KRAS,NRAS and BRAF Mutational Profile of Colorectal Cancer in a Series of Moroccan Patients

Abstract

Objectives

The present study aimed to evaluate the frequencies of KRAS, NRAS, and BRAF mutations and their possible associations with clinicopathological features in 249 Moroccan patients with colorectal cancer (CRC).

Methods

A retrospective investigation of a cohort of formalin-fixed paraffin-embedded tissues of 249 patients with CRC was screened for KRAS/NRAS/BRAF mutations using Idylla™ technology and pyrosequencing.

Results

KRAS, NRAS, and BRAF mutations were revealed in 46.6% (116/249), 5.6% (14/249), and 2.4% (6/249) of patients. KRAS exon 2 mutations were identified in 87.9% of patients (102/116). KRAS G12D and G12 C were the most frequent, at 32.8% and 12.93%, respectively. Among the patients with KRAS exon 2 wild-type (wt), 27.6% (32/116) harbored additional KRAS mutations. Concurrent KRAS mutations were identified in 9.5% (11/116); including six in codon 146 (A146P/T/V), three in codon 61 (Q61H/L/R), one in codon 12 (G12 A and Q61H), and one in codon 13 (G13D and Q61 L). Among the NRAS exon 2 wt patients, 64.3% (9/14) harbored additional NRAS mutations. Concurrent NRAS mutations were identified in 28.6% (4/14) of NRAS-mutant patients. Since 3.2% wt KRAS were identified with NRAS mutations, concomitant KRAS and NRAS mutations were identified in 2.4% (6/249) of patients. KRAS mutations were higher in the >50-year-old age-group (P = .031), and the tumor location was revealed to be significantly associated with KRAS mutations (P = .028) predominantly in left colon (27.5%) and colon (42.2%) locations. NRAS mutations were most prevalent in the left colon (42.8%) and in well-differentiated tumors (64.2%).

Conclusion

Detection of KRAS mutations, particularly the G12 C subtype, may be significant for patients with CRC and has possible therapeutic implications. However, rare KRAS concomitant mutations in CRC patients suggest that each individual may present distinct therapeutic responses. KRAS testing alongside the identification of other affected genes in the same patient will make the treatments even more personalized by contributing more accurately to the clinical decision process. Overall, early diagnosis using novel molecular techniques may improve the management of CRC by providing the most efficient therapies for Moroccan patients.

Keywords

colorectal cancer moroccan population

Introduction

Globally, colorectal cancer (CRC) is the third most prevalent cancer and the second leading cause of cancer mortality.¹ Due to the rapid rising of the global population size, aging and human economic development, the burden of CRC is predicted to increase by 2.2 million new cases and 1.1 million cancer mortalities by 2030,² and 3.2 million new incidence cases by 2040.³ The CRC incidence is higher in highly developed countries, and it is growing in middle- and low-income countries. In addition, increasing incidence of early-onset CRC is also appearing.³ CRC comprises 7.4% of all diagnosed cancer cases in the Middle East and North Africa, including Morocco, Algeria, Tunisia and Libya.⁴ When compared with sub-Saharan Africa (SSA), North Africa has the highest age-standardized incidence rates (ASR) of 8.66, while SSA has an ASR of 5.91,⁵ and an upward trend in their CRC mortality.⁶ With an incidence closer to the European data, Libya has the highest ASR for CRC, with rates of 17.5 and 17.2 for males and females, respectively, per 100,000 people.⁷ In addition, young patients with CRC are more common in North African countries, including Morocco, with a proportion of 11%.⁸

