Higher Peripheral Blood Blast Research Articles

Abstract B03: Acute megakaryoblastic leukemia is associated with poor overall survival and enriched in JAK2 variants

Abstract In this study, we aimed to characterize the molecular mechanisms of two very rare subtypes of AML: acute megakaryoblastic leukemia (AMKL) and acute panmyelosis with myelofibrosis (APMF). Our goal is to describe the clinical-pathologic features of APMF and explore the genomic landscape AMKL to further understand these rare malignancies. We identified 35 patients diagnosed with AMKL (n=27) and APMF (n=8) at Mayo Clinic between 1995 and 2015 by retrospective chart review. For 10 of the AMKL patients, tissue was available for whole-exome sequencing and was analyzed in comparison to a published AML reference population. The median overall survival was shorter in patients with AMKL (3.9 months, 95% CI 1.7-7.4) than in patients with APMF (14.3 months, 95% CI 0.7-NE, p = 0.026,). For both AMKL and APMF, patients who underwent allogeneic hematopoietic stem cell transplantation had better overall survival (14.3 months, 95% CI 1.9-NE) compared to those not undergoing transplantation (4.6 months, 95% CI 1.5-7.4, p = 0.003). Patients with AMKL presented more frequently with splenomegaly, with higher peripheral blood blast counts, and with higher serum lactate dehydrogenase concentrations. 9 out of 10 AMKL patients with available tissue for sequencing demonstrated variants in 22 of 35 AML driver genes, with JAK2 V617F as the most common mutation. Although our small study numbers preclude firm conclusions, it does reflect the rarity of these AML subtypes. Furthermore, inferior survival in AMKL (compared to APMF) was reflected in its proliferative disease features such as splenomegaly, high blast counts, and high lactate dehydrogenase. Interestingly, the presence of JAK2 V617F variants in 40% of the interrogated samples was higher than expected. Although JAK2 is a known molecular driver in myeloproliferative neoplasms, it is actually very rare in de novo AML (~1%). Despite improved survival with allogeneic stem cell transplant, we can conclude that treatment outcomes for AMKL and APMF with standard-of-care therapy were sobering and that patients with these rare AML subtypes should undergo molecular evaluation to qualify them for clinical trials with novel targeted therapies. Citation Format: Marissa Li, Moritz Binder, Basma Basha, Ben Zhang, Alejandro Ferrer, Yuanhan Liu, Mrinal Patnaik, David Viswanatha, Naseema Gangat. Acute megakaryoblastic leukemia is associated with poor overall survival and enriched in JAK2 variants [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B03.

Blastic transformation in Mexican population with chronic myelomonocytic leukemia treated in a tertiary referral center.

e18566 Background: Chronic myelomonocytic leukemia (CMML) is the most aggressive of chronic leukemias, with a short overall survival and a high transformation rate to acute leukemia. We investigated factors related to blastic transformation in Mexican population treated in a tertiary referral center Methods: Records of patients diagnosed with CMML between 2000-2015 were reviewed; patients with incomplete data were excluded. IBM SPSS Statics 21.0 software was used to perform statistical analysis. Results: 54 patients were included, with a median age of 71 years and an overall survival of 16 months. The rate of blastic transformation found was 33% (18 patients), with a time to progression of 9 (0-87) months. Comparing patients who didn’t underwent blastic transformation to those who did, those who progress to acute leukemia tend to be younger (58 vs. 71 years, p = 0.001), have a higher peripheral blood blast count (2% vs. 0%, p = 0.003), where more likely to have immature myeloid precursors circulating in peripheral blood (94% vs. 64%, p = 0.02). In multivariate analysis, age continued to be statistically significant (HR 0.97, 95% IC = 0.929-0.987). There where no statistical difference in the two groups regarding hemoglobin levels (8.2g/dL vs. 10.1g/dL) platelets count (115 X 109 vs. 93 X 109), absolute neutrophil count (5.83 X 109 vs. 5.18 X 109), absolute monocyte count (3.09 X 109 vs. 2.680 X 109), and bone marrow blast count (0 vs. 2%). Cytogenetic considered as high risk was not predictor of blastic progression. Intensive chemotherapy was offered to 7(38.9%) patients, with a complete response rate of 0%, the overall survival was 1.4 months (0-9). Conclusions: Contrary to other reported series;Mexican patients with CMML that progresses to overt acute leukemia were considerably younger, with a higher tumor burden and a very short overall survival. In this population, it is important to consider more aggressive treatments at diagnosis, focusing in high dose chemotherapy and hematopoietic stem cell transplantation in a short term ratter than watching and waiting or using agents that do not impact in disease natural history.

