miR‐582‐5p serves as an antioncogenic biomarker in intermediate risk AML with normal cytogenetics and could inhibit proliferation and induce apoptosis of leukemia cells

Abstract Abstract 4874 Introduction: Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults. About 50% of patients with AML have normal karyotype, and are categorized as intermediate risk group. However, the clinical behavior and response to treatment in this group is heterogeneous. As a result, there is strong interest in characterizing molecular genetic features in the intermediate-risk AML patients that might rectify their stratification risk. In this group, FLT3-ITD (Internal Tandem Duplication) and FLT3-TKD (Tyrosine Kinase Domain) mutations are known to confer unfavorable risk whereas NPM1 and CEBPA mutations are known to be favorable risk markers. The purpose of this study is to analyze the combination of NPM1 and CEBPA mutations in presence or absence of FLT3 mutations on prognosis of AML patients referred to the State's largest tertiary care center over a period of 10 years for the treatment of leukemia. Patients and Method: We performed a retrospective chart review of all patients with AML evaluated at University of Oklahoma Health Sciences Center between January 2000 and December 2010. Patient's age, gender, race, laboratory and clinical data as well as bone marrow biopsy and aspirate findings were reported. PCR and Fragment Analysis were conducted on all available DNA preserved bone marrow materials to test the FLT3, NPM1 and CEBPA mutations. For statistical analysis, Kaplan-Meyer curve was used. Results: A total of 239 patients were evaluated. Male to female ratio was 2/1. Median age at diagnosis was 46y. 21 out of the 239 patients were less than 18 year old. DNA samples were present on 132 patients and mutation analysis for FLT3, CEBPA and NPM1 was performed. Correlation between mutations and AML prognosis was determined. 67/132 (50.8 %) patients were categorized into intermediate risk group (majority of patients had normal cytogenetics). 14/67 (20.9%) pts were FLT3+ (FLT3-ITD or FLT3-TKD mutation). 17/67 (23.9%) were NPM1+. 7/67 (10.4%) were CEBPA +. Kaplan-Meier curve was used to identify cumulative proportion surviving over time. FLT3 presence or absence itself was not identified to be statistically significant (p 0.416) in terms of overall survival. Interestingly, presence or absence of combined NPM1/CEBPA mutation in FLT3 negative patients, among intermediate risk group, was found to be statistically significant (p<0.05) in determining overall survival. In this subgroup, presence of NPM1/CEBPA combination (NPM1+/CEBPA+) was associated with poor prognosis (figure 2, lower curve), while absence of NPM1/CEBPA combination (NPM1-/CEBPA-) carries a better prognosis (figure 2, upper curve). Conclusion: Results of our study highlight the importance of performing combinations of mutation analysis in evaluation of overall prognosis in AML patients. FLT3-/NPM1+ profile in patients with normal cytogenetics is thought to confer a favorable prognosis. We demonstrated in this study that using combination mutation analysis in patients with FLT3- can change the risk stratification in patients with intermediate risk group and might affect therapeutic interventions in this patient population. Larger prospective studies are needed in the future for further validation of our findings. Disclosures: No relevant conflicts of interest to declare.

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High BAALC Expression as an Independent Adverse Risk Factor in 309 Patients with Acute Myeloid Leukemia (AML) and Normal Cytogenetics: Results of the SHG’96 Study.

Blood ◽

10.1182/blood.v104.11.2009.2009 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2009-2009

Author(s):

Claudia D. Baldus ◽

Christian Thiede ◽

Clara D. Bloomfield ◽

Eckhard Thiel ◽

Gerhard Ehninger

Keyword(s):

