Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

2012 ◽  
Vol 425 (2) ◽  
pp. 333-339 ◽  
Author(s):  
Eleni Tholouli ◽  
Sarah MacDermott ◽  
Judith Hoyland ◽  
John Liu Yin ◽  
Richard Byers
Keyword(s):  
Gene Expression ◽  
Quantum Dot ◽  
Acute Myeloid Leukaemia ◽  
Gene Expression Profiling ◽  
Myeloid Leukaemia ◽  
Expression Profiling ◽  
In Situ Hybridisation ◽  
Tissue Microarrays ◽  
Acute Myeloid

Multiplex Quantum Dot ISH Measurement of Gene Expression in Tissue Microarrays Identifies HOXA9 and DNMT3A as Markers of Poor Response to Chemotherapy in Patients with Acute Myeloid Leukaemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2530-2530
Author(s):  
Eleni Tholouli ◽  
Sara A MacDermott ◽  
Judith A Hoyland ◽  
Caroline Glennie ◽  
Ric Swindell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multivariate Analysis ◽  
Quantum Dot ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Poor Response ◽  
Tissue Microarrays ◽  
High Expression ◽  
Response To Chemotherapy ◽  
Acute Myeloid

Abstract Microarray-based expression profiling has identified prognostic gene signatures for many cancers and validation is required in clinical samples. However, most clinical material is in the form of formalin fixed and paraffin embedded tissue (FFPET) in which gene expression analysis is problematic. We have developed a generic quantum dot (QD) based multiplexed in-situ hybridization (ISH) method enabling quantitative localization of multiple mRNA targets in FFPET. We expand on our previous work introducing a method for standardization of ISH signal, enabling comparative measurement of gene expression across multiple samples. This was applied to tissue microarrays (TMAs) using archived trephine biopsies from patients with acute myeloid leukaemia (AML) to identify prognostic genes. A total of 15 TMAs were prepared using FFPET samples from 240 patients with AML diagnosed and treated between 1994 and 2005 at Manchester Royal Infirmary (Manchester, UK). For the analysis, 192 patients were included as the remainder either died before, during or immediately after one course of chemotherapy or there was incomplete data collection. The median age was 52 years (range 17–77) and all patients received intensive chemotherapy according to standard UK MRC AML protocols. Three cores were taken from each sample for TMA preparation. A standard was prepared using a cell pellet obtained from whole blood white cells which was embedded, in triplicate, in each TMA. QD-ISH was performed for nine genes recognized to be of prognostic value in AML. Triplex QD-ISH using QD labeled anti-sense cDNA oligonucleotides was performed for the following targets: Bcl2, survivin and XIAP; DNMT1, DNMT3A and DNMT3B; HOXA4, HOXA9 and Meis1. Signal intensity for each gene was measured using spectral imaging. Scrambled sense cDNA oligonucleotides were used to measure the level of background staining for each gene in each core. Background noise was corrected for by dividing expression levels of anti-sense probes by that of the scrambled probe, for both samples and standards. This enabled direct comparison between TMAs as gene expression values of samples were normalized against the standard. The mean expression of each gene was calculated for each patient, divided into quartiles and correlated with clinical outcome data. Statistical analysis was performed using contingency tables, the chi-square test and Mann Whitney-U. Overall survival (OS) and disease free survival (DFS) were displayed using the Kaplan-Meier method and Cox regression was performed for univariate and multivariate analysis. The OS in this cohort of patients was 43% at 5 years with 80% achieving complete remission (CR) after induction chemotherapy. Patient age (<60 years), WCC (<100×109/l) at diagnosis, cytogenetics (good and intermediate risk) and low HOXA4 expression (median 577 [95%CI 325–828]) were all associated with improved OS (p<0.0001; p=0.02; p<0.0001; p=0.013) and DFS (p<0.0001; p=0.013; p<0.0001; p=0.025) on univariate and multivariate analysis. High expression of HOXA9 (median 0.843 [0.145–7.479]; p<0.0001) and DNMT3A (median 1.305 [0.073–5.477]; p=0.04) were associated with failure to achieve CR. High Meis1 expression was found to be of borderline significance for poor response to chemotherapy (median 0.716 [0.051–7.840]; p=0.05). Expression levels of the remaining 5 genes did not show any correlation with CR, DFS or OS. These findings are consistent with recently published data regarding the prognostic significance of various new markers. In line with others we have demonstrated low expression of HOXA4 is an independent good prognostic marker in adult AML. Although high expression of HOXA9, DNMT3A and Meis1 was associated with inferior CR rates in our study, OS and DFS were not adversely affected. This may be related to improvements and more aggressive clinical practice (eg stem cell transplantation) over recent years which can overcome potential deleterious gene effects. These results demonstrate that the application of a standardized, quantitative multiplex QD-ISH can be used for identification of prognostic markers in FFPET samples. The advantages of this method include its application to TMAs which allows high sample throughput, use of archived materials and its transferability across a spectrum of malignancies.

