non-M3 Acute Myeloid Leukemia Patients Research Articles

Prognostic Impact of Time from Diagnosis to Initial Chemotherapy for Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) has been considered as oncologic emergency and therefore, immediate initiation of chemotherapy is common in clinical practice. On the other hand, waiting until cytogenetic and molecular results return could offer individualized therapies according to the risk stratifications of AML based on cytogenetic or molecular abnormalities. There are few data on the impact of delaying induction chemotherapy on the prognosis of AML. According to one study, time from diagnosis to initial treatment (TDT) of more than 5 days had adverse impact on younger AML patients, but not older patients (Sekeres et al. Blood 2009). Another study showed TDT had no impact on overall survival (OS) at all age (Bertoli et al. Blood 2013). We retrospectively analyzed the impact of TDT on outcome of newly diagnosed AML patients in our institution.Methods: This study included 145 newly diagnosed non-M3 AML patients who were treated with induction chemotherapy in our institution between 2008 and 2017. Patients received various kinds of induction therapy including anthracycline, cytarabine, azacitidine and investigational drugs as initial therapy. Associations of TDT with patient characteristics available at diagnosis and complete remission (CR) rate and OS were examined.Results: The median age was 66 years (range, 18-88) and the median TDT was 1 day (range, 0-91). Cytogenetic risk group was favorable in 13 (9%), intermediate in 93 (64%) and adverse in 39 (27%) according to Medical Research Council cytogenetic classification. NPM1 mutation was detected in 11 of 38 (29%) evaluable patients and FLT3-ITD was in 8 of 45 (18%). Shorter TDT was significantly associated with progressive disease including younger age, poor performance status, de novo AML, elevated white blood cell (WBC) count, elevated percentage of bone marrow blasts, elevated lactate dehydrogenase levels (all P < 0.05). CR rate was 52% and median OS was 431 days. TDT had significant impact on CR rate (P = 0.004) but not OS (P = 0.978). However, after adjustment for age, cytogenetic risk groups and WBC count, the association with both CR rate and OS was not significant (P = 0.581 and P = 0593, respectively). Furthermore, TDT was not associated with early death in univariate analysis (p = 0.186).Conclusion: TDT is significantly associated with progressive disease, but not with outcome in newly diagnosed AML. These results suggest that, except for special situations, delaying initial treatment by a short period of time to wait for the results of laboratory tests may be acceptable, allowing for individual therapies. To verify the prognostic effect of TDT, further investigation in a larger cohort is required.Disclosures: No relevant conflicts of interest to declare. DisclosuresUsuki:Ono Pharmaceutical: Speakers Bureau; Novartis: Speakers Bureau; SymBio Pharmaceuticals Limited.: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; GlaxoSmithKline K.K.: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Shire Japan: Research Funding; Takeda Pharmaceutical: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau.

Association between TLR2 and TLR4 Expression and Response to Induction Therapy in Acute Myeloid Leukemia Patients

Background: Toll-like receptors (TLRs) are a family of transmembrane pattern-recognition receptors that play a crucial role in the realization of innate and adaptive immune response. TLRs may play a role in tumor development and growth because of expression or up-regulation of functional TLRs in some tumors and tumor cell lines. The participation of TLRs in the pathogenesis of acute myeloid leukemia (AML) remains unspecified. This study aimed to investigate the effect of TLR2 and TLR4 expression in peripheral blood mononuclear cells of AML patients in response to induction chemotherapy. Materials and Methods: Eighty- five patients with newly diagnosed AML were evaluated. Using quantitative reverse transcriptase PCR, the mRNA expression of genes TLR2 and TLR4 was measured before starting and after induction chemotherapy. The differences in the mean expression levels of TLR2 and TLR4 before and after chemotherapy were compared using a paired t-test. The mean expression levels of TLR2 and TLR4 regarding laboratory data were analyzed by one-way ANOVA and Chi-square test. Results: We found that the mRNA expression of TLR2 after induction chemotherapy was significantly lower as compared to before treatment (p=0.001). Also, we found a lower TLR4 gene expression level after chemotherapy as compared to before chemotherapy, albeit it was not statistically significant (p=0.21). Moreover, we observed significantly higher expression of TLR2 and TLR4 in AML-M3 cases compared to non-M3 AML patients. Conclusion: The decreased expression of TLR4 in leukemic samples after induction chemotherapy might indicate a novel potential prognostic role for this receptor, particularly in AML-M3 cases.

PARP-1 Overexpression as an Independent Prognostic Factor in Adult Non-M3 Acute Myeloid Leukemia.

