The Relationship Between Kirsten Rat Sarcoma Mutations and Mismatch Repair Status in Colorectal Cancer Patients: A Preliminary Study

Abstract

Background: Chromosomal instability (CIN) is a key pathway in colorectal cancer (CRC) tumorigenesis, occurring in 80%-85% of cases and leading to aneuploidy. CIN tumors often harbor mutations in APC, KRAS, and TP53, which drive tumor progression. Another crucial pathway, microsatellite instability (MSI), results from DNA mismatch repair (MMR) defects, leading to a hypermutable phenotype. MSI-CRC has distinct clinicopathologic features and a better prognosis than proficient MMR (pMMR) CRC. KRAS mutations, found in approximately 40% of CRC cases, drive tumor progression via the RAS/RAF/MAPK pathway. This study investigates the relationship between KRAS mutations and MMR status in CRC patients.

Methods: A preliminary cross-sectional study was conducted on CRC patients. KRAS mutations were analyzed using PCR, while MMR status was determined via immunohistochemistry and microsatellite testing. Clinical variables, including cancer stage, tumor category, and location, were analyzed.

Results: Among 55 patients, 91.3% had pMMR and 8.7% had deficient MMR (dMMR). KRAS mutations were more frequent in pMMR patients (80% of cases). Patients with pMMR and KRAS mutations exhibited aggressive tumors but responded to conventional therapy, whereas dMMR patients with KRAS mutations showed complex molecular profiles, potentially benefiting from immunotherapy.

Conclusion: KRAS mutations are primarily associated with pMMR in CRC, indicating distinct molecular pathways. Personalized treatment strategies should consider MMR status and KRAS mutations to optimize therapeutic outcomes. Further research is needed to explore their clinical implications.

Introduction

Colorectal cancer (CRC) is a major global health concern, with approximately 1.9 million new cases and nearly 1 million deaths annually [1]. In 2020, CRC accounted for 10% of all new cancer diagnoses worldwide. [2]. In Indonesia, CRC ranks among the most prevalent cancers, with 34,189 cases reported, [3, 4] highlighting the need for improved screening, treatment, and management strategies. Given its high incidence, understanding the molecular mechanisms underlying CRC is essential for optimizing patient outcomes [5].

CRC development is a complex, multifactorial process influenced by genetic and environmental factors [6-8]. It often progresses through the adenoma-carcinoma sequence, with key genetic alterations in APC, KRAS, TP53, and DCC playing crucial roles [1]. The two primary molecular pathways in CRC tumorigenesis are chromosomal instability (CIN) and microsatellite instability (MSI) [9]. CIN, present in 80%-85% of CRC cases, (1) is characterized by aneuploidy and mutations in genes such as APC, KRAS, and TP53 [10, 11]. MSI, resulting from defects in the DNA mismatch repair (MMR) system, leads to a hypermutable phenotype and distinct clinicopathologic features, often associated with a better prognosis [12-14].

KRAS mutations occur in approximately 40% of CRC cases and are predominantly linked to the CIN pathway [15].

These mutations activate the RAS/RAF/MAPK signaling cascade, promoting tumor proliferation and progression [16]. While KRAS mutations in proficient MMR (pMMR) CRCs are associated with poor response to anti-EGFR therapy, deficient MMR (dMMR) tumors exhibit different molecular profiles, including a higher prevalence of BRAF mutations and potential responsiveness to immunotherapy [17, 18]. Understanding the interaction between KRAS mutations and MMR status is crucial for refining CRC treatment strategies.

Despite extensive global research, limited studies have explored the relationship between KRAS mutations and MMR status in Indonesian CRC patients. Most existing studies have focused on other cancer types or international populations. This study aims to bridge this gap by analyzing KRAS mutations and MMR status in CRC patients in Makassar, providing valuable insights into the genetic landscape of CRC in Indonesia and contributing to more personalized treatment approaches.

