Detection of Aberrant TERT Promoter Methylation by Combined Bisulfite Restriction Enzyme Analysis for Cancer Diagnosis
2017; Elsevier BV; Volume: 19; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2017.01.003
ISSN1943-7811
AutoresSeungjae Lee, Sumit Borah, Armita Bahrami,
Tópico(s)Genomics and Chromatin Dynamics
ResumoAberrant CpG dinucleotide methylation in a specific region of the telomerase reverse transcriptase (TERT) promoter is associated with increased TERT mRNA levels and malignancy in several cancer types. However, routine screening of this region to aid cancer diagnosis can be challenging because i) several established methylation assays may inaccurately report on hypermethylation of this particular region, ii) interpreting the results of methylation assays can sometimes be difficult for clinical laboratories, and iii) use of high-throughput methylation assays for a few patient samples can be cost prohibitive. Herein, we describe the use of combined bisulfite restriction enzyme analysis (COBRA) as a diagnostic tool for detecting the hypermethylated TERT promoter using in vitro methylated and unmethylated genomic DNA as well as genomic DNA from four melanomas and two benign melanocytic lesions. We compare COBRA with MassARRAY, a more commonly used high-throughput approach, in screening for promoter hypermethylation in 28 formalin-fixed, paraffin-embedded neuroblastoma samples. COBRA sensitively and specifically detected samples with hypermethylated TERT promoter and was as effective as MassARRAY at differentiating high-risk from benign or low-risk tumors. This study demonstrates the utility of this low-cost, technically straightforward, and easily interpretable assay for cancer diagnosis in tumors of an ambiguous nature. Aberrant CpG dinucleotide methylation in a specific region of the telomerase reverse transcriptase (TERT) promoter is associated with increased TERT mRNA levels and malignancy in several cancer types. However, routine screening of this region to aid cancer diagnosis can be challenging because i) several established methylation assays may inaccurately report on hypermethylation of this particular region, ii) interpreting the results of methylation assays can sometimes be difficult for clinical laboratories, and iii) use of high-throughput methylation assays for a few patient samples can be cost prohibitive. Herein, we describe the use of combined bisulfite restriction enzyme analysis (COBRA) as a diagnostic tool for detecting the hypermethylated TERT promoter using in vitro methylated and unmethylated genomic DNA as well as genomic DNA from four melanomas and two benign melanocytic lesions. We compare COBRA with MassARRAY, a more commonly used high-throughput approach, in screening for promoter hypermethylation in 28 formalin-fixed, paraffin-embedded neuroblastoma samples. COBRA sensitively and specifically detected samples with hypermethylated TERT promoter and was as effective as MassARRAY at differentiating high-risk from benign or low-risk tumors. This study demonstrates the utility of this low-cost, technically straightforward, and easily interpretable assay for cancer diagnosis in tumors of an ambiguous nature. The telomerase reverse transcriptase (TERT) oncogene encodes the rate-limiting catalytic subunit of telomerase, the enzyme required by virtually all proliferative cells to maintain the integrity of chromosomal ends.1Nandakumar J. Cech T.R. Finding the end: recruitment of telomerase to telomeres.Nat Rev Mol Cell Biol. 2013; 14: 69-82Crossref PubMed Scopus (275) Google Scholar, 2Blackburn E.H. Collins K. 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Analysis of promoter CpG island hypermethylation in cancer: location, location, location!.Clin Cancer Res. 2011; 17: 4225-4231Crossref PubMed Scopus (120) Google Scholar and frequently occurs in medulloblastoma, medullary thyroid carcinoma, and prostate, esophageal, gastric, colorectal, and cervical cancer.17Wang N. Kjellin H. Sofiadis A. Fotouhi O. Juhlin C.C. Backdahl M. Zedenius J. Xu D. Lehtio J. Larsson C. Genetic and epigenetic background and protein expression profiles in relation to telomerase activation in medullary thyroid carcinoma.Oncotarget. 2016; 7: 21332-21346PubMed Google Scholar, 25Castelo-Branco P. Leao R. Lipman T. Campbell B. Lee D. Price A. Zhang C. Heidari A. Stephens D. Boerno S. Coelho H. Gomes A. Domingos C. Apolonio J.D. Schafer G. Bristow R.G. Schweiger M.R. Hamilton R. Zlotta A. Figueiredo A. Klocker H. Sultmann H. Tabori U. 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However, these assays have yet to be adopted in clinical laboratories mainly because applying high-throughput or difficult-to-interpret methylation assays to predict patient outcomes in routine clinical practice is challenging and cost prohibitive. Recent improvements in experimental protocols, ease of analysis, and cost may facilitate the use of such high-throughput assays in the clinic.35Yang Y. Sebra R. Pullman B.S. Qiao W. Peter I. Desnick R.J. Geyer C.R. DeCoteau J.F. Scott S.A. Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).BMC Genomics. 