The molecular and genomic analysis of patients with CRC provides data for the mutational status of Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS) and B-Raf murine sarcoma viral oncogene homolog B1 (BRAF).⁹ These oncogenes are the most frequently mutated genes found in patients with CRC.¹⁰ It is known that mutations in the downstream KRAS, NRAS and BRAF part of the epidermal growth factor receptor (EGFR) of the RAS/RAF/MAPK pathway are important in the process of carcinogenesis.¹¹ However, these mutations are associated with anti-EGFR monoclonal antibodies therapy resistance in patients with CRC;¹⁰ which, by binding to the extracellular domain of EGFR, inhibits and nullifies cell proliferation.¹¹ Recently, the National Comprehensive Cancer Network and the American Society for Clinical Oncology have posted guidance recommending testing of exon 2 (codons 12 and 13), 3 exon (codon 59 and 61) and 4 exon (codon 117 and 146) in both genes of KRAS and NRAS, and in BRAF gene of exon 15 (codon 600) prior to anti-EGFR therapy.^11,12 These alterations can behave as prognostic-predictive and driver mutations, which makes them a notable therapeutic target. In particular, the KRAS and BRAF V600 E mutations appear to be the lack of benefit of anti-EGFR mAbs, and they are biomarkers of poor prognosis and resistance to standard therapies.¹³ Therefore, in order to help physicians correctly select the best treatment strategy, the assessment of RAS and BRAF mutational status is the main diagnostic method for patients with CRC.¹⁴

The frequency of KRAS, NRAS and BRAF mutations varies in distinct populations. The present study aimed to evaluate the frequencies of KRAS/NRAS/BRAF mutations in a series of Moroccan patients with CRC and their possible associations with clinicopathological features in the Moroccan population.

Materials and methods

Subjects Study

This investigation is a bi-centric retrospective study on a total of 249 formalin-fixed paraffin-embedded (FFPE) blocks of Moroccan patients with CRC. The patients were selected consecutively from the Department of Pathology, the Nations-Unites Pathology Center of Rabat between January 2019 and December 2021 (N = 169), and from the Laboratory of Research and Biosafety P3, Mohammed V Military Teaching Hospital of Rabat, between September 2020 and December 2021 (N = 80). The study size was determined based on the availability of Formalin-fixed paraffin-embedded (FFPE) blocks during these specified time periods. All tissue samples were extracted from primary or metastatic tumors through surgical resection or endoscopic biopsy. All tissue sections were evaluated after staining using hematoxylin/eosin (H&E) by a histopathologist. Clinicopathological data were recorded from the medical records, including: age, gender, tumor type, tumor location, and tumor grade. In this study, we had categorized CRC cases based on the location of the tumor including left colon, right colon, colon and rectum. The term colon in this context refers to cases where tumor is located in the colon not especially in the left or the right sections. Thus, this colon category is distinct from the left and right colon cases. We have followed relevant Equator guidelines, and the reporting of this study conforms to the STROBE checklist.¹⁵

This study was approved by the Ethics Committee for Biomedical Research (CERB) of the Faculty of Medicine and Pharmacy in Rabat, with approval number; CERB 36-23 and approval date; January 22, 2024. Written informed consents were signed and obtained from all participants prior to their inclusion in the study. And we have de-identified all patient details.

Molecular analysis

Idylla platform

Allele-specific real-time PCR platform Idylla™ (Biocartis, Mechelen, Belgium) was performed in 169 FFPE tissue samples. FFPE tissue sections of 10 μm thick were sampled as close as possible (within the same FFPE block) to the sections used before to generate the reference result. FFPE tissue sections were placed directly into the Idylla™ cartridge following the assay instructions. The Idylla™ KRAS Mutation Test covers 21 KRAS mutations in exons 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146). The Idylla NRAS-BRAF mutation test uses allele-specific multiplex PCR reactions to amplify 23 mutations in exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146) of the NRAS oncogene and codon 600 of the BRAF oncogene.

A set of parameters describing the generated PCR curves are determined by the Idylla software, for example, ΔCq value (calculated as the difference between the quantification cycle value (Cq) of the gene control signal and the Cq of the mutant signal). A sample is classified as mutation positive if the parameters of the PCR curve generated are within the validated range. Otherwise the sample is reported as being mutation negative, that is, wild type (wt).

