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Neuro-Oncology Advance Access published online on October 15, 2009
Neuro-Oncology, doi:10.1093/neuonc/nop001
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© The Author(s) 2009. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Genome-wide profiling using single-nucleotide polymorphism arrays identifies novel chromosomal imbalances in pediatric glioblastomas

Hui-Qi Qu*, Karine Jacob*, Sarah Fatet, Bing Ge, David Barnett, Olivier Delattre, Damien Faury, Alexandre Montpetit, Lauren Solomon, Peter Hauser, Miklos Garami, Laszlo Bognar, Zoltan Hansely, Robert Mio, Jean-Pierre Farmer, Steffen Albrecht, Constantin Polychronakos, Cynthia Hawkins and Nada Jabado

Departments of Paediatrics and Human Genetics, Montreal Children's Hospital (H.-Q.Q., C.P.); Division of Hemato-Oncology, Departments of Paediatrics and Human Genetics, Montreal Children's Hospital Research Institute (K.J., D.B., D.F., N.J.); Molecular Diagnostic Laboratory, Department of Genetics, Montreal Children's Hospital Research Institute (R.M.); Division of Neurosurgery, Montreal Children's Hospital (J.-P.F.); and Department of Pathology, Montreal Children's Hospital, McGill University Health Centre, Montreal, Canada (S.A.); INSERM U 830, Institut Curie, Paris, France (S.F., O.D.); McGill University and Genome Quebec Innovation Centre, Montreal, Canada (B.G., A.M.); Division of Pathology, The Hospital for Sick Children, Toronto, Canada (L.S., C.H.); 2nd Department of Paediatrics, Faculty of Medicine, Semmelweis University, Budapest, Hungary (P.H., M.G.); Division of Neuro-Surgery, Division of Pathology, National Institute of Neurosurgery, Budapest, Hungary (L.B.); Department of Neurosurgery, Medical and Health Science Centre, University of Debrecen, Debrecen, Hungary (Z.H.)

Corresponding Author: Constantin Polychronakos, M.D., Montreal Children's Hospital, 2300 Tupper, Montreal, Que., Canada, H3H 1P3 (constantin.polychronakos{at}mcgill.ca).


   Abstract
Top
 Notes
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgments
 References
 
Available data on genetic events in pediatric grade IV astrocytomas (glioblastoma [pGBM]) are scarce. This has traditionally been a major impediment in understanding the pathogenesis of this tumor and in developing ways for more effective management. Our aim is to chart DNA copy number aberrations (CNAs) and get insight into genetic pathways involved in pGBM. Using the Illumina Infinium Human-1 bead-chip-array (100K single-nucleotide polymorphisms [SNPs]), we genotyped 18 pediatric and 6 adult GBMs. Results were compared to BAC-array profiles harvested on 16 of the same pGBM, to an independent data set of 9 pediatric high-grade astrocytomas (HGAs) analyzed on Affymetrix 250K-SNP arrays, and to existing data sets on HGAs. CNAs were additionally validated by real-time qPCR in a set of genes in pGBM. Our results identify with nonrandom clustering of CNAs in several novel, previously not reported, genomic regions, suggesting that alterations in tumor suppressors and genes involved in the regulation of RNA processing and the cell cycle are major events in the pathogenesis of pGBM. Most regions were distinct from CNAs in aGBMs and show an unexpectedly low frequency of genetic amplification and homozygous deletions and a high frequency of loss of heterozygosity for a high-grade I rapidly dividing tumor. This first, complete, high-resolution profiling of the tumor cell genome fills an important gap in studies on pGBM. It ultimately guides the mapping of oncogenic networks unique to pGBM, identification of the related therapeutic predictors and targets, and development of more effective therapies. It further shows that, despite commonalities in a few CNAs, pGBM and aGBMs are two different diseases.

Keywords: pediatric high-grade astrocytomas, brain tumors, SNP arrays, LOH

Received June 11, 2008; Accepted May 6, 2009


   Introduction
Top
 Notes
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgments
 References
 
Brain tumors are the largest group of solid neoplasms in children and are currently the leading cause of cancer-related mortality and morbidity in the pediatric years. Pediatric high-grade astrocytomas (HGAs), including grade IV astrocytomas (glioblastoma [GBM]) account for 15% of all brain neoplasms in children,1 have a dismal prognosis despite aggressive management, and have a high morbidity linked to current treatments. Although their diagnosis still relies mainly on pathology, little is known about the molecular mechanisms underlying their development.

