Microgenomics of Ameloblastoma

RESEARCH REPORTS Biological P. DeVilliers1, C. Suggs2, D. Simmons2, V. Murrah3, and J.T. Wright2* Microgenomics of 1Department of Pathology, University of Alabama at Ameloblastoma Birmingham; 2Department of Pediatric Dentistry, CB #7450, and 3Department of Oral Pathology, University of North Carolina at Chapel Hill, NC 27599, USA; *corresponding author, [email protected] J Dent Res 90(4):463-469, 2011 ABSTRACT INTRODUCTION Gene expression profiles of human ameloblastoma microdissected cells were characterized with the meloblastoma is an aggressive tumor of odontogenic epithelial origin, purpose of identifying genes and their protein Awith devastating morbidity if left untreated, due to its unlimited growth products that could be targeted as diagnostic and potential. It is characterized by a high rate of recurrence (up to 70%, depend- prognostic markers as well as for potential thera- ing on the treatment modality) and potential to undergo malignant transfor- peutic interventions. Five formalin-fixed, decalci- mation and to metastasize (up to 2% of cases). Malignant ameloblastoma is fied, paraffin-embedded samples of ameloblastoma defined as a histologically benign-appearing ameloblastoma with metastasis were subjected to laser capture microdissection, (Goldenberg et al., 2004; Cardoso et al., 2009). Surgical resection is the treat- linear mRNA amplification, and hybridization to ment of choice, which can cause further morbidity to the craniofacial com- oligonucleotide human 41,000 RNA arrays and plex, with loss of function and esthetics (Olasoji and Enwere, 2003). compared with universal human reference RNA, Gene expression profiling of tumor cell populations has advanced our to determine the gene expression signature. understanding of the pathogenesis of human tumors (Naderi et al., 2004). The Assessment of the data by Significance Analysis of identification of specific genes or groups of genes that are deregulated, and Microarrays (SAM) and cluster analysis showed thus potentially playing a role in initiation, proliferation, and morphological that 38 genes were highly expressed (two-fold determination of the tumor, could provide new diagnostic and therapeutic increase) in all samples, while 41 genes were approaches. Laser capture microdissection (LCM) allows for the isolation of underexpressed (two-fold reduction). Elements of single cells, with no detrimental effect on PCR amplification, and can be the sonic hedgehog pathway and Wingless type coupled to high-density oligonucleotide arrays to obtain expression profiles MMTV integration site family were validated by from cell populations of complex tumors (Luzzi et al., 2003). immunohistochemistry. We have identified the Knowledge of early events leading to and promoting tumorigenesis in expression of multiple genes and protein products ameloblastoma, and odontogenic tumors in general, remains limited, partly that could serve as potential diagnostic, prognos- because studies directed at identifying molecular factors and events that initi- tic, and therapeutic targets. ate and drive tumorigenesis are inconclusive. There have been two microarray studies of ameloblastoma (Heikinheimo et al., 2002; Carinci et al., 2004), one of them using cDNA microarrays containing 19,000 human cDNAs, and, KEY WORDS: ameloblastoma, gene expression more recently, an oligonucleotide analysis of whole-tissue ameloblastoma in profile, human, microarray. a 34,000 human RNA microarray (Lim et al., 2006). The objectives of our study were: (a) to characterize the gene expression profile of laser-captured microdissected ameloblastoma cells using oligonucleotide microarray tech- nology; (b) to compare the gene expression profile with universal human reference RNA; and (c) to identify genes or gene products that may have diagnostic, prognostic, or therapeutic potential. This study is the first to suc- cessfully perform microgenomics on formaldehyde-fixed, decalcified, and paraffin-embedded odontogenic tumor tissue. DOI: 10.1177/0022034510391791 MateriaLS & METHODS Received March 27, 2010; Last revision September 9, 2010; Case Selection Accepted September 9, 2010 This study was approved by the Institutional Review Board of the University A supplemental appendix to this article is published elec- of North Carolina at Chapel Hill (UNC). Ameloblastoma samples were tronically only at http://jdr.sagepub.com/supplemental. retrieved from the paraffin block archives of the departments of Surgical © International & American Associations for Dental Research Pathology and Oral and Maxillofacial Pathology at UNC; the diagnosis was 463 464 DeVilliers et al. J Dent Res 90(4) 2011 confirmed, based on the 2005 WHO Histologic Classification of cRNA from a single labeling reaction was used in all 