KRAS mutasyonlarının kolon ve akciğer kanseri eksozomlar aracılığıyla tayini ve primer dokuya kıyasla mutasyon durumlarının analizi

Yıl 2022, Cilt: 13 Sayı: 2, 253 - 259, 30.06.2022

Onur Tokgün , Nedim Karagenç , Pervin Elvan Tokgün , Kubilay Incı , Hakan Akça , Gamze Gokoz Dogu , Aydın Demiray

https://doi.org/10.18663/tjcl.1015438

Öz

Amaç: Kolorektal kanser ve akciğer kanserleri olan hastalarda epidermal büyüme faktörü reseptör hedefli tedavinin
etkinliği önemli ölçüde KRAS mutasyonu ile ilişkilidir. Bu çalışmanın amacı, Kolorektal kanser ve akciğer kanserli hastaların
serum ekzozom ve primer tümör dokusunda ki KRAS mutasyon durumlarının karşılaştırmaktır.
Gereç ve Yöntemler: Histolojik olarak doğrulanmış 19 adet kolorektal kanser ve 28 adet akciğer kanserli toplam 47
hastanın tümör dokularından genomik DNA izole edildi ve iki kür tedavi sonrasında ilgili hastalardan alınan periferik
kandan ekzomal RNA izole edildi. KRAS geninde üç bölgede gözlenen mutasyonlar (kodon 12, 13 ve 61) pyrosekanslama
yöntemi ile analiz edildi. Elde edilen sekans verileri doğrultusunda tümör dokuları ile serum ekozomlarında bulunan
nükleik asitlere ait KRAS mutasyon durumları ortaya kondu.
Bulgular: Doku örneklerinden KRAS mutasyon profilleri belirlenen hastaların eksozomlarında gözlenen KRAS
mutasyonları incelendiğinde 3 (%6,38) hastada dokuda belirlenen mutasyonun dışında yeni bir mutasyon saptanmış, 9
(%19,14) hastada doku örneğinde mutasyon var iken herhangi bir KRAS mutasyonu saptanamamış, 5 (%10,6) hastada
doku örneğinde mutasyon yok iken herhangi bir KRAS mutasyonu saptanmıştır. 30 (%63,8) hastada ise mutasyon olup
olmama durumu doku ve eksozom analizleri doğrultusunda değişmemiştir.
Sonuç: Elde ettiğimiz sonuçlar, kolorektal kanser ve akciğer kanserli hastaların hızlı ve non-invaziv bir materyal ile
genotiplenmesinde serum ekzomal mRNA'nın yeni ve güvenilir bir kaynak olarak kullanılabileceği ileri sürmektedir.

Anahtar Kelimeler

Likit biyopsi, KRAS, eksozom, DNA

Destekleyen Kurum

PAÜ Bilimsel Araştırma Projeleri Koordinasyon Birimİ

Proje Numarası

2018HZDP052

Detection of KRAS mutations by colon and lung cancer exosomes and analysis of mutational statuses compared to primary tissue

Öz

Aim: The efficacy of epidermal growth factor receptor-targeted therapy in colorectal cancer and lung cancers is significantly
associated with the KRAS mutation. Therefore, this study aims to compare the KRAS mutation status in serum exosome
and primary tumor tissue of colorectal and lung cancer patients.
Material and Methods: Genomic DNA was isolated from tumor tissues of 47 patients with histologically confirmed
19 colorectal cancers and 28 lung cancers, and exosomal RNA was isolated from peripheral blood from the respective
patients after two treatment cycles. Mutations (codons 12, 13, and 61) observed in three regions of the KRAS gene were
analyzed by pyrosequencing. The sequence data obtained revealed the KRAS mutation status of nucleic acids in tumor
tissues and serum exosomes.
Results: When the KRAS mutations were observed in the exosomes of the cases whose KRAS mutation profiles were
determined from the tissue samples, a new mutation other than the mutation determined in the tissue was detected in 3
(6.38%) cases. In comparison, there was a mutation in the tissue sample in 9 (19.14%) cases, and no KRAS mutation could
be detected in 5 (10.6%) cases. While there was no mutation in the tissue sample, any KRAS mutation was detected. In 30
(63.8%) cases, the presence or absence of mutation did not change in line with tissue and exosome analysis.
Conclusion: Our results suggest that serum exosomal mRNA can be used as a new and reliable source for genotyping
patients with colorectal and lung cancer with a rapid and non-invasive material.

