Microarray Analysis Detects Unique Expression Pattern for NPM1-Mutated AML with Normal Karyotype and Reveals Pathobiological Insights.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2382-2382
Author(s):  
Torsten Haferlach ◽  
Claudia Haferlach ◽  
Alexander Kohlmann ◽  
Lothar Wieczorek ◽  
Martin Dugas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Melting Curve ◽  
Normal Karyotype ◽  
Support Vector ◽  
Melting Curve Analysis ◽  
Study Cohort ◽  
Data Set ◽  
Npm1 Mutation ◽  
Mutation Status ◽  
Mutational Status

Abstract Recent data indicate that mutations in exon 12 of the nucleophosmin (NPM1) gene characterize a distinct subgroup of adult and pediatric acute myeloid leukemia (AML). AML carrying NPM1 mutations account for about one-third of all adult AML, exhibit distinctive biological and clinical features and show a strong association to AML with normal karyotype (55% mutated). However, the role of NPM1 in leukemogenesis still remains elusive. Here we present data on a cohort of n=66 AML cases with normal karyotype analyzed by high-density whole genome expression microarrays (Affymetrix HG-U133 Plus 2.0). In parallel melting curve analysis was used to assess NPM1 mutational status: 41 cases were characterized as mutated (NPM1+) and 25 cases were unmutated (NPM1−). We first investigated the gene signature that discriminated NPM1+ from NPM1− cases. Genes that were significantly overexpressed comparing NPM1+ against NPM1– cases included a strong homeobox genes signature (HOXA1, HOXA5, HOXA7, HOXA9, HOXA10, HOXA11, HOXB2, HOXB4, HOXB5, HOXB6, HOXB7, MEIS1, and PBX3). A functional analysis (Gene Ontology) revealed a clear association of the group of overexpressed genes with the cell components nucleosome, chromatin, and the nuclear envelope-endoplasmatic reticulum network as well as involvement in the biological processes of nucleosome and chromatin assembly, establishment of protein transport and localization, and Notch signaling pathway. In contrast, the cellular processes completely differed when genes were investigated that were significantly underexpressed in NPM1+ cases compared to NPM1− cases. This group of genes encoded membrane-related proteins (gap junction, intercellular junction, signalosome complex) and proteins involved in cellular morphogenesis and cell communication. The differences in gene expression signatures between NPM1+ and NPM1− cases permit a robust classification approach by gene expression profiling. Support Vector Machine analysis resulted in >92% prediction accuracy of NPM1 mutation status (10-fold cross-validation). The sensitivity was very high for the positive detection of NPM1+ cases (>97%). Using a 100-fold re-sampling approach and splitting the complete data set into a training set (n=44) and testing set (n=22) the following genes were most frequently selected as top discriminatory genes: HOXA5, HOXB4, HOXB5, HOXB6, MEIS1, PBX3, FGFR1, ADAM17, PRICKLE1, and TMPO. Interestingly, the classification was less accurate when also FLT3 internal tandem duplication mutation status was taken into account. The study cohort (n=66) then was distributed as follows: 19 NPM1+/FLT3+, 22 NPM1+/FLT3−, 4 NPM1−/FLT3+, and 21 NPM1−/FLT3− negative cases. Only 14 of 22 (64%) NPM1+/FLT3– cases were correctly predicted, with miscalls falling both into the group of NPM1+/FLT3+ and NPM1−/FLT3− cases. In conclusion, NPM1 mutations are the most frequent mutations in adult AML to date and their central prognostic role is increasingly recognized. Given the fact that they are nearly mutually exclusive with major recurrent genetic abnormalities and that they can be characterized by a distinctive gene expression program these data especially for of NPM1+/FLT3− AML with better outcome may support to classify this as a separate biological subgroup of AML with normal karyotype.