Risk Factors ◽

Risk Factor ◽

De Novo ◽

Intermediate Risk ◽

Free Survival ◽

Expression Levels ◽

Significant Difference ◽

Normal Cytogenetics ◽

The Impact ◽

Tandem Duplications

Abstract High mRNA expression levels of the human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) have been shown to be an adverse risk factor in newly diagnosed AML patients (pts; Blood2003;102:1613). The first study included 86 de novo AML pts under the age of 60 with normal cytogenetics and a more favorable FLT3 mutation status, which were uniformly treated on the Cancer And Leukemia Group B protocol 9621. An independent confirmation of these data is necessary, to confirm the impact of BAALC expression in intermediate risk AML pts. Here we present the results of a Süddeutschen Hämoblastosegruppe (SHG) study including 309 adult pts diagnosed with de novo (n=280) and secondary AML (n=29), normal cytogenetics, age <60 years that were uniformly treated on the SHG’96 protocol. Treatment consisted of 2 cycles of induction therapy followed by intensive consolidation including autologous as well as allogeneic stem cell transplantation. Median follow-up for patients alive was 28.7 months (range: 0–86 months). BAALC expression was measured in pretreatment peripheral blood blasts by comparative real-time RT-PCR. Pts having BAALC expression values within the lower 50% were classified as low BAALC and pts having BAALC expression values within the upper 50% were classified as high BAALC. Whereas no correlation was seen between BAALC expression and the prevalence of FLT3 internal tandem duplications (ITD; P=0.16), high BAALC patients had a significantly higher FLT3 mutant/wild-type (wt) ratio as determined by Genescan analysis (mean mutant/wt ratio: 0.59 vs. 0.17; P=0.012). There was no significant difference in the prevalence of MLL partial tandem duplications between the high and the low BAALC group. High BAALC expression was significantly more frequent in FAB groups M0/M1 as compared to the other subtypes (P=0.002). Pts with high BAALC expression levels had a significantly lower CR rate (62.3% vs. 73.5%; P=0.039) and a higher relapse rate (43.8% vs. 28.9%; P=0.030). The overall survival (OS) was significantly shorter for pts with high BAALC expression (OS at 4 years: 29.8% vs. 53.3%; P=0.0018), event-free survival (EFS, at 4 years: 21.6% vs. 45.8%; P=0.0001), and disease-free survival (DFS, at 4 years: 30.4% vs. 57.6%; P=0.0018). Pts with a high FLT3 mutant/wt ratio (greater than 0.8) had a significantly shorter OS and DFS. For patients with a low FLT3 mutant/wt ratio, high BAALC expression allowed identification of pts with a high risk of treatment failure. A multivariate analysis confirmed high BAALC expression and a high mutant/wt FLT3 ratio as the only independent adverse risk factors. For pts with high BAALC expression the hazard ratio of death was 1.7 (95% CI 1.18–2.48; P=0.005), and the hazard ratios for EFS and DFS were 1.8 (95% CI 1.3–2.6; P=0.0003) and 2.0 (95% CI 1.2–3.3; P=0.004), respectively. In conclusion, this independent and larger study strengthens high BAALC expression as one of the most relevant adverse prognostic risk factors in intermediate risk AML pts with normal cytogenetics. Thus, determination of BAALC expression should be considered for risk adaptive treatment strategies.

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Autologous Stem Cell Transplantation in Patients of High and Intermediate Risk Acute Myeloid Leukemia:-Perspective of a Developing Country.

Blood ◽

10.1182/blood.v114.22.4364.4364 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4364-4364

Author(s):

Velu Nair ◽

Satyaranjan Das ◽

Sanjeevan Sharma ◽

Ajay Sharma ◽

Deepak Kumar Mishra ◽

...

Keyword(s):

