Molecular Characterization of Acute Undifferentiated and Biphenotypic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1692-1692
Author(s):  
Sandra Heesch ◽  
Stefan Schwartz ◽  
Isabelle Bartram ◽  
Cornelia Schlee ◽  
Annette Sindram ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Leukemia ◽  
Mrna Expression ◽  
Scoring System ◽  
Specific Gene ◽  
Prognostic Impact ◽  
Acute Leukemias ◽  
Over Expression ◽  
Prognostic Features ◽  
Flt3 Mutations

Abstract Abstract 1692 Background: Acute leukemias of ambiguous lineage account for only 3–5% of all cases of acute leukemia and represent a heterogeneous group of rare, poorly characterized leukemia with adverse outcome. The aim of this study was to further molecularly characterize this rare subtype to gain insights into the pathogenesis of acute undifferentiated leukemia (AUL) and biphenotypic acute leukemia (BAL). These molecular insights may then lead to targeted treatment strategies for specific molecular subgroups. Design and Methods: The European Group for Immunologic Classification of Leukemia (EGIL) defines BAL as a single blast population with co-expression of myeloid- and lymphoid-associated antigens according to a scoring system. Cases without expression of lineage-specific antigens are designated as AUL. Based on the EGIL scoring system, we identified 11 and 20 adults with newly diagnosed AUL and BAL, respectively. In all samples, flow cytometry was performed using a panel of monoclonal antibodies for precursor cells (CD34, TdT), lymphoid-associated antigens, including surface and cytoplasmic antigens (CD79a, CD22, IgM, CD19, CD20, CD10, CD24, CD3, TCR, CD2, CD5, CD8, CD7, CD1a), and myeloid-associated antigens including myeloperoxidase, CD13, CD33, CD65s, CD117, CD14, CD15, CD64. In order to further molecularly characterize AUL and BAL, samples were studied for expression and genotype alterations of candidate genes with prognostic impact or with relevance in the pathogenesis of acute leukemias. The mRNA expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by quantitative real-time PCR. In addition, samples were studied for WT1 mutations in exons 7 and 9, for FLT3 mutations [internal tandem duplications (ITD) and mutations in the tyrosine kinase domain (TKD835)], as well as for BCR-ABL (breakpoints: e1/a2; b3/a2; b2/a2) and MLL-AF4 rearrangements. Results were than compared to data generated from acute myeloid leukemia (AML; n = 75), acute T-lymphoblastic leukemia (T-ALL; n = 242) and B-precursor ALL (n = 368) patients. Results: AUL patients were characterized by a specific gene expression pattern. In particular, in AUL over-expression was shown for IGFBP7 (P = 0.01) and MN1 (P = 0.04) compared to BAL; for BAALC (P = 0.001), IGFBP7 (P = 0.0001) and MN1 (P = 0.02) compared to T-ALL; for BAALC (P = 0.02) and IGFBP7 (P = 0.001) compared to AML; as well as for IGFBP7 (P = 0.0001) compared to B-precursor ALL. Genotype alterations (WT1, FLT3, BCR-ABL, MLL-AF4) were not detectable in AUL samples. The majority of BAL cases expressed either T-lineage plus myeloid antigens (n = 9; 45%) or B-lineage plus myeloid markers (n = 9; 45%), while co-expression of T/B lymphoid antigens was only seen in 2 samples of BAL. Cell surface expression of CD34 (≥ 20%) was most frequently found in BAL (17 of 20 cases; 85%). Gene expression studies revealed over-expression of BAALC and IGFBP7 in BAL compared to T-ALL, AML and B-precursor ALL, but less pronounced as shown for AUL. Moreover, BAL samples were characterized by frequent WT1 mutations in exon 7 (sufficient DNA was available in 14 samples of which 4 had WT1 mutations; 29%). In contrast, WT1 mutations were only found in 8% of T-ALL and have previously been reported in about 10% of AML patients with normal cytogenetics (Paschka et al, 2008). In concordance with published data, the BCR-ABL fusion gene was detected in 32% of BAL samples (19 samples studied of which 9 were positive for BCR-ABL). MLL-AF4 translocations and FLT3 mutations were not detectable in this BAL cohort. Conclusion: Here we present a comprehensive molecular study investigating mRNA expression and genotype alterations in the poorly characterized subgroup of acute leukemias with ambiguous or lacking lineage commitment. We show that AUL and BAL patients share important prognostic features of both myeloid and lymphoid leukemias. Over-expression of stem cell associated genes (BAALC and IGFBP7) and genes associated with an adverse prognosis (BAALC and MN1) was most prominent in AUL. As lineage-directed therapies are likely ineffective for these high-risk patients and the fact that AUL lack known specific molecular targets, further studies are warranted to unravel molecular aberrations, which might allow the development of more specific therapies. BAL patients however, were characterized by genetic aberrations including BCR-ABL rearrangements and WT1 mutations, which may imply targeted therapy options. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Biology of t(6;11) Fusion Proteins and Their Role in MLL-Rearranged Acute Leukemia Lineage Determination