Characterization of NPM1-Mutated/FLT3 ITD-Negative Acute Myeloid Leukemia with Normal Karyotype by Gene Expression Profiling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 155-155 ◽  
Author(s):  
Lars Bullinger ◽  
Konstanze Dohner ◽  
Raphael Kranz ◽  
Frank G. Rucker ◽  
Stefan Frohling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Normal Karyotype ◽  
Molecular Profile ◽  
Data Set ◽  
Mutational Status ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) with normal karyotype comprises a large number of molecularly distinct variants. For example the presence of internal tandem duplications (ITDs) of the FLT3 (fms-related tyrosine kinase 3) gene is associated with poor outcome, whereas mutations of the NPM1 (nucleophosmin) gene are prognostically favorable. However, this effect is mainly attributed to the NPM1-mutated/FLT3 ITD-negative AML cases. While NPM1-mutated cases are characterized by a distinct gene expression pattern, it remains unclear whether NPM1-mutated/FLT3 ITD-negative cases also display a characteristic signature, which might provide additional insights into the molecular basis for the good clinical outcome. Thus, we sought to identify a molecular profile for AML cases with NPM1-mutated/FLT3 ITD-negative normal karyotype disease. Towards this goal, we profiled gene expression of 138 samples of adult AML patients with normal karyotype using DNA microarray technology. All samples analyzed were derived from AML patients entered within the randomized multicenter treatment trial HD-98A of the German-Austrian AML Study Group (AMLSG). Based on supervised data analyses we were able to identify a 116-genes comprising expression pattern correlated with NPM1-mutated and FLT3 ITD-negative AML cases. In accordance with previous findings in NPM1-mutated cases (Alcalay et al. 2005, Verhaak et al. 2005), the NPM1-mutated/FLT3 ITD-negative pattern was also in part characterized by a prominent HOX gene cluster, which clearly separated the NPM1-wildtype from the NPM1-mutated cases. Similarly, the expression levels of BAALC and MN1 were correlated with the NPM1 mutational status, with NPM1-unmutated cases displaying higher BAALC and MN1 expression in our data set. However, as expected the newly defined signature also defined a NPM1-mutated group that did not contain many FLT3 ITD-positive samples. This group was characterized by several interesting genes including for example TLE1, which encodes a Groucho/TLE family protein. Groucho/TLE family proteins are transcriptional co-repressors, which mediate repression essential in embryonic development and are involved in regulation of Wnt signaling in adult tissue. Moreover, we identified several other genes of potential pathogenic relevance which also have been previously shown to be predictive in normal karyotype AML. Our findings support a distinct molecular mechanism associated with the favorable outcome of NPM1-mutated/FLT3 ITD-negative AML cases. Furthermore, the reported signature might contribute to improved risk stratification and clinical management of AML patients with normal karyotype disease.

Gene Expression Profiling in AML with Normal Karyotype: A Multicenter Study Investigating Molecular Markers in 252 Cases

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 751-751 ◽  
Author(s):  
Alexander Kohlmann ◽  
Lars Bullinger ◽  
Christian Thiede ◽  
Markus Schaich ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiling ◽  
Supervised Classification ◽  
Cross Validation ◽  
Hox Genes ◽  
Normal Karyotype ◽  
Npm1 Mutation ◽  
Mutation Status ◽  
Gene Expression Signatures ◽  
Fold Cross Validation

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease and AML with normal karyotype (AML-NK) is a subgroup with intermediate prognosis. Over the past years molecular analyses have led to the identification of multiple molecular biomarkers that will further allow to dissecting clinically meaningful subgroups in this disease. Here, we present a multicenter study investigating whole-genome expression profiles of 252 cases of AML-NK. In three centers (Dresden, n=78; Munich, n=97; Ulm, n=77) Affymetrix HG-U133 Plus 2.0 microarray analyses were performed according to a standardized protocol. In a first series of analyses we focused on nucleophosmin gene (NPM1) mutations, the most common genetic lesion described in adult de novo AML to date. Gene expression signatures for 138 NPM1-mutated cases were compared to the profiles of 114 NPM1-unmutated cases. Supervised classification analyses resulted in >95% prediction accuracy of NPM1 mutation status (10-fold cross-validation). The sensitivity was very high for the positive detection of NPM1-mutated cases (>97%). Using a resampling approach (100 iterations) and splitting the complete data into a training set (n=168) and test set (n=84) the following genes were amongst the most frequently selected with higher expression in NPM1-mutated cases: HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB9, MEIS1, and PBX3. Lower expression in NPM1-mutated cases was observed for ABCB1, BAALC, MN1, MLLT3, or SPARC. Furthermore, our signature also showed a strong overlap to predictive NPM1 mutation signatures as published by Verhaak et al., and Alcalay et al., with 16/20 common genes and 15/18 common genes, respectively. Interestingly, some few NPM1-unmutated cases were repeatedly classified as being NPM1-mutated suggesting mutations in gene sequences not routinely screened. A strong HOXA-cluster signature is clearly dominant as well in these cases, whereas HOXB-cluster genes demonstrated a similar expression as compared to NPM1-unmutated cases. With respect to the robustness of differential gene expression signatures in NPM1 mutated cases across the laboratories using the top 300 differentially expressed genes, an overlap of 67 (22.3%) probe sets was observed between all three laboratories (NPM1-mutated vs. NPM1-unmutated: Dresden: 36 vs. 42; Munich: 42 vs. 55; Ulm: 36 vs. 41). In a second series of analyses we investigated cases with CCAAT/enhancer binding protein alpha (CEBPA) gene mutations. Gene expression signatures for 26 CEBPA-mutated cases were compared to the profiles of 132 CEBPA-unmutated cases. Supervised classification analyses resulted in >90% prediction accuracy of CEBPA mutation status (10-fold cross-validation) and frequently selected genes included HOXA2, HOXA3, HOXA5, HOXA7, HOXA9, HOXA10, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, and HOXB9. Thus, several HOX genes were overlapping to the NPM1 signature, albeit with an inverse correlation, i.e., HOX genes with relative overexpression expression in NPM1-mutated AML-NK demonstrated low expression in CEBPA-mutated cases. Finally, all 252 cases have also been characterized according to their fms-related tyrosine kinase 3 gene (FLT3) internal tandem duplication mutation status and this data is currently being integrated with the obtained signatures from above to develop a multi-gene prognostic model for AML-NK. In conclusion, our gene expression profiling study demonstrates robust signatures for AML-NK with NPM1 or CEBPA mutations. As our knowledge of this heterogeneous disease is increasing it seems possible to refine the classification of AML by incorporating molecular risk-based gene expression signatures.