Stem Cell ◽

Myeloid Leukemia ◽

Intermediate Risk ◽

Trisomy 8 ◽

Leukemia Relapse ◽

Normal Cytogenetics ◽

Matched Donor ◽

Related Mortality ◽

Acute Myeloid

Abstract Abstract 4364 Introduction Most patients with acute myeloid leukemia (AML) who achieve a complete remission after induction chemotherapy will relapse if they do not receive effective consolidation therapy particularly patients of high and intermediate risk. Standard chemotherapy only achieves less than 30% overall survival at 2 years. The most effective consolidation for high risk and intermediate risk patients is allogeneic stem cell transplantation (allo-SCT) which may be related or unrelated. However in the developing world availability of a human leucocyte antigen (HLA) matched donor for those lacking a sibling match is rarely available due to minority of voluntary donors from Indian subcontinent in various registries. Hence autologous stem cell transplant (auto-SCT) is one option that has been studied extensively. The question as to whether auto-SCT after consolidation chemotherapy improves the probability of survival of patients with AML has not been settled. In view of the above considerations, we carried out a retrospective study aimed at analyzing the impact of disease, patient, and transplantation-related factors on relapse, non–leukemia-related death, and LFS of patients with AML. Patients and methods We present here retrospective data of 16 patients of AML who undergone auto SCT in our centre from Jan 2005 to Jan 2009. The median age of patients was 24 yrs with a range from 10 to 40 years. Male: Female ratio was 11:5. The diagnosis was established on both bone marrow morphology and immunophenotyping study. Morphologically there was 1 case each of AML-M1, AML-M4, AML-M5, AML-M6 and rest 13 cases were of AML-M2. Bone marrow karyotyping was done in 11 cases. Out of 11 cases where karyotyping was done, 2 were metaphase failures; 1 was deletion 7; 1 was trisomy 8 and rest had normal cytogenetics. All cases were classified as high risk or intermediate risk either by cytogenetics or by other standard criteria. All patients received standard induction chemotherapy (Idarubicin x 3days, Cytosine CI x 7days) followed by 2 or 3 high dose Cytosine. Fourteen patients were in first remission (CR-1) while 2 patients were in CR-2 (one case of AML-M5 with deletion 7 and one AML-M4 with normal cytogenetics). None of the patients had a related HLA matched donor and all patients underwent auto-SCT, the graft being peripheral blood stem cell. The conditioning regimen for initial 6 cases were Busulfan(16mg/kg over 4 days) and Cyclophosphamide(120mg/kg over 2 days)(BuCy) and the remaining 10 cases were adminstered Idarubicin (60mg/mt2 over 3days) and Busulfan (16mg/kg over 4 days) (IBu). All cases engrafted except one patient who died due to sepsis on 6th day post SCT prior to engraftment. The median engraftment for neutrophil was day10 (9-14) and platelet was on day 16 (13 - 20). Results Nine out of 16 patients are alive and free from leukemia on median follow up of 38 (18 to 56 months). Five patients died due to leukemia relapse; 1 patient had transplant related mortality (Sepsis) and 1 patient died due to unrelated cause (Severe heat exertion with multiorgan failure). The overall (OS) and leukemia free survival (LFS) on a median follow up of 38 months was 56.25% which is better than the chemotherapy group. The treatment related mortality (TRM) was 6.25%. Both patients transplanted in CR-2 relapsed and died. Three out of 6 patients from BuCy and 6 out 10 from IBu conditioning are alive. Out of 9 patients surviving, 5 had normal karyotype while in 4 same was not known. The patients with deletion 7 and trisomy 8 died due to leukemia relapse. Conclusion Auto-SCT is a viable option for AML patients who are in need of a allo-SCT but do not have a donor. However larger studies would be required to establish the exact role of auto-SCT in AML. Disclosures: No relevant conflicts of interest to declare.

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Proof-Of-Concept Study For Precision Medicine With Chromosome Genomic Array Testing (CGAT) For Drug Sensitivity Screening In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.2578.2578 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2578-2578

Author(s):

Min Fang ◽

Scott McElhone ◽

Xin Zhao ◽

Barry E. Storer ◽

Su-In Lee ◽

...