Pyrosequencing

Pyrosequencing was performed in 80 FFPE specimens using Qiagen, K-Ras Pyro®kit 24. V1, Ras-Extension Pyro®kit 24. V1 and BRAF® Pyro® Kit 24. V1. The target sequence covering the polymorphic site was amplified with one of the specific biotinylated primers. Briefly, PCR was used for amplifications of a full RAS (KRAS/NRAS) region containing codon 12, codon 13, and codon 61 in KRAS and NRAS, as well as codons 600 and 464-469 in BRAF gene. The PCR assay was performed in a reaction volume of 20 μL using 12.5 μL PyroMark PCR Master Mix, 2,5 μL CoralLoad Concentrate, 10X, 1 μL PCR Primer and 4 μL Water (H2O, supplied). The PCR conditions were as follows: Initial denaturation at 95°C for 15min, followed by 40 cycles of 95°C for 20 sec, 53°C for 30s and extension at 72°C for 20 sec and then final extension at 72°C for 5min). Unmethylated control DNA was incorporated in the product as a positive control for PCR and sequencing reactions. A negative control (without template DNA) was included. We subsequently immobilized, washed, and denatured the amplified products using the vacuum workstation and subjected those products to pyrosequencing using the PyroMark Q24 system (Qiagen, PyroMark Q24 MDx V2.0), Germany).

The pyrosequencing results were analyzed using the PyroMark-Q24 version 2.0.6 software (Qiagen, PyroMark Q24 MDx (version 2.0), Germany), which identifies the presence of a specific mutation and its percentage. Manufacturer-supplied Limits of detection (LOD) thresholds were used to call a mutation for LOD studies (≥% LOD is positive). Real-time curves and programs were interpreted according to the kit instructions and PyroMark ID software (Qiagen) allowed determination of mutant allelic frequency according to relative peak height.

Quality Control Measures

Comprehensive quality control measures have been meticulously implemented to ensure the accuracy and reliability of our molecular testing on CRC samples. For the Idylla™ platform, cartridge integrity checks and compliance with ΔCq value thresholds were paramount, while pyrosequencing procedures included positive and negative controls to validate each test. Cross-validation with alternative molecular methods was performed on a subset of samples, ensuring consistency of results. Certified technicians performed all analyses, with regular equipment calibration and rigorous monitoring of reagent quality, maintaining the highest standards of scientific rigor and data integrity throughout the study. To address potential sources of bias, comprehensive quality control measures were meticulously implemented, cross-validation with alternative molecular methods was performed on a subset of samples, and statistical analyses were conducted using established thresholds and categorizations.

Statistical Analysis

The results of KRAS, NRAS and BRAF mutational status were used as categorical variables (presence or absence of the mutation). The potential associations of KRAS, NRAS and BRAF mutations with the clinicopathological features of tumor cases, including age (patients <50 years old vs patients ≥50 years old), sex, tumor location and tumor grade, were analyzed using χ2 or Fisher’s exact tests to test for statistical significance. In our analyses, age was treated as a categorical variable, with patients grouped into those younger than 50 years old and those 50 years or older. This categorization was based on the established understanding that the risk of CRC increases significantly after age 50. The ΔCq value and the percentage of a specific mutation detected by pyrosequencing were analyzed as continuous variables to capture the full range of these measurements in our sample. The ΔCq value thresholds for the Idylla™ platform were strictly adhered to as per the manufacturer’s instructions. P ≤ .05 was considered to indicate a statistically significant difference. All analyses were conducted using the SPSS (Statistical Package for the Social Sciences) version 23 (IBM Corp.) software.

Results

Clinicopathological Characteristics

A total of 249 CRC cases were studied between January 2019 and December 2021. Overall, this comprised of tissues from 62.2% (155/249) men and 37.8% (94/249) women. The mean age was 61.4 years. The most frequent tumor location type was the colon (35.7%; 89/249) and the most common histological type and tumor state were adenocarcinomas well-differentiated (49.8%; 124/249). Demographics and clinicopathological data of all patients are summarized in Table 1.

Table 1.

Demographic and Clinical Characteristics of the CRC Patients Selected.

	Total N = 249	Mutated N = 130	Wild Type N = 119
Mean age	61.43	62.83	59.9
Sex
Male	155 (62.2%)	78 (60.0%)	77 (64.7%)
Female	94 (37.8%)	52 (40.0%)	42 (35.3%)
Tumor sample type
Biopsy	156 (62.7%)	80 (61.5%)	76 (63.9%)
Surgery	93 (37.3%)	50 (38.5%)	43 (36.1%)
Tumor location
Colon	89 (35.7%)	51 (39.2%)	38 (31.9%)
Left colon	69 (27.7%)	39 (30.0%)	30 (25.2%)
Right colon	35 (14.1%)	16 (12.3%)	19 (16.0%)
Rectum	56 (22.5%)	24 (18.5%)	32 (26.9%)
Tumor grade
ADK poorly differentiated	12 (4.8%)	2 (1.5%)	10 (8.4%)
ADK moderately differentiated	113 (45.4%)	65 (50.0%)	48 (40.3%)
ADK well differentiated	124 (49.8%)	63 (48.5%)	61 (51.3%)