scholarly journals Frequent Mutations and Their Influence on Prognosis in Patients with Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4949-4949
Author(s):  
Ekaterina Petrova ◽  
Irina Martynkevich ◽  
Luybov Polushkina ◽  
Luydmila Martynenko ◽  
Marina Ivanova ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Melting Curve ◽  
Normal Karyotype ◽  
Genetic Alterations ◽  
Melting Curve Analysis ◽  
Prognostic Impact ◽  
Age Related ◽  
Mutational Status

Abstract Background. Several genetic alterations such as translocations, gene mutations and deletions play an important role in myeloid leukemogenesis. The cytogenetic information is a very significant tool to classify pts at their initial diagnosis into prognostic categories. For pts with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3, CKIT, NRAS and DNMT3A. The aim of the research was to investigate the frequency and prognostic impact of FLT3, NPM1, CKIT, NRAS and DNMT3A mutations in AML pts and to analyze their interaction with other prognostic markers. Methods. This study was performed in 200 adult pts (190 pts with de novo and 10 pts with secondary AML), previously untreated, median age 55 years (18-86). According to the results of cytogenetic analyses pts were separated in four groups: with favourable (9,0%), unfavourable (14,0%) prognosis, with normal karyotype (NK) (48,5%) and other aberrations (28,5%). Mutations in FLT3, CKIT and NPM1 were analysed by PCR and in NRAS by sequencing. Mutation analysis of DNMT3A R882 was performed by high-resolution melting curve analysis. Cytogenetic studies were analysed on bone marrow samples using standard GTG-method. Results. Mutations in FLT3, CKIT, NRAS and NPM1 genes were detected in 105/200 (52,5%) pts. A total of 128 mutations were revealed in this group: 24,0% - FLT3-ITD, 6,5% - FLT3-TKD, 20,5% - in NPM1, 10,0% - in NRAS and 3,0% - in CKIT. 82 pts had single mutations and in 23 pts mutations occurred simultaneously: 17 with FLT3-ITD and in NPM1, 2 with FLT3-ITD and FLT3-TKD, 1 with FLT3-TKD and in NPM1, 3 with NPM1 and NRAS mutations. We found that mutations with the significantly higher incidence (p=0,001) were observed in the group of pts with NK (80/97), whereas there were only 8/28 pts with mutations in the group with complex karyotype. When analyzing the age-related features, it was shown that the majority of mutations were detected in the group of pts at the age from 60 to 69 years. Mutations FLT3-ITD and FLT3-TKD were associated with higher WBC count comparing with pts without mutations (p=0,001 and p=0,014, respectively). The median follow-up for overall (OS) and relapse-free (RFS) survival for pts with FLT3-ITD against ptswith FLT3-ITD- was: 5,4 vs 12,8 months and 4,9 vs 10,0 months (p=0,001 and p=0,001), respectively. Mutation FLT3-TKD was also found to be prognostically unfavorable, but only comparing with pts with FLT3-ITD- genotype. As the result of OS and RFS analyses in pts with and without NPM1 mutations we revealed the significant favorable influence of NPM1 mut on the prognosis (p=0,002 and p=0,020, respectively). However pts with genotype FLT3-ITD+/NPM1+ were found to get to the group with an intermediate risk. We detected the significant adverse influence of CKIT mut on RFS (p=0,041). Mutations in NRAS didn't impact on prognosis; we only showed the tendency towards worsening of OS and RFS in group of pts with favorable cytogenetics (p=0,214 and p=0,160, respectively). Mutations DNMT3A R882 were investigated in group of 143 AML pts and were detected in 23 (16,1%) pts. Pts with DNMT3A R882 had higher WBC (p=0,001) and platelets (p=0,020) count at diagnosis and more frequently belonged to FAB groups M5 (p=0,003), as compared with DNMT3A wt. Of 23 pts who had AML with DNMT3A mutations, 17 had tumors with NK profiles (24,3% of a total of 70 cytogenetically normal samples) (p=0,009). Pts with isolated DNMT3A mutations were seen in 4 cases, whereas in the rest of pts they were detected simultaneously with mutations in genes FLT3, NPM1, NRAS and CKIT. DNTM3A mutations were significantly more prevalent in NPM1 mut (p=0,005) and FLT3-ITD (p=0,005) positive cases than wild type. DNMT3A mutations were associated with negative influence on pts OS and risk of relapse, compared with DNMT3A wt (р = 0,031 and р = 0,045, respectively). Summary. Mutations in FLT3 and NPM1 had a significantly higher incidence in the group of pts with a normal karyotype. FLT3 mutations showed the adverse prognostic value. Insertions in NPM1 were shownto be the favorable factor, correlating with prolonged RFS in all pts excepting pts with FLT3-ITD+/ NPM1+ genotype. CKIT mut was associated with higher relapse incidence in AML pts, while NRAS mut showed lack of prognostic significance. AML with DNMT3A mut represent the group, homogeneous on a number of clinical and laboratory parameters, associated with adverse prognosis and a high risk of the relapse. Disclosures No relevant conflicts of interest to declare.

