scholarly journals "Detection of BCR-ABL1-like Subtype in Adult Acute Lymphoblastic Leukemia Using Digital Ncounter Nanostring Technology"

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-14
Author(s):  
Dikshat Gopal Gupta ◽  
Neelam Varma ◽  
Shano Naseem ◽  
Man Updesh Singh Sachdeva ◽  
Pankaj Malhotra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Fold Change ◽  
Lymphoid Tissues ◽  
Fusion Transcripts ◽  
Rna Transcripts ◽  
Ph Chromosome ◽  
Nanostring Technology

Introduction A new provisional entity of "B-lymphoblastic leukaemia/lymphoma, BCR-ABL1-like" has been introduced in the 2017 revised edition of WHO classification of tumours of haematopoietic and lymphoid tissues. BCR-ABL1-like cases are negative for Ph chromosome or t(9:22)(q34;q11.2) translocation, do not express the fusion BCR-ABL1 RNA transcripts and proteins resulting from the Ph chromosome,and are characterized by similar gene expression profiles as that of BCR-ABL1-positive acute lymphoblastic leukemia (BCR-ABL1-positive ALL).There is no 'short-cut approach' for making an accurate diagnosis of BCR-ABL1-like ALL. Two approaches namely Gene expression profiling (GEP) or Next-generation sequencing (NGS) and TLDA (TaqMan low-density array) are used for the detection of BCR-ABL1-like ALL cases. NGS is very costly and data interpretation requires a lot of bioinformatics skills and TLDA is not commercially available in India. Aims We planned to study the whole transcriptome of BCR-ABL1-positive ALL cases using microarray GEP, followed by customizing targeted gene panel using nCounter NanoString technology, for the detection of BCR-ABL1-like cases. METHODS Flow cytometric immunophenotying (FCM-IP) and multiplex RT-PCR were performed on 200 B-ALL cases to detect BCR-ABL1 chimeric fusion transcripts. Further, 12 BCR-ABL1-positive cases were subjected to transcriptome profiling using Affymetrix microarray (Gene Chip Human Genome U133 Plus 2.0 Array). The results were analyzed using TAC 4.0 software. Finally, a targeted panel of 50 differentially expressed genes [including 5 Housekeeping genes (HKGs)] was constructed according to our microarray findings and previously published data (Harvey RC et al.ASH 2013). A total of 96 B-ALL cases (16 BCR-ABL1-positive cases & 80 BCR-ABL1-negative cases) were subjected to GEP using nCounter Platform. The results were analyzed using nSolver4.0 software. RESULTS In the study cohort of 200 adult B-ALL cases, BCR-ABL1 chimeric fusion transcripts were detected in 34% (b2a2 and b3a2=18.05% & e1a2=15.5%), as revealed by multiplex assay. Global transcriptome profiling of 12 BCR-ABL1 RNA transcripts revealed a total of 1574 as DE genes (460 genes in e1a2, 515 genes in b2a2 and 599 genes in b3a2). DE genes were further filtered through hierarchical clustering analysis and a total of 45 DE genes with 10- to -86-fold change were identified. These genes were further analyzed using nCounter NanoString. To further identify the best classifier genes, log2 normalized expression values were analyzed using penalized logistic regression. Based on previous literature and regression coefficient values, 15 genes were finally selected whose performance was individually analyzed using receiver operating characteristic curve (ROC) and area under the curve (AUC). Optimal thresholds for these genes were estimated as the values with maximum sensitivity and specificity. Out of 78 examined BCR-ABL1-negative cases, 33(42.30%) BCR-ABL1-negative cases were clustered together with 15 BCR-ABL1-positive cases and were attributed as BCR-ABL1-like ALL cases in principal component analysis. Further, we categorized CRLF2 in two categories; high CRLF2 cases 25/33 (75.75%) & low 8/33 (24.24%) in BCR-ABL1-like ALL cases. JAK2p.R683G mutation was screened in CRLF2 high cases and showed positivity in 19/24 (79.16%) by the Amplification Refractory Mutation (ARMS) PCR. In 25 cases, the average log fold change of -0.80 &-5.83 was seen in P2YR8 & CSF2RA respectively by qPCR. In CRLF2 low expressing cases, the average log fold change of 11 kinase genes showed -0.75 in CENPC, -0.66 FOXP1, -0.16 NUP153, 1.04 RCSD1, 1.50 PAX5, 1.12 FLT3, -5.65 EPOR, -4.03 ILR2B, -3.46 PDGRFB, -7.49 NTRK3 &-2.83 ZNF274 respectively. The average log fold change of IKZF1 in 80 BCR-ABL1-negative cases was found to be 1.07. DISCUSSION & CONCLUSION We have devised a method that includes 15 genes according to AUC/ROC for the detection of BCR-ABL1-like ALL cases, using nCounter NanoString technology for the first time in Indian patients. Furthermore, we are planning to validate this model in future, on 50 BCR-ABL1-positive and 150 BCR-ABL1-negative cases and devise a simple, efficient, cost-effective qPCR method. It is very important to detect BCR-ABL1-like ALL cases to start the desirable TKI therapy & aid in treatment stratification, prognostication, and improve the overall survival of these patients. Figure Disclosures No relevant conflicts of interest to declare.