Methods

This preliminary study utilized an observational analytic cross-sectional design to investigate the relationship between KRAS mutations and MMR status in CRC patients. The research was conducted at Wahidin Sudirohusodo Hospital and its affiliated teaching hospitals between January and December 2023. The estimated number of participants was calculated using Lemeshow’s formula [19, 20]. Patients diagnosed with CRC were selected through consecutive sampling, with inclusion criteria comprising a confirmed pathological diagnosis, age of 18 years or older, and the availability of sufficient tissue samples for genetic analysis. Patients were excluded if they had systemic malignancies, metastases from other cancers, or conditions that could interfere with study participation or prognosis.

Detection of KRAS Gene Mutations

The detection of KRAS gene mutations in this study relied on the technique of Real-Time Polymerase Chain Reaction (RT-PCR), specifically targeting mutations in codons 12 and 13. Sample preparation commenced with the isolation of genomic DNA from patient tumor tissue. The amplification of the KRAS gene was performed using a master mix comprising template DNA, specific primers, a fluorescence probe, DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), and a reaction buffer. The thermal cycling process included denaturation, annealing, and elongation, with real-time detection of amplification products achieved through the measurement of fluorescence intensity during PCR cycles. Data analysis was based on the interpretation of amplification curves, where a significant increase in fluorescence signal indicated the presence of mutations in the KRAS gene.

Determination of Mismatch Repair (MMR) Status

The mismatch repair (MMR) status was determined using immunohistochemistry (IHC) to evaluate the expression of key proteins (MLH1, MSH2, MSH6, and PMS2) from Vantage Biosciences (London, UK; catalog numbers 285M-14, 286M-14, 287M-14, and 288M-14).

Paraffin-fixed tumor tissue sections underwent a series of steps, including deparaffinization, rehydration, and antigen retrieval. Subsequently, endogenous peroxidase blocking was performed before incubation with primary antibodies specific to MMR proteins. The detection of primary antibodies was carried out using secondary antibodies conjugated with horseradish peroxidase (HRP), followed by visualization with diaminobenzidine (DAB) substrate and nuclear counterstaining with hematoxylin. The MMR status of the tumor was classified based on the expression patterns of MMR proteins observed under a light microscope. Loss of expression of one or more proteins indicated a deficient MMR (dMMR) status, whereas intact expression of all proteins indicated a proficient MMR (pMMR) status.

Data were organized and analyzed using SPSS version 25.0 for windows. Continuous variables were presented as mean ± standard deviation or median with interquartile range, while categorical variables were expressed as frequencies.

Results

Data Overview and Patient Characteristics

Initially, data were collected from 55 patients; however, the analysis focused on 23 patients with complete KRAS mutation data, which is crucial for examining the relationship between KRAS mutations and MMR status. The sex distribution revealed a significant disparity, with females comprising 65.2% of the sample and males 34.8%. This imbalance suggests potential sex-related influences on the study’s outcomes, which should be considered in future analyses. The age distribution indicated that the majority of patients (37.0%) were within the 50–59 age group, consistent with the higher prevalence of CRC in this demographic. Additionally, 29.6% of patients were aged 60–69, indicating that CRC remains a significant concern in older populations. The frequency of patients decreased in the 70–79 (18.5%) and over 80 (3.7%) age groups, which may suggest either a lower disease prevalence at advanced ages or external factors such as increased mortality affecting patient numbers.

KRAS Mutation Frequency and Cancer Staging

The distribution of cancer stages among patients showed notable variation, with a substantial proportion diagnosed at an early stage. Specifically, 40.9% were in Stage I, suggesting that nearly half of the study population received early diagnoses (Figure 1).

Figure 1. Distribution of Cases by Staging.

Another 36.4% were in Stage II, reinforcing the effectiveness of current early detection efforts. However, only 13.6% of patients were diagnosed at Stage III, and 9.1% at Stage IV, the most advanced stage of the disease.

Tumor Characteristics and Location

Analysis of tumor histology showed that 87% of patients had non-mucinous colorectal cancer, while 13% had mucinous tumors (Figure 2).

Figure 2. Distribution of Mucinous and Non-Mucinous Colorectal Cancer Cases.

Regarding tumor burden, 90.5% of patients had single tumors, while 9.5% had multiple tumors, indicating either metastasis or multiple primary malignancies.