An FLT3 gene-expression signature predicts clinical outcome in normal karyotype AML

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4490-4495 ◽  
Author(s):  
Lars Bullinger ◽  
Konstanze Döhner ◽  
Raphael Kranz ◽  
Christoph Stirner ◽  
Stefan Fröhling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Outcome ◽  
Dna Microarrays ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Signature ◽  
Normal Karyotype ◽  
Mutation Status ◽  
Signature Genes ◽  
Starting Point

Abstract Acute myeloid leukemia with normal karyotype (NK-AML) represents a cytogenetic grouping with intermediate prognosis but substantial molecular and clinical heterogeneity. Within this subgroup, presence of FLT3 (FMS-like tyrosine kinase 3) internal tandem duplication (ITD) mutation predicts less favorable outcome. The goal of our study was to discover gene-expression patterns correlated with FLT3-ITD mutation and to evaluate the utility of a FLT3 signature for prognostication. DNA microarrays were used to profile gene expression in a training set of 65 NK-AML cases, and supervised analysis, using the Prediction Analysis of Microarrays method, was applied to build a gene expression–based predictor of FLT3-ITD mutation status. The optimal predictor, composed of 20 genes, was then evaluated by classifying expression profiles from an independent test set of 72 NK-AML cases. The predictor exhibited modest performance (73% sensitivity; 85% specificity) in classifying FLT3-ITD status. Remarkably, however, the signature outperformed FLT3-ITD mutation status in predicting clinical outcome. The signature may better define clinically relevant FLT3 signaling and/or alternative changes that phenocopy FLT3-ITD, whereas the signature genes provide a starting point to dissect these pathways. Our findings support the potential clinical utility of a gene expression–based measure of FLT3 pathway activation in AML.

