The Effect of KRAS and NRAS Variants on the Prognosis of Colorectal Cancer among the Western Saudi Population

Colorectal Cancer (CRC) is one of the most common cancer with high mortality rate globally and the second leading cancer in Saudi Arabia. RAS oncogenes play critical roles in the regulation of the cellular function and any mutation in these genes leads to develop CRC. Therefore, we identified the most common mutations in KRAS and NRAS genes by applying next generation sequencing (NGS) then, we assessed the correlation between these mutations and the clinicopathological features. KRAS-mutated carcinoma was significantly associated in patients who were older than 60 years old (83.3% vs 16.7%, P = 0.039) and it was associated with female patients as well, (83.3% vs 30%, P = 0.039). Also, KRAS-mutated carcinoma was significantly associated with mucinous differentiation (85.7% vs 14.3%; P = 0.012) and higher tumor grade (P = 0.014). In addition, the number of KRAS mutations per case was significantly associated with depth of the invasion (p = 0.049). The most common mutation was a missense mutation and it was highly associated with age and gender (both, p = 0.039). Also, it was highly associated with tumor grade and with mucinous differentiation (p = 0.014, p = 0.012), respectively. On the other hand, NRAS mutated carcinoma was associated only with distant metastasis however, this association was not significant (p = 0.064). For overall survival, KRAS-mutated carcinomas had a significantly worse overall survival (p = 0.025). While, no significant association was between NRAS mutation and overall survival (p = 0.985). We believe that KRAS and NRAS genes can be prognostic factors for CRC patients and the information obtained may contribute for better diagnosis and therapeutic effect.


Introduction
Colorectal Cancer (CRC) is the second leading cancer type of the estimated number of new cancer cases in 2016 in Saudi Arabis (12.6%) (Saudi Cancer registry, 2016) for both genders. According to the American Cancer Society, CRC remains one of the main causes of high rate of cancer related death in the world, regardless of the significant improvement in the prevention, screening and therapy. CRC arises due to the transformation of the normal colonic epithelium as a result of the alteration in the genetics and the epigenetics of these cells which promotes the tumor growth. The progression of the molecular level in the tumor cells will cause genetic instability and genetics defect which leads to the development of sporadic CRC. The incidence rate and the mortality of CRC differ based in etiological factors such as age, gender, genetic background and environmental factors [1] [2] [3] [4].
Approximately, 60% of CRC caries mutations in the RAS oncogene, either KRAS, NRAS or both. KRAS and NRAS play an important role in regulating many cellular functions including cellular proliferation, apoptosis, migrations and differentiation [5] [6]. The most commonly reported mutation in the KRAS gene is located on exon 2 and consequently changes the codons 12 and codon 13.
Also, another frequently reported mutation in codon 16 and 146 have been reported, however, it has lower effect on the development of the tumor cells. On the other hand, the most commonly reported mutation in the NRAS gene is located in codon 61 and 12 [7] [8]. Activating mutations in the KRAS gene has been found to be predictor of response to EGFR-targeted therapies, therefore, examining the KRAS and NRAS mutations are an essential step in clinical diagnosis and proper treatment.
Most tumors are formed from clones that carry different mutations which are called Tumor Heterogeneity. This heterogeneity has significant impact on cancer development and the response to a treatment. Since next generation sequencing becomes applicable, these genetic variations or tumor heterogeneity, can be clearly identified. Also, it can reveal any co-existing mutations in different genes [6] [9]. While defining the risk mutations and treatment approach, much needs to be done to correlate these mutations with the clinicopathological outcomes.
There are discrepancies in the literature regarding prognosis of KRAS mutation in colorectal cancer patients and the reports are contradictory.
To the best of our knowledge, no data has been published before concerning the effect of KRAS and NRAS mutations and their correlations with the clinicopathological variables and overall survival in Saudi patients with colon cancer.
Therefore, and to address the major gap in our knowledge of the different roles of these genetic variations and how they correlate with the pathological findings clinically, we aimed to identify the risk molecular variants and correlate them with the clinicopathological characteristics in Saudi patients with CRC. In addition, we assessed the role of these correlations in determining the prognosis, treatment personalization and overall survival.

Tumor Dissection and DNA Extraction
After evaluating the cases who were eligible for the current study, we included All the 16 cases were fulfilled the recommended DNA quality required for the molecular analysis.