Clinical Associations of Cytogenetic Abnormalities in Patients with Primary and Post-Essential Thrombocythemia and Post-Polycythemia Vera Myelofibrosis

Introduction: Several studies have evaluated the incidence and clinical relevance of cytogenetic abnormalities in primary myelofibrosis (PMF). However, little is known about the clinical correlation of these abnormalities in patients with post-essential thrombocythemia/polycythemia vera myelofibrosis (PET/PPV-MF).Objective: We aimed to measure the frequency of cytogenetic abnormalities (Abn) and describe their clinical significance in patients with PMF and PET/PPV-MF seen at our center.Methods: We retrospectively reviewed the charts of 1100 patients with MF (766 PMF, 354 PET/PV-MF) who were referred to our institution between 1984 and 2013. Diagnoses were made according to 2008 WHO criteria. Cytogenetic analysis done at the time of referral was reported according to the International System for Human Cytogenetic Nomenclature. Statistical analysis considered parameters at the time of cytogenetic studies. Complex karyotype (CK) was defined as 3 or more unrelated abnormalities (Abn) and monosomal karyotype (MK) was defined as 2 autosomal monosomies or a single monosomy with at least one additional structural abnormality. Fisher's exact, Kruskal-Wallis and Mann-Whitney tests were used for comparisons of categorical and continuous variables, respectively. The Kaplan-Meier analysis with log rank test were used for OS comparisons.Results: Cytogenetic data with > 10 analyzable metaphases were obtained in 981 (89%) patients. Overall, 360 (37%) patients had an abnormal karyotype. Frequency and types of cytogenetic Abn were similar between PMF and PET/PPV-MF (Table 1). Sixty one percent of patients (n=220) had a single Abn, 25% (n=89) had two, and 15% (n=52) had 3 or more Abn, including 6% with MK (n=26). Proportional distribution was similar between PMF and PET/PPV-MF patients.Comparison of patients with one, two, or ≥3 Abn (i.e. CK) revealed that patients with CK were more likely to be anemic (hemoglobin < 10 g/dL, p<0.001) and transfusion dependent (p<0.001) and had higher DIPSS risk scores (p=0.004) than patients with one or two Abn. Among patients with PMF, CK was also associated with thrombocytopenia (p<0.001), while among those with PET/PPV-MF, it was associated with advanced (MF-2, 3) bone marrow fibrosis (p=0.045). Among all patients with one or two Abn, those with Abn in chromosomes 17, 5, or 7 had lower hemoglobin and higher peripheral blood blasts, and were less likely to be JAK2 positive (all p<0.05).Estimated OS was 48 months (range, 43-52) for both patients with PMF and PET/PPV-MF. Among all patients, OS was shorter among those with CK (median 14 months, range 7-21) than in those with one (median 48, range 37-60) or two (median 58, range 43-73) Abn (p=0.000, chi-sq 26.6). Overall median follow up from the time of referral was 29 months (range, 0.5-251) and 61% (n=220) of patients had died. Among all patients, median survival from date of referral was 19 months (range, 0.5-251). During an observation period of 1105 persons-years, progression to AML occurred in 44 (12%) patients after a median time of 13 months (range, 0.5-175). The incidence of AML was higher among those with CK (23% vs 15% vs 13% with CK, two or one Abn, respectively; p=0.014, chi-sq 8.7). Among the known causes of death (in 109 patients), progression of MF/AML was the most common (24%, n=53).Conclusion: The frequency of cytogenetic abnormalities in patients with PMF and PET/PPV-MF and their clinical relevance appear to be similar. Patients with CK have shorter OS and a higher incidence of AML in both PMF and PET/PPV-MF. [Display omitted] DisclosuresCortes:ARIAD: Consultancy, Research Funding; Bristol-Myers Squib: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Predictors of Response Duration and Survival with Second-Line Bosutinib Therapy in Patients (pts) with Chronic Phase Chronic Myeloid Leukemia (CML) Resistant or Intolerant to Prior Imatinib