Expression Patterns of Hoxa4 and Meis1 Genes Are Regulated by Promoter Hypermethylation and When Combined Predict Survival in AML.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1204-1204
Author(s):  
Lykke Christina Grubach ◽  
Mike Zangenberg ◽  
Hans Beier Ommen ◽  
Anni Aggerholm ◽  
Peter Hokland
Keyword(s):  
Gene Expression ◽  
Overall Survival ◽  
Expression Patterns ◽  
Group Identification ◽  
Normal Karyotype ◽  
Promoter Hypermethylation ◽  
Melting Curve Analysis ◽  
Altered Expression ◽  
Expression Levels ◽  
Prognostic Group

Abstract INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous disease with varying survival rates depending mostly upon the molecular phenotype of the single leukemic clone. The most powerful predictor for the outcome of the individual patient is the cytogenetic profile at the time of diagnosis, dividing the patients into good, intermediate and adverse prognostic group. However, given that 40–60 percent of patients exhibits a normal karyotype and are assigned to an intermediate prognostic group, identification of biologic parameters, which either alone or in combination, predict disease outcome more precisely are needed. We have previously performed a gene expression profiling study (Grubach et al, Eur J. Hematol. 2008 Apr 10. [Epub ahead of print]) on a series of Polycomb, Hox and Meis genes expressed in hematopoietic cells. AIM: Based on the finding that HOXA4 could be used as a predictor for outcome in AML patients with a normal karyotype, we hypothesized that combining the gene expression of the HOXA4 gene and co-factor MEIS1 might unravel a leukemogenic impact in other cytogenetic prognostic groups (Grimwade et al. Blood. 1998 Oct 1;92(7):2322–33). In addition, given that epigenetic events might contribute to the regulation of these genes, we determined whether promoter hypermethylation of CpG islands in the promoter regions were of relevance to the expression levels of HOXA4 and MEIS1. MATERIALS & METHODS: Diagnosis samples from 248 AML patients were analyzed by RQ-PCR for expression levels of HOXA4 and MEIS1. 157 of these patients were further analyzed for promoter hypermethylation of the same genes by bisulphite treatment of DNA followed by methylation-specific melting curve analysis (MS-MCA). RESULTS: When combining the gene expression levels of HOXA4 with MEIS1 into the three main groups (low HOXA4/low MEIS1, low HOXA4/high MEIS1 and normal-high HOXA4/high MEIS1; (the latter pooled to enable statistical calculations)), clear differences in overall survival were found (Fig. 1). Thus, within the group of patients exhibiting low levels of HOXA4 transcript, those with a high expression of MEIS1 had a significantly worse outcome than those having low MEIS1 expression (p=0.025). Importantly, in a multiparameter regression analysis, the prediction was independent of the cytogenetic grouping, of mutations in NPM1 and FLT3 genes, WBC and age. Given the efficacy of demethylating therapy, we also considered the mechanism of HOXA4 and MEIS1 gene regulation. Thus, when promoter methylation of HOXA4 and MEIS1 in 157 patients was investigated, we found that 15 % of the patients had hypermethylation of the promoter region of MEIS1 and 77% of the patients showed hypermethylation of HOXA4. Importantly, a significant correlation for both of the genes between the expression level and methylation state was observed (MEIS1, p=0.001 and HOXA4, p=0.007). CONCLUSION: The altered expression levels of HOXA4 and MEIS1 in AML reflect, at least partly, an epigenetic regulation by virtue of promoter hypermethylation. The level of transcripts of HOXA4 and MEIS1 seem to contribute to the leukemogenesis in AML and can serve as independent prognostic variables regardless of their cytogenetic and molecular background. Fig. 1. Overall survival of AML patients-stratified by cytogenetics, mutations in NPM1 and FLT3, WBC and age. By combination of HOXA4 and Meis1 expression a significant better survival is linked to those with a low HOXA4/low MEIS1 compared to those with a low HOXA4/high MEIS1 expression. Fig. 1. Overall survival of AML patients-stratified by cytogenetics, mutations in NPM1 and FLT3, WBC and age. By combination of HOXA4 and Meis1 expression a significant better survival is linked to those with a low HOXA4/low MEIS1 compared to those with a low HOXA4/high MEIS1 expression.

Mutation in the Nucleophosmin Gene (NPM1) Is a Stable Marker for Minimal Residual Disease Monitoring in Acute Myeloid Leukemia Patients with Increased Sensitivity and Specificity Compared to Expression of the Wilms Tumor (WT1) Gene.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1602-1602
Author(s):  
Thomas Kristensen ◽  
Birgitte Strange Preiss ◽  
Lone Friis ◽  
Michael B. Møller
Keyword(s):  
Gene Expression ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Wilms Tumor ◽  
Normal Karyotype ◽  
Leukemia Cells ◽  
Mutational Load ◽  
Wt1 Gene ◽  
Npm1 Mutation ◽  
Minimal Residual

