Peripheral Blood Blast Percentage Research Articles

Reclassification of Acute Myeloid Leukemia According to the 2016 WHO Classification.

We reviewed our leukemia database to reclassify 610 patients previously diagnosed as having acute myeloid leukemia (AML) according to the updated 2016 WHO classification. Nine patients were categorized as having myelodysplastic syndrome and myeloid neoplasms with germline predisposition. AML with recurrent genetic abnormalities accounted for 57.4% (345/601) of the patients under the 2016 WHO classification. AML with mutated NPM1 was the most common form (16.5%), with the majority associated with monocytic differentiation (63.6%). AML with double CEBPA mutations accounted for 8.3% of these cases, and the majority were previously diagnosed as AML with/without maturation (78.0%). These newly classified mutations were mutually exclusive without overlapping with other forms of AML with recurrent genetic abnormalities. AML with mutated NPM1 and AML with myelodysplasia-related changes comprised the oldest patients, whereas AML with RUNX1-RUNX1T1 included the youngest patients. The leukocyte count was highest in AML with mutated NPM1, and the percentage of peripheral blood blasts was the highest in AML with double CEBPA mutations. Our results indicate that implementation of the 2016 WHO classification of AML would not pose major difficulties in clinical practice. Hematopathologists should review and prepare genetic tests for the new classification, according to their clinical laboratory conditions.

Percentage of Erythroid Cells in Bone Marrow at Diagnosis As a New Prognostic Factor in Patients with Myelodysplastic Syndrome

IntroductionRisk assessment is essential for guiding therapy in patients with myelodysplastic syndromes (MDS). Currently, the most widely used prognostic scoring models are the International Prognostic Scoring System (IPSS) and the revised IPSS (IPSS-R). The prognostic relevance of the percentage of erythroid precursors (EP) in bone marrow (BM) and its relationship with other biological characteristics has been poorly studied, although it has been proposed that a very low percentage of EP in BM may represent a cohort of patients with potentially adverse outcome. Our main aim was to analyze the biological and clinical features of MDS patients according to the percentage of EP in BM at diagnosis and evaluate its prognostic value on survival.Patients and MethodsData from 4,791 de novo MDS patients from the MDS Spanish Registry with available cytogenetics were collected. All patients included were diagnosed based on the 2008 WHO criteria and risk stratification was performed following the IPSS-R. Patients were distributed, according to the percentage of EP in BM, into three groups: less than 15% (EP<15%), between 15% and 49% (EP 15-49%) and equal or more than 50% (EP>49%).Proportions were compared by the Chi-square test. Survival curves were constructed by Kaplan-Meier method and differences between curves were evaluated by log rank tests. Multivariable analysis of survival was performed using Cox's proportional hazards regression model. P-values <0.05 were considered as statistically significant. All statistical analyses were performed by the R software.ResultsThe 4,791 cases were grouped according to the percentage of erythroid cells in BM at diagnosis. In Table 1 are displayed the clinical and biological characteristics of the patients in the whole series and in the three groups based on the EP percentage in BM. The group with EP<15% in BM showed a significantly greater number of cytopenias at diagnosis, a higher number of peripheral blood (PB) and BM blasts, and a higher percentage of patients classified in MDS subtypes with excess blasts. The EP>49% group presented higher rates of patients with refractory anemia with ring sideroblasts (RARS), lower number of PB and BM blasts, and more frequent high-risk cytogenetics. Conversely, the intermediate EP group with EP 15-49% showed less number of cytopenias, higher frequency of good-prognosis cytogenetics, and lower percentage of patients in higher-risk IPSS-R categories.Survival analyses confirmed that age, blast percentage in PB and BM, and IPSS-R cytogenetics are significantly associated with worse survival. A striking difference in survival within groups according to percentage of EP was also found, recognizing EP percentage as an independent risk factor for survival of MDS patients in multivariate analysis. Patients with EP<15% in BM experienced a significantly shorter OS (median, 28 months) in both univariable (P<0.0001) and multivariable analysis (P<0.0001) (Table 2 and Figure 1). The best survival was found for patients with EP 15-49% with a median survival of 58 months. The group with EP>50% in BM had also worse survival than the intermediate group, showing a median survival of 47 months (hazard ratio, 0.79; 95% CI, 0.67 to 0.93; P < 0.005).ConclusionsOur study proves the prognostic impact of the percentage of erythroid precursors in bone marrow in MDS patients, providing evidence that the presence of EP <15% in BM is an independent risk factor for survival. [Display omitted] DisclosuresDíez-Campelo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Augmentation of the Acute Myeloid Leukemia-Composite Model (AML-CM) with Performance Status and Secondary Leukemia

Background:To help optimize decision-making for acute myeloid leukemia (AML) treatment in the clinic, risk models comprising different variables were developed. A Treatment-Related Mortality (TRM) model was developed to predict 28-day mortality. The TRM model includes age, performance status, platelet count, albumin, secondary AML, peripheral white blood cell count, peripheral blood blast percentage, and creatinine (Walter et al, 2011 Nov 20; 29(33): 4417-4424). More recently, an AML-specific composite model (AML-CM) introduced the combination of cytogenetic/molecular risks per European Leukemia Network (ELN) classification with burdens of increasing age and comorbidities. The AML-CM can predict both early and late mortality after diagnosis of AML (Sorror et al, 2017; 3(12):1675-1682). All factors within the TRM model were tested during the development of the AML-CM, except for the impacts of Karnofsky Performance Status (KPS), and presence of secondary AML. To this end, we explored the possibility of augmenting the AML-CM by testing the prognostic value added by including KPS and presence of secondary AML.Methods:The study comprises two cohorts from two different institutions: 311 patients (pts) to be used for development of new scores and 187 pts to be used for independent validation. Both cohorts were seen for initial therapy for AML between 2008 and 2017. We collected patients' demographics and comorbidity information through patient charts. We defined secondary AML as an occurrence of AML after a previous malignancy or after hematological abnormalities for at least one month. Afterwards, Cox regression models were designed to test whether KPS and secondary AML have independent prognostic impacts that could be added to the AML-CM. Prediction of eight-week and one-year mortality were compared between the original versus augmented AML-CM using c-statistics and area under the curve (AUC) of the receiver-operator characteristic curve (ROC) in the independent validation cohort from the different institution.Results:In a multivariate model, after adjusting for AML-CM variables, both secondary AML (hazard ratio [HR] =2.19, p=0.0001) and worse KPS percentages (KPS 80-75%: HR=1.53, p=0.06; KPS ≤70%: HR=2.60, p=0.0001) were significantly associated with one-year mortality. Using the same equation used in the development of the AML-CM, we assigned 1 point to KPS 80-75%, 2 points to KPS ≤70% and 2 points to secondary AML. These points were added to those of age, comorbidities and ELN risks in order to calculate the augmented AML-CM scores.In the validation set, the augmented AML-CM had a c-statistic estimate of 0.71 vs. 0.69 for the original AML-CM. The true AUC for prediction of 1-year mortality was 0.75 vs. 0.72 while the AUC for prediction of 8-week mortality was 0.83 vs. 0.79 for the augmented AML-CM and original AML-CM, respectively.Figure 1 shows the discrimination of four risk groups per the new augmented AML-CM scores. Patients with scores of 1-3, 4-6, 7-9, and ≥10 had 1-year survival rates of 91%, 76%, 46% and 37%, respectively.Conclusions:In this study we found that worse KPS percentages add prognostic value to those of age and comorbidities, while secondary AML adds prognostic value to that of ELN cytogenetic/molecular risks for prediction of early (8-week) and late (1-year) mortality after diagnosis of AML. This suggests that KPS provides prognostic information above what is provided by age and comorbidities alone. Similarly, the classification of a patient's AML as secondary AML provides prognostic information beyond what is understood about the patient's disease risk as indicated by the ELN cytogenetic/molecular risk category.Combining these five factors: age, comorbidity burden, performance status, disease risk, and presence of secondary AML results in an augmented AML-CM that can be used in the clinic for improved risk-assessment before treatment decisions. [Display omitted] DisclosuresBecker:GlycoMimetics: Research Funding.

