High-Throughput Loss-Of-Heterozygosity Study of Chromosome 3P in Lung Cancer Using Single-Nucleotide Polymorphism Markers

Research Article

High-throughput Loss-of-Heterozygosity Study of Chromosome 3p in Lung Cancer Using Single-Nucleotide Polymorphism Markers

Amy L.S. Tai,1 William Mak,4 Phoebe K.M. Ng,4 Daniel T.T. Chua,1 Mandy Y.M. Ng,4 Li Fu,1 Kevin K.W. Chu,1 Yan Fang,5 You Qiang Song,2,4 Muhan Chen,1 Minyue Zhang,1 Pak C. Sham,3,4 and Xin-Yuan Guan1,5

Departments of 1Clinical Oncology, 2Biochemistry, and 3Psychiatry, 4Genome Research Center, The University of Hong Kong, Hong Kong, China and 5State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou, China

Abstract candidate TSGs, such as FHIT (7), RASSF1A (8), CACNA2D2 (9), and Loss of DNA copy number at the short arm of chromosome 3 is DLC1 (10), have been widely studied. However, comparative one of the most common genetic changes in human lung genomic hybridization results showed that the loss of 3p was often involved in the whole short arm in NSCLCs (5, 11). Therefore, cancer, suggesting the existence of one or more tumor suppressor genes (TSG) at 3p. To identify most frequently the existence of some unknown TSGs at loci other than 3p21.3 and deleted regions and candidate TSGs within these regions, 3p14.2 regions is likely. a recently developed single-nucleotide polymorphism (SNP)- Conventional LOH uses limited microsatellite polymorphism mass spectrometry-genotyping (SMSG) technology was ap- markers (12), which is a time-consuming process with low- pliedto investigate the loss of heterozygosity (LOH) in 30 resolution genotyping. In addition, it is difficult to automate this primary non–small-cell lung cancers. A total of 386 SNP method in large-scale LOH analysis. Because of these limitations, it markers that spanneda region of 70 Mb at 3p, from 3pter to is very hard to perform high-resolution LOH analysis using 3p14.1, were selectedfor LOH analysis. The average inter- conventional LOH method. The progress of the human genome marker distance in the present study is f180 kb. Several project makes it possible to apply single-nucleotide polymorphism (SNP) markers in LOH analysis. SNPs represent the most common frequently deleted regions, including 3p26.3, 3p25.3, 3p24.1, f 3p23, and 3p21.1, were found. Several candidate TSGs within form of sequence variation in the human genome, occurring 1 these frequently detected LOH regions have been found, every 1,200 bp (13). SNPs can be used as high-density polymorphic markers for studying genetic variations, including LOH (14), including APG7L at 3p25.3, CLASP2 at 3p23, and CACNA2D3 at 3p21.1. This study also showed that SMSG technology is a although their heterozygosity is relatively low compared with very useful approach to rapidly define the minimal deleted microsatellites. In this study, a high-throughput and high- resolution LOH study of chromosome 3p in 30 NSCLCs was done region andto identifytarget TSGs in a given cancer. (Cancer using SNP-mass spectrometry-genotyping (SMSG) technology. Res 2006; 66(8): 4133-8) Several high-LOH loci have been identified and three candidate TSGs were further studied. Introduction Lung cancer is one of the most common malignancies in the world and one of the leading causes of cancer death in the United Materials and Methods States, which accounts for f28% of all cancer death during 2001 Tumor samples. Thirty NSCLC samples were collected from the time (1). The overall 5-year survival rate of this prevalent cancer is <15% of surgical resection at the Cancer Institute, Sun Yat-Sen University (2). Lung cancer can be classified into two major types: small-cell (Guangzhou, China), during the period of 1998 to 2002. The specimens À j lung cancer and non–small-cell lung cancer (NSCLC), which is were snap frozen and kept at 80 C until DNA extraction. All the f25% and 75% of all lung cancers, respectively. The epidemiology samples were examined and macrodissected under a microscope by a pathologist to ensure the purity of tumor samples with <10% normal cell of lung cancer is multifactorial with cigarette smoking as the major contamination. cause (3). Deletion of chromosome 3p is one of the most frequent SNP marker selection andprimer design. A total of 386 SNP markers allelic imbalances in various human tumors, including NSCLC spanning over 0 to 70 Mb of 3p with minor allele frequency over 0.3 were (4, 5). Loss of heterozygosity (LOH) analysis is one of the most selected, including 326 SNPs with minor allele frequency over 0.45, 41 SNPs efficient methods to study chromosomal deletion and to narrow between 0.4 and 0.44, 16 SNPs with between 0.35 and 0.43, and 4 SNPs down the deleted region. High rates of LOH loci are commonly between 0.3 and 0.34 (see Supplementary Table S1). All minor allele found to harbor putative tumor suppressor genes (TSG). Previously frequencies were calculated from Caucasian populations. published LOH studies showed that 3p21.3 and 3p14.2 are the two MassARRAY AssayDesign software (Sequenom, San Diego, CA) was used most frequently deleted regions in lung cancer (4, 6). Several to design amplification and allele-specific extension primers for uniplexed or multiplexed assays. In the design, PCR primers have an additional 10-base tag (5V-ACGTTGGATG-3V) to prevent their interference in the resulting mass spectra. The designed assays were constrained to produce products of optimized size (90-120 bp) to maximize the PCR successful Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). rate. The extension primer was designed to hybridize to the amplicon near Requests for reprints: Xin-Yuan Guan, Department of Clinical Oncology, The the SNP locus for the extension of a single base or a few bases depending University of Hong Kong Medical Center, Room 109, Estate Building, 10 Sassoon Road, on the genotype of the allele (Supplementary Table S2). Pokfulam, Hong Kong, China. Phone: 852-25890458; Fax: 852-28169126; E-mail: PCR amplification anddephosphorylation. PCR reactions were done [email protected]. A I2006 American Association for Cancer Research. in a 384-well-plate format in a total volume of 6 L per reaction with 5 ng doi:10.1158/0008-5472.CAN-05-2775 genomic DNA, 0.3 pmol each of the specific forward and reverse primers, www.aacrjournals.org 4133 Cancer Res 2006; 66: (8). April 15, 2006