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5033-5033
Author(s):  
Arpita Kundu ◽  
Eric Kowarz ◽  
Jennifer Reis ◽  
Rolf Marschalek
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Acute Leukemia ◽  
Fusion Proteins ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Translocation ◽  
Fusion Genes ◽  
Disease Phenotype ◽  
Acute Leukemias

Chromosomal translocations are genetic rearrangements where a chromosomal segment is transferred to a non-homologous chromosome which give rise to novel chimeras. Chromosomal rearrangements play a significant role in the development of acute leukemias (acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)). Chromosomal translocation events occurring at 11q23 involving the KMT2A or Mixed-Lineage Leukemia (MLL) gene (n=102) can be diagnosed in about 5-10% of all acute leukemia patients (Marschalek Ann Lab Med 2016), especially prevalent in infant acute leukemias (up to 70% of cases). Different chromosomal translocation partner genes (such as AF4, AF6, AF9orENL and ELL) account for the majority of leukemia cases and have their genomic breakpoints within a major breakpoint cluster region (BCR intron 9-11; Meyer et. al. Leukemia 2018). Some rearrangements are specifically associated with particular disease phenotype e.g. the majority of ALL patients (~ 90%) are mainly caused by the following gene fusions, MLL-AF4, MLL-AF9, MLL-ENL. We are interested in a rare but yet drastic chromosomal translocation t(6;11)(q27;q23) which fuses KMT2A/MLL to Afadin (AFDN/AF6) gene. This chromosomal rearrangement has a very poor prognosis (survival-rate is ~10%) and is predominantly diagnosed in patients with high-risk AML. In this project, we investigate the molecular consequences of two different MLL-AF6 fusions and their corresponding reciprocal AF6-MLL fusions. MLL-AF6 fusions are mainly occurring within MLL intron 9 to 11 and are associated with an AML disease phenotype, while the same fusion occurring within the minor breakpoints region in MLL intron 21 until exon (ex) 24 are mainly diagnosed with T-ALL (T-cell acute lymphoblastic leukemia) disease phenotype. The molecular mechanism that determines the resulting disease phenotype is yet unknown. Therefore, we cloned all of these t(6;11) fusion proteins in order to investigate the functional consequences of the two different breakpoints (MLLex1-9::AF6ex2-30, AF6ex1::MLLex10-37; MLLex1-21::AF6ex2-30, AF6ex1::MLLex22-37). All 4 fusion genes were introduced into our inducible Sleeping Beauty system (Ivics et. al. Mobile DNA 2010; Kowarz et. al. Biotechnol J. 2015) and stably transfected reporter cell lines. Basically, these 4 fusion proteins differ only in the presence or absence of their Plant homeodomain 1-3/Bromodomain (PHD1-3/BD) domain (see Figure 1). The PHD domain regulates the epigenetic and transcriptional regulatory functions of wildtype MLL. Subsequently, we analyzed gene expression differences by the MACE-Seq (Massive Analyses of cDNA Ends). MACE data revealed fundamental differences in gene expression profiles when analyzing the two different sets of t(6;11) fusion genes. The resulting profiles have similarities to either AML or T-ALL and might give a rational explanation for the different lineages in these t(6;11) patients. Altogether, these results notably indicate that our study will provide a novel insight into this type of high-risk leukemia and subsequently will be useful for developing of novel and appropriate therapeutic strategies against acute leukemia. Disclosures No relevant conflicts of interest to declare.