Decitabine Versus Intensive Chemotherapy for Elderly Patients with Newly Diagnosed Acute Myeloid Leukemia

BackgroundElderly patients with acute myeloid leukemia (AML) has generally poor prognosis prognosis in accordance with their unfavorable clinical and biologic features. Hypomethylating agents have shown potential in the treatment of AML as well as myelodysplastic syndrome (MDS). In this retrospective study, we compared the outcomes of elderly AML patients according to induction treatment options: decitabine versus intensive chemotherapy. We also tried to identify specific subsets of patients who are most likely to benefit from decitabine or intensive chemotherapy.MethodsThis study included elderly patients aged 65 years or older who received induction treatment with decitabine or intensive chemotherapy for newly diagnosed AML at a single institute. The endpoints for this study were overall survival (OS), response, and event-free survival (EFS). Response included complete remission (CR), CR with incomplete hematologic recovery (CRi), and CR with partial hematologic recovery (CRh).ResultsA total of 107 patients, decitabine for 75 and intensive chemotherapy for 32, were analyzed. Decitabine was given as 20 mg/m2/day for 5 days every 4 weeks. Median 5 courses (range, 1-43) were delivered to the patients and 16 patients were still on decitabine treatment at the time of analysis. Intensive chemotherapy regimens included cytarabine plus daunoruribin (n=21) or idarubicin (n=10), and hyper-CVAD (n=1): 25 patients received one course and 7 received two courses for induction treatment. The rate for CR + CRi + CRh (CRR) was 38.6% (39 of 101 assessable patients). With a median follow-up duration of 14.8 months (95% confidence interval [CI], 12.0-22.8) among surviving patients, 79 patients died and 22 relapsed. The median OS and EFS were 12.3 months (95% CI, 10.0-14.7) and 4.1 months (95% CI, 2.5-5.7), respectively. Decitabine showed lower CRR (26.1% vs. 65.6, P<0.001) with similar EFS (median 3.4 vs. 6.1 months, P=.338) and OS (median 11.0 vs. 14.8 months, P=.124) compared to intensive chemotherapy (Figure 1). Multivariate analysis demonstrated that induction treatment option, peripheral blood (PB) blast percentage, and leukemia type (secondary vs. de novo) were independent risk factors for CRR. A presence of FLT3-ITD mutation, complex karyotype, and higher PB blast percentage were independently associated with shorter OS. Subgroup analysis for OS showed that intensive chemotherapy was superior to decitabine in patients with FLT3-ITD mutation (median 9.5 vs. 2.6 months, P=.025) and poor cytogenetic risk (10.8 vs. 6.1 months, P=.027), but decitabine showed tendency towards a longer OS compared to intensive chemotherapy in those with monosomy 7 or del(7q) (11.7 vs. 3.3 months, P=.093; Figure 2).ConclusionDecitabine showed similar OS to intensive chemotherapy despite of lower response rate in elderly AML patients. Clinical outcomes of specific subgroups seemed to be different according to induction treatment options. Further studies are warranted for selection of optimal treatment options for elderly AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Uniparental Disomies (UPD) of Chromosome 13q Is Associated with Shorter Disease-Free Survival in Adult Patients (Pts) with De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Approximately 45% of adult AML pts do not have cytogenetic abnormalities visible by microscopic karyotype analysis and are classified as CN-AML. However, these pts can still have one or more UPD (the loss of a chromosome or its segment and duplication of the remaining chromosome or segment), since UPDs are not detectable by typical karyotyping methods. Because UPDs have been shown to contribute to malignant transformation and outgrowth in various cancers and leukemias, we sought to identify recurrent UPDs in CN-AML pts and explore their associations with disease presentation, gene mutations and outcome. We used Infinium OmniExpress SNP arrays to detect UPDs in a cohort of 425 pts, who were similarly treated with intensive chemotherapy on Alliance for Clinical Trials in Oncology therapeutic trials. Pts having early death (<30 days) were excluded. The mutation status of 80 genes were previously assessed by targeted sequencing (Leukemia, 2017;31(10):2211-8).UPDs of 10 different chromosome arms were detected in >1% of pts (Figure A). The most commonly occurring UPDs were UPD of 13q (present in 7.5% of pts), UPD of 6p (in 2.4% of pts) and UPD of 11p (in 2.4% of pts). We examined associations between UPDs of 13q, 6p and 11p with demographic and clinical diagnostic characteristics. UPD of 6p was associated with higher peripheral blood (PB) blast percentage (median 86% vs 70%, P=.02); UPD of 11p was associated with lower platelet counts (41 vs. 60 x 109/L, P=.03) and higher PB blast percentage (85% vs 60%, P=.002); and UPD of 13q was associated with higher white blood cell count (53 vs 29 x 109/L, P=.02), higher PB blast percentage (77% vs 58%, P=.004) and higher bone marrow blast percentage (80% vs 69%, P=.005). We also examined the associations between UPDs and gene mutations present in >2% of CN-AML pts in our sample series. 13q UPD significantly co-occurred with FLT3-internal tandem duplication (ITD) (P<.001), and UPD of 11p significantly co-occurred with bi-allelic CEBPA mutations (P<.001). The co-occurrence of FLT3-ITD with 13q UPD likely has biologic relevance since FLT3 is located on 13q, whereas the association between 11p UPD with CEBPA mutation is less clear, as CEBPA is on 19q.Assessment of the association between UPDs and outcome was performed. Because of differences in induction therapies between pts younger and older than 60 years (y), outcome analyses must be performed separately in these two age groups. Based on sample size limitations we were only able to assess the impact of 11p UPD and 13q UPD on achievement of complete remission (CR), disease-free survival (DFS) and overall survival (OS) in the 315 pts younger than 60 y. UPD of 11p was associated with longer OS (3 y rates, 83% vs 45%, P=.02), and UPD of 13q was associated with shorter DFS and OS (3 y rates, 0% vs 38%, P<.001; 17% vs 49%, P<.001) (Figure B). To examine the combined effect of mutations and UPDs on outcome we performed multivariable analyses (MVAs). We found that UPD of 13q was significantly associated with shorter DFS (P=.009) after adjusting for hemoglobin levels (P=.02), FLT3-ITD status (P <.001), and mutation of DNMT3A (P<.001), RUNX1 (P=.002) and WT1 (P=.03) (Table). UPD of 13q did not impact on CR or OS when considered in a MVA, and UPD of 11p did not remain significant in MVA.Finally, since FLT3-ITD and UPD of 13q often co-occurred and were both found to be associated with shorter DFS in the MVA of adult younger pts, we examined younger pts who harbored FLT3-ITD for differences in DFS associated with UPD of 13q. We found those pts with 13q UPD had significantly shorter DFS (P=.003) compared to those without (Figure C), which further demonstrates 13q UPD status is useful for prognostic stratification independent from its association with the known prognostic marker FLT3-ITD.These data show the incorporation of 13q UPD might improve current genetic risk stratification models. Follow-up studies investigating the mechanistic roles of these recurrent UPDs, especially the relationship between UPD of 11p and bi-allelic CEBPA mutation, could shed light on novel pathways underlying leukemic transformation and proliferation in CN-AML. [Display omitted] DisclosuresPowell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria.