Keyword(s):

Myeloid Leukemia ◽

Drug Sensitivity ◽

Intermediate Risk ◽

Proof Of Concept ◽

Prognostic Effect ◽

Genomic Array ◽

Normal Cytogenetics ◽

Very High ◽

Acute Myeloid

Abstract Tremendous heterogeneity in acute myeloid leukemia (AML) poses a significant challenge to clinical management and effective therapy. Cytogenetics is one of the key prognostic factors for AML. New technologies, such as chromosome genomic array testing (CGAT) combining both SNP-based and non-SNP based array CGH, have allowed informative genomic profiling of chromosome abnormalities with very high resolution. Yet it remains unclear how these unique CGAT-identified clonal aberrations impact the outcome of AML patients and how they predict a patient’s response to a specific medication. In this proof-of-concept study, we aim to explore the correlation of genomic abnormalities identified by CGAT with in vitrodrug sensitivity data from 30 AML patients and their clinical responses. We performed CGAT on the pre-treatment bone marrow of these AML patients using CytoScanHD, which includes 2.5 million markers, 750K of which are SNP markers. Copy number aberrations (CNA) and copy-neutral loss of heterozygosity (cnLOH) were analyzed by ChAS and Nexus software. CGAT abnormalities were enumerated based on chromosome arms affected; i.e., multiple segmental CNAs and cnLOH on the same chromosome arm were counted as one aberration. Patients’ response to chemotherapy and clinical relapse were tracked. Drug sensitivity for individual patient leukemia blast samples was measured by a high-throughput in vitroassay consisting of a panel of 160 drugs, of which 45 are FDA approved and 115 investigational. Cell survival was measured by Cell Titer Glo, and data curves for 8 concentrations of each drug were generated to derive EC50 and IC50 values. Statistical correlation was analyzed by Fisher’s exact test, Cox regression, and regression analysis. CGAT detected all the chromosome abnormalities identified by cytogenetics except truly balanced translocations. CGAT also identified clonal aberrations in 7 of the 13 patients with normal cytogenetics. Of the 6 uniquely CGAT abnormal patients, 3 demonstrated cnLOH only and 2 CNA only. Overall, CGAT showed 11 complex karyotypes (CK, defined as >=3 aberrations; high-risk) and 19 normal or simple abnormal cases (low/intermediate-risk), as compared to the cytogenetics classification of 9 poor-risk, 15 intermediate-risk, and 6 low-risk karyotypes. cnLOH was detected in 9 patients (30%). Complete remission (CR) rate after the first induction course in the CGAT CK group was 18% versus 68% in the non-CK group (P=0.02). There was a very high concordance (90%) between CGAT risk based on CK and conventional cytogenetics. And when cytogenetics and CGAT disagree, as for the 2 patients who had normal cytogenetics but CGAT CK, CGAT risk stratification appeared better as neither patient achieved CR. CR duration longer than 1 year occurred more (P=0.04) in the CGAT normal patients (50%) than in the CGAT abnormal patients (8.3%). Among those who achieved CR, the hazard of relapse (HR) for CGAT CK patients was 9.9 (CI: 1.6-61; P=0.02); HR for cnLOH patients was 7.1 (CI: 1.2-43; P=0.03). Considering the total aberrations (median 1.5, range 0-16), the HR for relapse among CR patients was 1.3 (CI: 1.1-1.5; P=0.01) per aberration. Six of the 48 drugs that exhibited cytotoxicity in the in vitro assay, including clofarabine (r=0.45, P=0.01) and PKI-587 (dual PI3K/mTOR inhibitor) (r=0.48, P=0.008), showed significant correlation between CGAT aberrations and EC50; i.e, the more CGAT aberrations, the higher concentration is required for a given drug to kill these CGAT-abnormal leukemic cells in vitro. In conclusion, CGAT increased the diagnostic yield by 54% among AML patients with normal cytogenetics and altered the overall risk classification by 6.7%. Complex karyotype identified by CGAT carried the same poor prognostic effect as CK by cytogenetics. Although abnormal CGAT results did not appear to predict response, increasing CGAT abnormalities are associated with early relapse, as is the presence of cnLOH. Genomic complexity detected by CGAT appeared to associate with dosage-dependent resistance to selected drugs by in vitro drug sensitivity analyses. Characterization of prognostic effect and drug response associated with each type of CGAT abnormalities will require a larger data set. Nevertheless, this study demonstrated the clinical utility of CGAT for precision medicine in AML. Disclosures: Becker: Affymetrix: Research Funding.