253 GENE SILENCING OF CYCLOOXYGENASE-2 mRNA BY RNA INTERFERENCE IN BOVINE CUMULUS - GRANULOSA CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 234
Author(s):  
S.-I. Kobayashi ◽  
M. Sakatani ◽  
S. Kobayashi ◽  
K. Okuda ◽  
M. Takahashi
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Mrna Expression ◽  
Culture Medium ◽  
Cyclooxygenase 2 ◽  
Target Cells ◽  
Specific Gene ◽  
Cox 2 ◽  
Sirna Group ◽  
Interference Rna

Recently, interference RNA (RNAi), inducing inhibition of the specific gene expression, attracted a lot of attention. Many researchers have reported that the 21-mer small interference RNA (siRNA) is introduced into target cells and then siRNA can suppress the gene expression. RNAi is a useful tool for functional analysis of specific genes. However, there is little information about RNAi for the analysis of gene function in reproductive physiology in ruminants. Thus, this study was aimed at evaluating RNAi for the analysis of cyclooxygenase-2 (Cox-2) mRNA expression in bovine cumulus-granulosa (CG) cells as well as prostaglandin F2� (PGF2�) production. We investigated both the effective concentration of Cox-2 siRNA and the effect of the introduction time of siRNA on Cox-2 mRNA expression. Bovine CG cells were collected at slaughterhouse and cultured in 4-well dishes. After the cells reached confluency, two experiments were conducted. In the present study, Cox-2 siRNA was simply added to culture medium with lipofectamine" 2000 (Invitrogen Japan K. K., Tokyo, Japan) as the transfection reagent. In experiment 1, the concentration of 0, 100, 250, and 500 pM of Cox-2 siRNA was introduced into the CG cells. After 24 h of introduction, the amount of mRNA expression for Cox-2 was measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. In experiment 2, 250 pM siRNA for Cox-2 was introduced into CG cells for 0, 3, 6, 12, and 24 h. After culture, the amount of mRNA expression for Cox-2 was measured and the culture medium was collected to determine PGF2� concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by 100 pM siRNA introduced into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression (10% of that of the 0 pM siRNA group). Moreover, the suppressive effect of 250 pM siRNA was observed at 6 h after introduction (60% of that of the 0 pM siRNA group at 0 h) and the reduction of mRNA expression by RNAi became more obvious over 12 h (10% of that of the 0 pM siRNA group at 0 h). On the other hand, the PGF2� concentration in the culture medium was not significantly different at 12 h after siRNA introduction, however, the PGF2� concentration of the RNAi treatment group at 24 h was significantly lower than that of the 0 pM siRNA group at the same time point. These results suggest that gene silencing by Cox-2 siRNA is a means of analyzing the function and expression of specific genes in bovine CG cells. This study was supported by the Japan Society for the Promotion of Science for Young Scientists.

Gene Expression Profiling in Adult Acute Lymphoblastic Leukemia, Biphenotypic Acute Leukemia, and Acute Myeloid Leukemia M0: Confirmation of Immunophenotypic and Cytogenetic Diagnostic Findings.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 993-993
Author(s):  
Wolfgang Kern ◽  
Alexander Kohlmann ◽  
Claudia Schoch ◽  
Martin Dugas ◽  
Sylvia Merk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Diagnostic Criteria ◽  
Prediction Accuracy ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Support Vector ◽  
Specific Gene ◽  
Acute Leukemias ◽  
Acute Myeloid