Poly (ADP-ribose) polymerase-1 (PARP-1) plays an important role in the repair of damaged DNA and has prognostic significance in a variety of human malignancies. However, little is known about its expression levels and clinical implication in patients with acute myeloid leukemia (AML). Quantitative reverse transcription-polymerase chain reaction was done to evaluate PARP-1 expression levels in the bone marrow of 65 patients with non-M3 AML and 54 healthy counterparts. The correlation of PARP-1 expression with clinicopathological features of non-M3 AML patients was also analyzed. Non-M3 AML patients have higher PARP-1 expression than the healthy controls (p < 0.01). Patients with adverse cytogenetic risk have higher PARP-1 expression than other cytogenetic risk groups (p = 0.004). The PARP-1 median expression level divided AML patients into PARP-1 low-expressed and PARP-1 high-expressed groups. High expression levels of PARP-1 were associated with worse overall survival (OS) (p = 0.01) and relapse-free survival (RFS) (p = 0.005). Moreover, multivariate analysis revealed that high PARP-1 expression was an independent risk factor for both OS and RFS. Our results suggest that PARP-1 overexpression may define an important risk factor in non-M3 AML patients and PARP-1 is a potential therapeutic target for AML treatment.

Hyperleukocytosis is associated with distinct genetic alterations and is an independent poor-risk factor in de novo acute myeloid leukemia patients.

Acute myeloid leukemia (AML) with hyperleukocytosis (HL) is intuitively thought as a unique group with dismal prognosis. However, comprehensive studies regarding the genetic landscape and clinical outcome in this group of patients are limited. A total of 693 newly diagnosed de novo non-M3 AML patients were consecutively enrolled. We compared relevant mutations in 20 genes between AML patients with or without HL and exposed their prognostic implications. Hyperleukocytosis, defined as initial white blood cell counts above 50000/μL, occurred in 28.9% of AML patients. HL patients had higher incidences of FLT3-ITD, NPM1, DNMT3A, CEBPA, and TET2 mutations. Multivariate analysis demonstrated that HL was an independent poor prognostic factor for overall survival and disease-free survival in total patients, those with intermediate-risk cytogenetics and normal karyotype irrespective of genetic alterations. Intriguingly, HL predicted poor survival in CEBPA double mutated, NPM1+/FLT3-ITD- and NPM1-/FLT3-ITD- patients. Further, HL patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR) had a significantly longer overall survival and disease-free survival than those without allo-HSCT. Hyperleukocytosis is an independent poor prognostic factor irrespective of cytogenetics and mutation status. Allo-HSCT in first CR seems to ameliorate the poor prognostic impact of HL.

Low Expression of Long Noncoding RNA IRAIN Is Associated with Poor Prognosis in Non-M3 Acute Myeloid Leukemia Patients

Deregulation of the long noncoding RNA IRAIN has been identified in several cancers. However, the expression pattern of IRAIN and its clinical implication in acute myeloid leukemia (AML) are unknown. The purpose of this study was to investigate the expression status of IRAIN and its clinical significance in non-M3 AML patients. Quantitative reverse transcription-polymerase chain reaction was performed to examine IRAIN transcript levels in 64 de novo non-M3 AML patients and 51 healthy controls. The association of IRAIN expression with clinicopathological factors was statistically analyzed. Compared with the controls, IRAIN was significantly downregulated in non-M3 AML patients (p < 0.001). The median of IRAIN expression divided the non-M3 AML patients into IRAIN low-expressing (IRAINlow) and IRAIN high-expressing (IRAINhigh) groups. The IRAINlow group tended to have higher white blood cell count and blast counts and had markedly shorter overall survival (OS) and relapse-free survival (RFS) (p = 0.044 and 0.009, respectively). In addition, patients with refractory response to chemotherapies and those with subsequent relapse had lower initial IRAIN expression. Multivariate analysis further identified IRAIN transcript levels as an independent prognostic factor for both RFS and OS. Our finding suggests that IRAIN transcript levels may be a useful biomarker for the prognosis of non-M3 AML patients.

Complete Remission Rate and Adverse Ractions of Three Different Chemotherapy Regimens in the Treatment of Adult Patients with Newly Diagnosed Non-M3 AML

To compare the complete remission rate (CRR) and adverse reaction of the 3 different chemotherapy regimens (daunorubicin, idarubicin, imported idarubicin combined with cytarabine) for the treatment of adult patients with newly diagnosed non-M3 acute myeloid leukemia (AML). Seventy-one adult patients with newly diagnosed non-M3 AML were divided into 3 groups: 17 cases treated with daunorubicin plus cytarabine as group A, 24 cases treated with idarubicin plus cytarabine as group B, 30 cases treated with the imported idarubicin plus cytarabine as group C. The curative effects and adverse reactions were compared among the 3 groups after treatment. CCR in group C (86.67%) was significantly higher than that in group A (52.94%) and group B (70.83%), and the CRR in group B was significantly higher than that in group A (P<0.05). The incidence of adverse reaction such as nausea, vomiting, myelosuppression and infection among 3 groups were not statistically significantant (P>0.05). The curative effect of idarubicin for the treatment of non-M3 AML patients is better than that of daunorubicin, especially the curative efficiency of imported darubicin is much higher; the adverse reaction after treatment by daunorubicin and idarubicin can be controllable, so daunorubicin and idarubicin can be used as first-line drug for the patients with AML, and patients can choose more appropriate drug according to their own economic ability.