Next Generation Sequencing
For all 16 cases, 20ng of DNA extracted was prepared for sequencing. The as-

Detection of Variations
The sequenced reads were arranged and compared to the hg19/GRCh37 reference sequence and analyzed using the MiSeq reporter (Illumina

Statistical Analysis
Association between KRAS and NRAS gene polymorphism and the clinicopa-

Patients Characteristics
The details of clinicopathological features of the colon cancer cases who are included in the present study are summarized in Table 1

Mutation Profiles
While KRAS and NRAS mutations were identified 8/16 (50%) and 4/16 (25%) respectively of the tumor, all the patients in this study group had mutation in p53 gene (100%). Moreover, 5 patients were found to carry multiple mutations, including patients with co-existence of KRAS and/or NRAS genes mutations. One patient had a mutation in EGFR gene and another patient had a mutation in the PIK3CA gene. One of the patients had multiple mutations in the following genes: AKT1, NRAS, PDGFRA, EGFR, c-KIT, ERBB2, c-MET and TP53.

Correlation of KRAS and NRAS Mutations with Clinicopathological Variables
The frequency and the association of clinicopathological features of patients who harbored the mutant KRAS and NRAS tumors are shown in Table 2.
KRAS-mutated carcinomas were significantly more common in patients who  of invasion, and lymph node invasion were not significant predictors for NRAS mutational status.
We also noted that KRAS-mutated carcinomas were more common in the proximal colon. However, this association was not statistically significantly (63.6% vs 36.4%, P < 0.106). Besides that, NRAS mutations was found to be associated with distant metastasis but this association did not differ significantly between NRAS-mutated and NRAS-wild type carcinomas. (p = 0.064).

Type and Frequencies of KRAS and NRAS Gene Mutations
The type of the KRAS and NRAS gene mutations are presented in detail in Table   3. Eight cases were found to harbor at least one mutation in the KRAS gene. On the other hand, three cases with at least one mutation in the NRAS gene were detected. We found one case that had mutations in both KRAS and NRAS genes.
For the KRAS gene, the most common mutation in our study group was a missense mutation (87.5%) then synonymous mutation (12.5%). While, for the NRAS gene, the most common mutation is missense mutation (75%) then stop-gained mutation (25%).

Association of KRAS Polymorphisms with the Clinicopathological Features in Colon Cancer Cases
To study the association of KRAS polymorphism on colon cancer, we applied Pearson Chi-Square tests with different clinicopathological variables. The number of mutations per case was significantly associated with depth of the invasion (p = 0.049). The most common mutation in our study group was a missense mutation. A missense mutation was highly associated with age and gender (both,

Correlation of Mutations with Overall Survival
The mean follow-up time was 48.9 ± 16.3 months (19 -60 months). KRAS-mutated carcinomas had a significantly worse overall survival (p = 0.025) (Figure 1(a)). In addition, we found significant correlation between the number of mutations in KRAS in each case and the overall survival. Cases with high number of KRAS mutations had significantly the worst overall survival (p < 0.0001) (Figure 1(b)).
On the other hand, no significant association has been detected between NRAS mutation and overall survival (p = 0.985) (Figure 1(c)) in our study group.

Discussion
In this study, we investigated 16 patients with CRC by applying next generation sequencing in the population of the western region of the Saudi Arabia. Due to the main aim of the study, we focused on both genes KRAS and NRAS as prognostic factors. Furthermore, we assessed the association between these KRAS and NRAS mutations with the clinicopathological features which showed significant correlations. It is worth noting that, all the cases were muted for the P53 gene. For the KRAS mutation, the frequency was 50% of our study group. Several reports, including reports from Saudi, showed that the incidence of KRAS mutation ranged from 28.1% to 56% [10] [11] [12] [13]. This is consistent with the frequency of the KRAS mutation in our study [12] [14]. For NRAS mutation, the frequency in our study group was 25% of patients which was higher comparing the ranges of the previous reports (2.2% -7%) [14] [15]. Regardless of the small sample size in our study group, these finding are consistent due to the high accuracy of the next generation sequencing.
In this study, we found that KRAS-mutated carcinoma was significantly more  [25].
Concerning prognosis, we found that KRAS-mutated carcinomas had a significantly worse overall survival which is consistent with other published data [13] [20] [26]. On the other hand, we could not detect any correlation between NRAS mutation and overall survival.
The present study is believed to be the first to provide information on frequency and types of KRAS, NRAS, and p53 mutations in Saudi patients with CRC. However, it has some limitations although it is the first to examine the effect of the of KRAS, NRAS and P53 genes mutations on the clinicopathological findings of CRC in Saud population. The small sample size may limit the findings. This is due to the lack of funding. In addition, it is a retrospective research and selection bias inevitably exists, as well as the limited access to the data for prognosis. Given our limited sample size and data, we acknowledge that these observations are speculative. Additional studies are required to understand the correlation of the mutations in these three genes (KRAS, NRAS, and p53) with the development of CRC for better diagnosis and improve the therapeutic effect.