Abstract Diagnosis and classification of acute lymphoblastic leukemias (ALL) and their distinction from biphenotypic acute leukemias (BAL) and acute myeloid leukemias with minimal differentiation (AML M0) is largely based on immunophenotyping. The EGIL classification, adopted by the WHO classification, defines 4 different subtypes of both B-precursor and T-precursor ALL as well as detailed criteria for BAL. Specific cytogenetic features useful for classificationare found in some cases only. We analyzed gene expression profiles in 173 such patients (Pro-B-ALL n=25, c-ALL/Pre-B-ALL n=65 (with t(9;22) n=35, without t(9;22) n=30), mature B-ALL n=13, Pro-T-ALL n=6, Pre-T-ALL n=13, cortical T-ALL n=20, BAL (myeloid and T-lineage) n=17, AML M0 n=14). All cases were assessed by cytomorphology, immunophenotyping, cytogenetics, and molecular genetics. All cases with Pro-B-ALL had t(4;11)/MLL-AF4, all cases with mature B-ALL had t(8;14). Samples were hybridized to both U133A and U133B microarrays (Affymetrix). Top 300 differentially expressed genes were identified for each group in comparison to all other groups and individual other groups and used for classification by various Support Vector Machines (SVM) with 10-fold cross validation (CV). Prediction accuracy for discriminating T- from B-precursor ALL was 100%. Accordingly, principal component analysis (PCA) yielded a complete separation of both groups. PCA of B-precursor ALL cases showed distinct clusters for Pro-B-ALL, c-ALL/Pre-B-ALL, and mature B-ALL, however, c-ALL/Pre-B-ALL with t(9;22) were not completely discriminated from those without. Accordingly, classifying B-precursor ALL with SVM resulted in a 87.4% accuracy. Pre-T-ALL cases clustered distinct from cortical T-ALL with hte exception of two cases. The other Pre-T-ALLs clustered together with Pro-T-ALL. Analyzing T-precusor ALL with SVM and 10-fold CV resulted in an accuracy of only 56.4%. Including BAL and AML M0 into these analyses revealed significant overlaps between samples from these entities and T-ALL cases in PCA; prediction accuracy using SVM and 10-fold CV was 79.8%. This accuracy was confirmed applying 100 runs of SVM with 2/3 of samples being randomly selected as training set and 1/3 as test set which resulted in a median accuracy of 77.2% (range, 67.5% to 85.1%). A 100% prediction accuracy was achieved in Pro-B-ALL and mature B-ALL. Misclassifications were: c-ALL/Pre-B-ALL with t(9;22) as c-ALL/Pre-B-ALL without t(9;22) (6/35) and vice versa (6/30). Of the 13 Pre-T-ALL cases 4 were classified as BAL and 3 as cortical T-ALL. Of the 6 Pro-T-ALL cases 2 were classified as AML M0, 3 as BAL, and 1 as Pre-T-ALL. Of the 17 BAL cases 2 were classified as AML M0, 1 as c-ALL/Pre-B-ALL, 2 as Pre-T-ALL, and 1 as Pro-T-ALL. These analyses confirm that gene expression profiles allow the identification of Pro-B-ALL with t(4;11) and mature B-ALL with t(8;14) but do not unequivocally identify the presence of t(9;22) in c-ALL/Pre-B-ALL. Cortical T-ALL are characterized by a specific gene expression profile which is, however, shared by few cases currently diagnosed as Pre-T-ALL. Thus, diagnostic criteria (surface expression of CD1a only) should be optimized. The same applies to diagnostic criteria for more immature T-ALL, BAL, and AML M0. Loss of 5q is frequently observed in all of these latter entities and may be a future diagnostic marker superseding flow cytometry.

Prognostic Significance of Multidrug Resistance Gene 1 (MDR1), Multidrug Resistance-Related Protein (MRP) and Lung Resistance Protein (LRP) mRNA Expression in Acute Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4418-4418
Author(s):  
Hee Jin Huh ◽  
Chan-Jeoung Park ◽  
Seongsoo Jang ◽  
Eul-Ju Seo ◽  
Hyun-Sook Chi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multidrug Resistance ◽  
Acute Leukemia ◽  
Mrna Expression ◽  
Prognostic Significance ◽  
Multidrug Resistance Gene ◽  
Lung Resistance Protein ◽  
Mdr1 Mrna ◽  
Resistance Protein ◽  
Multidrug Resistance Related Protein

Abstract The prognostic significance of multidrug resistance (MDR) gene expression is controversial. We investigated whether multidrug resistance gene 1 (MDR1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) mRNA expression are associated with outcomes in acute leukemia patients. At diagnosis we examined MDR1, MRP and LRP mRNA expression in bone marrow samples from 71 acute leukemia patients (AML 39, ALL 32) using nested reverse transcription polymerase chain reaction (RT-PCR). The expression of each of these genes was then expressed as a ratio in relation to ß-actin gene expression, and the three genes were categorized as being either not expressed (0), weakly expressed (1+), moderately expressed (2+) or strongly expressed (3+). MDR1, MRP and LRP mRNA expression was detected in 23.9%, 83.1% and 45.1%, respectively, of acute leukemia patients. LRP mRNA expression was significantly associated with resistance to induction chemotherapy in acute leukemia patients, and in the AML proportion of these patients, compared to LRP-negative patients (p=0.02 and p=0.03, respectively). MRP and high MDR1 mRNA expression was associated with poorer 2-year survival (P=0.049 and p=0.04, respectively) compared to MRP-negative and non-high MDR1 mRNA-expressing patients, respectively. Patients expressing both MRP and LRP mRNA had poorer outcomes in response to induction chemotherapy and had worse 2-year survival compared to patients not expressing both genes. The present data suggest that MDR gene expression affects CR and survival rates in acute leukemia patients. Thus, determination of MDR gene expression at diagnosis appears likely to provide useful prognostic information for acute leukemia patients.