Methylation-associated DOK1 and DOK2 down-regulation: Potential biomarkers for predicting adverse prognosis in acute myeloid leukemia.

DOK-1 and DOK-2 (DOK1/2) are closely related members of downstream of tyrosine kinase (DOK) family genes, which are found to be frequently rearranged in several hematopoietic cancers. However, the clinical implications of DOK1/2 in acute myeloid leukemia (AML) remain largely unknown. To investigate the clinical significance, real-time quantitative PCR (RQ-PCR) was carried out to detect DOK1/2 expressions in 125 de novo AML patients and 28 healthy controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were applied to detect DOK1/2 methylation level and density. DOK1/2 expressions were significantly down-regulated in AML patients. The promoters of DOK1/2 were highly hypermethylated and negatively correlated with DOK1/2 expressions in AML patients. In addition, we also confirmed that DOK1/2 expressions could be restored by DOK1/2 demethylation using 5-aza-2'-deoxycytidine in leukemia cell line THP-1. Survival analyses showed that low-expressed DOK1/2 were associated with markedly shorter overall survival and leukemia free survival in both whole-cohort AML and non-M3 AML patients. Multivariate analyses further revealed that DOK1/2 were act as independent prognostic factors in AML patients. These findings indicate that decreased DOK1/2 expressions associated with their promoter hypermethylations predict adverse prognosis in AML.

Incorporation of Long Non-Coding RNA Expression Profile in the 2017 ELN Risk Classification Can Improve Prognostic Prediction of Acute Myeloid Leukemia Patients

Background: Expression of long non-coding RNAs (lncRNAs) has recently been recognized as a potential prognostic marker in acute myeloid leukemia (AML). However, it remains unclear whether incorporation of the lncRNAs expression in the 2017 European LeukemiaNet (ELN) risk classification can further improve the prognostic prediction. Methods: We enrolled 275 newly diagnosed non-M3 AML patients and randomly assigned them into the training (n=183) and validation cohorts (n=92). In the training cohort, we formulated a prognostic lncRNA scoring system composed of five lncRNAs with significant prognostic impact from the lncRNA expression profiling. Findings: Higher lncRNA scores were significantly associated with older age and adverse gene mutations. Further, the higher-score patients had shorter overall and disease-free survival than lower-score patients, which were also confirmed in both internal and external validation cohorts (TCGA database). The multivariate analyses revealed the lncRNA score was an independent prognosticator in AML, irrespective of the risk based on the 2017 ELN classification. Moreover, in the 2017 ELN intermediate-risk subgroup, lncRNA scoring system could well dichotomize the patients into two groups with distinct prognosis. Within the ELN intermediate-risk subgroup, we found that allogeneic hematopoietic stem cell transplantation could provide better outcome on patients with higher lncRNA scores. Through bioinformatics approach, we identified high lncRNA scores were correlated with leukemia/hematopoietic stem cell signatures. Interpretation: Incorporation of lncRNA scoring system in 2017 ELN classification can improve risk-stratification of AML patients and help clinical decision-making. Funding Statement: This work was supported Ministry of Science and Technology, and Ministry of Health and Welfare of Taiwan. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This study was approved by the Research Ethics Committee of the NTUH in accordance with the Declaration of Helsinki.

Bone marrow miR-10a overexpression is associated with genetic events but not affects clinical outcome in acute myeloid leukemia

BackgroundAccumulating studies have linked the disruptions of microRNA-10 (miR-10) to acute myeloid leukemia (AML) with NPM1 mutation. However, miR-10 expression and its clinical implication in AML remain poorly defined. Although a recent report showed high serum level of miR-10a was associated with adverse prognosis in AML, herein, we found bone marrow (BM) miR-10 overexpression was not a prognostic biomarker in AML. MethodsBM miR-10 expression was examined by real-time quantitative PCR in BM mononuclear cells in 115 de novo AML patients and 45 controls. ResultsBM miR-10 (miR-10a/b) expression was significantly up-regulated in AML patients, and was positively correlated with each other. Overexpression of miR-10a was associated with lower percentage of BM blasts, whereas miR-10b overexpression tended to correlate with higher percentage of BM blasts. Importantly, miR-10a overexpression was significantly associated with FAB-M3/t(15;17) subtypes and NPM1 mutation, meanwhile, overexpression of miR-10b was correlated with NPM1 and DNMT3A mutations. However, miR-10a/b overexpression was not associated with complete remission rate, and did not have an impact on both leukemia free survival and overall survival time in non-M3 AML patients without NPM1 mutation. ConclusionsBM miR-10 overexpression is associated with genetic events but not affects clinical outcome in AML.