Anahtar Kelimeler

liquid biopsy, KRAS, exosome, DNA

Proje Numarası

2018HZDP052

Kaynakça

• 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009; 59: 225-49.
• 2. Langer CJ. Roles of EGFR and KRAS Mutations in the Treatment Of Patients With Non-Small-Cell Lung Cancer. P T 2011; 36: 263-79.
• 3. Vogelstein B, Fearon ER, Hamilton SR et al. Genetic alterations during colorectal-tumor development. N Eng J Med 1988; 319: 525-32.
• 4. Arrington AK, Heinrich EL, Lee W et al. Prognostic and Predictive Roles of KRAS Mutation in Colorectal Cancer. Int J Mol Sci 2012; 13: 12153-68.
• 5. Lièvre A, Bachet JB, Boige V et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008; 26: 374-9.
• 6. Qiu LX, Mao C, Zhang J et al. Predictive and prognostic value of KRAS mutations in metastatic colorectal cancer patients treated with cetuximab: a meta-analysis of 22 studies. Eur J Cancer 2010; 46: 2781-7.
• 7. Linardou H, Dahabreh IJ, Kanaloupiti D et al. Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and metaanalysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol 2008; 9: 962-72.
• 8. Garassino MC, Borgonovo K, Rossi A et al. Biological and clinical features in predicting efficacy of epidermal growth factor receptor tyrosine kinase inhibitors: a systematic review and metaanalysis. Anticancer Res 2009; 29: 2691-701.
• 9. Mascaux C, Iannino N, Martin B et al. The role of RAS oncogene in survival of patients with lung cancer : asystematic review of the literature with meta-analysis. Br J Cancer 2005; 92: 131-9.
• 10. Schiller JH, Adak S, Feins RH et al. Lack of prognostic significance of p53 and KRAS mutations I primary resected non-small-cell lung cancer on E4592: A laboratory ancillary study on an eastern cooperative oncology group prospective randomized trial of postoperative adjuvant therapy. J Clin Oncol 2001; 19: 448-57.
• 11. Shepherd FA, Domerg C, Hainaut P et al. Pooled analysis of the prognostic and predictive effects of KRAS mutation status and KRAS mutation subtype in early-stage resected non-small-cell lung cancer in four trials of adjuvant chemotherapy. J Clin Oncol 2013; 31: 2173-81.
• 12. Pantel K, Alix-Panabières C. Liquid biopsy and minimal residual disease - latest advances and implications for cure. Nat Rev Clin Oncol 2019; 16: 409-24.
• 13. Babayan A, Pantel K. Advances in liquid biopsy approaches for early detection and monitoring of cancer. Genome Med. 2018;10(1):21. 3. Alix-Panabie` res C, Pantel K. Circulating tumor cells: liquid biopsy of cancer. Clin Chem 2013; 59: 110-8.
• 14. Mader S, Pantel K. Liquid Biopsy: Current Status and Future Perspectives. Oncol Res Treat. 2017; 40: 404-8.
• 15. Sluijter JP, Verhage V, Deddens JC et al. Microvesicles and exosomes for intracardiac communication. Cardiovasc Res 2014; 102: 302–11.
• 16. Kalluri R. The biology and function of exosomes in cancer. J Clin Invest. 2016; 126: 1208-15.
• 17. Kahlert C, Melo SA, Protopopov A, et al. Identification of doublestranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem 2014; 289: 3869-75.
• 18. Jahr S, Hentze H, Englisch S et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apop- totic and necrotic cells. Cancer Res 2001; 61: 1659–65.
• 19. Milane L, Singh A, Mattheolabakis G, Suresh M, Amiji MM. Exosome mediated communication within the tumor microenvironment. J Control Release 2015; 219: 278-94.
• 20. Thierry AR, Mouliere F, El Messaoudi S et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med 2014; 20: 430-435.
• 21. Perrone F, Lampis A, Bertan C et al. Circulating free DNA in a screening program for early colorectal cancer detection. Tumori 2014; 100: 115-21.
• 22. Skog J, Würdinger T, van Rijn S et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 2008; 10: 1470-6.
• 23. Castellanos-Rizaldos E, Grimm DG, Tadigotla V et al. ExosomeBased Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients. Clin Cancer Res. 2018; 24: 2944-50.