Association of bone marrow fibrosis with inferior survival outcomes in chronic myelomonocytic leukemia.

The impact of bone marrow fibrosis grade on the prognosis of patients with chronic myelomonocytic leukemia (CMML) remains controversial. Therefore, we examined the records of 82 patients diagnosed with CMML at our institution and summarized baseline characteristics and molecular profiles by subgroups of absent or mild (grades 0/1) and moderate (grade 2) fibrosis. Cox proportional hazards models were constructed to assess the prognostic significance of fibrosis grade. Grade 2 fibrosis was identified in 63 patients (76.8%), grade 1 in 16 patients (19.5%), and grade 0 in 3 patients (3.7%). Grade 2 fibrosis was associated with reduced hemoglobin levels (median 9.75 vs 11.0g/dL in grade 0/1; p = 0.04) and increased percentages of ringed sideroblasts (7.5 vs 0%; p = 0.008). In multivariable analysis, grade 2 fibrosis was an independent predictor of poor overall survival (OS; 95% CI 1.32-6.35; HR 2.90; p = 0.008), but not event-free survival (EFS; 95% CI 0.62-2.67; HR 1.28; p = 0.50). Absolute neutrophil count (ANC) was found to impact OS (95% CI 1.01-1.09; HR 1.05; p = 0.009), while both ANC (95% CI 1.00-1.07; HR 1.04; p = 0.04) and peripheral blood blast percentage (95% CI 1.02-1.32; HR 1.16; p = 0.02) impacted EFS. These results implicate fibrosis grade is an important indicator of prognosis, with high-grade fibrosis predicting inferior survival. Given the prevalence of marrow fibrosis in CMML, fibrosis grading should be incorporated into prognostic assessment and therapeutic decision-making.

Prognostic value of renin gene expression in acute myeloid leukemia

Background The majority of acute myeloid leukemia (AML) patients respond to initial chemotherapy, but only a minority of cases achieves long-term survival. Renin, as a part of the renin–angiotensin system, is directly linked to the activities of hematopoietic cytokines during normal hematopoiesis and in myeloproliferative neoplasms. In some hematological malignancies, blast cells have been observed to express renin, which is not found in normal marrow. Aim The aim of this was to study the expression of the renin gene as a prognostic marker in AML. Patients and methods Renin expression was measured in 45 AML patients and five controls by using quantitative real-time reverse-transcriptase PCR. Results Renin expression was detected in 93.3% of AML patients at diagnosis and 100% at relapse. Relapsed AML patients showed higher renin gene expression levels compared to those detected at complete remission and at diagnosis (P=0.000–0.001), respectively. None of our control subjects were positive for renin gene expression. At diagnosis, AML patients with haemoglobin less than 10 g/dl showed higher renin levels compared to those with haemoglobin of at least 10 g/dl. We used median renin gene expression levels to divide AML patients into high and low groups at diagnosis. At diagnosis, the group with higher renin expression showed higher total leukocyte count (TLC) than did the group with lower renin expression. At relapse, the higher renin expression group showed a higher peripheral blood (PB) blast percentage than did the lower renin expression group. Conclusion Renin expression can predict outcomes in AML patients and could be used as a therapeutic target.

Genetic Profiling Augments Prognostic Value of the EUTOS Long-Term Survival Score for Disease-Specific Mortality in Imatinib-Treated Asian Chronic Myeloid Leukaemia Patients

Introduction:Long-term survival rates among patients with chronic-phase chronic myeloid leukaemia (CP-CML) have remarkably improved since the introduction of imatinib, a BCR-ABL1 tyrosine-kinase inhibitor (TKI), as the standard first-line therapy. Several prognostic scores have been employed to predict clinical response and survival of CP-CML patients treated with TKIs. The EUTOS long-term survival (ELTS) score was recently introduced and shown to predict the probability of CML-specific death in long-term surviving patients on imatinib therapy, more effectively than the existing scores. The ELTS score was calculated by a formula that included age at diagnosis, spleen size below costal margin, platelet count and blast percentage in peripheral blood as prognostic factors. In our study, we evaluated the ELTS score in predicting the probabilities of CML-specific death, long-term overall survival (OS) and progression-free survival (PFS) rates in Asian CML patients treated with imatinib. As genetic differences, particularly the BCL-2 like 11 (BIM) deletion polymorphism, have been shown to confer intrinsic resistance to imatinib in East-Asian patients, we also explored the role of BIM deletion polymorphism profiling as a prognostic biomarker for CML-specific death among different risk groups stratified by the ELTS score.Methods:A retrospective analysis was performed on CP-CML patients treated with first-line imatinib within one year of diagnosis in Singapore General Hospital from June 2001 to November 2014. The ELTS score was obtained with online calculator at www.leukemia-net.org. Low-risk group was defined as a score ≤1.568, intermediate-risk group as a score >1.568 but ≤2.2185, and high-risk group as a score >2.2185. Progression was defined as transformation to accelerated or blast phase or death from any cause. OS and PFS were calculated with the Kaplan-Meier method and compared by the log-rank test. Cumulative incidence probabilities of CML-specific death were compared by the Gray test.Findings:134 patients were included for analysis. 63% were Chinese, 17% were Malays, 8% were Indians and 12% were of mixed ethnic origin. Median age at diagnosis was 45 years and 60% were male. Median follow-up was 7.7 years (range: 0.4 to 13.2 years). 17 deaths out of 134 patients (13%) were recorded, of which 11 were CML-specific (65%). 54% of patients were categorised as low-risk, 36% as intermediate-risk and 10% as high-risk by the ELTS score. The cumulative incidence probabilities of CML-specific death at 10 years were 43% in high-risk (hazard ratio (HR): 11.76, 95% confidence interval (CI): (2.32, 59.71), p=0.003) and 9% in intermediate-risk (HR: 2.24, 95% CI: (0.37, 13.49), p=0.38) compared to 3% in low-risk groups.10-year OS probabilities were 50%, 82% and 93% in high-, intermediate- and low-risk ELTS groups respectively (p=0.001). 10-year PFS probabilities were 50%, 84% and 89% in high-, intermediate- and low-risk ELTS groups respectively (p=0.004).Among 103 East-Asian patients with low- and intermediate-risk ELTS sub-groups, 15% harboured BIM deletion polymorphism. The probability of CML-specific death at 10 years in this subset was 16% with BIM deletion polymorphism, but 4% without polymorphism (HR 4.30, 95% CI: (0.76, 24.35), p=0.099). 10-year OS probabilities in the subset were 75% and 89% in patients with and without BIM deletion polymorphism respectively (p=0.014).Conclusions:The ELTS score was able to predict the probability of CML-specific death and identify high-risk patients in our multi-racial Asian CML patients treated with imatinib. Genetic profiling using BIM deletion polymorphism provided further stratification by identifying a subset of inferior long-term survivors with high probability of CML-specific death among otherwise non high-risk patients. [Display omitted] DisclosuresChuah:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chiltern: Honoraria.

