Clinical significance of frequent somatic mutations detected by high-throughput targeted sequencing in archived colorectal cancer samples

Journal of Translational Medicine Dallol et al. J Transl Med (2016) 14:118 DOI 10.1186/s12967-016-0878-9 Open Access RESEARCH Clinical significance of frequent somatic mutations detected by high‑throughput targeted sequencing in archived colorectal cancer samples Ashraf Dallol1,2*, Abdelbaset Buhmeida2, Mahmoud Shaheen Al‑Ahwal3,7, Jaudah Al‑Maghrabi4, Osama Bajouh1,5, Shadi Al‑Khayyat3, Rania Alam6, Atlal Abusanad3, Rola Turki5, Aisha Elaimi1,6, Hani A. Alhadrami1,6, Mohammed Abuzenadah1,6, Huda Banni1, Mohammed H. Al‑Qahtani2 and Adel M. Abuzenadah1,2,6 Abstract Background: Colorectal cancer (CRC) is a heterogeneous disease with different molecular characteristics associated with many variables such as the sites from which the tumors originate or the presence or absence of chromosomal instability. Identification of such variables, particularly mutational hotspots, often carries a significant diagnostic and/ or prognostic value that could ultimately affect the therapeutic outcome. Methods: High-throughput mutational analysis of 99 CRC formalin-fixed and paraffin-embedded (FFPE) cases was performed using the Cancer Hotspots Panel (CHP) v2 on the Ion Torrent™ platform. Correlation with survival and other Clinicopathological parameters was performed using Fisher’s exact test and Kaplan–Meier curve analysis. Results: Targeted sequencing lead to the identification of frequent mutations in TP53 (65 %), APC (36 %), KRAS (35 %), PIK3CA (19 %), PTEN (13 %), EGFR (11 %), SMAD4 (11 %), and FBXW7 (7 %). Other genes harbored mutations at lower frequency. EGFR mutations were relatively frequent and significantly associated with young age of onset (p = 0.028). Additionally, EGFR or PIK3CA mutations were a marker for poor disease-specific survival in our cohort (p = 0.009 and p = 0.032, respectively). Interestingly, KRAS or PIK3CA mutations were significantly associated with poor disease-specific survival in cases with wild-type TP53 (p = 0.001 and p = 0.02, respectively). Conclusions: Frequent EGFR mutations in this cohort as well as the differential prognostic potential of KRAS and PIK3CA in the presence or absence of detectable TP53 mutations may serve as novel prognostic tools for CRC in patients from the Kingdom of Saudi Arabia. Such findings could help in the clinical decision-making regarding thera‑ peutic intervention for individual patients and provide better diagnosis or prognosis in this locality. Keywords: Colon cancer, Next-generation sequencing, Mutational hotspots, Somatic, Hotspots Background Colorectal cancer (CRC) is major cause of morbidity and mortality around the world being the third most *Correspondence: 1 KACST Technology Innovation Center in Personalized Medicine, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Kingdom of Saudi Arabia Full list of author information is available at the end of the article common cancer type worldwide [1]. Localized statistics show that the age-standardized incidence rates (per 100,000) of CRC in KSA vary between 9.5 in females to 14.1 in males being the most common cancer type in Saudi males [1]. CRC is a heterogeneous disease affected by genetic and epigenetic variations acting as passengers or drivers of the tumor. However, common genetic features of CRC have emerged including © 2016 Dallol et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dallol et al. J Transl Med (2016) 14:118 Page 2 of 9 mutations affecting APC [2], activating mutations of KRAS or BRAF oncogenes [3], deletions of the 18q [4] and 17p [5] chromosomal regions, microsatellite instability (MSI) [6] with deleterious mutations affecting the tumor suppressor genes TP53 [7]. In terms of methylation, the CpG Island Methylator Phenotype (CIMP) pathway is the second most common pathway in sporadic CRC [8]. In terms of the application of precision medicine and personalized oncology, it is important to identify underlying variations as individually or in combination as such understanding can potentially affect treatment. For example, CRC tumors with high levels of chromosomal instability have a poor prognosis, especially if they are in stage II or III [9]. Conversely, tumors with high microsatellite instability have a better clinical outcome compared to microsatellite-stable tumors [6]. CIMP-positive CRC tumors are usually associated with the proximal colon of older females and often accompanied by BRAF mutations [10]. Male CRC patients who are CIMP negative and carry a polycomb target genes methylation signature have a favorable prognosis [11]. In terms of genetic mutations, KRAS mutations adversely affect patients’ response with anti-EGFR treatment modalities [3]. Furthermore, mutations in the EGFR itself may cause unpredictable responses to such treatments [3]. Mutations in the PIK3CA or BRAF downstream of EGFR signaling may also adversely affect treatment response [3]. We have used the cancer hotspot panel version 2 from Life Technologies in combination with the Ion Torrent personal genome platform in order to investigate the mutational status of 2800 COSMIC (catalogue of somatic mutations in cancer) mutations in 50 oncogenes and tumor suppressor genes in a cohort of CRC cases from Saudi Arabia. The results obtained will help us understand the genetic background of CRC from this population and help implement relevant modalities of precision medicine to the treatment of this disease. The pertinent clinicopathological data (gender, age, grade, and lymph node status), and follow-up results were retrieved from the patients’ records after obtaining the relevant ethical approvals. DNA was extracted from 10 μm-thin formalin-fixed paraffin-embedded slices using the Qiagen QIAMP Formalin-fixed Parafinembedded Tissue DNA extraction kit, following the manufacturer’s guidelines. Methods All statistical tests were performed using IBM SPSS Statistics version 19. Fisher’s exact test was used to identify statistical significance of correlation between mutational events and clinicopathological factors. The primary endpoints of the study included disease-specific survival (DSS) calculated from the date of diagnosis to the last recorded date of being alive or death caused by CRC. In calculating DSS, patients who died of other or unknown causes were excluded. All survival times were calculated by univariate Kaplan–Meier analysis, and equality of the survival functions between the strata was tested by log-rank (Mantel-Cox) test. Multivar (...truncated)