The Heterogeneous Fusion Gene Landscape in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4081-4081
Author(s):  
Yanara Marincevic-Zuniga ◽  
Johan Dahlberg ◽  
Sara Nilsson ◽  
Amanda Raine ◽  
Jonas Abrahamsson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
Gene Fusion ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Fusion Genes ◽  
Fusion Transcripts ◽  
Pediatric All

Abstract Background: Next generation sequencing allows for the detection of expressed fusion transcripts across the transcriptome and has spurred the discovery of many novel chimeric transcripts in various cancers. Structural chromosomal rearrangements that lead to fusion transcripts are a hallmark of acute lymphoblastic leukemia (ALL) and serve as markers for diagnosis and stratification of pediatric ALL patients into prognostically relevant subgroups. Improved delineation of structural alterations in ALL could provide additional information for prognosis in ALL and for improved stratification of patients into treatment groups. Methods: To identify novel fusion transcripts in primary pediatric ALL cells we performed whole transcriptome sequencing of 134 BCP and T-ALL patient samples collected at diagnosis. Our study include samples from patients with the well-known ALL subtypes t(12;21)ETV6-RUNX1, high hyperdiploid (51-67 chromosomes), t(9;22)BCR-ABL1, 11q23/MLL and dic(9;20), in addition to patients with undefined karyotype or non-recurrent cytogenetic aberrations ("undefined" and "other") (n=58). FusionCatcher was used for the detection of somatic fusion genes, followed by a stringent filtering pipeline including gene fusion validation by Sanger sequencing in order to reduce the number of false positives. Principal component analysis (PCA) of patients with fusion genes was performed using genome wide gene expression levels and DNA methylation levels (Infinium HumanMethylation450 bead array). Results: We identified and validated 60 unique fusion events in almost half of the analyzed patients (n=69). Of the identified fusion genes, 60% have not previously been reported in ALL or other forms of cancer. The majority of the fusion genes were found in a single patient, but 23% were recurrent, including known ALL fusion genes (n=10) and novel fusion genes (n=7). We found that BCP-ALL samples displayed a higher number of validated fusion genes (54%) compared to the T-ALL samples (28%) moreover in BCP-ALL patients with "other" and "undefined" karyotypes, we detected fusion genes in 71% and 61% of the samples, respectively. High hyperdiploid patients had the lowest rate of validated fusion genes (24%) compared to the other well-known subtypes, where we detected subtype-associated fusion genes in 97% of cases. We also identified promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5 and ZNF384 that fused with up to five different genes. Interestingly, PCA revealed molecularly distinct gene expression and DNA methylation signatures associated with these fusion partners. Conclusion: RNA-sequencing of pediatric ALL cells revealed a detailed view of the heterogeneous fusion gene landscape, identifying both known and novel fusion genes. By grouping samples based on recurrent gene fusion partners we are able to find shared gene expression and DNA methylation patterns compared to other subtypes of ALL, suggesting a shared molecular etiology within these distinct subgroups, offering novel insights into the delineation of fusion genes in ALL. Disclosures No relevant conflicts of interest to declare.