Physical changes in the DNA copy number of particular genomic regions, manifesting as loss of heterozygosity (LOH) or epigenetic changes such as loss of imprinting, have been shown to promote cancer formation and progression. In particular, LOH has been extensively used in the discovery of various tumor suppressor genes, including Rb1 and p53. A precise characterization of these genomic alterations in a given tumor may therefore increase our understanding of the oncogenic events promoting its growth and may provide more accurate means for its classification. However, most of the work done to date on nonrandom LOH in solid tumors has been based on searches for a small number of candidate loci. Comparative genomic hybridization (CGH), which identifies chromosomal segments with copy number changes (gain or loss),2–4 has an effective resolution that varies from ~20 Mb to ~100 kb and a far from optimal capacity in detecting chromosomal deletions, especially LOHs.5 More recently, a major breakthrough for the precise mapping of genomic aberrations in cancers has been made by the completion of the human genome sequence.6 Concurrently, high-density arrays for genotyping single-nucleotide polymorphisms (SNPs)6,7 have been made available (reviewed in Ref. 8). These arrays combine the genome-wide potential of hybridization arrays with higher-resolution (by an order of magnitude) detection of LOH, DNA copy number alterations, and other chromosomal aberrations compared to CGH.

Studies using these high-resolution arrays can be applied to unravel, with high precision, genomic imbalances in HGAs while providing an additional tool to classify these tumors. They have been used in adult HGAs (aHGAs, mainly high-resolution CGH arrays), providing more accurate tools for prognosis and the identification of therapeutic targets in this tumor.9–15 Genomic alterations involved in pHGAs are largely unknown, with only a few published studies using lower-resolution arrays.16–18 Moreover, pHGAs have distinct molecular profiles from aHGAs, indicating that results from adult studies cannot be applied to children.19,20 To identify genetic loci specifically involved in pHGAs, we analyzed 18 pediatric GBMs and 6 aHGAs using high-resolution Illumina 100K SNP arrays and an independent data set of 9 pHGAs using the Affymetrix 250K SNP arrays platform.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Supplementary Material
 Acknowledgments
 References
 
Sample Characteristics and Pathological Review
All samples were obtained under a protocol approved by the hospitals' institutional review boards and independently reviewed by senior pediatric neuropathologists (S.A. and C.H.) to ensure consistent classification based on contemporary guidelines from the World Health Organization. Eighteen pGBMs (average 10.3 ± 5.2 years) and 6 aGBMs (average 58.8 ± 18.2 years) were analyzed using the Illumina platform. Snap-frozen sections of areas immediately adjacent to the regions used for pathological diagnosis were provided and contained vascular tissue ranging from <10% to 30% of the full section. Normal tissue was not available for any of the samples. Tissues were obtained from the Pediatric Cooperative Human Tissue Network, the London/Ontario Tumor Bank, and from collaborators in Montreal and Toronto (Canada) and Hungary. Clinical findings of patients are provided in Table 1. Adult samples have previously been reported for gene expression analysis of pGBM.19


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  		Table 1. Clinical characteristics of GBM samples from adult and pediatric patients analyzed using the Illumina 100K SNP array