▪Bosutinib (BOS), a Src/Abl tyrosine kinase inhibitor (TKI), is approved for adults with Philadelphia chromosome-positive (Ph+) CML that is resistant/intolerant to prior therapy. In this retrospective analysis, baseline and on-treatment characteristics of chronic phase (CP) CML pts receiving second-line BOS following imatinib resistance (IM-R) or intolerance (IM-I) in an ongoing open-label, phase 1/2 study and a long-term extension study were examined to identify potential predictors of duration of major cytogenetic response (MCyR), overall survival (OS), and progression-free survival (PFS), using a backward-elimination multivariate Cox regression model.A total of 284 (IM-R, n=195; IM-I, n=89) pts who received BOS starting at 500 mg/d were included in this analysis. Median (range) age was 53 (18‒91) y; time from CML diagnosis was 3.7 (0.1‒15.1) y; treatment duration was 25.6 (0.2‒106.7) mo; follow-up duration was 53.7 (0.5‒106.8) mo. For the last enrolled patient, time from first BOS dose was ≥6 y.After ≥6 y of follow-up, median MCyR duration and OS were not yet reached. Kaplan-Meier estimated probability of maintaining MCyR at 6 y was 71%, OS rate was 83%, and cumulative incidence of on-treatment disease progression or death was 21%. Several factors were identified as predictive of MCyR duration, OS or PFS, including baseline Ph+ ratio ≥95% vs ≤35% and MCyR by week 12, which were significant predictors of all 3. Other significant predictors of decreased OS included: age ≥65, BOS-sensitive mutations vs no mutations and higher peripheral blood (PB) blasts at baseline (all P ≤0.031; Table). Other significant predictors of decreased PFS included: higher PB blasts and no dose reduction to 400 mg/d due to AEs (all P ≤0.025; Table). Prior IM response or resistance did not predict long-term outcomes, nor did any treatment-emergent adverse events examined except for abnormal liver function test (LFT), which was predictive of increased OS.In conclusion, pts with CP CML resistant/intolerant to IM treated with BOS were identified as having an increased risk of poorer outcomes if they had the following characteristics: baseline Ph+ ratio ≥95% vs ≤35%, higher PB blasts at baseline or no MCyR by week 12. Having a better understanding of factors that may be predictive of long-term patient outcomes with TKI therapies may aid healthcare providers in the future selection of optimal treatment regimens for pts with Ph+ CML. [Display omitted] DisclosuresCortes:ARIAD Pharmaceuticals Inc.: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Brümmendorf:Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Patent: Patents & Royalties: Patent on the use of imatinib and hypusination inhibitors. Kim:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ILYANG: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schafhausen:ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Nadanaciva:Pfizer Inc: Employment. Bardy-Bouxin:Pfizer Inc: Employment. Shapiro:Pfizer Inc: Employment, Other: Stock Ownership. Leip:Pfizer Inc: Employment. Lipton:Ariad: Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Gambacorti-Passerini:Pfizer: Consultancy, Research Funding; BMS: Consultancy.