Integrative Genome-Wide DNA Methylation and Gene Expression Analysis Reveals Biological and Clinical Insights In Adult Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 852-852
Author(s):  
Huimin Geng ◽  
Donna Neuberg ◽  
Elisabeth Paietta ◽  
Xutao Deng ◽  
Yushan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Inverse Correlation ◽  
Fold Change ◽  
Genome Wide ◽  
Core Set ◽  
Adult Acute Lymphoblastic Leukemia ◽  
Aberrant Dna Methylation

Abstract Abstract 852 Adult acute lymphoblastic leukemia (ALL) is an aggressive disease with <30% long-term survival. This relatively poor outcome can be explained in part by an increased frequency of high-risk molecular subtypes compared to childhood ALL, such as BCR/ABL (20%-40% in adults vs 2%-5% in children). We hypothesized that aberrant epigenetic gene regulation contributes to the pathogenesis and clinical features of adult ALL. We therefore performed genome-wide DNA methylation and gene expression microarray studies of 215 adult patients with B-lineage ALL enrolled in the ECOG E2993 phase III trial. Patients had a median follow-up 4.75 years (3.5 months to 13 years) and median age 39 years (17 to 63 years). BCR/ABL(+) cases (n=83) had a worse overall survival (OS, p=0.08) than BCR/ABL(-) cases (n=132). The smaller difference in survival in this series between BCR/ABL(+) and (-) cases is likely due to the use of imatinib in some of these patients. The HELP microarray assay was used to measure DNA methylation at 50,000 CpGs annotated to ∼22,000 RefSeq promoters. The accuracy of HELP was confirmed by extensive quantitative single locus validation studies. Supervised analysis revealed the presence of a markedly aberrant DNA methylation signature (166 genes) in BCR/ABL(+) ALL with the cutoff values of p<0.001 (FDR<0.002) and log fold change>1, and a differential gene expression profile of 416 genes at p<0.001 (FDR<0.004) and log fold change>1. Integrative analysis of expression and DNA methylation indicated that many of these genes are functionally connected within a gene network centered around IL8, which was over expressed and hypomethylated in BCR/ABL(+) cases, along with IL2RA(CD25), CEBPB, ABL1, ID1, IL15, BCL2L13, CD69, NOV, S100A8 and S100A9. KEGG and BioCarta pathway analysis also showed enrichment for IL8 signaling, NF-kB Activation, B Cell Development and Antigen Presentation pathways, suggesting these cytokine networks might play central and distinct roles in BCR/ABL(+) ALL. To identify a core set of functionally relevant genes, we explored the overlap of the DNA methylation and gene expression signatures. The overlap consisted of 13 genes, among which 11 showed inverse correlation, including CD200, GAB1, HLA-DQA1, HLA-DQB1, IL2RA (CD25), LST1, LTB, NOV, ROB04, S100A9, and CD38. Consistent with previous ECOG findings, CD25 positivity was a more dominant predictor than BCR/ABL, and could further stratify BCR/ABL(+) patients into a favorable (CD25-) and a poor (CD25+) outcome group (OS, p=0.07). When comparing CD25(+) and CD25(-) groups among BCR/ABL(+) cases, we found RhoH and UBE2J1 as the top differentially methylated and IL2RA(CD25) as the top differentially expressed genes, and all three genes showed concordant corresponding changes in expression and methylation. The aberrant DNA methylation signature of MLL/AF4 cases was even more dramatic, with 469 identified as differentially methylated (p<0.001 (FDR<0.015), log fold change>1) and 1108 genes differentially expressed (p<0.001 (FDR<0.009), log fold change>1). Integrated analysis of DNA methylation and expression implicated gene pathways centered around TNFA and MYC, respectively. A core set of 44 genes were identified overlapping between the methylation and gene expression signatures, among which 34 showed inverse correlation, including ANXA5, BRE, CAPG, CEBPA, FAIM, FLT3, FUT4, IGFBP7, IL1R2, ITGA7, ITGAE, MAP1A, MAP7, MRPL33, PARP8, RBKS, SLITRK4 and TFR2 with overexpression and hypomethylation, and BTBD3, CCR6, FYN, GAB1, GYPC, HPS4, IL2RA, KCNK3, LCK, LST1, LTB, PRKCH, QPCT, S100A13, SGPL1 and ZAP70 with underexpression and hypermethylation in MLL/AF4(+) ALL. All signatures were independent of B-ALL differentiation stage. Using univariate Cox Hazard Regression model adjusted by age, WBC, CD25 and BCR/ABL status, we furthermore identified 259 expression and 115 methylation markers which were significantly correlated with patient OS risk (p<0.01). Molecularly or immunophenotypically defined poor prognosis adult B- ALLs thus feature specific aberrant DNA methylation profiles with associated inversely correlated gene expression. These gene sets delineate specific biological functions that may contribute to disease phenotype and offer an opportunity for development of targeted therapy. Aberrantly methylated genes in adult B-ALL correlate with clinical risk independent of other clinical or molecular risk factors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia

2021 ◽  
Vol 22 (5) ◽  
pp. 2683
Author(s):  
Princess D. Rodriguez ◽  
Hana Paculova ◽  
Sophie Kogut ◽  
Jessica Heath ◽  
Hilde Schjerven ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Technological Developments ◽  
Cell Acute Lymphoblastic Leukemia

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

scholarly journals CDX2 gene expression in acute lymphoblastic leukemia

2014 ◽  
Vol 26 (2) ◽  
pp. 55-59 ◽  
Author(s):  
Hanaa H. Arnaoaut ◽  
Doha A. Mokhtar ◽  
Rania M. Samy ◽  
Sahar A. Khames ◽  
Shereen A. Omar
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia

Gene Expression Profiling Data in Lymphoma and Leukemia: Review of the Literature and Extrapolation of Pertinent Clinical Applications

2006 ◽  
Vol 130 (4) ◽  
pp. 483-520 ◽  
Author(s):  
Cherie H. Dunphy
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Clinical Applications

Abstract Context.—Gene expression (GE) analyses using microarrays have become an important part of biomedical and clinical research in hematolymphoid malignancies. However, the methods are time-consuming and costly for routine clinical practice. Objectives.—To review the literature regarding GE data that may provide important information regarding pathogenesis and that may be extrapolated for use in diagnosing and prognosticating lymphomas and leukemias; to present GE findings in Hodgkin and non-Hodgkin lymphomas, acute leukemias, and chronic myeloid leukemia in detail; and to summarize the practical clinical applications in tables that are referenced throughout the text. Data Source.—PubMed was searched for pertinent literature from 1993 to 2005. Conclusions.—Gene expression profiling of lymphomas and leukemias aids in the diagnosis and prognostication of these diseases. The extrapolation of these findings to more timely, efficient, and cost-effective methods, such as flow cytometry and immunohistochemistry, results in better diagnostic tools to manage the diseases. Flow cytometric and immunohistochemical applications of the information gained from GE profiling assist in the management of chronic lymphocytic leukemia, other low-grade B-cell non-Hodgkin lymphomas and leukemias, diffuse large B-cell lymphoma, nodular lymphocyte–predominant Hodgkin lymphoma, and classic Hodgkin lymphoma. For practical clinical use, GE profiling of precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, and acute myeloid leukemia has supported most of the information that has been obtained by cytogenetic and molecular studies (except for the identification of FLT3 mutations for molecular analysis), but extrapolation of the analyses leaves much to be gained based on the GE profiling data.

scholarly journals 583 Exploring the potential of T cell acute lymphoblastic leukemia (T-ALL) for generating leukemia-specific T cell responses that can be therapeutically harnessed with immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A613-A613
Author(s):  
Todd Triplett ◽  
Joshua Rios ◽  
Alexander Somma ◽  
Sarah Church ◽  
Khrystyna North ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Systemic Disease ◽  
Flow Cytometric Analysis ◽  
T Cell Responses ◽  
Lymphoid Tissues ◽  
Cell Responses ◽  
Cell Acute Lymphoblastic Leukemia

