Repressed Chromatin Drives Leukaemogenesis in Mutant IDH2 Acute Myeloid Leukaemia Via Inhibition of Granulocyte Differentiation and Cell Cycle Progression

Abstract Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets. We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, 'R'). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R. At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation. Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog. Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs. Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. Disclosures Silveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.

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Role of cell cycle regulatory molecules in retinoic acid- and vitamin D3-induced differentiation of acute myeloid leukaemia cells

Cell Proliferation ◽

10.1111/cpr.12100 ◽

2014 ◽

Vol 47 (3) ◽

pp. 200-210 ◽

Cited By ~ 13

Author(s):

X. T. Hu ◽

K. S. Zuckerman

Keyword(s):

Cell Cycle ◽

Retinoic Acid ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Vitamin D3 ◽

Induced Differentiation ◽

Acute Myeloid

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Finding a suitable library size to call variants in RNA-Seq

BMC Bioinformatics ◽

10.1186/s12859-020-03860-4 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Anna Quaglieri ◽

Christoffer Flensburg ◽

Terence P. Speed ◽

Ian J. Majewski

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cancer Biology ◽

Cancer Type ◽

Rna Seq ◽

Library Size ◽

Single Nucleotide ◽

Average Loss ◽

Acute Myeloid

Abstract Background RNA sequencing allows the study of both gene expression changes and transcribed mutations, providing a highly effective way to gain insight into cancer biology. When planning the sequencing of a large cohort of samples, library size is a fundamental factor affecting both the overall cost and the quality of the results. Here we specifically address how overall library size influences the detection of somatic mutations in RNA-seq data in two acute myeloid leukaemia datasets. Results We simulated shallower sequencing depths by downsampling 45 acute myeloid leukaemia samples (100 bp PE) that are part of the Leucegene project, which were originally sequenced at high depth. We compared the sensitivity of six methods of recovering validated mutations on the same samples. The methods compared are a combination of three popular callers (MuTect, VarScan, and VarDict) and two filtering strategies. We observed an incremental loss in sensitivity when simulating libraries of 80M, 50M, 40M, 30M and 20M fragments, with the largest loss detected with less than 30M fragments (below 90%, average loss of 7%). The sensitivity in recovering insertions and deletions varied markedly between callers, with VarDict showing the highest sensitivity (60%). Single nucleotide variant sensitivity is relatively consistent across methods, apart from MuTect, whose default filters need adjustment when using RNA-Seq. We also analysed 136 RNA-Seq samples from the TCGA-LAML cohort (50 bp PE) and assessed the change in sensitivity between the initial libraries (average 59M fragments) and after downsampling to 40M fragments. When considering single nucleotide variants in recurrently mutated myeloid genes we found a comparable performance, with a 6% average loss in sensitivity using 40M fragments. Conclusions Between 30M and 40M 100 bp PE reads are needed to recover 90–95% of the initial variants on recurrently mutated myeloid genes. To extend this result to another cancer type, an exploration of the characteristics of its mutations and gene expression patterns is suggested.

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ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia

Nature ◽

10.1038/nature21687 ◽

2017 ◽

Vol 543 (7644) ◽

pp. 265-269 ◽

Cited By ~ 84

Author(s):

Liling Wan ◽

Hong Wen ◽

Yuanyuan Li ◽

Jie Lyu ◽

Yuanxin Xi ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukaemia ◽

Histone Acetylation ◽

Myeloid Leukaemia ◽

Oncogenic Gene ◽

Acute Myeloid

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Gene Expression Profiling of Polycomb and Polycomb-Regulated Genes in Patients with Acute Myeloid Leukaemia - Significance and Relationship to Other Molecular Aberrations.

Blood ◽

10.1182/blood.v110.11.3165.3165 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3165-3165

Author(s):

Lykke C. Grubach ◽

Caroline Juhl-Christensen ◽

Anita Rethmeier ◽

Lene H. Olesen ◽

Peter Hokland ◽

...

Keyword(s):

Gene Expression ◽

Multidrug Resistance ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Expression Profile ◽