HnRNP K Overexpression Drives AML Progression By Altering Pathways Critical for Myeloid Proliferation and Differentiation

Acute myeloid leukemia (AML) is a disease largely defined by recurrent genetic and chromosomal abnormalities. As such, The Cancer Genome Atlas' recent detailed examination of the most common genetic abnormalities that drive AML uncovered relatively few novel alterations. However, within this list of recurrently mutated genes was heterogeneous nuclear ribonucleoprotein K (HNRNPK). While previously unknown to impact AML, we recently identified HNRNPK as a haploinsufficient tumor suppressor at the 9q21.32 locus. In contrast to its tumor suppressive roles, hnRNP K is most customarily overexpressed, rather than underexpressed, in several solid tumors.In this study, we show through FISH analyses, RNA expression profiling, and reverse phase protein array analyses that the HNRNPK gene is amplified with corresponding mRNA and protein overexpression in a sizeable cohort of patients with AML that do not carry a 9q.21.32 deletion. Together, this indicates that hnRNP K may be an uncharacterized oncogene in this context. Increased hnRNP K correlated with decreased event-free and overall survival, increased WBC, and increased bone marrow and peripheral blood blast percentage. High hnRNP K levels also correlated with elevated expression of histone modifiers (e.g., ASH2L and TRIM24), regulators of ribogenesis (e.g., nucleolin and EBP1), and potent oncogenes, such as c-Myc. Together, these observations indicate that increased hnRNP K expression significantly alters the landscape of myeloid biology and may thus contribute to the etiology of AML.To directly interrogate this possibility in vivo, we generated a Vav-Cre;HnrnpkTg mouse model. In addition to significant differences in hnRNP K expression compared to Vav-Cre mice, Vav-Cre;HnrnpkTg mice exhibited reduced survival, development of transplantable myeloid malignancies, and enhanced HSPC proliferation and differentiation potential. Reflecting data from our patients, Vav-Cre;HnrnpkTg mice also exhibited elevated expression of critical oncogenic pathways such as c-Myc.Currently, we are examining pharmacologic methods to abrogate the effects of aberrant hnRNP K expression on various activated pathways. Transplant models using diseased bone marrow from Vav-Cre;HnrnpkTg mice are being utilized to evaluate the efficacy of specific pathway inhibitors (i.e., bromodomain inhibitors or bortezomib) in prolonging survival and decreasing leukemic burden. These studies will provide the rationale for potential clinical trails that employ these agents for patients with hnRNP K-overexpressing AML. DisclosuresNo relevant conflicts of interest to declare.

Pilot Study of Combined Type I FLT3 Tyrosine Kinase Inhibitor, Crenolanib with Sorafenib in Pediatric Patients with Relapsed/Refractory FLT3+Ve AML

Background: Crenolanib is a novel type I pan-FLT3 inhibitor, which inhibits both FLT3-ITD, as well as FLT3 tyrosine kinase domain (TKD) activating mutations. Combination of crenolanib (Type I) and sorafenib (Type II) has been shown to have synergistic apoptotic effects in FLT3+ve AML blasts derived from patients (pts) who have relapsed after prior sorafenib treatment. We hypothesized that the combination of crenolanib and sorafenib may provide enhanced FLT3 inhibition by targeting both inactive as well as active FLT3 kinase conformations. We here report the results of a dose finding study of sorafenib given with fixed doses of crenolanib in pediatric pts with FLT3+ve AML.Design: This pilot study was designed to assess the tolerability of escalating doses of sorafenib with full doses of crenolanib (66.7 mg/m2 TID) in pediatric pts with relapsed or refractory FLT3+ve AML. Crenolanib was administered continuously at 66.7 mg/m2 TID starting on d1. Sorafenib was administered continuously starting from d8 at 150 mg/m2/d (dose level 1). The rolling-6 design was used and if no DLTs were seen, sorafenib was escalated to 200 mg/m2/d (dose level 2). Additionally, triple intrathecal therapy with methotrexate, hydrocortisone, and cytarabine was given on d8 and continued weekly in pts with CNS leukemia until CSF was free of blasts.Results: From April 2015 to March 2016, 7 children (median age, 9yrs; range, 5-14yrs) have been enrolled. All pts had progressed after a median of 3 prior AML therapies (range, 1-7). Four pts had progressed after frontline treatment, which included sorafenib maintenance; the other 3 pts had progressed after standard chemotherapy. Two pts had undergone prior allo SCT. Median WBC count prior to therapy was 1.7 x 109/L (range 0.7-7.5 x 109/L) with median blast percentage of 15% (range 0-60%). All 7 pts had a FLT3-ITD (mutant to wild-type FLT3 ratio ranged from 0.048 to 22, with one pt not having any wild type FLT3 allele). At the time of study entry, 2 pts had active CNS leukemia and required additional intrathecal therapy.Crenolanib 66.7 mg/m2 TID was well tolerated with no pt requiring crenolanib dose reduction. Sorafenib was administered to the first 3 pts at 150 mg/m2 QD from d8 onward. As no DLTs were observed at 150 mg/m2 QD dose, sorafenib was dose escalated to 200 mg/m2 QD for the next 4 pts. No DLTs were observed at either of the sorafenib doses. The combination of crenolanib/sorafenib was well tolerated with most AEs being grade 1 and 2 in severity. Rash was frequently seen in pts following the addition of sorafenib and one pt developed grade 2 hand-foot syndrome which resolved within 5d on the same dosing regimen. No grade 4 LFT elevations were seen in any pt.During cycle 1 of therapy, crenolanib/sorafenib combination resulted in clearance of peripheral blood blasts in 5/6 pts and 1 pt without blasts at baseline had resolution of extramedullary neck mass. Reduction of bone marrow blasts was seen in 4/7 pts. By Cheson criteria, one patient achieved a CR (with FLT3 negativity), two had a PR, and one had a marrow response of CRi but with persistent CNS disease. Three pts had NR although 2 had transient benefit from crenolanib on day 8 as determined by BM blast percentage. Peripheral blood and bone marrow blast percentages are summarized in Figure 1 and Table 1, respectively. Median overall survival was 221d (Figure 2) with 2 pts currently still alive.Conclusions: Crenolanib 66.7 mg/m2 TID can be safely combined with sorafenib at 200 mg/m2 QD. Even in pts with prior exposure to sorafenib, this combination showed clinical benefits, represented by rapid reduction of bone marrow or peripheral blasts, and was able to have improved quality of life. Although dual TKI combinations targeting the same receptor have not been tried in AML, such strategies have been shown to have efficacy in other diseases like HER2+ve breast cancer. Pharmacokinetics of crenolanib and sorafenib and FLT3-TKD mutation analysis will be presented. Accrual to this trial continues. [Display omitted] [Display omitted] [Display omitted] DisclosuresInaba:Arog: Research Funding. Eckardt:Arog: Employment, Equity Ownership.

