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

Öz

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

Abstract

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

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