Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer's disease.

BACKGROUND: Microglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer's disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs). METHODS: In this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo. RESULTS: We proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-ß plaques in vivo in a model of AD. CONCLUSIONS: This study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

A truncated HIV Tat demonstrates potent and specific latency reversal activity.

A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.

Is the forming of neuronal network activity in human-induced pluripotent stem cells important for the detection of drug-induced seizure risks?

BACKGROUND: Functional network activity is a characteristic for neuronal cells, and the complexity of the network activity represents the necessary substrate to support complex brain functions. Drugs that drastically increase the neuronal network activity may have a potential higher risk for seizures in human. Although there has been some recent considerable progress made using cultures from different types of human-induced pluripotent stem cell (hiPSC) derived neurons, one of the primary limitations is the lack of - or very low - network activity. METHOD: In the present study, we investigated whether the limited neuronal network activity in commercial hiPSC-neurons (CNS.4U®) is capable of detecting drug-induced potential seizure risks. Therefore, we compared the hiPSC-results to those in rat primary neurons with known high neuronal network activity in vitro. RESULTS: Gene expression and electrical activity from in vitro developing neuronal networks were assessed at multiple time-points. Transcriptomes of 7, 28, and 50 days in vitro were analyzed and compared to those from human brain tissues. Data from measurements of electrical activity using multielectrode arrays (MEAs) indicate that neuronal networks matured gradually over time, albeit in hiPSC this developed slower than rat primary cultures. The response of neuronal networks to neuronal active reference drugs modulating glutamatergic, acetylcholinergic and GABAergic pathways could be detected in both hiPSC-neurons and rat primary neurons. However, in comparison, GABAergic responses were limited in hiPSC-neurons. CONCLUSION: Overall, despite a slower network development and lower network activity, CNS.4U® hiPSC-neurons can be used to detect drug induced changes in neuronal network activity, as shown by well-known seizurogenic drugs (affecting e.g., the Glycine receptor and Na+ channel). However, lower sensitivity to GABA antagonists has been observed.

Differential gene expression, irrespective of circulating Hepatitis B Surface Antigen levels, between Inactive Carrier and Nucleos(t)ide Analogue-Treated Hepatitis B Virus patients.

Long-term viremia control in chronic HBV patients occurs either spontaneously in inactive carrier (IC) patients or therapy-induced by nucleos(t)ide analogues (NUC). To better understand the characteristics of viremia control, we evaluated gene expression in purified leukocyte subsets from IC versus NUC-treated patients, and evaluated the putative modulatory effects of hepatitis B surface antigen (HBsAg). We observed that gene expression in NUC-treated patients differed markedly from IC patients, especially in dendritic cells, monocytes, and CD8+ T cells, while serum HBsAg levels had little effect. Nevertheless, based on our findings it cannot be excluded that HBsAg may act locally in the infected liver or preferentially affects HBV-specific cells.

Effects of microglial depletion and TREM2 deficiency on Aß plaque burden and neuritic plaque tau pathology in 5XFAD mice.