 
DNA Extraction and Hybridization
DNA from frozen tumors was extracted as described previously.16 DNA (250 ng) from 25 samples was assayed with Infinium I whole genome genotyping, according to the recommendations of the manufacturer (Illumina, San Diego, CA, USA). The Illumina Sentrix Human-1 Genotyping BeadChip covers 109,365 gene-centric SNPs over the genome with a mean intermarker distance of 26 kb (13 kb median spacing). Image intensities were extracted using Illumina's BeadScan software. Data for each BeadChip were self-normalized using information contained within the array. For Affymetrix 250K SNP chips, 250 ng of tumor DNA was processed according to the manufacturer (Affymetrix, Inc., Santa Clara, CA, USA) and data were analyzed as described elsewhere.16 Genome profiles were created using the Illumina Genome Viewer and Chromosome Browser, which allow viewing, identifying, and manually annotating the chromosomal aberrations. For the initial analysis, the output by BeadStudio software was used to interpret the nature of the aberration. These variables include the normalized intensity of hybridization, expressed as its base-2 logarithm (log2 R), and the allelic ratio, expressed as the ratio of one allele over the sum of both alleles.21 Visualization of copy number and LOH in normal tissues is performed by plotting the log2R and the allele ratios across the genome. This algorithm, however, assumes single-lineage DNA and could not be used alone with these tumor tissue samples, which are unavoidably mixed with normal vascular tissue. Amplifications were therefore detected if there was an increase in the log2 R value (≥1, corresponding to tetraploid copy number ie ≥4). The SNPs with log2 R ≤ 2 in tumor tissues, but with normal log2 R in all CEU (CEPH [Centre de l'Etude du Polymorphisme Humain] European) DNA samples analyzed in the HapMap project were taken as homozygous deletions.21 If log2 R ≤ 2 in the CEU samples, the marker was discarded as a failed assay. To identify heterozygous deletions, we estimated the LOH score using the genotypes of all SNPs in a sequence window of 1 Mb, around any marker. This score is the base-10 logarithm of the ratio of the probability of observing the genotypes of all SNPs in the presence of LOH over the probability of observing the same genotype in the absence of LOH. It is based on the allele frequencies observed in the European-ancestry subjects used in the HapMap project (CEU set) and assigned to the marker in the middle of the window.22 We considered that a score of more than 10 is diagnostic of LOH. Four DNA samples from the CEU set were used as the normal control in the same assay.

BAC Arrays
The CGH-array analysis of tumor samples was performed as described previously.23 The V5S2 genomic array uses 3913 BAC markers from across the human genome with a mean resolution of 1 Mb. Normalization and data analyses were done with VAMP software (Bioinformatics Department, Curie Institute, Paris, France).23 Fluorescence ratios exceeding 1.2 were considered indicative of gains of chromosomal material, whereas losses were indicated by ratios lower than 0.8.

Validation of Copy Number Changes by Quantitative Real-Time PCR
Quantitative real-time PCR was done on an ABI-Prism 7000 sequence detector (Applied Biosystems) using a SYBR Green kit (Applied Biosystems). The target locus from each tumor DNA was normalized to the reference, Line-1 as previously described.16

Statistical Analysis
To be considered causative, a somatic copy-number change must be non-random, that is, recur at the same locus in different tumors more frequently than expected by chance alone. To assess the statistical significance of the occurrence of such overlaps in different tumors, their number was compared to a distribution generated by cyclically permuting the position of the LOH regions on each chromosome of each tumor 10,000 times. The false-detection rate (FDR) was calculated as the proportion of overlaps in any given number of tumors that would be expected to occur by chance alone (see Supplementary Material, Fig. S1).


   Results
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Supplementary Material
 Acknowledgments
 References
 
Genomic Alterations Identified in BAC and in SNP Arrays in 16 pGBM Samples
We used a previously validated BAC-array platform, which has provided numerous results in tumor LOH studies,23 and investigated the concordance of copy number aberration (CNA) detection between this platform and the Illumina 100K SNP arrays in 16 pHGAs (P1–P16; Table 1). BAC-array results validated data obtained by SNP arrays (see Supplementary Material, Table S1). However, because of the higher resolution of the SNP arrays, a higher number of alterations, left undetected by the BAC arrays, were uncovered. This is in keeping with previous findings on the higher sensitivity of SNP technology for the detection of CNAs.24

CNAs in pGBM and aGBMs Detected Using Illumina SNP Arrays
Analysis of the data set of 18 pGBMs and 6 aGBMs using the Illumina SNP arrays showed that heterozygous deletions, detected as LOH, are common phenomena mostly in pGBM and were seen for each chromosome in a number of samples (Tables 1GoGo–4; see also Supplementary Material, Table S1). Despite common LOH regions between pGBM and aGBM such as 9p24.3-9p13.1 and 17p13.3, there was little concordance between the CNAs detected in both settings, with most LOHs (Tables 3 and 4), amplifications (Table 5), and homozygous deletions (Table 6) found in children being different from the ones encountered in adults (Tables 2GoGoGo–6). Unsupervised hierarchical clustering analysis, using as input data regions with CNAs in at least one sample, clustered the aGBM samples separately from most pGBMs and the normal brain, confirming the presence of distinct molecular imbalances specific to pGBM.19 Interestingly, it also separated primary from secondary aGBMs (Fig. 1).