Expression Profiles ◽

The Other ◽

Expression Levels ◽

Healthy Donors ◽

Acute Myeloid

Abstract The Polycomb group (PcG) of genes is important for differentiation and X-chromosome silencing. Recently much attention has been afforded to the role of its aberrant expression in cancer, especially in relation to the inactivation of tumor suppressor genes. We hypothesized that a deregulation in the expression profile may contribute to the development of acute myeloid leukaemia (AML). To address this, we determined the RNA levels by RQ-PCR in diagnostic bone marrow samples from 126 patients and 20 healthy donors to delineate their expression profile of the PcG genes BMI-1, MEL18, SCML2, YY1 and EZH2. To address the interplay with downstream targets of PcG proteins, we also determined the expression of HOXA4, HOXA9 and MEIS1. These data were compared not only to the demographic and clinical data of the patients, but also to a large number of molecular assays already performed in these patients (Olesen LH et al. Br.J. Haematol. 2005 131(4):457–467; Rethmeier et al. Br.J. Haematol. 2006 133(3):276–283.). At first we noticed a striking heterogeneity in the expression profiles of the AML patients (Fig. 1). We also observed that HOXA9, MEIS1, SCML2, YY1, BMI-1 and EZH2 were significantly (p≤0.003) higher expressed in the patients compared to the healthy donors. Moreover, when patients were analyzed according to the three cytogenetic prognostic groups (normal, core-binding factor positive and complex), the expression profile of patients with the t(8,21) aberration was characterized by a significantly decreased expression of HOXA9 and MEIS1 and a higher one of SCML2, YY1 and BMI-1 than AML patients in general (p<0.003). When evaluating the impact of cytogenetic subgrouping, the expression levels of MEL18 and EZH2 significantly (p< 0.025) reflected highest expression in patients with adverse prognosis and lowest expression with patients exhibiting the most favourable prognosis. While the expression levels of the genes in focus did not correlate to course of disease, we observed that a direct relationship between transcript levels of PcG and PcG-related on the one hand and the DNA methyl transferases (DNMT’s), apoptosis and multidrug-resistance genes (p<0.001) on the other. In conclusion, in this study, which is the first to systematically analyze a series of PcG genes and genes regulated by PcG, we failed to demonstrate a correlation to the clinical outcome of patients with AML. On the other hand, our data strongly suggest that these genes might be involved in the leukaemogenic process by virtue of their relations to DNA methylation (DNMT1, DNMT3B), apoptosis (BAX, CASPASE 3) and multidrug resistance (MDR1, MRP1). Figure 1. Expression profiles of PcG or PcG-regulated genes in AML patients and healthy controls. A. Gene expression profile of all 126 AML patients included (black lines) compared to 20 healthy donors. B. Patients with CBF aberrations, t(8,21), n =7, or inv(16), n =12. The expression is calculated as 2−ΔCt *100), where ΔCt = CtTG−CtCG, CtTG is the Ct value of the target gene, and CtCG is the mean Ct value of the two control genes (B2M and ABL). Figure Figure

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2′–Cyano–2′–Deoxyarabinofuranosylcytosine Is Active in Acute Myeloid Leukaemia and Acts in Synergy with Cytarabine.

Blood ◽

10.1182/blood.v114.22.4153.4153 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4153-4153 ◽

Cited By ~ 1

Author(s):

Elisabeth J Walsby ◽

Chiara Ghiggi ◽

Ruth H Mackay ◽

Simon R Green ◽

Steven Knapper ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Lines ◽

Prolonged Exposure ◽

Nucleoside Transporters ◽

Deoxycytidine Kinase ◽

Dna Breaks ◽

Cytotoxic Effects ◽

Acute Myeloid

Abstract Abstract 4153 2′–Cyano–2′–deoxyarabinofuranosylcytosine (CNDAC) is the metabolic product of sapacitabine following hydrolysis of the palmitoyl sidechain from the pyrimidine analog primarily by plasma, gut and liver amidases. CNDAC is in turn phosphorylated into the active triphosphate form (CNDACTP) by deoxycytidine kinase (dCK). CNDACTP is incorporated into DNA resulting in single stranded DNA breaks during replication and inducing cell cycle arrest. Previously the cytotoxic effects of CNDAC have also been associated with intracellular accumulation of CNDAC triphosphate and chain termination. CNDAC and sapacitabine have overlapping cytotoxic effects. Acute myeloid leukaemia (AML) cell lines NB4 and HL-60 had an LD50 of 0.24μM (± 0.24) for CNDAC and 0.23μM (± 0.21) for cytarabine (AraC) following 24 hours treatment. Primary AML blasts isolated from patients at diagnosis (n = 15) had a higher mean LD50 (25.22μM ± 19.41) for CNDAC and AraC (8.09μM ± 8.93). This is thought to be due to the requirement of cells to be actively cycling in order to be susceptible to these agents. CNDAC induces apoptosis in NB4 and HL-60 cell lines with significant increases in the percentage of cells with increased Annexin V/propidium iodide staining at concentrations of 1.0μM and above (P < 0.04) and significant caspase-3 activation at concentrations of 0.1μM and above (P < 0.05). Treatment with CNDAC also results in a significant concentration-dependent accumulation in the G2 phase of the cell cycle after 24 hours in NB4 and HL-60 cells (P = 0.003 and 0.011 respectively). Synergy was observed in the AML cell lines when CNDAC was combined with AraC at a ratio of 2:1 The mean combination index for CNDAC and AraC was 0.67 (± 0.21). The activity of deoxycytidine kinase (dCK) was blocked by the addition of excess deoxycytidine, under these conditions the effects of CNDAC were abrogated (P < 0.05) in NB4 and HL-60 cells suggesting that CNDAC requires phosphorylation by dCK for its activation in the cells. The nucleoside transporters hENT 1 and 2 and hCNT3 transport a range of nucleoside analogues through the cell membrane into cells, the use of hENT inhibitors led to a 2.5 fold increase in the LD50 for CNDAC (P = 0.028) over 48 hours. This prolonged exposure to CNDAC could have resulted in some passive uptake of CNDAC into the cells potentially explaining why the agent retained some cell killing activity. Equivalent results have been obtained with dCK and hENT inhibitors in other cell lines indicating that there is a general requirement for these enzymes for CNDAC activity. Interestingly, when cells are treated with the parent drug sapacitabine in the presence of excess deoxycytidine the cytotoxicity is reduced, but when cells are treated in the presence of hENT inhibitors, sapacitabine's cytotoxicity is improved. This suggests that the presence of the palmitoyl side-chain allows membrane permeability even in the absence of the traditional nucleoside transporters. Disclosures: Green: Cyclacel Ltd: Employment.