expected molecular weights of all relevant peaks were calculated by the Table 1. Summary of the frequently detected LOH in 30 MassARRAY AssayDesign Software before analysis and identified from the NSCLC cases mass spectrum. In every assayed plate, one well for blank control and five wells for duplicate check on five samples for quality control were included. SNP ID Location No. LOH Mapped genes Twenty-four SpectroChips for 8-plexed reactions, in which each chip is composed of 3,072 genotypes, can be analyzed within 1 day. A high- rs3772313 3p26.3 10 CNTN6 throughput genotyping consisting of 73,728 genotypes is routinely done rs6794523 3p26.2 10 IL5RA, CNTN4 within 24 hours. rs6766718 3p26.1 11 To test the effect of the tumor purity in LOH detection using SMSG, rs7638724 3p25.3 9 APG7L DNA from one tumor sample that contained <5% normal cells was used rs3856803 3p25.3 11 VGLL4 to determine the accuracy of genotyping. DNA samples from tumor, rs6550072 3p24.3 10 matched normal tissue, and series of mixtures (containing 5%, 10%, 20%, rs3849542 3p24.3 9 FLJ22419 and 50% normal DNA, respectively) have been assessed with five SNP rs3856621 3p24.2 11 markers. rs2102801 3p24.1 10 Comparative genomic hybridization. Comparative genomic hybrid- rs987693 3p24.1 12 RBMS3 ization was done as previously described (15). Briefly, 1 Ag tumor test and rs6550226 3p23 13 CLASP2 normal reference DNA were labeled with Spectrum Green-dUTP and rs4389435 3p22.3 10 DLC1 Spectrum Red-dUTP (Vysis, Downers Grove, IL), respectively, by nick rs3816779 3p21.31 9 FLJ20211 translation at 15jC for 2 hours. The slide containing normal metaphase rs6797113 3p21.1 9 CACNA2D3* spreads was denatured and then hybridized with 200 ng of each probe at rs1449325 3p21.1 12 CACNA2D3 37jC for 2 days. After hybridization, the slide was washed and counter- rs1113042 3p21.1 8 CACNA2D3 stained with 1 Ag/mL 4V,6-diamidino-2-phenylindole in an antifade solution. rs589281 3p21.1 10 CACNA2D3 c The image of hybridized metaphases was captured using a Zeiss Axioplan 2 rs722070 3p14.2 9 FHIT microscope equipped with a Sensys cooled-charged device camera (Photo- rs2044613 3p14.2 9 FHIT metrics, Ltd., Tucson, AZ), analyzed using Quips comparative genomic rs832187 3p14.3 9 LOC132200 hybridization program (Vysis), and interpreted according to the fluores- rs264083 3p14.1 9 BAIAP1 cence intensity profile under program guidance. The threshold value for rs6548999 3p14.1 9 TAFA1 DNA copy number gain and loss was defined as intensity ratio of tumor/ normal >1.2 and <0.8, respectively.

