Identification of Genomic Classifiers That Distinguish Induction Failure in T-Lineage Acute Lymphoblastic Leukemia in COG Study 9404.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1826-1826
Author(s):  
Stuart S. Winter ◽  
Hadya Khawaja ◽  
Zeyu Jiang ◽  
Timothy Griffin ◽  
Barbara Asselin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Resistant Cell ◽  
Acquired Drug Resistance ◽  
Induction Failure

Abstract The clinical features of age, white count, and presence of extramedullary disease cannot predict risk for induction failure (IF) in patients who present with T-cell acute lymphoblastic leukemia (T-ALL). On the basis of recent observations that gene expression profiles can distinguish clinicopathologic cohorts of patients with acute leukemia, we hypothesized that microarray analyses performed on diagnostic T-ALL bone marrow samples might identify a genomic classifier for IF patients. Using a case-control study design for children and young adults treated for T-ALL on Children’s Oncology Group Study 9404, we analyzed 50 cryopreserved T-ALL samples using Affymetrix U133A Plus 2 genechips, which have 54,000 genes, ESTs and genomic classifiers. Following RMA normalization, we used Prognostic Multi-array Analysis (PAM) to identify a 116-member genomic classifier that could accurately identify all 6 IF cases from the 44 patients who achieved remission. Within the IF cohort, 37 genes were up-regulated and 79 were down-regulated in comparison to other outcome groups. To further investigate the genetic mechanisms governing IF, we developed four cell lines with acquired drug resistance: Jurkat and Sup T1; each having resistance to daunorubicin (DNR) and asparaginase (ASP). Using a comparative analysis for fold-change in gene expression among 6 IF patients and the T-ALL DNR and ASP-resistant cell lines, we identified seven genes that were up-regulated, and another set of seven genes that were commonly down-regulated. To validate the potential use of our 116-member gene set in predicting IF in T-ALL, we tested our genomic classifier in 42 cases which were treated on COG study 8704 and hybridized to the Affymetrix U133Av.2 chip. Because only 85 probes were shared between U133A Plus 2 and U133Av. 2 chips, we employed shrunken class centroids to constrain our classifier to 25 rank-ordered probes. This smaller classifier correctly identified the single IF case in 8704, as well as another patient who was an early treatment failure, indicating that similar genomic classifiers may identify IF patients in different clinical trials. These results indicate that genetic profiling may be useful in prospectively identifying IF patients in T-ALL. In addition, we identified genes that were commonly upregulated in IF patients and T-ALL cell lines with intrinsic drug resistance.

Changing Gene Expression Patterns during Early Treatment of B-Precursor Acute Lymphoblastic Leukemia May Be Predictive of Relapse.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 232-232 ◽  
Author(s):  
Valerie de Haas ◽  
Rob Dee ◽  
Goedele Cheroutre ◽  
Henk van den Berg ◽  
Huib Caron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Expression Level ◽  
Leukemic Cells ◽  
Diagnostic Samples