High bone marrow miR-19b level predicts poor prognosis and disease recurrence in de novo acute myeloid leukemia

Oncogenic role of miR-19 family has been identified in human cancers especially in lymphoid malignancies. However, to date, little studies investigated the role of miR-19 family in myeloid malignancies. Herein, we examined miR-19a/b expression and explored its clinical significance in de novo acute myeloid leukemia (AML). The detection of miR-19a/b expression was performed by real-time quantitative PCR in bone marrow mononuclear cells of 113 patients and 42 healthy donors. Both miR-19a/b levels were significantly increased in AML patients in contrast to controls. Patients with miR-19a/b overexpression were more frequently occurred in female, and had an older age. Moreover, cases with miR-19a overexpression had a higher frequency of U2AF1, C-KIT and CEBPA mutations, whereas miR-19b overexpressed cases harbored U2AF1 and IDH1/2 mutations. There was no significant association of miR-19a overexpression with complete remission (CR) rate and overall survival (OS) among whole-cohort AML, non-M3 AML, and cytogenetically normal AML (CN-AML). However, although miR-19b overexpression was not correlated with CR rate, patients with miR-19b overexpression presented significantly shorter OS in whole-cohort AML and a trend in non-M3 AML and CN-AML patients. Importantly, our data also showed that miR-19a/b expression level at CR phase was lower than diagnosis time, and was returned to primary level even higher when at relapse phase. Our findings revealed that miR-19a/b overexpression were frequent events in de novo AML patients. Moreover, up-regulation of miR-19b expression was associated with poor prognosis and disease recurrence in AML.

RASSF1A hypermethylation is associated with ASXL1 mutation and indicates an adverse outcome in non-M3 acute myeloid leukemia

ObjectiveThe purpose of this study was to evaluate the frequency of RASSF1A hypermethylation in patients with acute myeloid leukemia (AML), in an attempt to modify the current molecular model for disease prognosis.Materials and methodsAberrant RASSF1A promoter methylation levels were assessed in 226 newly diagnosed non-M3 AML patients and 30 apparently healthy controls, by quantitative methylation-specific polymerase chain reaction. Meanwhile, RASSF1A mRNA levels were detected by real-time quantitative polymerase chain reaction. Furthermore, hematological characteristics, cytogenetic abnormalities, and genetic aberrations were assessed. Finally, associations of RASSF1A hypermethylation with clinical outcomes were evaluated.ResultsRASSF1A hypermethylation was observed in 23.0% of patients with non-M3 AML (52/226), but not in controls. Meanwhile, hypermethylation of the RASSF1A promoter was significantly associated with ASXL1 mutation. Furthermore, the log-rank test revealed that RASSF1A hypermethylation indicated decreased relapse-free survival (RFS) and overall survival (OS) in patients with non-M3 AML (P=0.012 and P=0.014, respectively). In multivariate analysis, RASSF1A hypermethylation was an independent prognostic factor for RFS (P=0.040), but not for OS (P=0.060).ConclusionHypermethylation of the RASSF1A promoter is associated with ASXL1 mutation in non-M3 AML patients, likely indicating poor outcome. These findings provide a molecular basis for stratified diagnosis and prognostic evaluation.

Hypermethylation of secreted frizzled-related proteins predicts poor prognosis in non-M3 acute myeloid leukemia.