*CACNA2D3 covers four SNPs in this study and overall LOH involving CACNA2D3 was detected in 19 NSCLC cases. Results c FHIT covers two SNPs and overall LOH was detected in 15 NSCLCs. SMSG technology. In the SMSG system, a 90- to 120-bp DNA fragment containing the target SNP site was amplified by PCR with a pair of specific primers. The PCR product was then extended with another primer near the SNP site and extended sequence was A 200 mol/L of each of the deoxynucleotide triphosphates, 3.25 mmol/L automatically detected by MALDI-TOF. The diagrammatic repre- MgCl2, and 0.2 unit of HotStarTaq polymerase (Qiagen, Valencia, CA). A sentation of SMSG technology was shown in Fig. 1A.This total of 45 PCR cycles were carried out (95jC for 20 seconds, 56jCfor revolutionary technology provides a useful tool for high-through- 30 seconds, and 72jC for 1 minute) with an initial denaturation at 95jCfor 15 minutes and final extension at 72jC for 3 minutes. put and accurate genotyping analysis (16). A total of 386 SNP After PCR reaction, residual amplification nucleotides in the PCR markers, spanning from 1 bp to70 Mb of chromosome 3p (3pter- products were dephosphorylated with alkaline phosphatase. A mixture of p14.1), were selected for the LOH analysis in 30 primary NSCLC 0.2 AL hME buffer, 0.3 AL shrimp alkaline phosphatase (1 unit/AL; cases. The average intermarker distance in the present study is Sequenom), and 1.5 AL double-distilled water was added to the PCR f180 kb. products. The reaction solution was incubated at 37jC for 20 minutes, LOH. The overall positive call rate (the percentage of SNP loci followed by 85jC for 5 minutes to inactivate the enzyme. that genotypes can be assigned) was 91.0 F 2.1%. For a given Allele-specific primer extension. Mass extend reactions (MassEXTEND normal/tumor pair, each SNP can be identified as ‘‘LOH’’ Assay) were done in four groups of different terminations according to the (heterozygous in normal and LOH in tumor), ‘‘retention’’ (both design rationale (ddACG, ddACT, ddAGT, and ddCGT) in 10 AL reaction normal and tumor are heterozygous), and ‘‘noninformative’’ volume containing 1 unit of Thermosequenase (Sequenom), 50 Amol/L of the respective termination mix, and 0.6 pmol of each assay-specific (homozygous in normal; Fig. 1B). Among the 386 SNP markers extension primer. All the assays were done under the thermal cycle used in this study, 29 markers showed a very low informative rate conditions of initial denaturation at 94jC for 2 minutes followed by 55 (<5%), which were excluded for the data analysis. The average cycles of 94jC for 5 seconds, 52jC for 5 seconds, and 72jC for 5 seconds. heterozygosity for the remaining 357 SNP markers is 0.39. Genotype detection using matrix-assisted laser desorption/ioniza- To assess the effect of tumor purity on LOH detection, DNA tion-time of flight mass spectrometry. The final base extension products samples from one case, including tumor, matched normal tissue, were treated with SpectroClean resin (Sequenom) to remove the salts in the and series of mixtures (containing 5%, 10%, 20%, and 50% rnormal A reaction buffer. Briefly, 16 L resin per water suspension was added to each DNA, respectively), had been assessed with five SNP markers. base extension reaction and 10 nL of the reaction solution were dispensed Results indicated that LOH could be detected when the contam- onto a 384-well-plate format SpectroChip (Sequenom) prespotted with a ination of normal cell was <10%. Tumor samples with 80% tumor matrix of 3-hydroxypicolinic acid using the MassARRAY Nanodispenser (Sequenom). The modified Brucker Autoflex matrix-assisted laser desorp- purity gave an increased false ‘‘retention’’ (Fig. 2). tion/ionization-time of flight (MALDI-TOF) mass spectrometer (Brucker, The global pattern of LOH in 70 Mb of 3p in 30 NSCLC cases was Billerica, MA) was used for data acquisition. Data were automatically shown in Fig. 3. Consistent with the findings of others, 3p21.3 and imported into the SpectroTYPER database (Sequenom) for the robust data 3p14.2 are the two most frequently deleted regions in this study. analysis, including noise normalization and peak area analysis. The One of the most interesting findings is that several other frequently