Unsupervised Cluster Analysis of Antigen Expression Patterns Identifies Subgroups with Distinct Biological and Clinical Features in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Characterisation of antigen expression patterns is part of the standard diagnostic work-up in acute myeloid leukemia (AML). But the biological & clinical implication of such antigen expression patterns have not been studied extensively & remain unclear in AML patients (pts) undergoing allogeneic stem cell transplantation (SCT).We analyzed the diagnostic antigen expression patterns of 162 AML pts (median age 64.7 years [y, range 46.6-76.2y]) with available data who received allogeneic peripheral blood SCT after non-myeloablative conditioning (NMA-SCT) between 2001 & 2013 at our institution. Conditioning regimen was fludarabine 3x30mg/m2 & 2Gy total body irradiation. Donors were human leukocyte antigen (HLA) matched related (12%) or HLA matched (59%) or mismatched unrelated (29%). Mutation (mut) status of the NPM1, CEBPA, IDH1, IDH2 &DNMT3A gene, presence of FLT3 -ITD &FLT3-TKD & the expression status of BAALC, ERG, MN1, EVI1, miR-9 &miR-181a at diagnosis were accessed. Pts were grouped according to the European LeukemiaNet (ELN) genetic classification in 22% favorable (fav), 24% intermediate-I (int-I), 21% intermediate-II (int-II) & 32% adverse (adv). Median follow up was 3.2y. To assess antigen expression patterns at diagnosis for all pts, flow cytometric analysis utilizing a standard panel (CD2, CD7, CD11b, CD13, CD14, CD15, CD33, CD34, CD38, CD45, CD56, CD61, CD64, CD65, CD117 & Glycophorin A) of mononuclear cells in bone marrow (BM) was performed. Using R's gplot package we performed unsupervised hierarchical clustering of the antigen expression which revealed 4 subgroups with distinct antigen expression patterns (Figure 1).At diagnosis, pts grouped in cluster 1 (n=19) had higher white blood count (WBC, P =.004) & lower peripheral blood (PB) blast count (P =.03) & were more likely to have de novo AML (P =.05). They were also less likely to have trisomy 8 (P =.08) by trend & more likely to have normal karyotype (KT, P =.05), to have ELN fav risk (P =.04), to be NPM1 mut (P =.002) & to be DNMT3A mut by trend (P =.08) & had lower miR-181a (P =.04), lower BAALC (P<.001), lower ERG (P =.01) & lower MN1 expression (P<.001).Pts grouped in cluster 2 (n=35) had higher WBC (P<.001), PB blasts (P<.001) & BM blasts (P =.005) at diagnosis. They were less likely to have trisomy 8 (P =.008) & to have deletion (del) 7/7q (P =.07) by trend, were more likely to be NPM1 mut (P =.002) & to have FLT3 -ITD (P <.001) & had lower BAALC (P =.1) & lower EVI1 expression (P =.09) by trend.Pts grouped in cluster 3 (n=59) had lower WBC (P<.001), PB blasts (P<.001) & BM blasts (P<.001) at diagnosis & were less likely to have de novo AML (P<.001). They were more likely to have trisomy 8 (P =.05), del5/5q (P =.004), monosomal KT (P =.04), complex KT (P =.07) by trend & ELN adv risk (P =.04), were less likely to be NPM1 mut (P =.03) &FLT3 -ITD by trend (P =.08) & had lower ERG (P =.008) & higher miR-9 (P =.009) expression.Pts grouped in cluster 4 (n=49) had lower WBC (P =.03), higher PB blasts (P =.007) & BM blasts (P<.001) at diagnosis. They were less likely to have del5/5q (P =.008) &NPM1 mut (P <.001) & had lower miR-9 (P =.007) & higher BAALC (P<.001), ERG (P<.001) &MN1 (P<.001) expression.For the entire set of pts, belonging to one of the antigen expression clusters did not impact on outcome. However, when the ELN groups were regarded separately, within the ELN fav group, cluster 1 pts had a significantly shorter event free survival (EFS, P =.04, Figure 2A) & within the ELN int-I group, cluster 3 pts had a trend for better (P =.096) & cluster 4 pts for worse EFS (P =.087).In conclusion, the antigen expression patterns at diagnosis obtained by unsupervised cluster analysis associated with distinct biological & clinical features (Figure 2B): NPM1 mut were enriched in clusters 1 & 2. Cluster 1 was characterized by ELN fav risk, normal KT, de novo disease & lower BAALC, ERG, MN1 &miR-181a expression. Cluster 2 was characterized by a high incidence of FLT3-ITD. We found more pts with ELN adv risk, monosomal KT, secondary AML & low miR-9 expression in cluster 3 & higher miR-9 as well as lower BAALC, ERG &MN1 expression levels in cluster 4. Even though we did not observe a prognostic impact of the antigen expression patterns in the entire cohort, the patterns may help to refine the ELN risk classification for AML pts undergoing SCT. Assessing the diagnostic antigen expression patterns provides information on disease biology, clinical parameters and potentially disease aggressiveness in AML. [Display omitted] [Display omitted] DisclosuresFranke:BMS: Honoraria; MSD: Other: Travel Costs; Novartis: Other: Travel Costs. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

