Ahmet Uludağ, Sevilhan Artan, Emre Tepeli, Nur Büyükpınarbaşılı, Muhammed Hamza Müslümanoğlu, Muhsin Ozdemir, Murat Oznur, Hüseyin Aslan, Oguz Cilingir

Küçük Hücreli Olmayan Akciğer Kanserleriyle İlişkilendirilmiş Tumor Süpressör Genlerin Multiplex Ligation-Dependent Probe Amplification (MLPA) Yöntemi ile Metilasyon Paternlerinin İncelenmesi

Akciğer kanseri, tüm dünyada mortalitesi en yüksek kanser türüdür ve kardiyovasküler hastalıklardan sonra ölüm nedenleri arasında 2. sırada yer almaktadır. Metilasyon bir epigenetik mekanizma olup DNA düzeyinde herhangi bir mutasyon olmamasına rağmen ilgili genin eksprese olmasını engelleyen bir moleküler mekanizmadır. Çalışmada henüz çok yeni bir yöntem olan Multiplex Ligation-dependent Probe Amplification (MLPA) tekniği ile akciğer kanserleriyle daha önce ilişkilendirilmiş 24 ayrı tümör süpressör genin promoter bölgelerinin metilasyon paternlerinin SALSA MS-MLPA ME001 tumorsuppressor probemix kiti kullanılarak incelenmesi amaçlanmıştır. Bu çalışmaya İstanbul Yedikule Göğüs Hastalıkları Hastanesinde, histopatolojik olarak incelenerek “küçük hücreli olmayan akciğer kanseri” tanısı almış 100 olgu dahil edildi..Her hastaya ait doku örneklerinden kanser dokusu içeren 5 mikronluk kesitlerden elde edilen DNA örnekleri çalışma grubu olarak, aynı hastaya ait kanser dokusu içermeyen kesitlerden elde edilen DNA örnekleri de kontrol grubu olarak çalışmaya alındı. Çalışmada, akciğer kanserli tümöral ve çevre akciğer dokularında birbirlerine yakın oranlarda en sık CDKN2B, BRCA1, CDH13 ve HIC1 prob bölgelerinde metilasyon görüldü. Yine tümöral ve çevre dokularda PTEN, TIMP3, ATM, VHL, CD44, CDKN1B, RASSF1, IGSF4 ve ESR1 prob bölgelerinde de metilasyon saptandı. APC, CDKN2A, MLH1, RARB, CHFR ve GSTP1 prob bölgelerinde farklı oranlarda, tümöral dokularda metilasyon görülürken, çevre akciğer dokularında bu prob bölgelerinde metilasyon saptanmadı. Sonuç olarak MS MLPA yönteminin akciğer kanserlerinin metilasyon profillerinin taramasında kullanılabilecek, ucuz ve hızlı sonuç verebilen bir teknik olduğu görülmüştür. MS MLPA yönteminin diğer metilasyon tarama yöntemleriyle karşılaştırılarak bir an önce senstivite ve spesivitesinin belirlenmesi ve akciğer kanserinin erken tanısında rutin kullanıma geçirilmesi gerektiğini düşünmekteyiz.

Anahtar Kelimeler:

Akciğer Kanseri, Tümör Süpressör Gen, Metilasyon, MS-MLPA

Analysis of Methylation Patterns of Some Tumor Suppressor Genes in Non-Small Cell Lung Cancer Using the Multiplex Ligation-Dependent Probe Amplification [MLPA] Method