Abstract Abstract 1602 Poster Board I-628 Mutation in exon 12 of the nucleophosmin (NPM1) gene occurs in approximately 60% of acute myeloid leukemia (AML) patients with normal karyotype. To date, molecular minimal residual disease (MRD) monitoring in this patient group has primarily been based on expression of the Wilms tumor gene (WT1), although expression of WT1 in non-leukemia cells limits the specificity of this marker. Mutation in the NPM1 gene is potentially a superior MRD marker compared to WT1 gene expression by being specific to the malignant clone. The use of NPM1 mutation as a MRD marker would furthermore be in line with the widespread use of leukemia cell specific fusion-genes as MRD markers in AML patients with balanced translocations. In the present study, we therefore evaluated NPM1 mutation as a MRD marker with respect to stability, sensitivity and specificity in direct comparison to WT1 gene expression. A total of 13 relapsed AML patients with normal karyotype that were positive for mutation in NPM1 and WT1 gene expression at the time of diagnosis were included in the study. The NPM1 mutational load and WT1 gene expression was analyzed by real-time qPCR in up to 22 peripheral blood mononuclear cell samples per patient from the time of primary diagnosis to latest follow-up to compare the kinetics of the two markers during periods of morphological remission and relapse events. The 13 patients experienced a total of 18 morphological relapses which were all accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of NPM1 mutation during relapse in the present material. During periods of complete morphological remission, the NPM1 mutational load was below detection limit (< 1/1000 cells) in all samples. In contrast, WT1 gene expression was detectable in 70% of these samples, thus demonstrating the limited specificity of this marker. This background WT1 expression in non-leukemia cells reached levels of up to 1% of the levels detected at the time of diagnosis thus limiting the de facto MRD marker sensitivity of WT1. All samples with detectable levels of NPM1 mutation after a period of complete molecular remission were followed by a morphological relapse within weeks. The present study therefore shows that mutation in NPM1 is a stable and more sensitive and specific, and therefore superior, molecular MRD marker than WT1. Disclosures No relevant conflicts of interest to declare.

Global and HOX Gene DNA Methylation In Normal Karyotype Acute Myeloid Leukemia: Clinical Implications and Molecular Correlations

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 231-231
Author(s):  
Stefan Deneberg ◽  
Andreas Lennartsson ◽  
Bertil Uggla ◽  
Verena Gaidzik ◽  
Andrea Corbacioglu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Hox Genes ◽  
Normal Karyotype ◽  
Global Methylation ◽  
Hox Gene ◽  
Mutational Status ◽  
Average Methylation ◽  
Wbc Count

Abstract Abstract 231 Background: DNA methylation induces gene silencing in a nonrandom fashion in many types of malignancies. In AML there is no consensus regarding the clinical implications of DNA methylation on a global level. Normal karyotype AML (NK AML) can be prognostically stratified by molecular mutations in genes such as FLT3 and NPM1. We have previously reported that increased gene promoter methylation levels may have positive prognostic implications in AML. Now we have focused and expanded our analysis in a homogenous group of NK AML in an effort to illuminate these issues. Methods and patients: We analyzed genome wide DNA methylation signatures from the diagnostic bone marrow samples of 58 de novo NK AML with the IlluminaHuman27 Methylation array, covering 27000 CpG sites, mainly located in the promoter regions of 15000 individual genes. Global methylation was defined as the average methylation values of all measured CpG sites in the specific sample. All patients were between 18 and 67 years of age and received standard induction chemotherapy. All were eligible for intensive consolidation therapy including allogeneic transplantation. FACS sorted normal bone marrow separated into four stages of myeloid differentiation were analyzed as normal controls. Methylation data were correlated to clinical outcomes and molecular mutational status of NPM1 and FLT3. Functional annotation analyses were performed on differentially methylated genes to find epigenetically perturbed pathways. Further molecular analysis of CEBPA, IDH1 and IDH2 is currently performed. Results and discussion: Global methylation levels varied substantially between AML samples but remained mainly unchanged during normal myeloid differentiation. Methylation levels were significantly higher in AML cases than in the normal myeloid progenitors (p<0.001). There were no correlations between methylation levels, age, white blood cell (WBC) count or bone marrow blast percentage at diagnosis. Increased global methylation correlated significantly to increased in vivo chemosensitivity and to patient survival. CR rates after one course of induction chemotherapy were 64% in the quartile of patients with the highest level of global DNA methylation compared to 32% in others (HR 3.9, p=0.04). Median overall survival of patients of the most methylated quartile was not reached and significantly longer than the 16 months median survival of the least methylated quartile (p=0.001) (see figure below). In a Cox Regression model stratified for FLT3-mutational status, methylation level was an independent prognostic factor for survival (p=0.01) together with WBC count (p=0.01). A functional annotation analysis revealed that NPM1 wild type samples had an enrichment of Homeobox (HOX) gene methylation as compared to NPM1 mutated cases (p=1.7×10−11), providing a mechanism for the previously described difference in HOX gene expression in NPM1 wt vs. NPM1 mutated AML. The average methylation levels of all HOX genes were higher in wild type NPM1 cases compared to NPM1 mutated cases (p=0.02). Additional gene expression array data in our cohort of patients also showed methylation levels of individual HOX genes to be correlated inversely with expression. HOX genes were also enriched in non-FLT3ITD cases compared to FLT3ITD-cases, however, not to a statistically significant degree. In conclusion we show that global DNA methylation levels are predictive of response to chemotherapy and an independent prognostic factor for survival in normal karyotype AML. Furthermore, our results suggest that HOX gene methylation may be the mechanism underlying the previously known difference in HOX-gene expression between NPM1 mutated and unmutated AML cases. Disclosures: Nahi: Jansen-Cilag: Honoraria; Celgene: Honoraria.

