, suggesting aberrant kinase activation. We previously demonstrated aberrant JAK/STAT and PI3K/mTOR signaling in CRLF2-overexpressing ALL cell lines and primary human samples in vitro, and thus hypothesize that inhibition of these hyperactive signaling networks has therapeutic relevance.To further characterize this high-risk subset of ALL, we have established multiple xenograft models of CRLF2-rearranged and JAK2-mutated ALL, providing a robust platform for preclinical testing of signal transduction inhibitors. In this model, primary human ALL samples are intravenously injected into NOD/SCID/γc null (NSG) mice, and engraftment is determined by flow cytometry of peripheral blood for human CD19+/CD45+ blasts. Eighteen of 21 primary cryopreserved specimens provided by the Children’s Oncology Group engrafted successfully. In order to biochemically characterize the xenografts, we measured phosphorylation of relevant signal transduction proteins by phosphoflow cytometry. Spleens of mice xenografted with CRLF2-rearranged ALL had uniformly increased surface staining of human CRLF2, a component of the heterodimeric receptor complex for the cytokine, thymic stromal lymphopoeitin (TSLP). In vitro stimulation of the CRLF2-overexpressing ALL xenograft spleens with TSLP induced phosphorylation of STAT5, Akt, S6, and 4EBP1, but not of ERK 1/2. In additon, in vitro JAK inhibition with INCB018424 abrogated TSLP-induced JAK/STAT and PI3K/mTOR signaling. The mTOR inhibitor sirolimus, mTORC1/2 inhibitor PP242, and PI3K/mTOR inhibitor PI-103 potently inhibited phosphorylation of S6 and 4EBP1 in these xenograft specimens. These data suggest that the JAK/STAT and PI3K/mTOR pathways may interact in these CRLF2-overexpressing leukemias.These data led us to hypothesize that inhibition of the PI3K/mTOR or JAK/STAT pathways may represent potential therapeutic targets; therefore, we utilized these very high-risk ALL xenograft models to study novel, targeted therapies. Once xenografts had engrafted with sufficient disease burden to detect >5% peripheral CD19+/CD45+ blasts, mice were randomized to receive the mTOR inhibitor sirolimus, the JAK inhibitor INCB018424, or vehicle for three to four weeks. Disease burden was assessed weekly by flow cytometric determination of CD19+/CD45+ blast count in peripheral blood, and at sacrifice, by spleen CD19+/CD45+ blast count. To assess potential differential efficacy based on CRLF2 overexpression (CRLF2+) and/or JAK2 activating mutations (JAK2mut), we treated mice with each subtype of ALL. Sirolimus induced a significant decrease in peripheral blast count in 7 of 7 primary ALL xenografts tested (2 JAK2mut/CRLF2+ samples, 1 JAK2mut/CRLF2- sample, 2 JAK2wt/CRLF2+ samples, and 2 JAK2wt/CRLF2- samples) and a significant decrease in spleen blast count in 6 of 7 samples tested. The most profound reduction of disease burden was seen in the JAK2mut/CRLF2+ leukemias. In addition, the JAK inhibitor INCB018424 decreased peripheral blast count and spleen blast count in a JAK2mut/CRLF2+ xenograft. We next determined if sirolimus conferred a survival advantage in xenografts of 2 ALL specimens, a robust responder and an intermediate responder to sirolimus by blast count. Sirolimus treatment significantly prolonged survival of both xenografts (63 days vs. 23 days, p=0.0015; 91 days vs. 58 days, p=0.0027). Additional human ALL xenograft studies of INCB018424 and other kinase inhibitors are ongoing.The preclinical in vivo efficacy of sirolimus and INCB018424 suggests that novel, targeted therapies have therapeutic potential in CRLF2-overexpressing ALL. Based in part on these data, both INCB018424 and temsirolimus (a parenteral ester of sirolimus) are currently being investigated in multi-center early phase clinical trials for children with relapsed or refractory leukemias. Disclosures:No relevant conflicts of interest to declare.