Tumor location also played a critical role in disease presentation and management. The study found that 61.9% of patients had left-sided colon tumors (descending colon), while 38.1% had right-sided tumors (ascending colon) (Figure 3).

Figure 3. Tumor Location Distribution.

Association Between KRAS Mutations and MMR Status

Data analysis revealed that 91.3% of patients with KRAS mutations had pMMR status, while only 8.7% had dMMR (Figure 4).

Figure 4. Status Distribution Patients with KRAS Mutations had MMR Mutation.

Discussion

This study found that 91.3% of CRC patients had pMMR status, while only 8.7% had dMMR, with KRAS mutations predominantly occurring in pMMR cases. Among the patients, 77.3% were diagnosed at early stages (I and II), while 22.7% were in advanced stages (III and IV). Additionally, 61.9% of tumors were located in the left colon, and 87% were non-mucinous.

Previous research suggests that the presence of KRAS mutations, particularly in conjunction with pMMR status, may influence the effectiveness of certain cancer treatments and the overall prognosis of patients [1, 21]. The high proportion of early-stage diagnoses (77.3%) reflects the success of screening programs; however, the presence of advanced-stage cases underscores the need for continued efforts in early detection and timely intervention. Delays in diagnosis and disparities in healthcare access remain critical factors in advanced-stage presentations [6]. The detection of patients at later stages (22.7%), however, highlights persistent gaps in early diagnosis, potentially due to disparities in healthcare access or delays in symptom recognition [22]. In this study, mucinous cancers were found to be less common. Mucinous cancers are often associated with more aggressive biological behavior and variable responses to chemotherapy. Although less common, mucinous tumors may require more individualized treatment approaches due to their distinct molecular characteristics and potential resistance to standard therapies.

In this study, the predominance of single tumors suggests that most cases presented with localized disease, which is generally associated with better prognoses and higher responsiveness to localized treatments such as surgery or radiation. In contrast, patients with multiple tumors are more likely to require aggressive therapeutic strategies, including systemic chemotherapy or targeted therapies.

In examining the relationship between KRAS mutations and MMR status, we found that 91.3% of patients with KRAS mutations had pMMR, while only 8.7% had dMMR. This suggests that KRAS mutations are more prevalent in cases where DNA repair mechanisms, though not flawless, remain largely intact [23]. Conversely, the lower prevalence of KRAS mutations in dMMR patients may indicate that CRC in this subgroup primarily progresses through microsatellite instability rather than KRAS-driven pathways [7].

These findings have significant clinical implications. The association between KRAS mutations and pMMR status suggests that pMMR patients may benefit from different therapeutic approaches than those with dMMR, who are more likely to respond to immune-based therapies [9]. Further research is needed to explore the underlying molecular pathways and refine treatment strategies that incorporate both KRAS mutation status and MMR proficiency [24].

This study has several limitations. First, the sample had an uneven distribution of demographic and clinical variables, with females comprising the majority (65.2%). This imbalance may influence the interpretation of the relationship between KRAS mutations and MMR status. Additionally, the proportion of patients with advanced- stage cancer (stages III and IV) was relatively low (22.7%), which may limit our understanding of the role of KRAS mutations in advanced disease. Second, this study focused solely on KRAS mutations and MMR status, without considering other molecular factors that contribute to CRC development, such as BRAF mutations or the expression of proteins involved in alternative signaling pathways. Third, the study was conducted at a single research site Wahidin Sudirohusodo Hospital and its affiliated hospitals in Makassar. As a result, the findings may not be generalizable to the broader CRC population in Indonesia.

In conclusion, KRAS mutations are more frequently associated with pMMR in CRC, indicating distinct molecular pathways. Personalized therapeutic approaches should consider both KRAS mutation status and MMR proficiency to optimize treatment outcomes. However, due to the limited number of dMMR patients in this study, further research with a larger sample size is necessary to confirm these findings.

Acknowledgments

Statement of Transparency and Principals:

• Author declares no conflict of interest

• Study was approved by Research Ethic Committee of author affiliated Institute.

• Study’s data is available upon a reasonable request.

• All authors have contributed to implementation of this research.