ObjectiveSecreted frizzled-related proteins (SFRPs) as Wnt signaling antagonists have been found to be dysregulated by promoter hypermethylation in several cancers including acute myeloid leukemia (AML). This study aimed to investigate the methylated status of SFRPs promoter region and its clinical relevance in Chinese non-M3 AML patients.MethodsSFRPs methylation in 139 primary non-M3 AML patients was determined using methylation-specific real-time quantitative polymerase chain reaction.ResultsThe frequency of aberrant methylation was as follows: 30.2% for SFRP1, 27.3% for SFRP2, 5.0% for SFRP4, and 1.4% for SFRP5. Hypermethylation of at least one SFRP gene occurred in 51.8% (72/139) of non-M3 AML patient samples, which was significantly higher compared to normal control (0/21) (P<0.001). Hypermethylation of SFRP1 was potentially associated with N/K-RAS mutations (P=0.043), and the frequency of SFRPs methylation was higher in patients ≥50 years compared to those <50 years, especially for SFRP2 (P<0.05). Furthermore, both whole cohort and cytogenetically normal (CN) patients with high SFRPs-methylated group showed a shorter overall survival (OS) compared to those with low group (P=0.036 and P=0.035, respectively). Moreover, Cox regression multivariate analysis revealed that SFRPs hypermethylation acts as an independent prognostic biomarker among both whole cohort (hazard ratio =1.804, P=0.026) and CN (hazard ratio =2.477, P=0.023) patients. In leukemic cell line HL60 treated with 5-aza-2′-deoxycytidine, the alteration of SFRP1/2 expression inversely correlated with change in SFRP1/2 methylation (r=−0.975, P=0.005 and r=−0.975, P=0.005, respectively). A tendency of negative correlation was observed between SFRP1 expression and its promoter methylation in AML patients (r=−0.334, P=0.038).ConclusionThese findings suggested that hypermethylation of SFRP1/2 was a frequent event and silenced SFRP1/2 expression in AML. Moreover, hypermethylation of SFRPs promoter was an adverse risk factor for OS in Chinese non-M3 AML patients.

Marker Chromosomes Can Arise from Chromothripsis and Predict Adverse Prognosis in Acute Myeloid Leukemia

Introduction: Cytogenetic testing is routinely performed in newly diagnosed acute myeloid leukemia (AML) for risk stratification. Elaborate risk classifications based on karyotyping are provided by both the European Leukemia Net (ELN) and the Medical Research Council (MRC). Complex aberrant, monosomal and abnl(17p) karyotypes confer a poor prognosis. In cytogenetic studies, chromosome aberrations that cannot be identified due to gross rearrangement, thereby preventing the allocation to a specific chromosome, are designated as "Marker Chromosomes" (MC). The significance of MC as prognostic factor for AML has remained elusive so far. In this study we have assessed frequency, cytogenetic characteristics and prognostic impact of MC as well as their underlying biological origin. Given the gross structural chromosomal damage inherent to MC we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event.Patients and Methods: Patients recruited intwo large consecutive, prospective, randomized, multicenter clinical trials for newly diagnosed non-M3 AML patients from the German Study Alliance Leukemia (SAL) were analyzed (AML96, NCT00180115; AML2003, NCT00180102). All karyotypes were retrospectively screened for MC. For the detection of chromothripsis array-CGH was used. For each sample 50 ng of DNA were hybridized to an Affymetrix® CytoScan HD Oligo/SNP-array and scanned with the Affymetrix GeneChip® Scanner 3000 7G. Chromothripsis was defined according to the criteria of Rausch et al., which require at least 10 switches in segmental copy number involving two or three distinct copy number states on a single chromosome.Results: MC were detectable in 165/1026 (16.1%) of aberrant non-CBF karyotype cases. Adverse-risk karyotypes displayed a higher frequency of MC (40.3% in complex aberrant, 26.5% in adverse-risk as defined by MRC criteria and 41.2% in abnl(17p) karyotypes, p<.001 each). MC were associated with a poorer prognosis compared to other non-CBF aberrant karyotypes as well as with lower remission rates (CR+CRi; 36.0% vs. 55.8% in AML96 ≤60 years, p=0.01; 14.3% vs. 44.1% in AML2003, p<0.001), inferior event-free survival (2.24 vs. 6.54 months, p<0.001; 3.45 vs. 8.03 months, p<0.001) and overall survival (5.72 vs. 11.87 months, p<0.001; 8.68 vs. 20.78, p=0.01). In multivariate analysis with co-variables age, prior MDS, therapy-related AML and adverse-risk cytogenetics according to MRC criteria, MC independently predicted poor prognosis in AML96 ≤60 years but not in AML2003 with its higher allogeneic transplantation rate.As detected by array-CGH, in about one third of MC karyotypes (18/49, 36.7%, including 3 cases with 8 or 9 copy number switches) MC had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of complex aberrant karyotypes without MC (1/34) (p<0.001). Chromothripsis in MC karyotypes typically involved one single chromosome (n=11), with two or three chromosomes affected in 5 and 2 patients, respectively. There was no predilection for a particular chromosome. MC karyotypes positive for chromothripsis were characterized by a particularly high degree of karyotype complexity as compared to those that were negative for chromothripsis (complex aberrant 100% vs. 64.5% p<0.01; abnl(17p) 50.0% vs. 16.1%, p=0.01). In 12/18 (66.7%) cases, at least one of the chromothriptic chromosomes was reported as loss in the karyotype formula, suggesting that the grouping of a chromothriptic chromosome as a marker is paralleled by a putative loss of the affected chromosome. The chromothripsis positive MC karyotype subgroup had a particularly dismal prognosis with a combined CR+CRi rate of 2/16 vs. 10/31 (p=0.14). It also displayed inferior event-free and overall survival, though statistical significance was not reached for either endpoint, likely due to the already poor prognosis of the entire MC positive group.Conclusion: This is the first study showing that MC are a frequent finding predominantly in adverse-risk AML and associated with particularly poor prognosis. Our data provide evidence that a substantial portion of MC arise from chromothripsis. DisclosuresThiede:AgenDix: Employment, Other: Ownership.