Mutations frequency

The full RAS and BRAF gene mutations for 249 patients with CRC (169 patients using Idylla™ platform and 80 with pyrosequencing) were screened. Out of the 169 patients who were screened by Idylla™ platform, full RAS gene mutations were detected in 46.1% (78/169) with 42% (71/169) in KRAS, and 4.1% (7/169) in NRAS, and BRAF mutations were present in 3.5% (6/169). Out of the 80 patients who were screened by pyrosequencing, RAS mutations were identified in 57.5% (46/80) with 56.2% (45/80) in KRAS, and 8.8% (7/80) in NRAS) and no detected mutations in BRAF gene.

Overall, full RAS gene mutations were detected in 49.8% (124/249) of tumor cases examined; concurrent mutations were identified in 2.4% (6/249) between KRAS and NRAS genes. In the KRAS gene, the total mutation rate was 46.6% (116/249), while the exon 2 mutation rate was 87.9% (102/116). Exon 2 codons 12 and 13 constituted 69.0% (80/116) and 19.0% (22/116) of all mutations, respectively. Mutations identified in codon 12 included G12D (32.8%; 38/116), G12 C (12.9%; 15/116), G12 V (10.3%; 12/116), G12S (6.9%; 8/116), G12 A (4.3%; 5/116) and G12 R (1.7%; 2/116). Mutations in codon 13 included G13D (19.0%; 22/116).

Outside exon 2, the mutation rate of exon 3 was 8.6% (10/116); including Q61H (3.4%; 4/116), Q61 L (2.6%; 3/116) and Q61 R (2.6%; 3/116) at codon 61, while no mutation was detected at codon 59.

In exon 4, the mutation rate was 19.0% (22/116) within K117 N at codon 117 (1.7%; 2/116) and at codon 146 (17.2%; 20/116) including A146 T (6.9%; 8/116), A146P (5.2%; 6/116) and A146 V (5.2%; 6/116). The rate of concurrent KRAS mutations was 9.5 % (11/116); including six in codon 146 (A146P/T/V), three in codon 61 (Q61H/L/R), one in codon 12 (G12 A and Q61H) and one in codon 13 (G13D and Q61 L).

In the NRAS gene, the total mutation rate was 5.6% (14/249), while the mutation rate of exon 2 was 64.3% (9/14). Exon 2 codons 12 and 13 constituted 14.3% (2/14) and 50% (7/14) of all mutations, respectively. Mutations identified in codon 12 included G12D (7.1%; 1/14) and G12 R (7.1%; 1/14). Mutations in codon 13 included G13 A (21.4%; 3/14), G13 R (14.3%; 2/14) and G13 V (14.3%; 2/14). Outside exon 2, the mutation rates of exon 3 were 50% (7/14) at codon 61, including Q61H (28.6%; 4/14), Q61 K (14.3%; 2/14) and Q61 L (7.1%; 1/14), while no mutation was detected at codon 59. The mutation rate in exon 4 included K117 N 14.3% (2/14) at codon 117. Concurrent NRAS mutations were identified in 28.6% (4/14); two in codon 13 (G13 R/V) and two in codon 117 (K117 N and G13 A). Since 3.2% (8/249) of wt KRAS were identified with a mutation in the NRAS gene, concurrent mutations were identified between KRAS and NRAS genes (2.4%; 6/249).

BRAF mutations were present in 2.4% (6/249) of wt patients and the patients with BRAF mutations harbored V600 E mutation in exon 15. The full RAS gene mutation frequencies and subtype distributions are detailed in Table 2.

Table 2.

Mutations by Location and Type in KRAS and NRAS Genes.