2015; 16: 350Crossref PubMed Scopus (54) Google Scholar Combined bisulfite restriction enzyme analysis (COBRA) is a simple, sensitive, and low-cost assay for detecting DNA methylation at specific genetic loci. In this study, we demonstrate the effectiveness of COBRA in detecting TERT promoter UTSS hypermethylation in tumor samples, and compare it to detection by the high-throughput MassARRAY approach. In COBRA, hypermethylation of CpG dinucleotides results in their protection from cytosine to uracil conversion by sodium bisulfite that results in PCR restriction fragment length polymorphisms (PCR-RFLPs). PCR-RFLPs can distinguish DNA amplified from methylated and unmethylated UTSS. The appearance of short restriction fragments after BsiW1, Hpy188I, or Hpy99I digestion indicates the presence of hypermethylated UTSS in a sample and, thus, cancer. We demonstrate that this approach is highly robust, easy to perform and interpret, and reliable in distinguishing high-risk malignant tumors from low-risk and benign lesions. Tumor samples from two cohorts—the melanoma cohort and the NBL cohort—were analyzed with institutional review board approval. For the melanoma cohort, samples included four malignant melanomas arising in a giant congenital nevus and two benign proliferative nodules in the giant congenital nevus. For this cohort, tumor characteristics and UTSS methylation status by next-generation bisulfite sequencing have been previously described.36Fan Y. Lee S. Wu G. Easton J. Yergeau D. Dummer R. Vogel P. Kirkwood J.M. Barnhill R.L. Pappo A. Bahrami A. Telomerase expression by aberrant methylation of the TERT promoter in melanoma arising in giant congenital nevi.J Invest Dermatol. 2016; 136: 339-342Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar For the NBL cohort, samples included 28 tumors, classified as 12 high-risk, 5 intermediate-risk, and 11 low-risk NBL samples by the International Neuroblastoma Risk Group classification system.37Brodeur G.M. Pritchard J. Berthold F. Carlsen N.L.T. Castel V. Castelberry R.P. De Bernardi B. Evans A.E. Favrot M. Hedborg F. Kaneko M. Kemshead J. Lampert F. Lee R.E.J. Look A.T. Pearson A.D.J. Philip T. Roald B. Sawada T. Seeger R.C. Tsuchida Y. Voute P.A. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment.J Clin Oncol. 1993; 11: 1466-1477Crossref PubMed Scopus (1848) Google Scholar, 38Cohn S.L. Pearson A.D. London W.B. Monclair T. Ambros P.F. Brodeur G.M. Faldum A. Hero B. Iehara T. Machin D. Mosseri V. Simon T. Garaventa A. Castel V. Matthay K.K. The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report.J Clin Oncol. 2009; 27: 289-297Crossref PubMed Scopus (1231) Google Scholar Supplemental Table S1 gives clinicopathologic characteristics of the NBL cohort. Genomic DNA from formalin-fixed, paraffin-embedded tissue was isolated by using the Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega, Madison, WI). From each sample, 500 ng of genomic DNA was modified with sodium bisulfite by using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA), according to the manufacturer's protocol. Sodium bisulfite–treated DNA was used for bisulfite PCR amplification, as previously described.36Fan Y. Lee S. Wu G. Easton J. Yergeau D. Dummer R. Vogel P. Kirkwood J.M. Barnhill R.L. Pappo A. Bahrami A. Telomerase expression by aberrant methylation of the TERT promoter in melanoma arising in giant congenital nevi.J Invest Dermatol. 2016; 136: 339-342Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar PCR amplification of the UTSS was performed by using GoTaq Long PCR Master Mix (Promega), as previously described,36Fan Y. Lee S. Wu G. Easton J. Yergeau D. Dummer R. Vogel P. Kirkwood J.M. Barnhill R.L. Pappo A. Bahrami A. Telomerase expression by aberrant methylation of the TERT promoter in melanoma arising in giant congenital nevi.J Invest Dermatol. 2016; 136: 339-342Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar with primer set 1 (forward: 5′-GGAAGTGTTGTAGGGAGGTATTT-3′ and reverse: 5′-AAAACCATAATATAAAAACCCTAAA-3′), generating bisulfite amplicons of 244 bp, or primer set 2 (forward: 5′-AGGAAGAGAGGGGAAGTGTTGTAGGGAGGTATTT-3′ and reverse: 5′-CAGTAATACGACTCACTATAGGGAGAAGGCTAAAAACCATAATATAAAAACCCTAAA-3′),24Castelo-Branco P. Choufani S. Mack S. Gallagher D. Zhang C. Lipman T. Zhukova N. Walker E.J. Martin D. Merino D. Wasserman J.D. Elizabeth C. Alon N. Zhang L. Hovestadt V. Kool M. Jones D.T. Zadeh G. Croul S. Hawkins C. Hitzler J. Wang J.C. Baruchel S. Dirks P.B. Malkin D. Pfister S. Taylor M.D. Weksberg R. Tabori U. Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study.Lancet Oncol. 