High expression of Midkine (MK) indicates poor prognosis in childhood acute lymphoblastic leukemia

Objectives: Midkine (MK) expression has been reported to be correlated with the poor prognosis of patients with various tumors. However, there are no data available about the prognostic value of MK expression in childhood acute lymphoblastic leukemia (ALL).Methods: In this study, MK mRNA expression was determined by real-time polymerase chain reaction in 120 childhood ALL and 30 healthy volunteers. Patients were dichotomized at the median value and divided into two groups: MKlow group and MKhigh group.Results: MKhigh patients had higher white blood cell counts, higher peripheral blood blasts percentages, and higher minimal residual disease levels than MKlow patients. Moreover, the MK gene was expressed significantly higher in patients with relapsed ALL than in patients who maintained complete remission or at diagnosis. MKhigh patients harbored inferior relapse-free survival (RFS, P = 0.047) and overall survival (OS, P = 0.022) than MKlow patients, and high expression of MK was found to be independently predictive of inferior OS (P = 0.032) but not RFS (P = 0.077) in the overall cohort.Conclusion and discussion: MK high expression is an independent adverse prognostic factor in childhood ALL. Its level may be incorporated into an improved risk classification system for ALL and suggest the need of alternative regimens.

Molecular and Prognostic Correlates of Cytogenetic Abnormalities in Chronic Myelomonocytic Leukemia: A Mayo Clinic-French Consortium Study

Background:The prognostic significance of cytogenetic abnormalities in chronic myelomonocytic leukemia (CMML) was recently revisited (AJH, 89; 813-818, 2014 and Blood April, 2013). Using a large Mayo Clinic-French Consortium database, we analyzed the molecular and prognostic correlates of cytogenetic abnormalities in CMML.Methods:CMML diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies.Results:Spectrum and frequency of cytogenetic abnormalities: A total of 409 patients participated in this study including, 268 (66%) from the Mayo Clinic and 141 (34%) from the French CMML consortium. Of these, 396 (97%) had ≥20 metaphases and 13 (3%) had ten to 19, analyzed. One hundred and fifteen (30%) patients displayed an abnormal karyotype, including 82 (71%) sole, 20 (17%) two and 13 (11%) complex abnormalities. The most common abnormalities were; +8 (23%), -Y (20%), -7/7q- (14%), 20q- (8%), +21 (8%) and der (3q) (8%). Other cytogenetic abnormalities included 5q-, 12p-, 13q- and i(17q), present at a much lower frequency (0.9-4%).Phenotypic correlates: Abnormal vs normal karyotype was associated with older age (p=0.03), hemoglobin<10 g/dL (p=0.0009), white blood cell count (WBC) >15 x 109/L (p=0.02), absolute neutrophil count (ANC) >10 x 109/L (p=0.03), absolute lymphocyte count (ALC) >2.5 x109/L ( p=0.04), peripheral blood (PB) blast ≥1% (p<0.0001), bone marrow (BM) blast ≥10% (p<0.0001) and circulating immature myeloid cells (IMC) (p=0.0003). +8 (p=0.01), +21 (p=0.03) and der (3q) (p=0.03) were associated with hemoglobin <10 g/dL. -Y was associated with older age (p=0.04), lower PB (p=0.04) and BM (p=0.02) blasts. -7/7q was associated with leukocytosis (p=0.005), neutrophilia (p=0.04), and higher PB blasts (p=0.004). 20q- was associated with thrombocytopenia (p=0.04).Molecular correlates:ASXL1 mutations were associated with abnormal karyotype (p=0.04) and SRSF2 with normal karyotype (p=0.02). In comparison to other abnormal karyotypes, the incidence of ASXL1 mutations was lower in –Y (P=0.04) and der(3q) (p=0.03). U2AF1 mutations were associated with monosomal karyotype (p=0.03) and SF3B1 with der (3q) (p<0.0001).Prognostic relevance: Median follow-up was 1.8 years with 244 (60%) deaths and 79 leukemic transformations (19%). A step-wise survival analysis resulted in three distinct cytogenetic risk categories (Figure 1): high (complex and monosomal karyotype), intermediate (all abnormalities not in high or low risk) and low (normal, sole -Y and sole der (3q)); the corresponding median survivals were 0.2 (HR 8.1, 95% CI 4.6-14.2), 1.7 (HR 1.7, 95% CI 1.2-2.3). In multivariable analysis, the particular cytogenetic risk stratification remained significant in the context of Mayo molecular model (p<0.0001), MDAPS (p<0.0001), and the GFM risk model (P<0.0001). The Mayo-French cytogenetic risk model was also effective in predicting leukemic transformation with HR of 10.9 (95% CI 4.2-27.8) for high and 2.2 (95% CI 1.3-3.7) for intermediate risk groups.Conclusion:Cytogenetic abnormalities are seen in approximately 30% of patients with CMML and display significant associations with certain molecular and phenotypic characteristics. We describe a novel cytogenetic prognostic model for both over-all and leukemia free survival in CMML. [Display omitted] DisclosuresNo 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.