Gene Location	No. Mutated Cases per Gene
Exon 2	Codon	KRAS (n = 116, %)	NRAS (n = 14, %)
		102 (87.93)	9 (64.28)
	Codon 12	80 (68.96)	2 (14.28)
	G12 V	12 (10.34)	0 (0)
	G12D	38 (32.75)	1 (7.14)
	G12 C	15 (12.93)	0 (0)
	G12 A	5 (4.31)	0 (0)
	G12S	8 (6.89)	0 (0)
	G12 R	2 (1.72)	1 (7.14)
	Codon 13	22 (18.96)	7 (50)
	G13 A	0 (0)	3 (21.42)
	G13D	22 (18.96)	0 (0)
	G13 R	0 (0)	2 (14.28)
	G13 V	0 (0)	2 (14.28)
3		10 (8.62)	7 (50)
	Codon 61	10 (8.62)	7 (50)
	Q61H	4 (3.44)	4 (28.57)
	Q61 K	0 (0)	2 (14.28)
	Q61 L	3 (2.58)	1 (7.14)
	Q61 R	3 (2.58)	0 (0)
4		22 (18.96)	2 (14.28)
	Codon 117	2 (1.72)	2 (14.28)
	K117 N	2 (1.72)	2 (14.28)
	Codon 146	20 (17.24)	0 (0)
	A146P	6 (5.17)	0 (0)
	A146 T	8 (6.89)	0 (0)
	A146 V	6 (5.17)	0 (0)

Correlations between KRAS/NRAS/BRAF mutations and clinicopathological features

The correlations between the molecular features at KRAS, NRAS and BRAF genes in association with the clinicopathological characteristics of patients are presented in Table 3. Compared with <50 year-old patients, KRAS mutations in ≥50-year-old patients were more common (11 vs 89%; P = .031). The tumor location was revealed to be significantly associated with KRAS mutations (P = .028) predominantly in the left colon (27.5%) and colon (42.2%) locations.

Table 3.

Associations Between Genetic and Clinicpathological Features.

	KRAS		NRAS		BRAF		WT
	N(%) 116	P value	N(%) 14	P value	N(%) 6	P value	N(%) 119	P value
Age. Median	62.36		67.71		67		59.91
Age. Range		.031		.885		.946		.062
<50	12 (10.3)		2 (14.2)		1 (16.6)		24 (20.1)
≥50	104 (89.6)		12 (85.7)		5 (83.3)		95 (79.8)
Sex (n = 249)		.402		.686		.822		.446
Female	47 (40.5)		6 (42.8)		2 (33.3)		42 (35.2)
Male	69 (59.4)		8 (57.1)		4 (66.6)		77 (64.7)
Tumor location		.028		.518		.395		.59
Colon	49 (42.2)		3 (21.4)		2 (33.3)		38 (31.9)
Left colon	32 (27.5)		6 (42.8)		3 (50)		30 (25.2)
Right colon	14 (12.0)		1 (7.14)		1 (16.6)		19 (15.9)
Rectum	21 (18.1)		4 (28.5)				32 (26.8)
Tumor sample		.860		.663		.519		.706
Biopsy	72 (62.0)		8 (57.1)		3 (50)		76 (63.8)
Surgery	44 (37.9)		6 (42.8)		3 (50)		43 (36.1)
Tumor grade		.859		.426		.833		.586
ADK poorly differentiated	1 (.86)		1 (7.14)				10 (8.40)
ADK moderately differentiated	61 (52.6)		4 (28.5)		3 (50)		48 (40.3)
ADK well differentiated	54 (46.5)		9 (64.2)		3 (50)		61 (51.2)

The KRAS mutation frequency of colon location tumors is higher (42.2%, 49/116) than that of rectum location tumors (17.2%, 20/116). The NRAS mutation frequency of colon is lower (21.4%, 3/14) than that of rectum location (28.5%, 4/14). However, there is no significant difference for this comparison.

The NRAS mutated specimens were more frequently found in the left colon 42.8% (6/14) and in well-differentiated tumors 64.2% (9/14), but with no statistically significant difference. No significant differences in clinicopathological features were observed between NRAS-mutated tumors and wt tumors of all patients with CRC in the current study. Regarding BRAF analysis, there was no significant difference.

Discussion

The oncogenes RAS and BRAF are frequently mutated in CRC and have been associated with therapy resistance in patients with CRC.¹⁶ In a large cohort of patients with CRC in Morocco, the current study revealed the mutation frequencies of KRAS, NRAS and BRAF to be 46.6, 5.6 and 2.4% of patients, respectively. Overall, combined mutational analysis of KRAS, NRAS and BRAF was able to identify 54.6% of patients with CRC as likely non-responders to anti-EGFR therapy.