EZH2 Mutations Can Be Detected in 23% of t(10;11)(p13;q14)/PICALM-MLLT10 Positive Acute Leukemias,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3440-3440
Author(s):  
Vera Grossmann ◽  
Ulrike Bacher ◽  
Valentina Artusi ◽  
Hans-Ulrich Klein ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Leukemia ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Gene Expression Signature ◽  
Mutation Load ◽  
Employment Equity ◽  
Acute Leukemias ◽  
Equity Ownership ◽  
Mixed Phenotype

Abstract Abstract 3440 The t(10;11)(p13;q14)/PICALM-MLLT10 (CALM-AF10) rearrangement is most frequently associated with T-lineage acute lymphoblastic leukemia/lymphoma (T-ALL), and is rarely observed in AML. The EZH2 gene, located on 7q36.1, is a highly conserved histone H3 lysine 27 methyltransferase that influences stem cell renewal. EZH2 mutations were observed in 10% of patients with myelofibrosis, myelodysplastic/myeloproliferative neoplasms, or chronic myelomonocytic leukemia. In a previous study, we had investigated AML patients for EZH2 deletions using FISH. 6/20 (30%) of cases had been detected to carry a deletion. Additionally, we had screened these 6 cases for molecular mutations in EZH2 (transcript-ID: ENST00000320356) using an amplicon-based deep-sequencing assay, and one of the 6 patients was harboring both an EZH2 deletion and an EZH2 mutation. More interestingly, this double-mutated case was carrying a PICALM-MLLT10 rearrangement. Therefore, in this study, we were interested to investigate an expanded cohort of 13 cases (T-lineage ALL and AML) harboring a PICALM-MLLT10 rearrangement. Our cohort comprised 12 adults and one pediatric patient (7 males, 6 females) and was characterized by a predominant T-cell origin: 11 patients had T-ALL, 1 patient had mixed phenotype T/myeloid acute leukemia, and 1 patient had AML. EZH2 alterations were detected in 3/13 (including the index case). In more detail, the EZH2 mutation carriers were characterized as follows: Patient #1 (male, 26 years, AML) had a splice site mutation in exon 14 with a mutation load of 13% in a cysteine-rich region. Patient #2 (male, 19 years, T-ALL) harbored a missense mutation (Phe136Leu) with a mutation load of 93%. Patient #3 (female, 53 years, T-ALL) showed three concomitant EZH2 missense mutations in exon 5: His120Gln, Tyr124His, and Gly150Arg. The mutation load detected was 17% for each alteration. A fourth patient had a 1459G>A base substitution (corresponding to Ala487Thr) which to our knowledge had not been described before. However, this alteration had to be interpreted as germline as it was still detectable in the remission state. In contrast, in an independent cohort of 12 patients with PICALM-MLLT10 negative T-ALL (7 females, 5 males) analyzed for comparison no EZH2 mutation was detected. Interestingly, in patients #2 and #3, the mutations were located in exon 5 in the region which interacts with the DNMT1, DNMT3A, and DNMT3B DNA methyltransferase genes (D1). Moreover, DNMT3A mutations were recently identified in patients with AML and MDS in association with poor outcomes. Therefore, we additionally performed investigation for DNMT3A mutations in all 13 patients with PICALM-MLLT10 positive leukemias but detected no mutation. To investigate further molecular associations, we analyzed these cases also for RUNX1 mutations and FLT3-ITD, but we did not detect any mutation in these molecular genes. Further, we compared the gene expression profiles of 8 patients with PICALM-MLLT10 positive T-ALL to the profiles of 21 PICALM-MLLT10 negative T-ALL patients. Hierarchical clustering revealed a distinct gene expression signature of the PICALM-MLLT10 positive cases. Significant upregulation was found for HOXA5 and HOXA9 genes. Other differentially overexpressed HOX were HOXA3, A4, A6, A7, A10. Genes with a function for cell differentiation and regulation of apoptosis (ZAK) as well as for signal transduction (AKT3) were significantly underexpressed. Subsequently, we compared the gene expression profiles of 2 EZH2 mutated patients to 6 EZH2 wild-type patients in the PICALM-MLLT10 positive cohort. By hierarchical clustering, both EZH2 mutated cases showed a distinct gene expression signature. Increased expression was observed for genes with a role for the regulation of transcription (ZNF207, KDM5B, or CASZ1) or for intracellular transport (SARB1). In summary, we detected EZH2 mutations in 3/13 cases in this series of PICALM-MLLT10 positive malignancies, comprising mostly T-ALL, but also AML or mixed phenotype acute leukemia. This further emphasizes a cooperative effect of EZH2 mutations with the PICALM-MLLT10 fusion in acute leukemias of different lineages. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Artusi:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Multidrug resistance (mdr1) gene expression in adult acute leukemias: correlations with treatment outcome and in vitro drug sensitivity