Prognostic Factors in the Blastic and Accelerated Phase of Patients with Chronic Myeloid Leukemia (CML) in the Tyrosine Kinase Inhibitor (TKI) Era

AbstractAbstract 3786With the advent of TKIs, significant improvement has been made in the survival outcome of CML patients. Many prognostic factors have in identified in chronic phase CML patients. However, prognostic factors in blastic phase (BP) and accelerated phase (AP) of CML patients have not been previously investigated, especially in the era of TKIs.We have analyzed CML patients either been diagnosed in or progressed into BP or AP from 2000 to 2010 enrolled into the clinical trials with different TKIs such as imatinib, nilotinib, dasatinib, and bosutinib at MD Anderson Cancer Center. All demographic, clinicopathologic variables and medication data including TKIs and other medication were collected at the time of the diagnosis of BP or AP. Primary outcomes; progression free survival (PFS) and overall survival (OS) were defined as time from diagnosis of either BP or AP to treatment failure and death, respectively.Among a total of 260 CML patients, mean age was 51.4 (SD 14.1). Males were 59%. Caucasians were 72%; African-Americans, 12%; and Hispanics, 14%. Only 17% reached complete hematologic remission and 62% eventually died during the follow up (mean 4.1 yrs, SD 4.0 yrs). 26% were in BP and 74% in AP. 38 patients were in BP (n=9) or AP (n=29) at initial diagnosis. Mean time from initial diagnosis in chronic phase to BP or AP was 4.1 yrs (0 to 23 yrs). Among BP, 17% had lymphoid BP and 84% myeloid BP. Cytogenetic analysis at diagnosis demonstrated 38% had only Philadelphia chromosome (Ph) present while 62% had other chromosomal abnormalities besides Ph. 30% had received one or more prior TKI by the time of transformation. 64% started on monotherapy imatinib (no prior TKI), 15% nilotinib (86% prior TKI), 14% dasatinib (93% prior TKI), and 3% bosutinib (100% prior TKI); 3.5% received imatinib combined with other agents (57% prior monotherapy with imatinib). Median PFS was 0.7 yr and median OS, 2.4 yrs.Factors linked with worse PFS were higher bone marrow (BM) blast % (HR=1.01, p<0.001), higher peripheral blood (PB) blast % (HR=1.01, p<0.001), higher PB basophil % (HR=1.01, p=0.02), lower PB polymononuclear cells (HR=0.98, p<0.001), lower hemoglobin (HR=0.91, p=0.02), lower platelet counts (HR=0.99, p=0.045), and higher number of prior TKI treatments (HR=1.52, p<0.001). In AP, compared to other TKIs, imatinib was associated with superior PFS (HR=0.63, p=0.03) while nilotinib was linked with inferior PFS (HR=1.81, p=0.04). Dasatinib (HR=1.17, p=0.57) and bosutinib (HR=1.85, p=0.18) did not show difference in PFS. In BP, there was no difference in OS by TKIs. Concurrent use of ACE inhibitors, ARBs, statins, aspirin, or metformin with TKIs was not associated with change in PFS. In multivariate analysis adjusting for the above variables, only prior TKI use was associated with worse PFS (adjusted HR [AHR]=1.57, p=0.001). Imatinib (AHR=0.70, p=0.36) nor dasatinib (AHR=0.67, p=0.11) were no longer associated with favorable or unfavorable PFS.Factors associated with inferior OS were old age (HR=1.02, p<0.001), bigger spleen size (HR=1.03, p=0.001), higher BM blast % (HR=1.02, p<0.001), higher PB blast % (HR=1.02, p<0.001), lower PB polymononuclear cells (HR=0.98, p<0.001), lower hemoglobin (HR=0.80, p<0.001), lower platelet counts (HR=0.99, p=0.008), higher LDH (HR=1.0002, p<0.001), presence of other cytogenetic abnormality besides Ph (HR=1.45, p=0.03),and higher number of prior TKI treatments (HR=1.64, p<0.001). In