5 hybrid- Odontogenic Tumors (Barnes et al., 2005). Five ameloblastoma izations to minimize technical variance (Novoradovskaya et al., samples were selected (1 unicystic, 1 recurrent tumor, and 3 2004). Precautions were taken to avoid small-size aRNA hybrid- solid/multicystic), all of which were originally decalcified in ization to gene oligos, by removing free nucleotides as previ- Richard Allan Scientifics’ decalcifying solution containing ously described (Coudry et al., 2007). water, hydrochloric acid, EDTA, tetrasodium tartrate, potassium sodium, and tartrate. Data Analysis The hybridized arrays were scanned at 488 nm in a G2500 Scanner Laser Capture Microdissection (Agilent). A normalization factor was estimated from ratios of the Serial 7-µm-thick sections were cut from each block in an medians. The log2 ratios for all the targets on the array were cali- RNAse-free lab environment for a total of 75 slides (15 from brated by the normalization factor, and log2 ratios outside the each block). In addition, tissue scrapes from each block of the 5 99.7% confidence interval were determined as significantly samples were processed for the assessment of total RNA fidel- changed in the ameloblastoma cells. Cluster analysis of microarray ity, following the protocol of Optimum™ FFPE RNA Isolation data and statistical analysis were performed on the Agilent Kit (Ambion, Inc., Austin, TX, USA) optimized for RT-PCR GeneSpring Analysis Platform™ as well as by Significance analysis. For the purposes of the present study, tissue blocks that Analysis of Microarrays (SAM) (Larsson et al., 2005). were one year old or less were selected, since they provide bet- ter fidelity than older blocks. Once the quantity and quality of Validation of Protein Expression total RNA were established, the ameloblastoma sections were subjected to laser capture microdissection with the AutoPix™ Immunohistochemistry was performed on sections of the amelo- with infrared diode laser (Arcturus Engineering, Santa Clara, blastomas used for LCM and microarray analysis as well as on CA, USA). The cells from the basal cell layer of the ameloblas- additional samples from the Pathology laboratory archives. toma samples, which have a uniform histologic architecture, Standard protocols were used to evaluate expression and local- were microdissected. For correct identification of the cells of ization of sonic hedgehog pathway members (SHH, Gli, Ptch, interest, H&E sections were scanned in the Aperio ScanScope™ and SMO) (R&D Systems Inc., Minneapolis, MN, USA) and system and viewing software (Vista, CA, USA). Scanned images WNT10 (ProSci Incorporated, Poway, CA, USA). Positive and were viewed simultaneously with the LCM procedure. negative controls were included for each antibody. Microdissected cells were captured with a CapSure™ cap (Arcturus Engineering), and the cells were placed in RNA RESULTS extraction buffer. Total RNA was isolated from the microdissected cells with the PicoPure RNA Isolation kit (Arcturus Laser capture microdissection yielded an estimated 500 cells Bioscience, Santa Clara, CA, USA). The quality and yield of per sample (Table 1) and approximately 10-15 ng total RNA. total RNA were assessed on an Agilent Bioanalyzer 2100 The 260/280 ratio for the 5 samples was between 2.12 and 2.14. (Agilent Technologies, Palo Alto, CA, USA). Anti-sense RNA generated from the ameloblastoma samples had an average size of 500 nucleotides. The microarray hybridization was performed with Agilent microarrays including more RNA Amplification than 41,000 human genes and transcripts; the raw data have The TargetAmp™ 2-Round Aminoallyl-aRNA Amplification been submitted to the Gene Expression Omnibus (GEO) micro- Kit (Epicentre Biotechnologies, Madison, WI, USA) was used. array database (accession number GPL1708). We considered This protocol is based on an improved “Eberwine” (Van Gelder genes based on Bonferroni’s inequality, which limits the chance et al., 1990) linear amplification process. In parallel with the of a false-positive result to be no more than by multiplying each laser-captured and microdissected samples, 2 additional samples nominal p-value by N (with a maximum of 1). Hierarchical were included in the amplification, a positive control (human cluster analysis of genes is depicted in Fig. 1. In total, 21 genes universal RNA) and a negative control (no RNA template). were highly expressed, with a two-fold increase over the reference RNA in all 5 samples (Table 2), while 41 genes were underexpressed (two-fold) in 4 out of 5 samples; among these Oligonucleotide Microarray Analysis was RAB31, a member of the RAS oncogene family (Entrez Whole Human Genome Oligo Microarrays (G4112A) (Agilent Gene ID 11031) T-cell acute lymphocytic leukemia 1 (Entrez Technologies

Load more