Dystrophic neuronal processes harboring neuritic plaque (NP) tau pathology are found in association with Aß plaques in Alzheimer's disease (AD) brain. Microglia are also in proximity to these plaques and microglial gene variants are known risk factors in AD, including loss-of-function variants of TREM2. We have further investigated the role of Aß plaque-associated microglia in 5XFAD mice in which NP tau pathology forms after intracerebral injection of AD brain-derived pathologic tau (AD-tau), focusing on the consequences of reduced TREM2 expression and microglial depletion after treatment with the colony-stimulating factor 1 (CSFR1) inhibitor, PLX3397. Young 5XFAD mice treated with PLX3397 had a large reduction of brain microglia, including cortical plaque-associated microglia, with a significant reduction of Aß plaque burden in the cortex. A corresponding decrease in cortical APP-positive dystrophic processes and NP tau pathology were observed after intracerebral AD-tau injection in the PLX3397-treated 5XFAD mice. Consistent with prior reports, 5XFAD × TREM2-/- mice showed a significant reduction of plaque-associated microglial, whereas 5XFAD × TREM2+/- mice had significantly more plaque-associated microglia than 5XFAD × TREM2-/- mice. Nonetheless, AD-tau injected 5XFAD × TREM2+/- mice showed greatly increased AT8-positive NP tau relative to 5XFAD × TREM2+/+ mice. Expression profiling revealed that 5XFAD × TREM2+/- mice had a disease-associated microglial (DAM) gene expression profile in the brain that was generally intermediate between 5XFAD × TREM2+/+ and 5XFAD × TREM2-/- mice. Microarray analysis revealed significant differences in cortical and hippocampal gene expression between AD-tau injected 5XFAD × TREM2+/- and 5XFAD × TREM2-/- mice, including pathways linked to microglial function. These data suggest there is not a simple correlation between the extent of microglia plaque interaction and plaque-associated neuritic damage. Moreover, the differences in gene expression and microglial phenotype between TREM2+/- and TREM2-/- mice suggest that the former may better model the single copy TREM2 variants associated with AD risk.

Tales of 1,008 small molecules: phenomic profiling through live-cell imaging in a panel of reporter cell lines.

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC ≥ 0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current study's conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.

Role of Kv7.2/Kv7.3 and M1 muscarinic receptors in the regulation of neuronal excitability in hiPSC-derived neurons.

The Kv7 family of voltage-dependent non-inactivating potassium channels is composed of five members, of which four are expressed in the CNS. Kv7.2, 7.3 and 7.5 are responsible for the M-current, which plays a critical role in the regulation of neuronal excitability. Stimulation of M1 muscarinic acetylcholine receptor, M1 receptor, increases neuronal excitability by suppressing the M-current generated by the Kv7 channel family. The M-current modulation via M1 receptor is well-described in in vitro assays using cell lines and in native rodent tissue. However, this mechanism was not yet reported in human induced pluripotent stem cells (hiPSC) derived neurons. In the present study, we investigated the effects of both agonists and antagonists of Kv7.2/7.3 channel and M1 receptor in hiPSC derived neurons and in primary rat cortical neuronal cells. The role of M1 receptors in the modulation of neuronal excitability could be demonstrated in both rat primary and hiPSC neurons. The M1 receptors agonist, xanomeline, increased neuronal excitability in both rat cortical and the hiPSC neuronal cells. Furthermore, M1 receptor agonist-induced neuronal excitability in vitro was reduced by an agonist of Kv7.2/7.3 in both neuronal cells. These results show that hiPSC derived neurons recreate the modulation of the M-current by the muscarinic receptor in hiPSC neurons similarly to rat native neurons. Thus, hiPSC neurons could be a useful human-based cell assay for characterization of drugs that affect neuronal excitability and/or induce seizure activity by modulation of M1 receptors or inhibition of Kv7 channels.

Genetically Engineered iPSC-Derived FTDP-17 MAPT Neurons Display Mutation-Specific Neurodegenerative and Neurodevelopmental Phenotypes.

Tauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND AND PURPOSE: In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. EXPERIMENTAL APPROACH: Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. KEY RESULTS: Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. CONCLUSION AND IMPLICATIONS: We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. LINKED ARTICLES: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System.

Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro.

Functional and Transcriptional Characterization of Histone Deacetylase Inhibitor-Mediated Cardiac Adverse Effects in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

UNLABELLED: Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile. SIGNIFICANCE: Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various cardiac adverse events such as hearth rhythm disorders. This study found that human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can predict cardiac adverse events in patients caused by HDAC inhibitors. Furthermore, transcriptional changes at the level of gene expression supported the effects on the beating properties of hiPS-CMs and highlight targets that might cause these cardiac adverse effects. hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and to support development of safer HDAC inhibitors.

MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells.

Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.

Integrating High-Dimensional Transcriptomics and Image Analysis Tools into Early Safety Screening: Proof of Concept for a New Early Drug Development Strategy.