2013; 14: 534-542Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar generating bisulfite amplicons of 285 bp. Cycling parameters were as follows: 95°C for 15 seconds, 53°C for 20 seconds, and 68°C for 30 seconds for 45 cycles. For COBRA, 3 to 5 μL of each 285- or 244-bp PCR amplicon was digested with BsiW1 or Hpy188I, according to the manufacturer's instructions (New England BioLabs, Ipswich, MA). Samples were electrophoresed on Novex 20% polyacrylamide TBE gels (Thermo Fisher Scientific, Waltham, MA), which were then stained with GelRed (Biotium, Fremont, CA) before visualization using an Odyssey Fc Imaging System (LI-COR, Lincoln, NE). To characterize the specificity of RFLP detection by COBRA, commercially prepared High Methylated (>85% methylation) and Low Methylated Human Genomic DNA (<5% methylation) samples were used as templates (EpigenDx, Hopkinton, MA). To determine the sensitivity of COBRA to detect methylated UTSS, premixed methylation calibration standards for human genomic DNA were used as templates (EpigenDx). For sequencing analysis, amplicons were cloned into the PCR2.1 vector, using blue/white selection (TOPO TA Cloning Kit with PCR2.1; Thermo Fisher Scientific), as per the manufacturer's instructions. Incorporation of insert of the expected length was verified by using T7 and M13 reverse-amplification primers, and 9 or 10 clones from each sample were randomly selected for sequencing. The UTSS PCR product was amplified from bisulfite-treated sample DNA, or from completely methylated or unmethylated control DNA, using primer set 2.24Castelo-Branco P. Choufani S. Mack S. Gallagher D. Zhang C. Lipman T. Zhukova N. Walker E.J. Martin D. Merino D. Wasserman J.D. Elizabeth C. Alon N. Zhang L. Hovestadt V. Kool M. Jones D.T. Zadeh G. Croul S. Hawkins C. Hitzler J. Wang J.C. Baruchel S. Dirks P.B. Malkin D. Pfister S. Taylor M.D. Weksberg R. Tabori U. Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study.Lancet Oncol. 2013; 14: 534-542Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar These PCR products were used as templates for in vitro transcription reactions by using T7 RNA polymerase. The ensuing RNA transcripts were specifically cleaved at uracil residues, and differences in fragment sizes and charges from each sample were measured by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MassARRAY System; Agena Bioscience, San Diego, CA). Data were analyzed by the EpiTYPER software version 1.0 (Agena Bioscience), using a 10% cutoff value. Building on our recent study of TERT promoter hypermethylation in melanoma,36Fan Y. Lee S. Wu G. Easton J. Yergeau D. Dummer R. Vogel P. Kirkwood J.M. Barnhill R.L. Pappo A. Bahrami A. Telomerase expression by aberrant methylation of the TERT promoter in melanoma arising in giant congenital nevi.J Invest Dermatol. 2016; 136: 339-342Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar we sought to use COBRA as a diagnostic screen for distinguishing malignant tumors from benign or low-grade tumors. First, the nucleotide sequence of this region, amplified from bisulfite-treated genomic DNA of both melanoma and benign samples, was analyzed to identify PCR-RFLPs that would be useful for differentiating hypermethylated from unmethylated UTSS.36Fan Y. Lee S. Wu G. Easton J. Yergeau D. Dummer R. Vogel P. Kirkwood J.M. Barnhill R.L. Pappo A. Bahrami A. Telomerase expression by aberrant methylation of the TERT promoter in melanoma arising in giant congenital nevi.J Invest Dermatol. 2016; 136: 339-342Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Restriction sites for BsiWI, Hpy188I, and Hpy99I were present in a PCR product amplified from hypermethylated but not unmethylated UTSS, contingent on bisulfite treatment of the sample DNA (Figure 1A). For example, BsiW1 digestion of a 285-bp PCR product amplified from a hypermethylated UTSS is expected to yield a 63-bp restriction fragment, although no such fragment will be generated if this template was originally unmethylated. Similarly, Hpy188I digestion of a PCR product amplified from the hypermethylated UTSS should yield both 21- and 26-bp fragments, both of which would be absent if this template was unmethylated. To confirm these predictions, commercially available methylated and unmethylated genomic DNA was treated with sodium bisulfite and used as a template for UTSS amplification. Each resulting PCR product was digested with either BsiW1 or Hpy188I (Figure 1B). As expected, the PCR product obtained from the methylated DNA was almost completely digested by each of these restriction endonucleases to yield smaller fragments of expected lengths. No such fragments were detected when the unmethylated DNA was used as a template, confirming that bisulfite treatment of the methylated, but not the unmethylated, promoter generated restriction sites recognized by these enzymes. Next, to measure the sensitivity of this approach for detecting methylated UTSS in an excess of unmethylated template, as is usually the case in actual patient samples, different ratios of bisulfite-treated methylated and unmethylated genomic DNA were mixed together before amplification. A mixture containing as little as 5% methylated DNA was successfully detected by this assay (Supplemental Figure S1). We then tested the effectiveness of COBRA in accurately detecting hypermethylated UTSS in four cases of malignant melanoma, using two cases of benign proliferative nodules arising from the giant congenital nevus that had unmethylated UTSS as negative controls. Hypermethylated UTSS had prev
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