By comparing the KRAS mutation prevalence in different countries, the KRAS mutation rate among Americans (40.0%) is the highest, followed by Europeans (36.0%), then Asians (24.0%),¹⁷ and Africans (21%).¹⁸ Moreover, the prevalence of KRAS mutations among patients with CRC with primary tumors in the region of the East Mediterranean, Europe, the Americas, Southeast Asia and the West Pacific was 30.23, 35.12, 31.83, 33.17 and 32.64%, respectively. Corresponding rates for mutation among metastatic cases were 42.20, 38.46, 36.6, 42.80 and 33.05%, respectively.¹⁹ However, the overall frequency (46.6%) revealed in the present study was close to or higher compared with that observed in the different countries. Various factors could impact this difference in KRAS mutation status, including the percentage of tumor cells found in the formalin-fixed paraffin-embedded tissue samples, the extracted DNA quality, the testing methods and the testing target as well as different molecular pathogenetic mechanisms and environmental exposures.

Rates or spectrum of KRAS mutation are influenced not only by race/ethnicity,^20,21 but also by regional factors. For instance, Africans have a 21% frequency of CRCs with KRAS mutations,²² a higher detection rate of KRAS mutation (54.4%) in Europeans CRC patients, while Asians predominantly have wt KRAS (51.4%) in CRC patients.²³ A study on mCRC patients in Jordan showed a distinct mutational profile with a higher frequency of KRAS (83.69%) and NRAS (6%) mutations compared to Western populations, suggesting an ethnic influence.²⁴

Within the same population, the prevalence of KRAS mutation can also vary. In Japan, mutation frequencies ranged from 9%-71% across different ethnic groups.^25,26 Similarly, in U.S., non-Hispanic Black (48%) and Hispanic patients (44%) had a higher KRAS mutation rate compared to non-Hispanic White (39%) and Asian or Pacific Islander patients (37%).²⁷ Assessing the impact of KRAS mutation within each race/ethnic group revealed a 7% increased survival risk for Hispanic whites and a 15% increased risk for non-Hispanic blacks.²⁷

Differences in mutational profiles across ethnic groups may be influenced by unique genetic factors, potentially affecting disease management and testing standards. Hence, ethnicity plays a crucial role in influencing the mutational profile of CRC.

In previous Moroccan studies, the KRAS mutation rate has been reported to be in the range of 23.9%–51%,^28–33 which is close to the findings of the present study. However, in neighboring countries, the reported KRAS frequency in patients with CRC varies from 31.5%–86.6% in the Tunisian population,^34–37 and 31.4%–50.0% in the Algerian population.^38,39 In the Libyan population, the KRAS mutation frequency has been identified to be 38.2% of patients with CRC.⁴⁰ This epidemiological variation indicates the importance of gathering local CRC KRAS mutation data in different North African populations.

KRAS mutations are often found in exons 2, 3 and 4, with exon 2 mutations being the most common, accounting for 93%–96% of all KRAS mutations.^24,41,42 Similarly, in the present study, 87.9% (102/116) of KRAS mutations were in exon 2; whereas 69% (80/116) of the mutations were detected in codon 12 and 19% (22/116) in codon 13. Outside exon 2, mutation detection rates in exon 3 and exon 4 were 8.6 and 19.0%, respectively. The distribution of KRAS mutations in codon 12 and 13 varies in different studies. The current study revealed that the most frequent amino acid substitution in codon 12 was G12D (32.8%), then G12 C (12.9%) and G12 V (10.3%), which is in accordance with the Catalogue of Somatic Mutations in Cancer (>90%).⁴³ Similarly to the present study, in North Africa, G12D and G12 C have been revealed in 31.6 and 10.2%, respectively, of all KRAS mutations in patients with CRC.⁴⁴ In addition, the G12D mutation is estimated to impact 180,000 patients in the U.S. and Europe, and occurs in 12% of patients with CRC, with ∼80,000 patients in the U.S. and Europe,⁴⁵ while G12 C is estimated to impact >70,000 patients in the U.S. and Europe and occurs in 3%–4% of patients with CRC, with ∼20,000 patients in the U.S. and Europe.^45,46