CTNNA1 hypermethylation, a frequent event in acute myeloid leukemia, is independently associated with an adverse outcome.

The aim of this study is to evaluate the frequency of CTNNA1 hypermethylation in acute myeloid leukemia (AML) patients in an attempt to improve molecular prognostic model. CTNNA1 promoter methylation levels in 319 newly diagnosed AML patients were detected using quantitative methylation-specific polymerase chain reaction (qMS-PCR). Furthermore, hematological characteristics, cytogenetic abnormalities, and genetic mutation status were analyzed, followed by assessment of clinical impact. Our findings demonstrated that CTNNA1 hypermethylation was observed in 25% AML patients. Hypermethylation of the CTNNA1 promoter was associated with unfavorable karyotype, and also possessed the higher frequency of coexisting with ASXL1 and RUNX1 mutations. Patients with CTNNA1 hypermethylation exhibited the shorter relapse-free survival (RFS) and overall survival (OS) in the whole AML and non-M3 AML patients. Moreover, patients with the higher methylation levels had more aggressive course than those with relative lower levels. In multivariate analyses, CTNNA1 hypermethylation was an independent factor predicting for poor RFS, but not for OS. In conclusion, CTNNA1 hypermethylation may be a reliable factor for improving prognostic molecular model for AML.

Reduced miR-215 expression predicts poor prognosis in patients with acute myeloid leukemia.

Abnormal expression of microRNA-215 has been identified in a variety of solid cancers. However, little is known about the expression pattern of microRNA-215 in acute myeloid leukemia. This study was to investigate the status of microRNA-215 expression and further analyze its clinical significance in acute myeloid leukemia. Real-time quantitative polymerase chain reaction assay was performed to evaluate the expression level of microRNA-215 in 113 patients with acute myeloid leukemia. Besides, the relationship between microRNA-215 levels and clinical and pathological factors was explored. Compared with the healthy individuals, microRNA-215 expression in acute myeloid leukemia patients was significantly down-regulated (P= 0.001). MicroRNA-215 low-expressed patients had higher white blood cells than microRNA-215 high-expressed patients (P= 0.014). The incidence of FLT3/ITD mutation in the patients with low microRNA-215 expression was significantly higher than those with high microRNA-215 expression (P= 0.025). MicroRNA-215 low-expressed patients had significantly shorter overall survival than microRNA-215 high-expressed patients in both non-M3 acute myeloid leukemia patients and cytogenetically normal patients (P= 0.017 and P= 0.044, respectively). Meanwhile, multivariate analysis confirmed the adverse prognostic value of microRNA-215 expression in acute myeloid leukemia patients with non-M3 subtypes. Our study demonstrates that reduced microRNA-215 expression is a common event and is associated with poor clinical outcome in acute myeloid leukemia.

High Incidences of Invasive Fungal Infections in Acute Myeloid Leukemia Patients Receiving Induction Chemotherapy without Systemic Antifungal Prophylaxis: A Prospective Observational Study in Taiwan.

Invasive fungal infections (IFIs) is an important complication for acute myeloid leukemia (AML) patients receiving induction chemotherapy. However, the epidemiological information is not clear in Southeastern Asia, an area of potential high incidences of IFIs. To clarify it, we enrolled 298 non-M3 adult AML patients receiving induction chemotherapy without systemic anti-fungal prophylaxis from Jan 2004 to Dec 2009, when we applied a prospective diagnostic and treatment algorithm for IFIs. Their demographic parameters, IFI characters, and treatment outcome were collected for analysis. The median age of these patients was 51 years. Standard induction chemotherapy was used for 246 (82.6%) patients, and 66.8% of patients achieved complete remission (CR) or partial remission. The incidence of all-category IFIs was 34.6% (5.7% proven IFIs, 5.0% probable IFIs and 23.8% possible IFIs). Candida tropicalis was the leading pathogen among yeast, and lower respiratory tract was the most common site for IFIs (75.4%, 80/106). Standard induction chemotherapy and failure to CR were identified as risk factors for IFIs. The presence of IFI in induction independently predicted worse survival (hazard ratio 1.536 (1.100–2.141), p value = 0.012). Even in those who survived from the initial IFI insults after 3 months, the presence of IFIs in induction still predicted a poor long-term survival. This study confirms high incidences of IFIs in Southeastern Asia, and illustrates potential risk factors; poor short-term and long-term outcomes are also demonstrated. This epidemiological information will provide useful perspectives for anti-fungal prophylaxis and treatment for AML patients during induction, so that best chances of cure and survival can be provided.