Gene expression profiling of Polycomb, Hox and Meis genes in patients with acute myeloid leukaemia

2008 ◽  
Vol 81 (2) ◽  
pp. 112-122 ◽  
Author(s):  
Lykke Grubach ◽  
Caroline Juhl-Christensen ◽  
Anita Rethmeier ◽  
Lene Hyldahl Olesen ◽  
Anni Aggerholm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukaemia ◽  
Gene Expression Profiling ◽  
Myeloid Leukaemia ◽  
Expression Profiling ◽  
Acute Myeloid

Gene expression profiling in acute myeloid leukaemia (AML)

2009 ◽  
Vol 22 (2) ◽  
pp. 169-180 ◽  
Author(s):  
Ulrike Bacher ◽  
Alexander Kohlmann ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukaemia ◽  
Gene Expression Profiling ◽  
Myeloid Leukaemia ◽  
Expression Profiling ◽  
Acute Myeloid

scholarly journals Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia

2020 ◽  
Vol 191 (5) ◽  
pp. 935-938
Author(s):  
Nya D. Nelson ◽  
Christine M. McMahon ◽  
Farah El‐Sharkawy Navarro ◽  
Craig W. Freyer ◽  
Jacquelyn J. Roth ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Induction Therapy ◽  
In Situ Hybridisation ◽  
Fluorescence In Situ Hybridisation ◽  
Acute Myeloid

scholarly journals Gene expression profiling in normal cytogenetic acute myeloid leukaemia of Malay patient

2021 ◽  
Vol 20 (3) ◽  
pp. 556-562
Author(s):  
Najah Kamarudin ◽  
Imilia Ismail ◽  
Rosline Hassan ◽  
Muhammad Farid Johan ◽  
Marini Ramli
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Expression Profiling ◽  
Medical Science ◽  
Newly Diagnosed ◽  
Significant Difference ◽  
Age Range ◽  
Pan Cancer ◽  
Acute Myeloid

Background: Insight into recent molecular analyses of patients with acute myeloid leukaemia (AML) and a normal cytogenetic have revealed a striking heterogeneity with regard to the presence of acquired changes in gene expression. Nano String technology which is aimed to identify descriptive profiling of normal cytogenetic AML in differentially expressed genes involved in the different pathways in normal cytogenetic AML patients. Materials and method: Blood samples were obtained from eight at diagnosis patients with normal cytogenetics AML, two follow-up patients and two normal healthy controls prior to RNA extractions. RNA gene expression assay was performed using Nano Stringn Counter® Pan Cancer Pathway Panel (Nanostring Technologies, Seattle, Washington). Briefly, 100 ng of each total RNA sample was used as input into then Counter Pan Cancer Pathway Panel sample preparation. Data was extracted using then Counter RCC Collector and raw data output was imported into nSolver v2.6 analysis software for data analysis. Results: The age range was from 13-69 years, with a median age-range of 41 years. We found the most enriched up regulated genes in newly diagnosed normal cytogenetic AML enlisted MPO (7.25 log FC), FLT3 (5.02 log FC), MYCN (4.99 log FC), MYB (4.74 log FC), ITGA9 (4.48 log FC), KIT (4.41 log FC), MCM2 (3.47 log FC), RAD51 (3.40 log FC), CCNA2 (3.37 log FC) and PROM1 (3.24 log FC) which those commonly be found in AML cases. For highly expressed down regulated genes were GZMB (-5.80 log FC), IL8 (-5.79 log FC), TNFRSFSF10C (-5.03 log FC), LEF1 (-4.82 log FC), IL2RB (-4.70 log FC), IL7R (-4.44 log FC), BCL2A1 (-4.15 log FC), RASGRP1 (-4.13 log FC), IL1R2 (-4.00 log FC) and HSPA6 (-3.99 log FC). Discussion and conclusion: Nano String required smaller amounts of starting material, and can perform mRNA expression profiling with digital precision, therefore the results do not require further validation by another method. MPO expression was significantly associated with disease-free survival (DFS). Previous report demonstrated that the groups by the MPO expression in the intermediate cytogenetic risk group showed a significant difference in DFS (p<0.001). A study by Valk et al., (2004) showed FLT3, a hematopoietic growth factor receptor, is the most common molecular abnormality in AML. The presence of such mutations in FLT3 and elevated expression of the transcription factor EVI1 confer a poor prognosis. N-MYC expression levels in AML samples from patients with favorable, intermediate, or unfavorable prognosis were compared with that in CD34+ cells from four healthy bone marrow donors. The most highly down regulated gene in newly diagnosed AML goes to Granzyme B (GZMB) which involved in cytolytic activity that showed high correlation with other transcripts expressed in activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells as well as lymphocyte activation-related gene validating it as a robust and specific metric of active cellular immunity. Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment. Bangladesh Journal of Medical Science Vol.20(3) 2021 p.556-562

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