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Figure 1. A, diagrammatic representation of SMSG technology. DNA sequence containing the target SNP site is PCR amplified with a pair of specific primers (P1 and P2). After PCR amplification, P3 primer is used for the allele- specific extension of a single or few bases near the SNP loci. The extension products are dispensed onto a 384-well-plate format SpectroCHIP and a MALDI-TOF mass spectrometer is used for data acquisition. B, representative examples of LOH, retention, and noninformative SNPs detected by MALDI-TOF. LOH, if a heterozygote in normal and a homozygote in tumor specimen is detected. Retention, if both normal and tumor are heterozygotes. Noninformative, if both normal and tumor are homozygotes.

deleted regions at 3p, including 3p26.3, 3p25.3, 3p24.1, and 3p23, were identified. Validation of LOH results. LOH results in this study are generally agreed with the comparative genomic hybridization data. In comparative genomic hybridization study, 22 NSCLC cases showed DNA copy loss at 3p (first 22 cases in Fig. 3) and 8 cases showed nearly no loss (last 8 cases in Fig. 3). Figure 4A compared three comparative genomic hybridization results and their matched LOH results. Interestingly, an interstitial deletion of 3p at 3p14.3-p14.1 detected by SNP mass array in case L22 was perfectly confirmed by comparative genomic hybridization. LOH results in this study were also confirmed by sequencing analysis. Ten SNP markers were randomly selected and PCR products of a pair of tumor (with LOH) and its matched normal tissue (without LOH) for each SNP were sequenced. LOH was detected in all 10 tested tumor samples, whereas it was not observed in 10 matched normal tissues (Fig. 4B). About 10% to 38% of allelic loss was found at 357 SNP loci for the 22 cases with comparative genomic hybridization changes, whereas <7% of allelic loss was detected in those eight cases without comparative genomic hybridization change. Candidate TSGs. The most frequently detected LOH was found at 22 SNP loci, which was shown in Fig. 5A and summarized in Table 1. Several candidate TSGs within these frequent LOH regions have been found, including APG7-like gene (APG7L) at 3p25.3, Figure 2. The effect of tumor purity on LOH detection. DNA from tumor, matched normal tissue, and series of mixtures (containing5%, 10%, 20%, CLIP-associated protein 2 (CLASP2) at 3p23, and calcium channel, and 50% normal DNA, respectively) were assessed by MALDI-TOF with three voltage-dependent, a-2/d subunit 3 (CACNA2D3) at 3p21.1, besides SNP markers. www.aacrjournals.org 4135 Cancer Res 2006; 66: (8). April 15, 2006