ITD mutation burden for the prognosis in FLT3-ITD positive acute myeloid leukemia patients

目的探讨FLT3-ITD突变数量、重排碱基长度和比例与急性髓系白血病（AML）患者的总体生存（OS）时间及持续完全缓解时间（CRD）的关系。方法采用PCR-毛细管电泳法检测130例AML（除外M3）患者的FLT3-ITD突变。根据电泳峰型数量分为单或多个（片段）突变；FLT3-ITD突变比例为突变型峰面积/野生型和突变型峰面积之和；分析FLT3-ITD突变患者临床特征并随访患者预后。结果FLT3-ITD多个突变常见于60岁以上患者；FLT3-ITD单个突变患者初诊骨髓原始细胞比例高于多个突变患者（0.758对0.638，P=0.028）；FLT3-ITD突变数量不影响患者预后。FLT3-ITD突变重排碱基长度亦对患者预后无明显影响。FLT3-ITD突变比例<10％患者的OS时间和CRD与同期C-KIT突变的中危组AML患者相似，均显著长于突变比例≥10％患者（中位OS时间分别为未达到、未达到、9.9个月，P<0.05；中位CRD分别为未达到、未达到、6.7个月，P<0.05）。FLT3-ITD突变比例≥10%的AML患者中，FLT3-ITD突变伴随NPM1或CEBPA突变患者的中位CRD显著长于单纯FLT3-ITD突变的患者（25.0个月对5.1个月，P=0.003），但两组中位OS时间差异无统计学意义（11.4个月对8.0个月，P>0.05）。结论FLT3-ITD突变阳性的AML（除外M3）患者中，FLT3-ITD突变比例<10％的患者预后好于突变比例≥10％的患者。

Significance of GRP78 expression in acute myeloid leukemias

Abstract The GRP78 (glucose-regulated protein 78) is a major endoplasmic reticulum (ER) chaperone facilitating proper folding of the newly synthesized proteins. By the interaction with caspases, GRP78 has antiapoptotic properties allowing cells to survive under stress condition. GRP78 expression and its association with tumor proliferation, metastasis and resistance to chemotherapy were observed in solid tumors. There are limited data on the expression and impact of this protein on the clinical course and treatment response in acute myeloid leukemia (AML). The aim of this study was to evaluate the expression of GRP78 mRNA in patients with de novo AML. These results were compared to healthy controls, blast phenotype, molecular and cytogenetic status and clinical features of AML. 101 non-M3 AML patients and 26 healthy individuals were included in this study. The expression of GRP78 mRNA in bone marrow was analyzed by real-time quantitative polymerase chain reaction (RQ-PCR). We demonstrated increased GRP78 mRNA expression in AML patients compared to healthy controls, although this difference was statistically significant only in CD34+ leukemias. There was also no significant correlation between GRP78 mRNA expression and complete remission rate, relapse-free survival and overall survival. These results indicate that GRP78 expression is increased in CD34+ leukemias and has no prognostic impact on clinical outcome in AML.

Sample Cryopreservation Does Not Affect Functional Read Outs In SCNP Assays: Implications for Biomarker Development