BackgroundT cell Acute Lymphoblastic Leukemia (T-ALL) is a devastating malignancy found primarily in pediatric populations. Unfortunately, standard of care for T-ALL has not progressed from highly toxic, intensive regimens of chemotherapy, which fails to cure all patients. Immunotherapies designed to activate patients‘ leukemia-specific T cells may provide a new therapeutic avenue to increase complete response rates, reduce toxicity without the need to engineer (e.g. CAR) cells. However, it is unknown whether T-ALL is capable of being recognized by T cells due given its relatively low mutation-rate. These studies therefore sought to investigate whether signs of leukemia-specific T cell responses are generated by T-ALL. Because T-ALL results in systemic disease and infiltrates multiple lymphoid and non-lymphoid tissues, these studies also determined how the divergent immune contextures of these TMEs impacts T cell responses to T-ALL. From this, we aim to identify immunotherapeutic targets capable of activating T cells across tissues to eradicate leukemia systemically.MethodsPrimary leukemia cells isolated from a spontaneous murine model (LN3 mice) into immune-competent, congenic (CD45.1) recipient mice. Tissues were harvested at distinct stages of disease for analysis by flow cytometry or utilizing NanoString Technologies’ GeoMX Digital Spatial Profiling (DSP) platform.ResultsFlow cytometric analysis of T cells revealed extensive changes in response to T-ALL that included multiple features of exhaustion typically associated with anti-tumor responses as determined by upregulation of co-inhibitory receptors and TOX. This included a surprisingly high-frequency of PD1+ T cells, which was accompanied by PDL1- and PDL2-expressing myeloid cells that likely are restraining these subsets. Importantly, combination immunotherapy with OX40 agonists while inhibiting PD1 resulted in drastically reduced tumor burden and concomitant expansion of proliferating granzyme-expressing CD8 T cells. To gain better insight into T cell responses within distinct organs, we analyzed tissue sections using DSP. This technique enabled us to evaluate T cells in direct contact with leukemia infiltrates compared to T cells in regions without T-ALL, which further revealed an enrichment of activated subsets. Importantly, these studies have provided critical insight needed to better understand how T cells responding to T-ALL diverge between distinct types of tissues.ConclusionsThe results from these studies collectively suggest that T cells are activated by T-ALL and that they can be therapeutically harnessed despite relatively low mutation-rates. Future studies will continue analysis of individual organs and use these results to rationally design combinations of immunotherapies by tailoring to activate T cells in all tissue types.AcknowledgementsSpecial thanks to all the support and analysis from everyone at NanoString, along with financial support provided by a SITC-NanoString DSP Fellowship awarded to Dr. Todd Triplett used for DSP analysis of all frozen tissues in these studies. Salary support for Dr. Triplett and pilot funding was provided by departmental funds via a Cancer Prevention and Research Institute of Texas (CPRIT) Scholar Award (Grant #RR160093; awarded to Dr. Gail Eckhardt).

scholarly journals Evidence for a multipotential stem cell disease in some childhood Philadelphia chromosome-positive acute lymphoblastic leukemia

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1458-1461 ◽  
Author(s):  
N Tachibana ◽  
SC Raimondi ◽  
SJ Lauer ◽  
P Sartain ◽  
LW Dow
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Myelocytic Leukemia ◽  
Complete Clinical Remission ◽  
Ph Chromosome ◽  
Cytogenetic Analyses ◽  
Marrow Cells

Children with Philadelphia chromosome (Ph+) acute lymphoblastic leukemia (ALL) have a poorer prognosis than do most pediatric patients with ALL. Because of this poor prognosis and the presence of the Ph chromosome, we have asked whether or not Ph + ALL involves a multipotential stem cell. We cultured hematopoietic progenitors from two children with Ph+ ALL and examined individual BFU-E and CFU-GM colonies for the Ph chromosome. We studied cells from two patients after 18 to 34 months of first complete clinical remission; direct cytogenetic analyses showed 26% and 13% Ph+ metaphases in these patients' marrow cells. BFU-E colonies were obtained from light density marrow cells cultured in methylcellulose supplemented with erythropoietin and CFU-GM colonies from agar or methylcellulose cultures stimulated with leukocyte feeder layers. Fifty-seven G-banded metaphases were recovered from 33 colonies. Ten metaphases from seven colonies were Ph+. Ph+ metaphases were found in three of 12 and three of five BFU-E colonies from the two patients. One of 16 CFU-GM colonies from one patient had the Ph+ chromosome; analyzable metaphases were not obtained from CFU-GM of the other patient. No colonies contained both Ph+ and Ph- cells. These results indicate that Ph+ ALL with persistence of Ph+ cells in remission involves a multipotential stem cell for erythroid and granulocyte/macrophage as well as lymphoid lineages. Multipotential stem cell involvement in the pathogenesis of some childhood Ph+ ALL suggests similarities to Ph+ chronic myelocytic leukemia and may contribute to the poor prognosis of these patients.

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