FHIT and DLC1 (Table 1). The genomic size of CACNA2D3 gene is enzyme essential for the APG12 conjugation system that mediates f1 Mb, which was covered by four SNP markers in this study. The membrane fusion in autophagy (17). LOH involving CACNA2D3 overall LOH involving CACNA2D3 was detected in 19 cases. gene was the most frequent genetic alteration in the present study. Similarly, FHIT was covered by two SNPs and the overall LOH of Although CACNA2D3 has not been associated with cancer FHIT was observed in 15 cases. The expression levels of these development, exogenous expression of its family member CAC- candidate TSGs were studied by reverse transcription-PCR (RT- NA2D2 could significantly inhibit tumor cell growth and induce PCR) in 12 primary NSCLCs. Absent or down-regulated expressions apoptosis (18). Because both CACNA2D2 and CACNA2D3 are of APG7L, CLASP2, and CACNA2D3 were observed in 10, 5, and 6 subunits of the Ca2+ channel complex, it is reasonable to believe cases, respectively (Fig. 5B). that CACNA2D3 might also be correlated with pathogenesis of lung cancer. CLASP2 interacts with CLIP, binds to microtubules, and has microtubule-stabilizing effects (19). Increasing microtubule insta- Discussion bility may cause genetic instability and loss of CLASP2 function It is believed that the pathogenesis of lung cancer is a long-term may induce genetic instability and lead to the development of process that involves multiple genetic alterations, including the cancer. loss of function of TSGs at 3p. Isolation and characterization of High-throughput LOH study using SMSG provides a very useful TSGs at 3p will significantly improve our knowledge in the tool for a rapid and high-resolution LOH investigation of interesting pathogenesis of lung cancer. In the present study, a recently chromosomal regions. Recently, Lindbland-Toh et al. (20) applied a developed high-throughput and high-resolution technology, SNP whole-genome HuSNP array (Affymetrix, Santa Clara, CA) with mass array, was applied to identify frequent LOH loci at 3pter-p14.1 nearly 1,500 SNP markers for the LOH detection in small-cell lung and isolate candidate TSGs within these loci. cancer. Later, HuSNP array was used to study LOH in prostate (21) Three candidate TSGs, including APG7L, CACNA2D2,and and bladder cancers (22). More recently, GeneChip human mapping CLASP2, have been identified. APG7L is the human homologue of 10 K array, which contains over 10,000 SNPs, has been used for yeast Apg7, a key autophagy gene encoding an ubiquitin-E1-like genomewide genotyping and linkage analysis (23, 24).

Figure 3. Summary of the global pattern of LOH in 70 Mb of 3p in 30 NSCLC samples. Each sample is represented in columns and SNP markers are arranged accordingly in rows. Gray, SNP marker detected with LOH; white, retention; light gray, noninformative.

Cancer Res 2006; 66: (8). April 15, 2006 4136 www.aacrjournals.org

Figure 4. A, comparison of 3p deletions detected using comparative genomic hybridization and SMSG technology. Left, comparative genomic hybridization results; right, SMSG results. Vertical red bars on the left of chromosome ideograms, DNA copy number loss detected by comparative genomic hybridization. In LOH detection, each five consecutive SNP markers were arranged in a group and different levels of LOH were represented in different colors. B, sequencingresults of two randomly selected LOH cases usingSNP markers rs3849582 ( left) and rs1435715 (right). LOH was observed in the tumor parts in both cases.

Figure 5. A, summary of LOH detected in individual SNP markers. SNP markers are represented accordingly in their base position of chromosome 3p. B, RNA expressions of candidate TSGs detected by RT-PCR. RNA from normal lungs (N) and tumor (T) specimens. h-actin was amplified as RT-PCR internal control.

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Comparing with currently available HuSNP array, the SMSG regions with high resolution (20-100 kb) and less cost comparing technology used in this study is more rapid, less expensive, and with genomewide genetic characterization. In conclusion, the more versatile. For example, investigation of 60 samples (30 pairs of method described in this study extends the limits of conventional NCSLCs) with 386 SNP markers (60 Â 386 = 23,160 genotypes) can LOH analysis by providing a rapid source of high-throughput be finished within 24 hours. As no hybridization is included in the method and thus enabling the high-resolution LOH investigation of process, the bias toward genotype interpretation can be avoided. In chromosomal regions. Also, this highly reproducible method allows addition, only a small amount of DNA (5 ng for each reaction) is the rapid identification of candidate TSGs in a given cancer. required and that makes it possible to use purer DNA from tumor cells captured by laser dissection, which is the major confounded factor in LOH study. Another advantage of this method is its Acknowledgments capability to provide a high-resolution analysis within a given Received 8/5/2005; revised 1/16/2006; accepted 2/10/2006. chromosomal region. In most solid tumors, recurrent deletions are Grant support: Chinese State Key Program for Basic Research grant G1998051207, only detected in a few chromosomal regions by comparative Research Grant Council grant HKU7393/04 M, and Leung Kwok Tze Foundation. The costs of publication of this article were defrayed in part by the payment of page genomic hybridization studies in a particular cancer. The strategy charges. This article must therefore be hereby marked advertisement in accordance described here can focus on some of the given chromosomal with 18 U.S.C. Section 1734 solely to indicate this fact.

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Amy L.S. Tai, William Mak, Phoebe K.M. Ng, et al.

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