Lung cancer has the leading mortality rate among all cancer types and is the second most common cause of death after cardiovascular diseases. Apart from being an epigenetic mechanism, methylation is also a molecular mechanism which inhibits cancer-related gene expression although there is no mutation at DNA level. The aim of this study was to analyze a probe panel interrogated DNA for methylation patterns in 24 tumor suppressor genes in non-small cell lung cancer using the Methylation Specific- Multiplex Ligation-dependent Probe Amplification [MS-MLPA] method the Multiplex Ligation-dependent Probe Amplification [MLPA] method. Previously examined clinically and histopathologically diagnosed a hundred cases with “non-small cell lung cancer”were included into the study. The DNAs were extracted samples from both cancerous tissues of the cases and their corresponding control tissues. The relations of the methylation profile to clinicopathological factors in NSCLC were evaluated. The genes frequently methylated in NSCLC including CDKN2B, BRCA1, CDH13 and HIC1 were also hypermethylated in surrounding nontumorous lung tissues. However, hypermethylated APC, CDKN2A, MLH1, RARB, CHFR and GSTP1 probe regions were only specific to the tumorous tissues of the cases. The aberrant methylation profile of the CDH13 probe region was only detected in the surrounding normal tissues and the difference was statistically significant. Methylation rates for ATM, RARB, CDKN2B, HIC1, CDKN1B, PTEN, VHL and APC were different between squamous and adenocarcinomas. Almost all hypermethylated probe regions were detected in higher grade tumors but CDKN2B hypermethylation seemed to be an early event in NSCLCs. In conclusion, MS-MLPA can be used to determine aberrant methylation patterns of specific genes in lung tumors. However, since MS-MLPA is a screening test, the confirmation and expression analysis by using different approaches are necessary.

Keywords:

Lung cancer, tumor-cuppressor gene, methylation, MS-MLPA,

PDF

___

The Information Centre for Health and Social Care and RCP. National Lung Cancer Audit, Report for the audit period 2005 December 2006
Liloglou T, Bediaga NG, Brown BRB, Field JK, Davies MPA. Epigenetic biomarkers in lung cancer. Cancer Letters (2012), article in press.
A. Portela, M. Esteller. Epigenetic modifications and human disease, Nat. Biotechnol. 28 2010 1057–1068.
Kondo Y, Issa JPJ. Epigenetic changes in colorectal cancer. Cancer Metastasis Rev. 2004; 23: 29–39.
Lin CH, Hsieh SY, Sheen I. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res. 2001, 61: 4238–4243.
Zhu ZZ, Sparrow D, Hou L, Tarantini L, Bollati V, et al. Repetitive element hypomethylation in blood leukocyte DNA and cancer incidence, prevalence, and mortality in elderly individuals: the Normative Aging Study.Cancer Causes Control 2011, 22: 437–447.
Vaissiere T, Hung RJ, Zaridze D, Moukeria A, Cuenin C, Fasolo V, Ferro G, Paliwal A, Hainaut P, Brennan P, Tost J, Boffetta P, Herceg Z. Quantitative analysis of DNA methylation profiles in lung cancer identifies aberrant DNA methylation of specific genes and its association with gender and cancer risk factors. Cancer Res 69(1):243252, 2009.
Hawes SE, Stern JE, Feng Q, et al. DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type. Lung Cancer. 2010;69(2):172– 179.
Sriram KB, Larsen JE, Yang IA, Bowman RV, Fong KM. Genomic medicine in non-small cell lung cancer: paving the path to personalized care. Respirology. 2011 Feb;16(2):257-63.
Herbst RS, Heymach JV, Lippman SM. Lung cancer. N. Engl. J.Med. 2008; 359: 1367–80.
Esteller M. Epigenetics provides a new generation of oncogenes and tumour- supressor genes. Br. J Cancer,2006, 94(2):179-83.
Esteller, M. Cancer epigenomics: DNA methylomes and histonemodification maps. Nav. Rev Genet. 2007, 8: 286-98.
Esteller M. Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum. Mol.Genet. 2007, 6:1, R50-R59.
Belinsky SA. Gene-promoter hypermethylation as a biomarker in lung cancer. Nature Reviews Cancer. 2004, 4: 707-717.
Shames DS, Girard L, Gao B, Sato M, Lewis CM, Shivapurkar N, Jiang A, Perou CM,Kim YH, Pollack JR, Fong KM, Lam CL, Wong M, Shyr Y, Nanda R, Olopade OI, Gerald
W, Euhus DM, Shay JW, Gazdar AF, Minna JD. A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies. Plos Med. 2006, 3(12):e486.
Jacinto FV, Esteller M. Mutator pathways unleashed by epigenetic silencing in human cancer. Mutagenesis. 2007, vol. 22 no. 4 pp. 247–253.
Grote HJ, Schmiemann V, Gedder t H, et al. Aberrant promoter methylation of p16INK4a, RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer. Int JCancer 2005;116:720^ 5.
Guo M, House MG, Hooker C, et al. Promoter hypermethylation of resected bronchial margins: a field defect of changes ? Clin Cancer Res 2004; 10:5131 ^ 6.
Belinsky SA, Palmisano WA, Gilliland FD, et al. Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. Cancer Res 2002;62:2370^ 7.
Zochbauer-Muller S, Fong KM, A.K. Virmani AK, Geradts J, A.F. Gazdar A.F,
Minna JD. Aberrant promoter methylation of multiple genes in nonsmall cell lung cancers. Cancer Res. 61 (2001) 249–255.
Paluzczak J, Misiak P. et al. Frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT in larengeal squamous cell carsinomas and adjacent normal mucoza. Oral Oncology 47 (2011) 104–107.
Bowman RV, Yang IA, Semmler ABT, Fong KM. Invited review series: lung cancer, Respirology. 2006, 11, 355-365.
Eunice CC, Lam SY, Fu KH, Kwong YL. Polymorphisms of the GSTM1, GSTP1, MPO, XRCC1 and NQO1 genes in Chinese patients with non small cell lung cancers: relationship with aberrant promoter methylation of the CDKN2A and RARB genes, Cancer Genetics and Cytogenetics. 2005, 162, 10-20 p.
Fronaka O, Takeshima Y, Awaya H, et al. Aberrant methylation of p14, p15 and p16 genes and location of the primary site in pulmonary squamous cell and carcinoma, Pathology International. 2004,54, 549-555.
Kim SD, Cha SI, Lee JH, et al. Aberrant DNA Methylation profiles of non small cell lung cancer in a Korean population, Lung Cancer. 2007, 2749, 1-6.
Sato K, Morishita Y, Fukasawa M, et al. Anthracotic index and DNA methylation status of sputum contents can be used for identifying the population at risk of lung carcinoma, Cancer Cytopathology. 2004, 102, 6, 348-354.
Ulivi P, Zoli W, Calistri D, Fabbri F, et al. p16 and CDH13 hypermethylation in tumor and serum of non small cell lung cancer patients, Journal of Cellular Physiology. 2006, 206, 611-615.
Heller G, Zielinski CC, Müler SZ. Lung cancer: From single-gene methylation to methylome profiling. Cancer Metastasis. 2010, 29:95–107.
Kim SD, Cha SI, Lee JH, et al. Aberrant DNA Methylation profiles of non small cell lung cancer in a Korean population, Lung Cancer, 2007, 2749, 1-6.
Zhang Y,Wang R, Song H, Huang G, Yi J, Zheng Y, Wang J, Chen L. Methylation of multiple genes as a candidate biomarker in nonsmall cell lung cancer . Cancer Letters. 2011, 303, 21–28.
Castro M, Grau L, Puerta P, Gimenez L, Venditti J, Quadrelli S, Sánchez-Carbayo M. Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in lung Cancer. Journal of Translational Medicine 2010, 8:86.
Kontic M, Stojsic J, Jovanovic D, Bunjevacki V, Ognjanovic S, Kuriger J, Puumala S, Nelson HH. Aberrant Promoter Methylation of International Journal of Clinical Research 2013;1(2):58-66 66 CDH13 and MGMT Genes is Associated With Clinicopathologic Characteristics of Primary Non–Small-Cell Lung Carcinoma Clinical Lung Cancer, 2011,Dec, 12.
Arvind, K, Tsou, JA, Siegmund KD, Shen YC, et al. Hierarchical clustering of lun cancer cell lines using NA methylation marker. Cancer Epidemiology, Biomarkers and prevention. 2002, 11, 291297.
Zöchbauer-Müller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in nonsmall cell lung cancers. Cancer Res, 2001,1; 61(1): 249-55.