Clinical Implications of TP53 Mutations in Adult Patients with Newly Diagnosed Acute Lymphoblastic Leukemia (ALL) Treated with the Hypercvad-Based Regimens

Introduction -TP53 is the most extensively studied gene in cancer and is associated with a poor outcome. The clinical implication of TP53 gene mutations in adult ALL has not been evaluated.Methods - We screened for TP53 mutations in 119 newly diagnosed patients with ALL who were treated with HCVAD-based regimens (2012-2016) and evaluated the predictive and prognostic value of TP53mutations. Bone marrow samples underwent targeted amplicon-based next-generation sequencing (NGS) based mutation analysis.Results - Among 119 patients with adult ALL, we identified 23 patients (19%, 22 B-cell; 1 T-cell) with TP53 mutations (TP53mut); 96 had wild type TP53 (TP53wt; 83 B-cell; 13 T-cell). BCR/ABL1 rearrangement was noted in 2/23 TP53mut ALL and 43/96 TP53wt ALL (p=0.001). Of the 23 patients with TP53mut, 20 were missense, 2 were nonsense mutations and 1 was an insertion/ deletion, spanning exons 2-10. The most common pattern of amino acid substitution,in 10 of the 23 patients, was a substitution of arginine to histidine or proline on different codons. The median TP53 mutant allelic frequency was 42.2% (range, 1.4 - 93.8). Seven patients (30%) with TP53mut had concurrent mutations: NRAS in 2, BRAF, NOTCH1, TET2, DNMT3A, and EZH2 - 1 each. Among patients with TP53wt, 40 patients (42%) had mutations in other genes: 9 JAK2, 7 NRAS, 5 KRAS, 5 NOTCH1, 3 IDH2, 2 each with DNMT3A, ASXL1, FLT3, PTPN11, and 1 with TET2. The clinical characteristics, pattern of mutations, response to therapy, and outcomes of patients with TP53mut ALL (n=23) vs. TP53wt ALL(n=96) were compared (Table 1 and Figure-1). Patients with TP53mutALL were significantly older at presentation (median age 60 years [24-81] versus 46 years [18-81]; p=0.01) and had significantly lower platelet counts (29 x 109 /L versus 45 x 109/L; p=0.01) and lower peripheral blood blast percentages (10% versus 43%; p=0.005). Distribution of chromosomal aberrations was significantly different among the two groups: t(9;22) was observed at a much lower rate in TP53mut ALL, while hyper and hypodiploidy were more commonly encountered in TP53mut ALL There was no significant difference in the complete remission (83% in TP53mut ALL versus 91% in TP53wt ALL, p=0.25) and minimal residual disease (66% in TP53mut ALL versus 59% in TP53wt ALL, p=0.23) rates between the 2 groups. Overall, the median follow-up was 11.3 months (range, 0.2 to 41 months). There was no statistically significant difference in outcome between patients with and without TP5mutation. (Figure -1 A-B). The 3-year event-free (EFS) and overall survival (OS) rates were 45% and 42% in patients with TP53mutand 46% and 59% in those with TP53wt. The median EFS was 18 and 26.7 months (p=0.37) respectively. The median OS was 27.5 and 37.2 months (p=0.20), respectively. Furthermore, no significant difference was observed when only patients with Philadelphia-negative B-ALL were assessed.Conclusions -TP53 mutations are seen in about 19% of adult with newly diagnosed ALL. Patients tend to be older with higher incidence of hypodiploidy. TP53 mutations had no significant negative impact on outcome in patients treated with Hyper-CVAD based regimens. Further studies are underway at our institution to expand the cohort and identify the relevance of TP53mutations in adult ALL in the era of novel chemoimmunotherapy.*3 patients in TP53 - Mutated group had 1 patient each with miscellaneous/insufficient metaphases/ not done while 6 patients had miscellaneous aberrations and 4 with insufficient metaphases TP53wild type respectively [Display omitted] [Display omitted] DisclosuresKonopleva:Calithera: Research Funding; Cellectis: Research Funding. Jain:Incyte: Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novimmune: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Servier: Consultancy, Honoraria; BMS: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Novartis: Consultancy, Honoraria. Wierda:Novartis: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Abbvie: Research Funding. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy.

Higher EZH2 expression is associated with extramedullary infiltration in acute myeloid leukemia.

Accumulating evidence indicates that enhancer of zeste homolog 2 (EZH2) promotes the metastatic ability of solid tumors, but the role of EZH2 in extramedullary infiltration (EMI) in acute myeloid leukemia (AML) has not been thoroughly explored. In the present study, we investigated the possible association between EZH2 and EMI. We found that the messenger RNA (mRNA) and protein expression levels of EZH2 in AML patients were both significantly higher than in idiopathic thrombocytopenic purpura (ITP) patients. Furthermore, a positive correlation between EZH2 mRNA expression and percentage of peripheral blood blasts wa s found in AML patients (r = 0.404, p = 0.009). The migratory capacities of Kasumi-1 and HL-60, which both show a high level of EZH2 expression, were markedly higher than those of U937 and KG-1α. In contrast, silencing of EZH2 resulted in reduction in proliferation and migration ability and an increase in apoptosis. The latter observation was accompanied by reduced expression of associated proteins p-ERK, p-cmyc, and matrix metalloproteinase 2 (MMP-2) and an increase in epithelial cadherin (E-cadherin). These data suggest that higher expression of EZH2 may be associated with extramedullary infiltration in acute myeloid leukemia and affect pathogenesis via activation of the p-ERK/p-cmyc/MMP-2 and E-cadherin signaling pathways.