The KRAS mutation spectrum reported in CRC vary widely among populations. This variability in results may be due to the high sensitivity of the RAS mutation analysis used in recent studies compared with previous studies. More sensitive tests can detect mutations <1%. Additionally, the majority of previous studies have reported mutations only in KRAS exon 2 codons 12 and 13. It is important to note that ∼20% of patients with CRC whose wt test based on KRAS exon 2 analyses may actually harbor undetected extended KRAS mutations in exons 3 and 4.^20,21

The treatment of CRC has evolved to include a combination of surgery, radiation, chemotherapy, and more recently, targeted therapies. A significant advancement in this field is the identification of the KRAS G12 C mutation as a druggable target in CRC. The Code Break 100 trial has underscored the potential of targeted therapies by revealing the potent antitumor effects of Sotorasib (AMG510), a novel KRAS G12 C inhibitor, against KRAS G12C-mutant solid tumors, including metastatic CRC. Furthermore, Phase 1/2 clinical trials have shown that Adagrasib (MRTX849), another KRAS G12 C inhibitor, when combined with Cetuximab, an EGFR inhibitor, exhibits greater anti-tumor activity in CRC patients, with an objective response rate of 43%–46%.^47–49 These findings highlight the implications of extended mutational profile testing in CRC, paving the way for more personalized treatment options and improved patient outcomes.

In our study, concurrent KRAS mutations were identified in 9.5 % (11/116); including six in codon 146 (A146P/T/V), three in codon 61 (Q61H/L/R), one in codon 12 (G12 A and Q61H) and one in codon 13 (G13D and Q61 L), this revealed the presence of rare KRAS concomitant mutations in CRC patients. The presence of concomitant mutations may affect the response to targeted therapies. Further investigation on the importance of these concomitant KRAS mutations in CRC patients’ prognosis and treatment response is warranted.

NRAS mutations are found in 5%–10% of patients with CRC. NRAS is mutated in the same codon of KRAS, particularly in exon 2 (found in 3%–5% of patients with CRC) and in exon 3 (2%–6% of patients with CRC). Therefore, these positions are hot-spot mutations. In the present study, the total NRAS mutation rate was 11.0%, with a prominence of mutation rates of exon 2 that represented 64.3%, while the mutation frequency of exon 3 was 50.0% of all mutated NRAS. The present results demonstrated that the frequency of NRAS mutations was slightly higher compared with that previously reported in the Moroccan population. In addition, NRAS exon 4 mutation in CRC appears to be a rare event with a frequency of <.2%,⁵⁰ while the current study demonstrated that 14.3% patients carried a mutation in the NRAS exon 4. Moreover, 8/124 (6.4%) of patients with CRC had a KRAS wt mutation, which was identified with mutation in the NRAS gene.

The present results indicated that analysis of NRAS mutations is necessary in patients with CRC and the KRAS wt gene. These findings suggested that extended RAS mutation testing, including KRAS exon 3 and 4 and NRAS exon 2, 3 and 4, should be undertaken before the administration of anti-EGFR mAbs in the Moroccan population. Coexistence of KRAS and NRAS gene mutations was revealed in 6/124 (4.8%) of patients with CRC. The fact that >1 mutated gene can coexist in the same tumor potentially reflects that multiclonal presentation leads to intratumor heterogeneity. Therefore, instead of assessing single gene alterations from MAPK signaling, assessing co-occurrence of mutations within ≥1 of those genes should also be performed. This may impact future treatments and should be considered in the diagnostic workflow.⁵¹

The BRAF wt is also required for response to cetuximab or panitumumab, suggesting that BRAF analysis should be used with KRAS for the selection of the patients with CRC. BRAF mutations have been found in 8%–15% of patients with CRC.⁵² The T1779 A point mutation in BRAF exon 15, which results in a V600 E amino acid substitution, is the most common mutation and accounts for >90% of all mutations found in the BRAF gene.⁵³ In the present study, BRAF mutations were revealed in 2.4% of patients with CRC. The V600 E mutation was identified in all patients with the BRAF mutant, and all patients with the V600 E BRAF mutation possessed a wt KRAS genotype. Based on these results, it was hypothesized that BRAF mutations were exclusively present in patients with wt KRAS.