AbstractAbstract 4843 Background:All major molecular technology platforms have used cryopreserved samples as a source in biomarker development studies. Such samples enable the conduct of prospectively designed studies using banked samples with relevant clinical annotations, thus potentially reducing the duration of biomarker development. Single Cell Network Profiling (SCNP) is a new technology platform requiring viable cells. It uses multi-parametric flow cytometry to measure biological pathways focusing on intra-cellular signaling and post-translational modulations in response to a variety of modulators (growth factors, cytokines, drugs, etc.). As part of a collaboration between industry, academic institutions, and cooperative groups to improve acute myeloid leukemia (AML) management, we are developing clinical tests based on SCNP to predict induction chemotherapy response or risk of relapse. To date, all the development studies have been performed on DMSO cryopreserved specimens of ficoll fractionated bone marrow (BM) or peripheral blood (PB) mononuclear cells. Demonstrating a correlation of SCNP readouts between fresh and cryopreserved preparations is essential to applying these proteomic signatures to the clinical setting. Objectives:To compare the results of SCNP assays between paired fresh and cryopreserved samples in a multicenter prospective study. Methods:To date, 13 fresh BM and PB samples were prospectively collected from pediatric or adult non-M3 AML patients at 3 academic centers and shipped over night to the Nodality CLIA lab. Samples were required to have 2 million viable cells per aliquot for SCNP assays, and underwent ficoll separation and mononuclear cells were divided into 2 aliquots - one processed fresh, and the second cryopreserved for 1 month, and then thawed and processed for the SCNP assay. 70 SCNP node-metrics (i.e. proteomic readouts in the presence or absence of modulator), identified previously as candidate proteomic signatures for several assays in development (including PIK3, Jak/STAT and DNA damage/apoptosis pathways) were investigated. The assay readouts for blast cells from a fresh aliquot were compared to the results from a cryopreserved aliquot by linear regression, Bland-Altman, and Lin's concordance analysis. Results:The analysis of paired aliquots from 13 patients, with median WBC of 27.9 (3-60) 10e3/ul, showed that cryopreservation did not affect sample quality as measured by percent of cells that were negative for cleaved PARP expression (R2 = 0.92 cryopreserved vs. fresh). The majority of unmodulated node-metrics (59%) and modulated node-metrics (68%, see Table) had a good correlation between the two preparations as measured by linear regression i.e., R2 > 0.64. The node-metrics with a lower R2 were using either a dim fluorophore (i.e. Alexa-647) and/or were within the low signal range (e.g., Erk basal); and therefore were not good candidates for future test development. Results using both Bland Altman and Lin's Concordance methods showed good concordance. Assessment of aliquots with up to 4 years in cryopreservation is ongoing and data from the 6 month time-point will be presented. Conclusions:These studies highlight the importance of cryopreservation of AML samples at clinical sites and by cooperative groups. These results demonstrate that cryopreservation maintains the activation signaling potential of AML blasts. SCNP assays developed and validated using cryopreserved samples can be applied to fresh samples and integrated prospectively into frontline clinical trials and clinical practice.Table 1:Goodness of fit (R2) values from regressing Cryo against Fresh for modulated node-metrics. Fold and Uu (rank based) metrics measure changes in signaling protein levels due to modulation. A= AlexaModulatorAssay Read-outColorR2 for FoldR2 for UuCytarabine + DaunorubicincPARPFITC0.710.63pChk2A. 6470.380.37EtoposidecPARPFITC0.780.49pChk2A 6470.520.37FLT3LpAktA 6470.130.09pErk 1/2PE0.460.55pS6A 4880.890.94G-CSFpStat1A 4880.730.72pStat3PE0.880.94pStat5A 6470.890.85H2O2pAktA 4880.790.85pPLCy2PE0.830.89pSlp76A 6470.800.82IL-27pStat1A 4880.920.93pStat3PE0.940.90pStat5A 6470.930.92PMApCrebPE0.920.93pErk 1/2A 6470.940.90pS6A 4880.930.92SCFpAktA 6470.490.09pErk 1/2PE0.150.18pS6A 4880.860.75 Disclosures:Cesano: Nodality Inc.: Employment, Equity Ownership. Gotlib: Nodality Inc.: Honoraria. Lacayo: Nodality Inc.: Honoraria. Putta: Nodality Inc.: Employment, Equity Ownership. Lackey: Nodality Inc.: Employment, Equity Ownership. Gayko: Nodality Inc.: Employment, Equity Ownership. Kornblau: Nodality Inc.: Honoraria.

WT1 mutation in 470 adult patients with acute myeloid leukemia: stability during disease evolution and implication of its incorporation into a survival scoring system

AbstractThe impact of WT1 mutations in acute myeloid leukemia (AML) is not completely settled. We aimed to determine the clinical implication of WT1 mutation in 470 de novo non-M3 AML patients and its stability during the clinical course. WT1 mutations were identified in 6.8% of total patients and 8.3% of younger patients with normal karyotype (CN-AML). The WT1 mutation was closely associated with younger age (P < .001), French-American-British M6 subtype (P = .006), and t(7;11)(p15;p15) (P = .003). Multivariate analysis demonstrated that the WT1 mutation was an independent poor prognostic factor for overall survival and relapse-free survival among total patients and the CN-AML group. A scoring system incorporating WT1 mutation, NPM1/FLT3-ITD, CEBPA mutations, and age into survival analysis proved to be very useful to stratify CN-AML patients into different prognostic groups (P < .001). Sequential analyses were performed on 133 patients. WT1 mutations disappeared at complete remission in all WT1-mutated patients studied. At relapse, 3 of the 16 WT1-mutated patients who had paired samples lost the mutation and 2 acquired additional mutations, whereas 3 of 110 WT1-wild patients acquired novel mutations. In conclusion, WT1 mutations are correlated with poor prognosis in AML patients. The mutation status may be changed in some patients during AML progression.