GATA2 Expression Level in Chronic Myeloid Leukemia (CML) Patients Correlates with Their Prognostic Scores and Is Associated with Disease Stage at Diagnosis

The Sokal, Hasford and EUTOS scoring systems have been successively proposed to predict the outcome of CML patients in chronic phase (CP-CML), both in terms of disease response and survival. The EUTOS score, which is the only one developed for patients treated with tyrosine kinase inhibitors, considers 2 risk groups based on spleen size and peripheral blood basophil percentage. No clear link has been identified between the covariates used to calculate those scores and CML molecular pathophysiology. To better delineate the molecular mechanisms underlying those scoring systems, a gene expression profiling study was performed on 48 CP-CML patients included in the Novartis ENEST1st study, for whom good quality peripheral blood total RNA was available at diagnosis. First, patients with low-risk (n=21) and high-risk (n=12) Sokal scores were compared, leading to identify 13 genes differentially expressed between those groups (q<.05). GATA2, which encodes a critical hematopoietic transcription factor, was among the 10 genes significantly overexpressed in high Sokal score CP-CMLs. Robust regression analyses confirmed that GATA2 expression level was correlated to the 3 prognostic scores, when considering those as continuous covariates (R2=.39, p<10-4 [Sokal]; R2=.16, p=.007 [Hasford]; R2=.16, p=.007 [EUTOS]). Among the covariates used to calculate those scores, platelet count (R2=.35, p<10-4), basophil (R2=.22, p=.0012), eosinophil (R2=.14, p=.01) and peripheral blood blast (R2=.13, p=.017) percentages were correlated to GATA2 expression level. Using a quantitative RT-PCR assay, GATA2 expression was subsequently assessed on an independent set of 80 CP-CML patients followed at Angers University Hospital from 2005 to 2014 (Sokal low, n=32; int, n=30; high, n=18 of which 7 EUTOS high). This validation set confirmed that GATA2 expression level was significantly correlated to the 3 prognostic scores (R2=.31, p<10-4 [Sokal]; R2=.28, p<10-4 [EUTOS]; R2=.11, p=.003 [Hasford]). Among the covariates used in those scores, basophil percentage (R2=.44, p<10-4), platelet count (R2=.31, p<10-4) and peripheral blood blast percentage (R2=.22, p<10-4) were significantly correlated to GATA2 expression level in this second cohort. The ENEST1st gene expression dataset and a linear regression approach were then used to better define the role of GATA2 in CP-CMLs. This analysis identified 12 genes significantly correlated to GATA2 in terms of expression levels (q<10-5). Half of those - CPA3, FCER1A, ENPP3, HDC, IL1RL1, SLC45A3 - were basophil-related genes. Interestingly, in normal umbilical cord blood-derived myelopoiesis, the expressions of GATA2, CPA3, FCER1A, and HDC were significantly correlated and coexpressed in common myeloid progenitors (CMP), granulocyte/monocyte progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) (GEO Microarray, GSE24759). Transcription factor ChIP-Seq data from the ENCODE project confirmed that, in K562 cell line, among the 12 genes identified above, GCSAML, EPAS1, FCER1A, HDC, IL1RL1, MGAT3, RDH11 and SLC45A3 were direct GATA2 targets. To extend this analysis, the gene expression dataset from Radich et al study (Proc Natl Acad Sci USA 2006;103:2794-9 - GEO Microarray, GSE4170), based on 108 peripheral blood samples from patients with CP-CML (n=57), accelerated phase (AP)-CML (n=17) or blastic crisis (BC)-CML (n=22 myeloid BC, n=12 B-cell lymphoblastic BC) were reanalyzed, and confirmed that GATA2 expression level was significantly correlated to the one of the basophil-related genes FCER1A, HDC and SLC45A3 in CP-CMLs. It also showed that GATA2 expression level significantly increased from CP-CMLs to myeloid BC-CMLs, whereas it was expressed at the lowest levels in lymphoblastic BC-CMLs. Similar results were observed when analyzing the dataset from Cramer-Morales et al study (Blood 2013;122:1293-304 - GEO Microarray, GSE47927), in which GATA2 was expressed at significantly higher levels in the hematopoietic stem cell, multipotent progenitors, CMP, GMP, and MEP compartments in myeloid BC-CMLs, when compared to the same compartment in AP-CMLs, CP-CMLs or in healthy volunteers. Therefore, GATA2 is a key gene affecting the outcome of CP-CMLs and the evolution into myeloid BC-CMLs. GATA2 is associated with a basophil-related gene expression signature that might explain the prognostic influence of the basophil percentage within the EUTOS score. DisclosuresGardembas:Novartis: Speakers Bureau. Spentchian:Novartis: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Radich:Gilliad: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Rousselot:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Speakers Bureau.

Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance.Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors.In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008).Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. DisclosuresNo relevant conflicts of interest to declare.

P38MAPK Inhibition Blocks Inflammatory Signaling in Acute Myeloid Leukemia

Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients.Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 < 1000nM were considered drug responsive.Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity.Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. DisclosuresWinski:Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells.

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs =0.317, P=0.006), hemoglobin levels (rs =0.210, P=0.049), and percentage of peripheral blood blasts (rs =0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs =–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Blast Transformation in Chronic Myelomonocytic Leukemia: Risk Factors and Outcome