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Disulfiram/copper causes ROS levels alteration, cell cycle inhibition, and apoptosis in acute myeloid leukaemia cell lines with modulation in the expression of related genes

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2018.01.109 ◽

2018 ◽

Vol 99 ◽

pp. 561-569 ◽

Cited By ~ 16

Author(s):

Saeed Hassani ◽

Parisa Ghaffari ◽

Bahram Chahardouli ◽

Kamran Alimoghaddam ◽

Ardeshir Ghavamzadeh ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Lines ◽

Leukaemia Cell ◽

Cell Cycle Inhibition ◽

Acute Myeloid Leukaemia Cell ◽

Acute Myeloid

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An Unusual Expression: The Tumour Antigen SSX2IP Is Preferentially Expressed on the Surface of Acute Myeloid Leukaemia Cells during Early Mitosis.

Blood ◽

10.1182/blood.v108.11.4305.4305 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4305-4305

Author(s):

Frances A.K. Denniss ◽

Nicola R. Hardwick ◽

Ghulam J. Mufti ◽

Barbara A. Guinn

Keyword(s):

Cell Cycle ◽

Confocal Microscopy ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

K562 Cells ◽

Serum Starvation ◽

Normal Donor ◽

Normal Tissues ◽

Acute Myeloid ◽

Early Mitosis

Abstract The SSX family includes five functionally active and highly homologous members which are only expressed in the thyroid and testis in normal tissues. However these genes are expressed in several cancers including melanoma. Of note SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. Previous investigators used yeast two-hybrid systems to elucidate the SSX2 interacting protein (SSX2IP) through its association with SSX2. SSX2IP has been shown to be ubiquitously expressed in many normal tissues. We found SSX2IP through the immunoscreening of a testis cDNA library with pooled presentation M4 and M5 acute myeloid leukaemia (AML) sera. SSX2IP was found to be preferentially recognized by 62% of acute myeloid leukaemia (AML) sera (n=22) compared to 21% of normal donor sera (n=20). By RT-PCR SSX2IP was found to be expressed by four of 12 (30%) AML patient samples at presentation but none of eight normal donor haematopoietic samples (bone marrow and peripheral blood). By immunocytochemistry (ICC) we found SSX2IP protein expression in three of six myeloid leukaemia cell lines (K562, P39 and HL60), three of nine AML samples at presentation but none of three normal donor haematopoietic samples (peripheral blood). By confocal microscopy we noticed SSX2IP was predominantly expressed on the surface of K562 cells in early mitosis. We then synchronized K562 cells (as demonstrated by flow cytometry) using either serum starvation (0.1% fetal calf serum for 5 days) in the G0 phase or using 0.3mM hydroxyurea for 3 days at the G1/S interface of the cell cycle. On release back into the cell cycle (as shown by cell counts and flow cytometry) we observed a 3hr period, post-synchronisation, at 24–26 hours for serum starvation or 14–17 hours for hydroxyurea treatment during which time SSX2IP expression peaked, as detected by ICC and confocal microscopy. We transfected K562 cells with the cell surface expressed costimulatory molecule CD80 and demonstrated that SSX2IP was almost entirely restricted in its expression to the cell surface, mimicing the expression pattern of CD80. SSX2IP has a murine counterpart with 87% amino acid sequence similarity called ADIP. ADIP is a novel Afadin- and alpha-actinin-binding protein which localises at cell-cell adherens junctions. ADIP is at least partly involved in the physical association of nectins and cadherins and has more recently been implied to play a role in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex. The mouse protein ADIP has been shown to interact with AF6, the human equivalent of which is involved in the human MLL-AF6 translocation in AML and acute lymphocytic leukaemia. However AF6 was not found to colocalise with SSX2IP in K562 cells as determined by confocal microscopy in our studies although both were expressed. P39 cells only expressed detectable levels of SSX2IP and not AF6. We have demonstrated a novel expression of the tumour antigen SSX2IP on the surface of AML cells in early mitosis. As such SSX2IP may provide a novel antibody target for the depletion of the AML cells from harvests prior to autologous transplant, when no suitable allogeneic donor can be found.

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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 ◽

10.1182/blood.v112.11.2530.2530 ◽

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.

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