AP, imatinib was associated with more favorable OS (HR=0.65, p=0.07) compared to others (nilotinib: HR=1.28, p=0.47; dasatinib: HR=1.70, p=0.077; bosutinib: HR=1.09, p=0.88). In BP, there was no difference in OS by TKIs. Concurrent use of ACE inhibitors, ARBs, statins, aspirin, or metformin with TKIs was not associated with change in OS. In multivariate analysis adjusting for the above variables, only age (AHR=1.01, p=0.002) and BM blast% (AHR=1.02, p=0.005) were associated with worse OS. Imatinib was no longer associated with favorable outcome (AHR=1.27, p=0.57).In conclusion, we identified spleen size, blast counts, and number of prior TKI use as major prognostic factors in CML patients in BC or AP. Imatinib was associated with favorable outcome. This is likely due to the fact that patients treated with imatinib had received no prior TKIs while most patients treated with 2nd generation TKI had. Further studies are required to validate our findings in a larger and prospective cohort. [Display omitted] [Display omitted] Disclosures:Kantarjian:Novartis: Research Funding; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Jabbour:Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Cortes:Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

Serial assessment of regulatory T cells (Tregs) in pediatric AML: A prospective study.

9549 Background: Data of Tregs in pediatric AML is lacking. The study objectives were determining Tregs in pediatric AML at diagnosis and follow up; and correlating with outcome. Methods: From Nov 2010-May 2011, 30 consecutive AML patients ≤ 18 years were prospectively enrolled with 6 healthy controls. All patients received daunorubicin / cytarabine induction and 3 courses of cytarabine. Tregs (CD4+CD25+FoxP3+) were assessed at diagnosis, post-induction, post-consolidation, 3 and 6 months follow up and relapse. Results: 30 cases with median age 9.5 years; male/female ratio 14:16 had significantly higher baseline Tregs than healthy controls (12.36±4.65% vs 3.16±1.49%; p=0.0001). Patients with high Tregs frequency were females (p=0.044), WBC&gt;50,000/mm3 (p=0.023), hypoalbuminemia (p=0.002), absence of lymphadenopathy (p=0.04) and linear correlation with peripheral blood blast% (r=0.55, p=0.0014). CR rate was 83.3% (25/30); EFS 38% and OS 73% at 14 months follow up. When Tregs were categorized as high and low based on median value, CR rate (86.6% vs 80%, p=NS), EFS [17% vs 60%, HR 1.46 (0.51-4.14) p=0.46] and OS (66% vs 80%, HR 0.58 (0.13-2.44) p=0.45) were not different. Amongst those who achieved CR, there were 7/13 relapses in those with high Tregs versus 3/12 in those with low Tregs (p=0.226).Tregs reduced post-induction (12.1±4.6 vs 6.32±2.8, p&lt;0.000) from baseline. Using generalized estimating equation regression model, average Tregs reduction from post-induction till 3 month follow up was 0.785 units/visit (p&lt;0.005) in those with continuous CR(n=15) and 1.25 units/visit (p&lt;0.001) in those whom relapsed (n=10). Difference between these two groups was not significant. Tregs significantly increased at relapse as compared to post consolidation value (15.3±5.2 vs 4.5±1.9, p=0.003). Conclusions: This first study in pediatric AML demonstrates that Tregs is increased at diagnosis and is significantly associated with females, high WBC count, hypooalbuminemia, absence of lymphadenopathy and high peripheral blood blast%. Tregs significantly reduced post-induction on follow up; however, it increased at relapse. Baseline Tregs did not predict CR, EFS, or OS. However, there were more relapses in those with high baseline Tregs.