Background: Prognosis in chronic myelomonocytic leukemia (CMML) is dire and blast transformation (BT) is a main cause of death. Current risk factors and prognostic models are mostly designed to predict overall (OS) and not leukemia-free (LFS) survival. Established risk factors for OS in CMML include anemia, thrombocytopenia, leukocytosis, monocytosis, presence of circulating immature myeloid cells (IMC), peripheral blood (PB) and bone marrow (BM) blasts, lymphocytosis, ASXL1 mutations, high risk abnormal karyotype and advanced age. In the current study, we examined risk factors for BT in CMML and survival outcome after BT.Methods: All CMML patients seen at our institution and with complete presentation data were considered. Clinical and laboratory variables, including cytogenetic information, at time of CMML diagnosis and also at time of BT were collected. DNA analysis for SRSF2, SF3B1, U2AF1, ASXL1 and SETBP1 mutations was carried out on specimens obtained at time of CMML diagnosis. We used both conventional and revised (see accompanying ASH 2014 abstract) cytogenetic risk models to query their influence in the current study population.Results:Clinical and laboratory features at time of CMML diagnosis: 277 consecutive patients (median age 71 years, range 18-91; 67% males) with WHO-defined CMML were considered. 236 (85%) patients had CMML-1 and the remainder CMML-2. According to the molecular Mayo risk model, 86 (33%) were high, 77 (29%) were intermediate-2, 77 (29%) were intermediate-1 and 24 (9%) were low risk. Mutational frequencies were 40% for ASXL1 (n evaluable =264), 44% for SRSF2 (n evaluable =263), 5% for SETBP1 (n evaluable =263). According to the revised Mayo-French consortium cytogenetic risk model, karyotype was high risk in 5%, intermediate in 21% and low risk in 74%.Risk factors for blast transformation (BT): At a median follow-up of 16.2 months for all 277 patients from time of CMML diagnosis, 204 (74%) deaths and 38 (14%) BTs were documented. Median time from CMML diagnosis to BT was 13.3 months. In univariate analysis, female sex, younger age, lower hemoglobin, higher leukocyte count, higher absolute monocyte count, higher lymphocyte count, presence of circulating immature myeloid cells (IMC), increased peripheral blood (PB) blast %, increased bone marrow (BM) blast %, BM blasts ≥10%, CMML-2, abnormal karyotype, high risk karyotype per the Spanish or Mayo-French consortium, and higher risk disease per Mayo, MDACC or Dusseldorf models were all associated with higher risk of BT. In contrast, ASXL1 (p=0.84), SRSF2 (p=0.28) or SETBP1 (p=0.97) mutations were not found to be significant. In multivariable analysis, only PB blast % (p<0.0001), presence of PB IMC (p=0.004) and younger age (p=0.03) remained significant.Outcome of blast transformation: At time of BT, karyotype was abnormal in 58% and clonal evolution from time of CMML diagnosis was documented in 13 (36%) of 36 evaluable patients. Among the 38 patients with BT, induction chemotherapy was documented in 12 patients and allogeneic stem cell transplant (ASCT) in 6. At a median follow-up of 4.2 months from time of BT, 34 (89%) of the 38 patients with BT had died. Among the four patients who were alive at the time of this writing, 3 had received induction chemotherapy and two ASCT. Median survival of patients with BT, from time of disease transformation, was 4.5 months with 1, 2 and 3 year survival rates of approximately 30%, 16% and 3%, respectively. In univariate analysis, higher leukocyte count (p=0.01) and not receiving induction chemotherapy (p=0.03) were associated with shortened survival. Neither ASXL1 nor SRSF2 mutations affected survival after BT.Conclusions: Blast transformation in CMML occurs in approximately 14% of patients followed for a median of less than 1.5 years. Presence of circulating immature cells and percentage of peripheral blood blasts are the major determinants of BT in CMML. Genetic risk factors for overall survival did not appear to influence leukemia-free survival. Prognosis of post-CMML BT is dismal with median survival of less than 6 months and a 3-year survival of less than 5%. Outcome was better in patients receiving induction chemotherapy followed by ASCT. DisclosuresNo relevant conflicts of interest to declare.

Multigene Mutational Clinical Profiling Using Next Generation Sequencing in a Cohort of 451 Patients with MDS: Impact on Clinical Outcomes

Background: Analysis of genomic alterations can play an important role in clinical and pathological evaluation of patients with MDS. Most studies have assessed the role of only a few genes. It is difficult to analyze a large number of genes in a large patient cohort in the context of a clinical molecular diagnostic laboratory.Aim: We sought to characterize the role of 56 candidate genes in MDS outcomes in 451 consecutive patients referred to one institution.Methods: DNA extracted from bone marrow aspirates were analyzed in a clinical molecular diagnostic laboratory underCLIA regulations between 9/2012 and 1/2014. Mutation analysis of 56 cancer-related genes was performed using a next generation sequencing (NGS) panel. Briefly, DNA extracted from bone marrow sample was tested for mutations in 56 genes using Illumina TruSeq chemistry on MiSeq (Illumina Inc.) NGS instrument. Sequence variants were detected using MiSeq Reporter software and verified by pathologists’ review. Summary statistics were used to describe the study population. Overall survival (OS) was calculated as number of months from diagnosis to death or last follow-up date. Patients alive at their last follow-up were censored on that date. Event-free survival (EFS) was calculated as the number of months from diagnosis to relapse, transformation to AML, death, or last follow-up date. The Kaplan-Meier product limit method was used to estimate the median OS for each clinical/demographic factor. Univariate Cox proportional hazards regression was used to identify association with each of the variables and survival outcomes. For these univariate analyses, some variables were transformed using the natural log scale. If the transformed variable showed a narrowed 95% CI (confidence interval) for the hazard ratio (HR), the natural log of the variable was used for analysis.Results: Median age was 66.6 (18-90) years. Fifty-nine patients (13%) were considered to have MDS/MPN, and 128 (28%) patients received prior therapy at the time analysis. IPSS-R cytogenetics were very good for 10 patients (2.4%), good for 198 (47%), intermediate for 98 (23%), poor for 16 (4%), and very poor 96 (23%). Most patients (62%) received HMA-based therapy. Complete response to any therapy was seen in 82 patients (38%). The median number of mutations was 1 (range 0 to 6), and 228 patients (50%) had no mutations, 154 (34%) had 1 mutation, 45 (10%) had 2 mutations, and 24 (5%) 3 or more mutations. Only 16 out 56 genes had frequencies >5: ASXL1, DNMT3A, EZH2, IDH1, IDH2, JAK2, KRAS, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, CEBPA, and FLT3-ITD. These genes were used for further analysis. There was no significant association between any clinical or demographic parameters and mutations in JAK2, NOTCH1, RUNX1, or CEBPA. All significant associations between clinical or demographic parameters and mutated genes are summarized in Fig 1. To further characterize the role of genetic mutation in outcomes, we created a “gene score” parameter based on univariate Cox regression models for each of the 16 genes. The gene score was the sum of all mutational contributions for a given survival parameter. Using stepwise backward selection in a multivariate model, we identified age (HR=1.08, p<0.001), bone marrow blast percentage (HR=1.44, p=0.012), and the gene score (HR=2.47, p=0.001) as associated with worse event-free survival. Peripheral blood blast percentage (HR=0.92, p<0.001), log of white blood cell count (HR=0.76, p<0.001), and hemoglobin (HR=0.64, p<0.001), were significant factors for better event-free survival. Age (HR=1.05, p<0.001) and gene score (HR=2.77, p<0.001) were associated with worse OS, and hemoglobin (HR=0.77, p=0.001), log of platelet count (HR=0.7, p=0.005), and peripheral blood blasts (HR=0.98, p=0.023) were associated with better OS.Conclusions: We identified a group of 12 genes that are significantly associated with clinical and demographic variables in a large cohort of MDS patients. Further characterization of the results of analysis and role of gene score as predictor of response may allow for more accurate stratification of patients based on genomic parameters.Abstract 4658. Table 1ASXL1DNMT3AEZH2IDH1IDH2KRASNPM1NRASPTPN11TET2TP53FLT3-ITDAgeBM blastsPB blastsMean WBCMean neutrophilsMean plateletsMean HgbIPSS CG riskComplex CGPrior chemotherapyPrior radiotherapyDiagnosis DisclosuresNo relevant conflicts of interest to declare.