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Ferritin-Conjugated Antibody for the Demonstration of Isoantigens on the Surface of Murine Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099982 ◽

1968 ◽

Vol 26 ◽

pp. 48-49

Author(s):

T. Aoki ◽

J. Izard ◽

U. Hämmerling ◽

E. de Harven ◽

L. J. Old

Keyword(s):

Cell Surface ◽

Gamma Globulin ◽

Leukemia Cells ◽

Labelling Technique ◽

Direct Labelling ◽

Labelling Method

Although a variety of viral and cellular antigens have been demonstrated by ferritin-labeled antibody, this technique has not been used to locate isoantigens on the surface of nucleated cells. The recognition of several systems of isoantigens on the surface of thymocytes, lymphocytes and leukemia cells of the mouse and the ease with which these cells can be obtained in free suspension led us to consider the ferritin-labelling method to determine the amount and location of these isoantigens on the cell surface. Because of the problems involved in the direct labelling of mouse gamma globulin by ferritin, we have chosen an indirect labelling technique (i.e. ferritin-conjugated rabbit anti mouse γG)to detect localization of mouse isoantibody.

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The distribution of antigen on the surface of RBL-2H3 rat basophilic leukemia cells observed by back-scattered electron imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142992 ◽

1986 ◽

Vol 44 ◽

pp. 274-275

Author(s):

R.F. Stump ◽

J.R. Pfeiffer ◽

JC. Seagrave ◽

D. Huskisson ◽

J.M. Oliver

Keyword(s):

Cell Surface ◽

Dynamic Properties ◽

Leukemia Cells ◽

Antigen Binding ◽

Scattered Electron ◽

Ige Receptor ◽

Receptor Complexes ◽

Rat Basophilic Leukemia Cells ◽

Electron Imaging ◽

Basophilic Leukemia

In RBL-2H3 rat basophilic leukemia cells, antigen binding to cell surface IgE-receptor complexes stimulates the release of inflammatory mediators and initiates a series of membrane and cytoskeletal events including a transformation of the cell surface from a microvillous to a lamellar topography. It is likely that dynamic properties of the IgE receptor contribute to the activation of these responses. Fewtrell and Metzger have established that limited crosslinking of IgE-receptor complexes is essential to trigger secretion. In addition, Baird and colleagues have reported that antigen binding causes a rapid immobilization of IgE-receptor complexes, and we have demonstrated an apparent increase with time in the affinity of IgE-receptor complexes for antigen.

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Involvement of GER in Friend leukemia virus assembly in high-pressure frozen cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160509 ◽

1990 ◽

Vol 48 (3) ◽

pp. 590-591

Author(s):

W. Djaczenko ◽

M. Müller ◽

A. Benedetto ◽

G. Carbone

Keyword(s):

High Pressure ◽

Virus Assembly ◽

Leukemia Virus ◽

Leukemia Cells ◽

Serial Sections ◽

Myeloma Cells ◽

Virus Particles ◽

Friend Leukemia ◽

Carbon Coated ◽

Friend Leukemia Virus

A thickening of ER membranes in murine myeloma cells was attributed by de Harven to the assembly of intracisternal virus particles. We observed similar thickening of GER membranes in Friend leukemia cells (FLC) apparently associated with Friend leukemia virus (FLV) assembly. We reinvestigated the problem of GER involvement in FLV assembly using high pressure cryofixed FLC.FLC (745A clone growing in suspension and FF clone growing in monolayer) were immersed in Hexadecene (Fluka, Switzerland) and rapidly frozen in Balzers HPM 010 freezing machine working at 2200 bar. All cells were freeze substituted at -90°C in 2% OsO4 in absolute acetone. Serial sections cut to avoid misinterpretations due to the geometry of sections, were collected on carbon coated 100 mesh grids.

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