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 586-592 ◽  
Author(s):  
JP Marie ◽  
R Zittoun ◽  
BI Sikic
Keyword(s):  
Gene Expression ◽  
Multidrug Resistance ◽  
Complete Remission ◽  
Acute Leukemia ◽  
Remission Rate ◽  
Leukemia Cells ◽  
Acute Leukemias ◽  
Mdr1 Gene Expression ◽  
Mdr1 Expression

Resistance to multiple chemotherapeutic agents has been related to the production of P-glycoprotein, a trans-membrane drug efflux pump that is encoded by the multidrug resistance (MDR) gene mdr1. To investigate whether mdr1 could be involved in clinical resistance to chemotherapy in acute leukemias, we have analyzed retrospectively the RNA from adult acute leukemia cells by slot-blot hybridization with a human mdr1 probe. Units of mdr1 expression were defined by reference to drug- sensitive human sarcoma and K562 leukemia cell lines (1 U) and the highly resistant doxorubicin selected leukemia cells K562/R7 (50 U). We studied 41 adult patients with acute leukemias: 5 acute lymphoblastic leukemias, 23 acute myeloid leukemias, and 13 secondary leukemias or blast crisis of chronic myelogenous leukemia. Expression of 10 U or more of mdr1 was found in 6 of 31 (19%) leukemias at diagnosis, versus 5 of 10 (50%) after relapse from therapy, P = .06. The complete remission rate and in vitro sensitivity to daunorubicin were both correlated with low expression (1 U, v 2 U or more) of mdr1. Among 36 evaluable attempts to induce remission, the complete remission rate was 67% (8 of 12) for patients with undetectable or minimal mdr1 expression (1 U), versus 29% (7 of 24) in patients with 2 U or more of expression, P = .03. In vitro resistance to daunorubicin or other MDR-related drugs was associated with expression of 2 U or more of mdr1 in 11 of 11 cases, while specimens that were sensitive to these agents were negative for mdr1 expression in 5 of 11 cases, P = .03. These data suggest that mdr1 expression contributes to chemoresistance in acute leukemia. Determination of mdr1 gene expression may be useful in designing therapy for patients with leukemia.

scholarly journals CRLF2 over-Expression Is a Poor Prognostic Marker in Children with High Risk T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1071-1071
Author(s):  
Chiara Palmi ◽  
Daniela Silvestri ◽  
Ilaria Bronzini ◽  
Gunnar Cario ◽  
Angela Savino ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Risk ◽  
Prognostic Marker ◽  
Prognostic Value ◽  
Cox Model ◽  
Lymphoblastic Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Prognostic Impact ◽  
Over Expression ◽  
Increased Risk