Figure 1
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  		Fig. 1. Unsupervised hierarchical clustering of 24 GBMs (18 pediatric and 6 adult) and 1 control normal brain (CB). We used statistically significant CNAs present in at least one sample as data input (Welsch t-test, p < 0.001) and separated our data set accordingly. Pediatric (P) samples clustered separately from adult samples (A) and from the CB. DNA from one pediatric sample P1 was extracted from two separate parts of the same tissue sample. DNA from both extractions was independently subjected to whole-genome amplification prior to analysis on the 100K SNP array platform. P1a and P1b clustered similarly showing the reproducibility of the analysis of genome profiling. Primary adult glioblastoma (Ap) clustered separately from secondary adult glioblastoma (As).

 


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  		Table 2. CNAs identified in 18 pGBMs and aGBMs using the 100K SNP Illumina array

 


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  		Table 3. LOH analysis showing distinct imbalances between pGBM and aGBM

 


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  		Table 4. Identification of statistically overrepresented gene ontologies (GOs) in the genes harbored in LOH regions analyzed using the GOstat tool (http://gostat.wehi.edu.au/cgi-bin/goStat.pl)

 


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  		Table 5. Amplifications in pGBM and aGBM

 


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  		Table 6. Homozygous deletions in pGBM and aGBM

 
Analysis of Recurrent Regional LOH in GBMs
Several overlapping regions of LOH in tumors were identified in pGBM samples (Tables 3 and 4). By permutation analysis, the 95% confidence interval of the FDR for seven tumor-overlaps is 0–0.096, which makes it unlikely that more than one of these overlaps is the result of chance alone (see Supplementary Material, Fig. S1). Some of these LOHs found in more than seven samples have been previously described, but most are novel (Tables 2Go–4). The highest LOH frequency peak in pGBM was from a 103-kb cluster located in Chr15q15 (10/18) (Tables 3 and 4). Novel genes, including protein p53 binding protein 1 (TP53BP1) and Cyclin D-type binding-protein 1 (CCNDBP1), and two potential tumor suppressor genes that regulate the p53 and the Rb pathways are within this LOH peak.

Analysis of Regional Chromosomal Amplification and Homozygous Deletions in GBMs
Only a small number of amplifications (defined as 4 copies or more) and homozygous deletions were found in the pGBM samples. These were mainly in regions close to the centromeres of Chrs 5, 7, 11, 19, and 20, where typically no genes are found (Table 3). In contrast, chromosomal amplifications were more frequent in aGBMs (Tables 1 and 5). For example, all aGBMs had amplification of 4p16, 5p11, 11q11, and 20p11.2, and five of six had amplification in 8p23 and 19p12-13.3. Further, as expected and previously shown, epidermal growth factor receptor (EGFR) maps to the largest SNP cluster of amplification in Chr7p12 in two-thirds of the primary aGBMs included in this study (Table 5).25,26 In pGBM, a previously described amplification of 7p12 including EGFR was observed with a similar incidence to what is reported in the literature (2 of 18; 10.5%). However, a new amplification in 7q21-22 including CDK6 (Cyclin D Kinase 6) was seen in 5 of 18 pGBMs. All tumors were supratentorial. Interestingly, the latter is a known copy number variant with a seemingly higher incidence in pGBM than in the general population (5 of 18 versus 7 of 270; 26% versus 3%, p < 0.001, Fisher exact test). For regional homozygous chromosomal deletion, the most significant finding was the deletion of an SNP cluster in aGBMs containing interferon genes (IFNB1, IFNW1, IFNA21) that are important in the induction of p53 gene expression (Table 6).27 No homozygous deletions were observed in pGBM.