Prediction of Treatment-Related Mortality in Patients with Relapsed and Refractory Acute Myeloid Leukemia

IntroductionDespite improvements in supportive care, treatment-related mortality (TRM) remains a significant problem for patients with acute myeloid leukemia (AML). In order to quantify fitness for intensive AML therapies, we recently developed a multivariate model for predicting TRM, defined as death within 28 days of treatment initiation in patients undergoing intensive induction chemotherapy for newly-diagnosed AML. Here, we examine the performance of this TRM model in patients with relapsed or refractory AML and assess whether the model can be improved for this population.MethodsUsing a database of patients treated for AML at our institution since 2003, we identified patients with relapsed or refractory disease treated with intensive chemotherapy, defined as regimens that were at least as intense as typical “7+3” regimens; patients who received treatment protocols with cytarabine <100 mg/m2/day or demethylating agents alone were excluded. We collected the following variables required to calculate the TRM score: ECOG performance status, age, platelet count, albumin, presence or absence of secondary AML, WBC, peripheral blood blast percentage, and creatinine. TRM scores were then calculated as described in J Clin Oncol 2011;29:4417. For a subset of these patients, the following additional variables were collected: duration of prior complete remission, number of prior chemotherapy regimens, number of prior hematopoietic cell transplants, and the number of antibiotics prescribed for treatment of presumed or documented infections at the time of chemotherapy initiation. For this smaller subset, we used a logistic regression model to create a preliminary updated TRM model that included these clinical variables in addition to the variables in the original TRM score. Finally, we used the area under the receiver operator characteristic curve (AUC) to quantify the ability of a model to predict TRM; in this approach, an AUC of 1 indicates perfect prediction of TRM while an AUC of 0.5 indicates no prediction; AUC values of 0.6-0.7, 0.7-0.8, and 0.8-0.9 are commonly considered as poor, fair, and good, respectively.ResultsA total of 270 patients met our study inclusion criteria. Fifteen (5.6%) died within 28 days of starting chemotherapy, i.e. experienced TRM. The AUC for the previously published TRM score in predicting early death in this population was 0.66. For comparison, in our original study of 2,238 patients with newly-diagnosed AML, we obtained an AUC of 0.82. Expanding the definition of TRM to include those patients who died within 60 (N = 47, 17.4%) or 90 days (N = 75, 27.8%) of starting chemotherapy did not improve the original model’s performance in our cohort (AUCs of 0.62 and 0.59, respectively). The additional variables described above were available for 133 of the 270 patients in our cohort. Within this subset, the AUC for the original TRM score was 0.65 in predicting death within 28 days. Incorporation of these new covariates in a preliminary TRM model yielded an improved AUC of 0.81 for this smaller subset.ConclusionsOur data indicate that the TRM score, originally developed for patients with newly-diagnosed AML, only has a fair ability to predict TRM in patients with relapsed and refractory AML. However, with inclusion of additional covariates, such as prior CR duration and number of prior chemotherapies, TRM can be predicted quite well, to a degree similar to the accuracy demonstrated by the original model in newly-diagnosed patients. While our findings need to be confirmed in larger, independent cohorts of patients, they suggest the possibility of developing an objective measure to determine fitness for intensive salvage chemotherapy in AML. DisclosuresNo relevant conflicts of interest to declare.

The Family Members of Mrps Expression and significance in Childhood Acute Lymphoblastic Leukemia

Objective Multidrug resistance-associated proteins 1 to 6 have been reported involved in a large number of tumors and have a close correlation with tumor multi-drug resistance. In this work, we detect the MRP2-6 genes expression in childhood acute lymphoblastic leukemia (ALL) by Q-RT-PCR and explore their clinical significance.Methods 156 patients at different stages of ALL were enrolled in this study and treated by the protocol (CCLG-2008) during 2012 to 2013, including 67 cases at initial stage, 70 cases at complete remission, and 9 cases at relapse, 10 patients diagnosed as idiopathic thrombocytopenic purpura (ITP) as control. MRP1-6 genes’ expressions were detected using real-time quantitative PCR (QRT-PCR)and their clinical significance was analyzed by the SPSS software 16.0. P value below 0.05 was regarded as statistic significance.Results The median expression of MRP1 was 5.82 and 8.49 for initial and relapse group,respectively, which was statistic higher than that at complete remission, the latter was 1.99. MRP1 expression level had close correlation with ALL risk, the median of MRP1 expression was 4.28, 5.62 and 7.56 for standard-risk group (SR), intermediate-risk group (IR) and high-risk group (HR), respectively. Initial ALL children were divided into two groups including high expression group and low expression group by the median expression, the rate of sensitivity of blast cells to prednisone on 7th day was 70.6% in high expression group (n=34), which was statistic lower than that in low expression group which was 90.9% (n=33, P=0.035). The rate of complete remission on 33th day in high expression group was 64.7%, while 87.9% in low expression group, which showed a significant difference between them (P=0.026). The rate of complete remission on 15th day in high expression group was 68.8%, and 69.7% in low expression group, which showed no significance between them (P=0.664). The transcription level of MRP1 in initial group of T-ALL (median=7.71) was statistic higher than that in B-ALL (median=5.18) (P=0.007). Correlation analysis indicated that mRNA expression level of MRP1 didn’t show any relationship with gender, age, WBC count, hemoglobin, platelet and blast percentage in bone narrow and peripheral blood at diagnosis.The median expression of MRP2 at initial stage was highest, higher than that at relapse and complete remission, but did not reach statistic significance. However, the median expression of MRP3 at initial stage was highest and statistic higher than complete remission. MRP4 and MRP5 showed a similar pattern in their expression, namely, high expression for relapse, intermediate expression for initial stage and low expression at complete remission which reached statistic significance. The median expression of MRP6 for relapse was 2.003, which was higher than initial and complete remission group, but the differences among them were not significant. The median of MRP2 and MRP5 expression for intermediate-risk group (IR) (Median MRP2=4.622, Median MRP5=1.712) was higher than standard- risk group (SR) (Median MRP2=3.279, Median MRP5=1.277) and high risk group (HR) (Median MRP2=2.145, Median MRP5=1.673). However, there was no statistical significance among them. The median of MRP4 expression was increase continually with the risk of ALL, but did not reach statistical significance among them; the median of MRP6 expression for high-risk group (HR) was higher than standard-risk group (0.8812 vs 0.6205) and intermediate-risk group (0.8812 vs 0.4053), but the differences among them were not significant either. Initial ALL children were divided into two group including high expression group and low expression group by median expression, and evaluated their prediction on the treatment response on 7th, 15th and 33th day. The results revealed no significant difference between them, neither between B-ALL and T-ALL. Single factor analysis showed that MRP2 has relationship with platelet count at diagnosis and MRP4 has relationship with gender.Conclusions In children ALL, the expression of MRP1 is closely related with immunophenotyping, treatment response, hazard level and disease relapse which was a poor biomarker for ALL prognosis. MRP2-6 has a different expression pattern. MRP4 and MRP6 mRNA expression showed a close relation with relapse. DisclosuresNo relevant conflicts of interest to declare.