Abstract Treatment of pediatric ALL is based on the concept of tailoring the intensity of treatment to a patients risk. Clinical studies have shown that it is possible to stratify patients according to the levels of minimal residual disease after induction therapy and early during further treatment, since it has been demonstrated that the MRD level is the best predictive level for disease outcome. More recently, it has been shown that gene expression profiles of leukemic cells at diagnosis might be correlated with outcome. In previous studies we reported that slow responding subclones represent the clones causative for a leukemic relapse in oligoclonal disease. Based on these results, we hypothesized that the gene expression profile of the slow responding subclones present after the first weeks of chemotherapy might be more predictive than the profiles of all leukemic cells at diagnosis. Twenty-four genes were selected; most signalling molecules, transcription factors and functions relevant for oncogenesis, drug resistance and metastasis. Selection of genes was based on the presently available data on prognostic cDNA microarry studies of cytogenetically defined subgroups of childhood ALL. In particular, we analyzed results of recently published studies that compared gene expression levels between diagnosis and relapse in B-precusor acute lymphoblastic leukemia. (Staal, 2003 and Beesley, 2005). Gene sequences were obtained from public databases. Genes were tested on different leukemic cell lines. For all cell lines differences in gene expression level were demonstrated. The same panel of genes was tested on diagnostic samples of 16 ALL patients, subsequently followed by investigation of paired diagnosis - day 15 - relapse samples of 3 relapsed ALL patients. Leukemic material was obtained from cryopreserved bone marrow samples. All leukemic cells were purified by MACS purification based on markers expressed on the tumour, i.e. CD34, CD19 and CD10. RNA extraction and cDNA synthesis was performed according to the TRIZOL protocol. Expression levels were determined in a SYBR Green based real-time PCR assay. We were able to show different gene expression profiles in the 16 tested diagnostic samples. For the paired samples from relapsed B-precursor ALL patients, the expression level of several genes at day 15 was different (ΔCT>1) in regard to diagnosis. Moreover, the changed expression at day 15 was comparable to the expression level of this gene at relapse. We conclude that indeed we were able to demonstrate that some of the genes have a changing pattern of expression during early therapy (day15), a pattern which is comparable to the pattern of gene expression at relapse and which is different from the pattern at diagnosis. We also demonstrated that purification of the bone marrow samples is necessary to be certain that the gene expression shown is relevant for the leukemic cells and not contaminated by other cells, i.e. T-cells. Figure Figure

Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 71-71 ◽  
Author(s):  
Richard B Lock ◽  
Jennifer Richmond ◽  
Laura High ◽  
Hernan Carol ◽  
Kathryn Evans ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Wild Type ◽  
Induction Type ◽  
P53 Status

Abstract Introduction While the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. Methods In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. Results Unsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). Conclusions RG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation. This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification and Validation of Novel Reference Genes in Acute Lymphoblastic Leukemia for Droplet Digital PCR

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 376 ◽  
Author(s):  
Vanessa Villegas-Ruíz ◽  
Karina Olmos-Valdez ◽  
Kattia Alejandra Castro-López ◽  
Victoria Estefanía Saucedo-Tepanecatl ◽  
Josselen Carina Ramírez-Chiquito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Reference Genes ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cellular Processes

Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.

Histone Deacetylase Inhibition Using LBH589 Is Effective in Lymphoma and Results in Down-Regulation of the NF-KB Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3730-3730 ◽  
Author(s):  
Jason L. Smith ◽  
Amee Patel ◽  
Siyao Fan ◽  
Cassandra L. Jacobs ◽  
Katherine J. Walsh ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Expression Profiles ◽  
Hdac Inhibitors ◽  
Gene Expression Profiles ◽  
Resistant Cell ◽  
Hdac Inhibition ◽  
Down Regulation ◽  
Highly Sensitive