Validation of CNAs Identified in This Study
In the absence of control DNA from the same individual allowing confirmation that the CNAs we observed are tumor-derived and not a normal variant, we cross-referenced our data with the Database of Genomic Variants and the CEU study.28,29 We additionally cross-referenced our data for the most relevant CNAs with data from a set of 1,363 control DNA analyzed with the Illumina Human-Hap 300K platform.30 This allowed us to identify some previously reported regions of common structural polymorphisms in the general population, further validating the other regions as tumor-specific LOHs (Tables 2GoGoGo–6; see also Supplementary Material, Tables S1 and S2). We also compared our data with other available genotyping studies on pHGAs16–18 and on aHGAs9,31,32 as the number of adult cases used for comparison in this study is too few to make a meaningful conclusion. We also compared our data set to two publicly available 100K SNP array data sets on aHGAs (NCBI GEO DataSets as GSE9635 [NCBI GEO] and GSE6109 [NCBI GEO] Records).13,33 Cross-referencing our results with these data sets on aHGAs showed several commonalities with previously published studies, mainly for the adult patients included in our study (Pearson correlation, r: 0.83 and Tables 1GoGoGoGo–6; see also Supplementary Material, Table S1); however, only few previously published CNAs were common with pGBM (Pearson correlation, r: 0.21). We further validated the data set against an independent data set of 9 pGBMs analyzed using another SNP platform, the Affymetrix 250K SNP array that interrogates more than 200,000 loci and has a similar average resolution of 15 kb,34 and obtained concordant results for several CNAs, further validating data obtained using the Illumina SNP arrays (see Supplementary Material, Fig. S2).

We also arbitrarily chose a set of genes involved in regions identified as homozygous deletions, LOH, or DNA amplification in our data set and validated these CNAs using quantitative real-time (qRT)-PCR on DNA extracted from the same tumors (Tables 5 and 6). ELAVL2 (embryonic lethal, abnormal vision, Drosophila-like 2), a gene located on 9p21, was within a region of LOH in 5 of 18 pGBMs and potentially deleted in 1 of 18. We confirmed these results using qRT-PCR (Table 7). Chromosomal amplification in pGBMs on 7q21-22, which contains CDK6, was identified in 5 of 18 tumors. CDK6 amplification was also confirmed using qRT-PCR (Table 7).


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  		Table 7. Validation of Illumina Infinium 1 SNP-array data using qRT-PCR on selected genes

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Supplementary Material
 Acknowledgments
 References
 
Understanding the molecular pathogenesis of pHGAs requires a detailed cataloguing of all the genetic lesions in the somatic lineage of this cancer. This study includes the largest cohort of pGBMs analyzed for genomic imbalances in tumor DNA and is the first to use high-resolution SNP arrays. While further confirming that aGBM and pGBM are genetically distinct cancers, we provide a comprehensive whole-genome map of CNAs for pGBM and identify, within regions of CNAs, altered genes and pathways for further analysis (Tables 1GoGoGoGoGo–7).

Our data show findings in common with available array-based genomic studies on pGBM, including ELAVL2, CDKN2A, and CDKN2B deletions that have been reported in both pGBM and aGBM, supporting the validity of our data set. However, previous studies on pHGAs used arrays that had a lower resolution of at least one log than the 100K and 250K arrays used in this study.16 In these former studies, as for BAC arrays, this resulted in a vast number of genetic abnormalities going undetected. We uncover herein several previously unidentified CNAs specific to pediatric tumors that help provide insight into molecular pathways involved in the genesis of pGBM. These CNAs include LOH in 15q15.1-15q23 and 17p13-17p11.2 and amplification of 7q21-22 and 1q43-44. Even if some CNAs are common to the pediatric and adult setting, their overall incidence will differ whether they occur in children or in adults. When we reviewed published data sets on aHGAs, commonalities with our findings on pGBM included LOH of 22q17p and 9p, whereas LOH of 3p21 and 10q21 and amplification of 7q, 2p, 9p, and 10q seemed specific to pGBM (see Supplementary Material, Table S2). As an added example, amplification of 7p12 including EGFR is a frequent event in primary aGBMs (~45%).9,35 We and others16,36 show amplification of 7p12 including EGFR in only rare cases of pediatric GBMs (less than 10%). Conversely, amplification of 7q21-22 including CDK6 is a rare event in aHGAs (one case report), whereas it was present in 5 of 18 pGBMs. In addition, as a consequence of the higher resolution of the arrays we used, the genetic interval subject to alteration in tumors will differ in pediatric and adult samples in many cases. For example, 10q LOH/deletion will include the PTEN gene (10q23.31) in adults whereas the genetic interval will be different in children and LOH on 10q will not include it (10q21.3-10q22.1).