Abstract Introduction: Although risk-adapted therapy improved the prognosis of pediatric T-ALL in the last decades, patients with T-ALL still have a worse outcome compared to B Cell Precursor (BCP)-ALL, and therefore they would benefit from the identification of new prognostic markers for a better treatment stratification and novel strategies to complement current chemotherapy regimens. Among newly reported genomic abnormalities, a subset of BCP-ALL patients is characterized by the over-expression of the Cytokine Receptor-like Factor 2 (CRLF2) gene, due to either an intra-chromosomal deletion causing the P2RY8-CRLF2 fusion or the IGH@-CRLF2 translocation. These two CRLF2 rearrangements were shown to correlate with poor outcome in BCP-ALL patients. In T-ALL, alterations of CRLF2 were not reported yet, but recently, mutations in its partner IL7Rα have been identified in about 10% of T-ALL patients. Aim: To estimate the incidence of CRLF2 aberrations and their prognostic value in pediatric T-ALL. Methods: We analyzed CRLF2 gene expression in 120 T-ALL patients, consecutively enrolled in the AIEOP-BFM ALL2000 study in Italian centers (AIEOP) from September 2000 to July 2005, and, as a validation cohort, in 92 consecutive patients treated with the same protocol in German centers (BFM-G), from July 1999 to December 2004. CRLF2 transcript levels were analyzed by RQ-PCR. Relative gene expression of CRLF2 was quantified by the 2-DDCt method. The DDCt for AIEOP and BFM-G samples was referred to the median DCt of their respective cohort. Results: An heterogeneous expression of CRLF2 was observed among AIEOP T-ALL patients (range: 0.06 to 82 fold change). Seventeen patients (14.2%) presented an expression 5 times higher than the median (‘CRLF2-high’). Interestingly, none of the CRLF2-high cases resulted to be positive for P2RY8-CRLF2 fusion, 1/5 was positive for the IGH@ translocation and 1/7 showed a supernumerary X chromosome. JAK2 and CRLF2 mutations were absent in all 120 cases, while IL7R mutations were detected in 5/107 patients (4.7%), but unexpectedly they were not associated to CRLF2 over-expression. CRLF2-high patients had a significantly inferior 5-y EFS (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and an increased, although not statistically significant, cumulative incidence of relapse (CIR) compared to CRLF2-low patients (41.2%±11.9 vs. 26.3%±4.3, p=0.17). The prognostic value of CRLF2 over-expression was confirmed in the BFM-G cohort (5-y EFS in 12 CRLF2-high patients was 50.0%±14.4 vs 83.8%±4.1, p-=0.008; CIR: 33.3%±13.6 vs. 11.3%±3.5, p= 0.06). Interestingly, CRLF2-high patients were more frequently allocated to the high risk (HR) T-ALL subgroup (20.9% in HR vs.8.3% in no-HR in the two cohorts analyzed together). In this subgroup, CRLF2 over-expression was significantly associated to a poor prognosis (5-y EFS: 31.6%±10.7 vs. 62.5%±5.7, p-value=0.008; CIR: 47.4%±11.5 vs. 29.2%±5.4, p=0.14). When analyzed according to prednisone (PDN) response, 17/28 (61%) were prednisone poor responders (PPR), and in the ‘PPR-only’ subgroup (non-HR by other features) 4/9 (44%) CRLF2-high patients relapsed versus 4/36 (11%) CRLF2-low. Cox model analysis adjusted by risk group showed that CRLF2-high expression had a relevant prognostic impact with a 2-fold increased risk of relapse (Hazard ratio 2.12; 95% CI 1.07-4.21; p=0.03). The mechanisms responsible for CRLF2 over-expression in T-ALL and its contribution to the pathogenesis of the disease is still being investigated. Notably, gene set enrichment analysis (GSEA) showed an inverse correlation between the expression of CRLF2 and positive cell cycle regulators, thus suggesting that CRLF2-high blasts have a low proliferating activity and may therefore be less sensitive to conventional chemotherapy. Conclusions: CRLF2 over-expression is a poor prognostic marker not only in BCP-ALL patients, but also in T-ALL, identifying a subset of HR T-ALL patients with extremely severe outcome. Specifically, this marker would allow identifying T-ALL patients that could benefit from alternative therapy, potentially targetting the CRLF2 pathway. Disclosures No relevant conflicts of interest to declare.

Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 567-578 ◽  
Author(s):  
A.P. Monaghan ◽  
K.H. Kaestner ◽  
E. Grau ◽  
G. Schutz
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Expression Patterns ◽  
Primitive Streak ◽  
Definitive Endoderm ◽  
Specific Gene ◽  
Axis Formation ◽  
Specific Gene Expression ◽  
Mouse Tissues ◽  
Alpha Beta

The HNF-3 alpha, beta and gamma genes constitute a family of transcription factors that are required for hepatocyte-specific gene expression of a number of genes, e.g. transthyretin, alpha-1 antitrypsin and tyrosine aminotransferase. These genes share a highly conserved DNA-binding domain first found in the Drosophila gene, forkhead, which is required for the normal patterning of the developing gut and central nervous system in Drosophila. In adult mouse tissues, transcripts from HNF-3 alpha and beta have been localised to the liver, intestine and lung, whereas HNF-3 gamma is found in the liver, intestine and testis. In light of the early developmental significance of forkhead in Drosophila, we have compared the patterns of expression of HNF-3 alpha, beta and gamma mRNAs during murine embryogenesis. We find that these genes are sequentially activated during development in the definitive endoderm. HNF-3 beta mRNA is expressed in the node at the anterior end of the primitive streak in all three germ layers and is the first gene of this family to be activated. Subsequently, HNF-3 alpha is transcribed in the primitive endoderm in the region of the invaginating foregut and HNF-3 gamma appears upon hindgut differentiation. These genes have different anterior boundaries of mRNA expression in the developing endoderm and transcripts are found in all endoderm-derived structures that differentiate posterior to this boundary. Therefore, we propose that these genes define regionalization within the definitive endoderm. Furthermore, differential mRNA expression of HNF-3 alpha and beta is detected in cells of the ventral neural epithelium, chordamesoderm and notochord. In the neural epithelium, expression of HNF-3 alpha and beta mRNA becomes localised to cells of the floor plate. We propose that, in addition to their characterised requirement for liver-specific gene expression, HNF-3 alpha and beta are required for mesoderm and neural axis formation. We also conclude that HNF-3 beta is the true orthologue of the Drosophila forkhead gene.