A gene expression signature that distinguishes malignant from benign thyroid nodules.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21011-e21011
Author(s):  
Song Tian ◽  
John DiCarlo ◽  
Jiaye Yu ◽  
George J Quellhorst ◽  
Raymond K Blanchard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Random Forest ◽  
Thyroid Nodule ◽  
Thyroid Nodules ◽  
Gene Expression Signature ◽  
Support Vector ◽  
Data Set ◽  
Expression Signature ◽  
Benign Thyroid Nodules ◽  
Benign Thyroid

e21011 Background: Thyroid nodules can be detected in as high as 67% of the population. Distinguishing thyroid cancers from benign lesions is crucial for determining an appropriate treatment plan. For years a gene expression signature for discriminating malignant from benign thyroid nodules has been sought by clinicians. In this study, multivariate bioinformatics tools were used to generate a qPCR based gene expression signature for determining malignancy in thyroid nodules. Methods: Multiple mathematical models, such as Random Forest, Support Vector Machine (SVM), and Nearest Shrunken Centroid (NSC), were used to analyze published microarray data sets and select 366 putative classifier (biomarker) mRNA targets. The selected 366 genes were further evaluated for their expression pattern by real-time PCR using a panel of 49 pathology assessed thyroid nodule samples (fresh frozen, 23 malignant and 26 benign). Results: Using the qPCR data set, Random Forest was compared with SVM and NSC classifier methods and was found to be more successful in finding genes with better discriminative powers. A Random Forest method identified a panel of 7 genes together with 5 reference genes as a gene expression signature for thyroid malignancy, which led to the development of a companion classifying algorithm to provide a probability score to assess malignancy of thyroid nodules. In our limited sample set, this signature was shown to distinguish malignant and benign thyroid nodules with 92% accuracy and 100% specificity. Conclusions: Our results suggest that a combination of multiple bioinformatics analysis tools is the proper approach for biomarker candidate selection from high-throughput gene expression data. As demonstrated here, panel of 12 genes and a companion classification algorithm has the potential to successfully discriminate malignant thyroid nodule with high accuracy and specificity. This panel of twelve genes is for molecular biology applications only.

Changes in Type I Mutations Between Initial and Relapse Samples of Pediatric AML Patients Occur Frequently and Are Associated with Time to Relapse

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2540-2540
Author(s):  
Costa Bachas ◽  
Gerrit J. Schuurhuis ◽  
Lê D Huynh ◽  
Bianca F. Goemans ◽  
D. Reinhardt ◽  
...  
Keyword(s):  
Melting Curve ◽  
Melting Curve Analysis ◽  
Type I ◽  
Type Ii ◽  
Mutation Status ◽  
Pediatric Aml ◽  
Exon 11 ◽  
Time To Relapse ◽  
Mean Time