Cell-cycle abnormalities, including alterations of the p53 and the RB1 pathways, play an important role in the genesis of HGAs, including pHGAs.20,26 As shown in the analysis of genes included in the CNA regions (Tables 3GoGo–6) and by the gene ontology analysis (Table 4), genes associated with the cell-cycle checkpoints and genes involved in the regulation of cell-cycle and cell-death pathways are the major targets of LOHs in pGBM. For example, we identified recurrent LOHs in several tumors that encompass several genes including H76p, TP53BP1, and CCNDBP1 on 15p (Table 3). H76p is associated with the {gamma}-tubulin complexes and may participate in the nucleation process.37 TP53BP1 is a conserved checkpoint protein with properties of a DNA double-strand break sensor. It binds to the central domain of p53, enhancing transactivation, and its deletion may play an important role by impairing function of tumor protein p53 (TP53).38 CCNDBP1 is a helix–loop–helix leucine zipper protein, recently identified as a novel tumor suppressor in epithelial cancers, showing LOH and/or deletions on Chr15.39 It decreases the levels of Cyclin D expression, reducing the phosphorylation of RB1, thus regulating the RB1 pathway and cell growth.39

The rare gene containing regions showing genomic amplification in pGBM included 7q21-22 in 5 pGBMs (Table 5). This region is interesting from two standpoints. It includes CDK6, which encodes for a kinase also regulating the RB1 pathway. Amplification of CDK6 in this subset of pGBM was validated by qRT-PCR (Table 7) and has not been previously reported in pHGAs. In aHGAs, amplification of CDK6 has been reported in only one patient, whereas its over-expression, without genetic amplification, is more common (~40%). Secondly, a CNA found with statistically higher frequency in a given tumor is likely a germline predisposition to the tumor. 7q21-22 seems to be amplified in a small percentage of the normal population, and the higher incidence of its amplification in pGBM, when further confirmed, may indicate an association of a common variant with susceptibility to these tumors (Table 5).

Little is known on the genetics of pGBM and our study fills an important gap in understanding this pediatric brain tumor. Moreover, the considerable wealth of information on the molecular genetics of aHGA is often projected onto the pediatric population without critical comparison between these two different disease contexts. Our data document how profound these differences are. We propose that analyses such as the one presented in this study may ultimately guide mapping of oncogenic signaling networks unique to pGBM, identification of the related therapeutic predictors and targets, and development of more effective therapies. Alteration of copy number for genes involved in p53 and the RB pathways including, for example, amplification of CDK6, or LOH of CCNDBP1 or TP53BP1, are some of the interesting findings unraveled in our data set that will help shed light on the unique molecular pathogenesis of this disease, providing hope of developing new therapeutic strategies to improve survival in a devastating cancer.


   Supplementary Material
Top
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
Acknowledgments
 References
 
Supplementary material is available at Neuro-Oncology Journal online.


   Acknowledgments
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 Acknowledgments
References
 
This work was supported by the Canadian Institutes of Health Research, the Cole Foundation, One Day at a Time, and the Vinchiaturese Association (N.J.), the Hungarian Scientific Research Fund (OTKA) Contract No. T-04639, and the National Research and Development Fund (NKFP) Contract No. 1A/002/2004 (P.H., M.G., L.B., and Z.H.). H.Q. and K.J. are the recipients of a fellowship and a studentship, respectively, from the Canadian Institutes of Health Research. N.J. is the recipient of a Chercheur Boursier Award from Fonds de la Recherche en Sante du Quebec.

Conflict of interest statement. None declared.


   Notes
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 Acknowledgments
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* These authors contributed equally to the manuscript. Back


   References
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 Discussion
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 Acknowledgments
 References
 

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