High Rate of FLT3 Mutations In Adult ETP-ALL.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1031-1031 ◽  
Author(s):  
Martin Neumann ◽  
Ebru Coskun ◽  
Ouidad Benlasfer ◽  
Sandra Heesch ◽  
Stefan Schwartz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Expression Pattern ◽  
Mirna Expression ◽  
Lymphoblastic Leukemia ◽  
Specific Gene ◽  
Wild Type ◽  
Specific Gene Expression ◽  
Flt3 Mutation ◽  
Flt3 Mutations

Abstract Abstract 1031 Introduction: Mutations of FLT3 are recognized as important prognostic factor in acute myeloid leukemia (AML) with an impact on new therapeutic strategies. However, FLT3 mutations are rarely (1-3%) found in acute T-lymphoblastic leukemia (T-ALL). Recently, a new T-ALL subgroup defined by a specific gene expression profile and a characteristic immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers) and with poor outcome has been described in pediatric T-ALL patients. This subgroup likely originates from early thymic progenitors (ETP) and has therefore been termed ETP-ALL. To unravel the molecular alterations of this distinct subgroup with myeloid characteristics, we studied adult ETP-ALL patients for FLT3 mutations. Moreover, we analyzed miRNA expression to explore their potential involvement in the specific gene expression signature observed in ETP-ALL. Patients and methods: We screened 1241 peripheral blood and bone marrow samples of T-ALL patients that were sent to the central diagnostic reference laboratory of the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL). Of the immunophenotypically identified ETP-ALL patients (n=142), sufficient material was available in 70 cases. FLT3 mutations (ITD and TKD) were assessed using the FLT3 mutation assay (InVivoScribe Technologies, San Diego, USA). Expression of miRNAs was first screened by TaqMan low density arrays including 667 miRNAs in ETP-ALL (n=8) and compared to non-ETP T-ALL (n=6). Subsequently, expression of miR-221 and miR-222 was examined by real-time PCR in all 70 ETP-ALL. Results: In our cohort of 1241 T-ALL, 142 (11.4 %) patients showed an immunophenotype of an ETP-ALL, which is in the range of reported frequencies in pediatric patients. Among the 70 ETP-ALL samples analyzed, we identified 9 patients with a FLT3 D835 mutation and 15 patients with a FLT3 ITD. In total, 34 % (24 of the 70) patients carried a FLT3 mutation, which represents approximately 4 % of all T-ALL patients. In contrast, only one patient was identified with a FLT3 mutation within an arbitrarily selected control group of 107 T-ALL patients with a non-ETP immunophenotype. Interestingly, ETP-ALL with FLT3 mutations showed a distinct immunophentype compared to ETP-ALL with a wild type FLT3: ETP-ALL with FLT3 mutations had a higher rate of positivity for CD2 (88% vs. 30%, P<0.001), CD117 (83% vs. 28%, P<0.001), and CD13 (100 % vs. 37%, P<0.001). On the other hand, ETP-ALL with wild type FLT3 were characterized by a more frequent positivity of CD5 (54% vs. 4%, P<0.001) and CD33 (54% vs. 4%, P<0.001). Based on these findings a specific immunophenotype may be linked to FLT3 mutated ETP-ALL: in 73% (21/29) of patients with CD2/CD13 positivity a FLT3 mutation was present, compared to only 7% (3/41) of the remaining patients with ETP-ALL. In addition, we generated miRNA expression profiles in ETP-ALL and identified 7 miRNAs that were differentially expressed compared to non-ETP T-ALL. Among these miR-221 and miR-222, which were significantly overexpressed in ETP-ALL, are targeting genes characterizing the ETP-ALL phenotype (e.g. ETS1, KIT). Examination of miR-221 and miR-222 in 70 ETP-ALL revealed their particular overexpression in FLT3 mutated samples (miR-221 2.45-fold, P=0.012; miR-222: 2.05-fold, P=0.008) compared to ETP-ALL with wild type FLT3. Conclusion: We identified a yet unreported high frequency of FLT3 mutations in T-ALL, which are nearly exclusively found within the subgroup of ETP-ALL. In this group the rate is comparable high to the rate of FLT3 mutations in AML. Therefore, on the basis of established flow cytometry analysis we identified T-ALL patients that should be now prospectively and routinely screened for FLT3 mutations. Moreover, the distinct miRNA expression pattern may impact on the specific gene expression pattern of ETP-ALL. Thus, patients of this molecular distinct ETP-ALL entity may benefit from tyrosine kinase inhibitors in relapse situations or with presence of minimal residual disease as a bridging therapy to allogeneic stem cell transplantation. Disclosures: Baldus: Novartis: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document