Cytoplasmic localization of nucleophosmin in bone marrow blasts of acute myeloid leukemia patients is not completely concordant with NPM1 mutation and is not predictive of prognosis

Nucleophosmin (NPM1) gene mutations are reported to predict a favorable prognosis in acute myeloid leukemia (AML) patients. Aberrant cytoplasmic localization of nucleophosmin (NPM) protein is reported be a surrogate for NPM1 gene mutation. Using immunohistochemical (IHC) analysis, we assessed for NPM (clone 376) expression in formalin-fixed, formic acid-decalcified bone marrow biopsy specimens. DNA sequencing of exon 12 of NPM1 gene was performed in 104 patients. The study included 252 AML patients: 192 de novo AML, 33 AML preceded by either myelodysplastic syndrome or chronic myelomonocytic leukemia, and 27 therapy-related AML. The median age was 62 years and 115 patients were <or=60 years old. All patients received intensive chemotherapy. Cytoplasmic NPM was detected in 59 of 252 (23%) patients, including 48 of 192 (25%) de novo AML and 33 of 94 (35%) with a normal karyotype. DNA sequencing identified NPM1 mutations in 30 of 38 cases with cytoplasmic NPM and 10 of 66 cases with nuclear NPM. Cytoplasmic NPM was associated with young patient age (P=.024), FLT3/ITD (P=.005), CD34 negative blasts (P<.001), high peripheral blood blast count (P=.041), and high serum albumin level (P=.028). No statistical differences in overall or event-free survival were found on the basis of NPM localization. Similar results were obtained in patients<or=60 years old with normal karyotype and wild-type FLT3 (P=.768). IHC assessment for NPM localization did not predict prognosis in this patient cohort. The discordance between immunohistochemistry and DNA sequencing results indicates that DNA sequencing cannot be replaced by IHC assessment.

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells.

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.

Adriamycin in combination chemotherapy of adult acute lymphoblastic leukemia: A southwest oncology group study

Eighteen (72%) of 25 evaluable and previously untreated patients with adult acute lymphoblastic leukemia entered complete remission (CR) following induction therapy with adriamycin, vincristine, and prednisone in a Southwest Oncology Group study. Remission maintenance therapy with methotrexate and 6-mercaptopurine resulted in a median duration of CR of 10.2 months. The addition of Adriamycin to prednisone and vincristine may be beneficial in slow responders or nonresponders to these two drugs and in patients with initially high peripheral blood blast counts.

T-lymphoblastic leukaemia: a distinct variant of acute leukaemia.

Two cases of acute lymphoblastic leukaemia (A.L.L.) of T-cell type are reported. Clinically they were characterized by very high peripheral blood blast cell counts at presentation, the early development of meningeal leukaemia, and relative resistance to treatment with combination chemotherapy. Leukaemic cells from both patients lacked all the B-cell markers investigated, but 60-65% of their cells formed rosettes with sheep red cells. Cytochemical and surface structure studies helped to define additional features of these cells and confirmed their T-cell nature.It seems that this variant of A.L.L. may not be uncommon and that it can be distinguished on clinical and immunological grounds from the usual type of A.L.L. which runs a less aggressive course and lacks B- or T-cell markers.