During drug discovery and development, the early identification of adverse effects is expected to reduce costly late-stage failures of candidate drugs. As risk/safety assessment takes place rather late during the development process and due to the limited ability of animal models to predict the human situation, modern unbiased high-dimensional biology readouts are sought, such as molecular signatures predictive for in vivo response using high-throughput cell-based assays. In this theoretical proof of concept, we provide findings of an in-depth exploration of a single chemical core structure. Via transcriptional profiling, we identified a subset of close analogues that commonly downregulate multiple tubulin genes across cellular contexts, suggesting possible spindle poison effects. Confirmation via a qualified toxicity assay (in vitro micronucleus test) and the identification of a characteristic aggregate-formation phenotype via exploratory high-content imaging validated the initial findings. SAR analysis triggered the synthesis of a new set of compounds and allowed us to extend the series showing the genotoxic effect. We demonstrate the potential to flag toxicity issues by utilizing data from exploratory experiments that are typically generated for target evaluation purposes during early drug discovery. We share our thoughts on how this approach may be incorporated into drug development strategies.

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment.

PURPOSE: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. EXPERIMENTAL DESIGN: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALK(F1174L) or ALK(R1275Q) hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALK(F1174L) double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. RESULTS: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALK(F1174L) and ALK(R1275Q) regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. CONCLUSIONS: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies.

Microarray profiling of DNA extracted from FFPE tissues using SNP 6.0 Affymetrix platform.

High-density oligonucleotide microarrays are commonly used for GWAS studies as well as for tumor genome alteration identifications. The recent Affymetrix Genome-Wide SNP 6.0 microarray generation has two major advantages: (1) showing high genome coverage and (2) starting with very small amount of DNA material. The hybridization protocol needs to be standardized and highly reproducible, as DNA is first digested by restriction enzymes and then PCR-amplified to reduce genome complexity. Especially the restriction digestion step is highly sensitive to degradation of the initial material. The stronger the sample is degraded, the lower the number of restriction sites still present in the genome, and hence the less-efficient amplification step.Paraffin-embedded material generally only allows to extract partially degraded DNA, and therefore is difficult to analyze using SNP array technology. We and others (Jacobs et al., Cancer Res 67:2544-2551, 2007; Tuefferd et al., Genes Chromosomes Cancer 47:957-964, 2008) have shown that target preparation protocol can be adjusted to improve hybridization performances. The final in silico data analysis procedure should be modified accordingly to extract most of the biological information from the signal measured. By optimizing these crucial steps, it is possible to use Affymetrix SNP array 6.0 -technology in the context of genome variation, even for FFPE partially degraded material. This opens a lot of potential for large retrospective series of samples.

The ontogeny of drug metabolizing enzymes and transporters in the rat.

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals. The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1-3 and 6, Bsep and Bcrp, Oct1-2, Oat1-3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague-Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR. There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression. Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood-brain barrier. Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.

Genome-wide copy number alterations detection in fresh frozen and matched FFPE samples using SNP 6.0 arrays.

SNP arrays offer the opportunity to get a genome-wide view on copy number alterations and are increasingly used in oncology. DNA from formalin-fixed paraffin-embedded material (FFPE) is partially degraded which limits the application of those technologies for retrospective studies. We present the use of Affymetrix GeneChip SNP6.0 for identification of copy number alterations in fresh frozen (FF) and matched FFPE samples. Fifteen pairs of adenocarcinomas with both frozen and FFPE embedded material were analyzed. We present an optimization of the sample preparation and show the importance of correcting the measured intensities for fragment length and GC-content when using FFPE samples. The absence of GC content correction results in a chromosome specific "wave pattern" which may lead to the misclassification of genomic regions as being altered. The highest concordance between FFPE and matched FF were found in samples with the highest call rates. Nineteen of the 23 high level amplifications (83%) seen using FF samples were also detected in the corresponding FFPE material. For limiting the rate of "false positive" alterations, we have chosen a conservative False Discovery Rate (FDR). We observed better results using SNP probes than CNV probes for copy number analysis of FFPE material. This is the first report on the detection of copy number alterations in FFPE samples using Affymetrix GeneChip SNP6.0.