Previously, in Moroccan studies, the BRAF V600 E mutation incidence has been reported to be in the range of 3.9%–5.0%.^30,33 The frequency of the BRAF V600 E mutation in CRC tumors has been variable in different studies, ranging from 1%–22% and from a lower prevalence in certain Asian populations to a higher prevalence in certain Western countries.^54,55 This difference may be partially due to the fact that every patient is screened for KRAS, but only those that have KRAS wt are screened for BRAF, since KRAS and BRAF mutations are generally mutually exclusive.⁵⁶ These findings demonstrate that the incidence of important oncogenic mutations can vary in populations of different ancestral backgrounds. Whether and to what degree these variants have an impact on incidence, response to treatment or survival is yet to be determined.⁵⁷

In the present study, there was no significant difference in the clinicopathological features between patients with KRAS mutant and KRAS wt, except for the age of the patients, where KRAS mutation rates were higher in patients >50 years old compared with those ≤50 years old (P = .031), and in tumor location (P = .028). Comparing the present results with existing literature, previous studies demonstrated the same results;⁵⁸ while others indicate that the mutation rate of the KRAS gene is not associated with clinicopathological factors such as sex, age, degree of differentiation, tumor location and type of specimen. In the present study, no significant differences in clinicopathological features were observed between NRAS and BRAF mutated tumors and wt tumors in all patients with CRC.

The limitations of our study encompass a variety of factors. These include the participant group size, the study’s retrospective nature, and reliance on available FFPE blocks. The study also acknowledges potential selection and detection bias, the latter arising from the use of the Idylla™ platform and pyrosequencing, which may not detect all possible mutations. Information bias could also be present due to potential errors or omissions in the clinicopathological data recorded from medical records, and the binary categorization of age. Importantly, the study also highlights that full RAS and BRAF mutations, which are clinically relevant, are not consistently screened across various clinical institutions.

Furthermore, the study’s findings may lack generalizability as it is based on Moroccan patients with CRC. The suggested gene mutation testing strategy, developed based on clinicopathological differences such as sex, age, degree of differentiation, tumor type histology, tumor location, tumor stage, and type of specimen, is not routinely screened in several clinical institutions. Lastly, the sample size, determined by the availability of FFPE blocks, might not represent the broader CRC patient population. These limitations highlight the need for further large-scale studies.

Conclusion

In conclusion, the identification of KRAS, NRAS, and BRAF mutation frequencies in CRC patients has enhanced our understanding of CRC’s molecular characteristics and emphasized the need for more molecular studies to refine treatment strategies. The detection of KRAS mutations, especially the G12 C subtype, is significant for CRC treatment. These findings may support personalized medicine, where specific mutations inform tailored treatment plans, potentially improving outcomes and minimizing side effects. For example, patients with the G12 C mutation may benefit from targeted therapies using the KRAS G12 C inhibitors such as Sotorasib or Adagrasib. The use of novel molecular techniques for early diagnosis may transform CRC management, allowing for the implementation of the most effective therapies at an earlier disease stage. This is particularly crucial for Moroccan CRC patients, where such advancements may significantly improve prognosis and quality of life.

Therefore, the present study revealed the presence of rare KRAS concomitant mutations in CRC patients, which suggests that each individual may present distinct therapeutic vulnerabilities depending on their tumor’s mutational spectrum. KRAS testing alongside the identification of other affected genes in the same patient will make the treatments even more personalized by contributing more accurately to the clinical decision process.

Footnotes

Acknowledgments

This study was partially supported by the team of the Department of Pathology, Nations- Unites Pathology Center, the Laboratory of Research and Biosafety P3 and the Department of Pathology, Mohammed V Military Teaching Hospital, whom we thank a lot.

Author contributions

SElZ, AL, MG, Designed, Conceived the study, wrote the manuscript, made the analysis and the interpretation of the data. AB, TB, SB, MJ, WB, HElA, RA, MRT, SElK, contributed to the collection of samples. FK, YS, MO, IAL, RAElH, KE, confirmed and validated the authenticity of all the data.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical Statement

ORCID iDs

Sara El Zaitouni

Soukaina Benmokhtar

Data of Availability Statement

The data involving participants or patients cannot be publicly shared. Individual requests for further information on the study can be sent to the corresponding author.*

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