Abstract Abstract 3730 Poster Board III-666 Background Histone deacetylase (HDAC) inhibition has emerged as a promising therapeutic approach in malignancies. HDAC inhibition has proved to be a particularly effective option in patients with lymphoma. The HDAC inhibitor vorinostat is approved for the treatment of patients with cutaneous T-cell lymphomas and is being tested in patients with B cell lymphomas. More recently, a number of other HDAC inhibitors have entered preclinical and clinical testing. The mechanisms through which HDAC inhibitors exert their downstream effects are currently unknown. As the number of HDAC inhibitors in development increases, it is unclear if they share a class effect or display unique mechanisms of action. Recently, LBH589 has been described as an orally available, highly potent inhibitor of HDAC. We decided to explore whether LBH589 would be an effective therapeutic option for patients with lymphoma. Methods and Results In order to evaluate whether LBH was efficacious and potent in B cell lymphomas, we tested both vorinostat and LBH589 in the same cell line(s). We found that LBH589 was over 10 times more potent than vorinostat (mean IC50 7.4nM versus 830nM). We decided to further test LBH589 in an expanded panel of 18 cell lines derived from 5 different lymphoid malignancies: Burkitt lymphoma, mantle cell lymphoma, Hodgkin lymphoma, multiple myeloma and diffuse large B cell lymphoma. LBH589 was found to be lethal in each of these cell lines at IC50 concentrations varying from 5.6-31.5 nM (mean 11.2nM), suggesting that this drug may be effective at physiologically achievable concentrations. Based on the IC50 cut-off of 10nM, we assigned the treated cell lines to 2 groups: highly sensitive (IC50 < 10nM, N=11) and less sensitive (IC50> 10nM, N=8). We performed gene expression profiling on 12 of these cell lines and compared the gene expression profiles of the highly sensitive versus less sensitive cell lines. Further, we performed time course experiments in which we evaluated the effects of LBH589 at its IC50 on cell lines at 6 and 12 hours post-treatment. Gene expression profiling was performed on the treated cells at each time point. We also engineered resistant cell lines by incremental dose escalation over a period of months to a concentration greater than or equal to the IC50. The resistant cell lines were also profiled for gene expression and compared to the wild type cell lines. The gene expression profiles of LBH589 treated cells at 6 and 12 hours demonstrated a clear and progressive down regulation of genes associated with the NF-KB pathway (Figure 1). Furthermore, cell lines with high expression of genes in the NF-KB pathway were uniformly highly sensitive to LBH589 with IC50<10nM in all cases. Conclusion NF-KB activation is a common feature of many different lymphoma types. Our data suggest that HDAC inhibition using LBH589 could provide a potent method for treating lymphomas and that HDAC inhibitors may exert their effects through the down-regulation of the NF-KB pathway. Our data also suggest a rationale for dual inhibition of HDAC and NF-KB in the treatment of lymphoma. Disclosures: Rizzieri: Merck & Co., Inc.: Consultancy.

scholarly journals Mutation, methylation, and gene expression profiles in dup(1q)-positive pediatric B-cell precursor acute lymphoblastic leukemia

Leukemia ◽  
2018 ◽  
Vol 32 (10) ◽  
pp. 2117-2125 ◽  
Author(s):  
Rebeqa Gunnarsson ◽  
Sebastian Dilorenzo ◽  
Kristina B Lundin-Ström ◽  
Linda Olsson ◽  
Andrea Biloglav ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Cell Precursor

Bioinformatics analysis of gene expression profiles in childhood B-precursor acute lymphoblastic leukemia

Hematology ◽  
2014 ◽  
Vol 20 (7) ◽  
pp. 377-383 ◽  
Author(s):  
Jun Li ◽  
Xiaowen Zhai ◽  
Hongsheng Wang ◽  
Xiaowen Qian ◽  
Hui Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Bioinformatics Analysis ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles

Identification of Gene Expression Profiles in Acute Lymphoblastic Leukemia Cells That Discriminate Intracellular Thioguanine Nucleotide Accumulation in ALL Cells after In Vivo Treatment with Mercaptopurine.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 453-453
Author(s):  
Gianluigi Zaza ◽  
Meyling Cheok ◽  
Wenjian Yang ◽  
Pei Deqing ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Post Treatment ◽  
Leukemia Cells ◽  
True Positive

Abstract Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (< 2.46 and > 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (< 0.55 and > 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p<0.001) for MP alone and rho=0.65 (p<0.001) for MTX+MP, with false discovery rate (FDR) computed using Storey’s q-value (MP: 50% true positive, MTX+MP: 70% true positive respectively). Genes highly associated with the post-treatment TGN level in ALL patients treated with MP alone encode transporters, enzymes involved in the MP metabolic pathway and cell proliferation. Genes associated with post-treatment levels of TGN after combined therapy have been implicated in protein and ATP biosynthesis. Together, these in vivo data provide new insights into the basis of inter-patient differences in TGN accumulation in ALL cells, revealing significant differences between treatment with MP alone or in combination with MTX.

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