Abstract Activating type I mutations provide cells with proliferative and survival advantages. Together with type II abnormalities, which cause a differentiation arrest and an increase in self renewal properties, they cooperate to cause acute myeloid leukemia (AML). Most studied type I mutations for their presumed prognostic relevance are the family of RAS oncogenes (e.g. N-RAS, K-RAS) and receptor tyrosine kinases (FLT3 and KIT). Relevant type II abnormalities include for example the NPM1 and CEBPα genes. We previously showed in paired initial and relapsed samples of 80 AML patients (40 children and 40 adults) and reported a shift from FLT3/ITD positive status at diagnosis to negative at relapse, associated with prolonged time to relapse, whereas a shift from FLT3/ITD negative to positive to be associated with a shorter time to relapse (Cloos et al., Leukemia, 2006). Here we extended the mutation analysis of paired initial and relapsed pediatric samples to study the stability of other mutations, besides FLT3. Samples of 34 pediatric AML patients were analyzed. Patients were treated with protocols of the BFM-AML Study Group or Dutch Childhood Oncology Group between 1992 and 2004. Using capillary gel electrophoresis based fragment analysis, we analyzed the patient samples for insertions/deletions in exons 14,15 and 20 of FLT3, exon 11 of KIT, exon 12 of NPM1 and 3 hotspots in the CEBPα gene. FRET based melting curve analysis or high resolution melting curve analysis were used to detect point mutations in exons 8, 9 and 17 of KIT, exons 3 and 13 of PTPN11, codon 12/13 and 61 mutations of N-RAS and codon 12/13 mutations of K-RAS. The frequencies of mutations and instabilities are summarized in Table 1. We found no mutations in CEBPα and PTPN11. Instabilities were found in 14 out of 34 (41%) patients (11/34 when excluding FLT3 mutations). Patients with and without a type I mutation at initial diagnosis had no significantly different mean time to relapse (17.2 (n=20) vs 18.6 (n=14) months, p=0.80). However, when patients are stratified according to the presence of a type I mutation at relapse (Table 2), independent of the presence at diagnosis, they had a significantly shorter mean time to relapse than patients without a type I mutation at relapse (8.3 vs 21.7 months, p=0.016). We defined poor prognosis mutation status as presence of type I mutation at relapse whereas a favorable prognosis as no type I mutation or presence of NPM1 mutation at relapse. Remarkably, patients who acquired a poor prognosis mutation status at relapse showed a similar short time to relapse as compared to those who had the mutation at diagnosis and retained the mutation at relapse (Table 2). Patients with an acquired favorable mutation status had a time to relapse comparable to those who already had a favorable prognosis at diagnosis. In conclusion, nearly 50% of pediatric AML patients show instability of type I mutations, with both gains and losses from diagnosis to relapse. This indicates that mutation profiling at diagnosis is not always sufficient for accurate risk assessment. Therefore, additional molecular characterization of AML at minimal residual disease and relapse might improve prognostics and guide targeted therapy. Possible explanations for mutational shifts are: expansion of minor leukemic sub-clones, present, but not detected at diagnosis; appearance of new leukemic sub-clones during therapy due to genomic instability. Future research in a larger patient group will focus on the detection and characterization of malignant sub-clones. Table 1: Frequencies of mutations in genes of interest and their instabilities (N=34) Gene of interest Mutated at diagnosis Mutated at relapse Gain Loss FLT3/ITD 5 5 1 1 FLT3 D835 1 0 0 1 N-RAS codon 12/13 4 4 1 1 N-RAS codon 61 3 1 0 2 K-RAS 12/13 2 3 3 2 KIT exon 11 ITD 1 0 0 1 KIT D816 1 0 0 1 NPM1 1 0 0 0 Table 2: Relation between mutation status at relapse and time to relapse Risk Groups: categorized according to mutations at relapse Number of patients (total: n=34) Mean time to relapse in months (percentiles 25–75) Analysis of variance p-value I Favorable mutation status at relapse 24 21.7 (14.8–28.6) P=0.016 (I–II)     a) Retained favorable mutation status 18 20.3 (12.0–28.5) P=0.094 (a–d)     b) Acquired favorable mutation status 6 26.1 (8.9–43.4) II Poor mutation status at relapse 10 8.3 (5.9–10.8)     c) Retained poor mutation status 7 8.6 (4.9–12.3)     d) Acquired poor mutation status 3 7.6 (3.2–12.2)

Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia

Blood ◽  
2011 ◽  
Vol 118 (22) ◽  
pp. 5905-5913 ◽  
Author(s):  
Anna Staffas ◽  
Meena Kanduri ◽  
Randi Hovland ◽  
Richard Rosenquist ◽  
Hans Beier Ommen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Prognostic Markers ◽  
Npm1 Mutation ◽  
Mutation Status ◽  
Expression Levels ◽  
Pediatric Aml ◽  
Gene Expression Levels ◽  
Acute Myeloid

Abstract Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.

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