Molecular Diversity and Characteristics of KRAS Mutations in Colorectal Cancers

Background: KRAS oncogene is involved in colorectal carcinogenesis in 22 to 45% of cases. KRAS mutations can render Epidermal Growth Factor Receptor (EGFR) inhibitors ineffective. The present study is aimed to determine the molecular diversity, frequency, and characteristics of KRAS mutations in metastatic Colorectal Cancer (mCRC). The gene KRAS contains 4 coding exons and 1 non-coding exon, of which exon 2 has the highest mutation rate, which is directly associated with the occurrence of poor prognosis and drug resistance. Codon 12, 13, 61 are common KRAS mutations. The most prevalent KRAS mutation observed was codon 12-G12D mutation where Glycine is changed to Aspartic acid.

Materials and Methods: A retrospective study was conducted on both in-situ and metastatic colorectal cancers that were registered for KRAS gene mutation testing in Department of Molecular Pathology and Genomics, Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute, from January 2016 to January 2022. KRAS mutations studies were carried out in primary and metastatic 87 patients of colorectal cancers. The samples contained 37 women aged 25-80 years and 50 men aged 29-77 years. Formalin fixed and paraffin embedded tissue samples were evaluated using Sanger sequencing and statistical analysis was carried out.

Results: KRAS mutations were demonstrated in 26 cases of 87 patients (29.88%). Of the 26 cases, the substitution nucleotide variations as point mutations were as follows: Codon 12-18 cases (69%), codon 13-4 cases (15%), codon 61-2 cases (8%), codon 146-1 case (4%), and codon 10-1 case (4%). The Codon 12 mutations were detected as follows: G12D-9 cases (50%), G12V-3 cases (17%), G12A-3 cases (17%), G12S-2 cases (11%), G12C-1 case (5%); Codon 13 showed all 4 mutations (4%) in G13D. KRAS mutations were found to be more frequent in elderly age group. Most m-KRAS tumors were located in sigmoid colon and rectum (left sided/distal colon), although, the mutations were spread across including ascending colon (right sided/proximal colon) and rarely in jejunum too. The TNM stage showed majority (50%) of KRAS mutations in stage IV, and 20% of cases in stage II and III each.

Conclusion: In this series, 29% of colorectal cancer tissue samples had KRAS mutations. Their frequency and distribution showed majority mutations on left sided/distal colon, elderly population, more common in females and presented at an advanced stage. There was no clinicopathological significance to histomorphology. Understanding KRAS mutations in different aspects can give a better comprehensive understanding of KRAS associated CRCs.

KRAS: Kirsten Rat Sarcoma viral oncogene; EGFR: Epidermal Growth Factor Receptor; mCRC: metastatic Colorectal Cancer; TNM stage: T: Size of the tumor and any spread of cancer into nearby tissue, N: Spread of cancer to nearby lymph nodes, M: Metastasis (spread of cancer to other parts of the body); FDA: Food and Drug Administration; MSS: Microsatellite Stable; PCR: Polymerase Chain Reaction; FFPE: Formalin-Fixed Paraffin-Embedded; GTPase: Guanine Tri Phosphatase

Colorectal Cancer (CRC) is a third most common worldwide and usually presents at an advanced stage and shows high mortality [1]. Recent advances in molecular targeted therapeutic approaches to CRC has led to the approval of Cetuximab and Panitumumab, Antiepidermal Growth Factor Receptor (EGFR) targeted therapies by US Food and Drug Administration (FDA) in 2009 [2,3]. Kirsten Rat Sarcoma viral oncogene homolog (KRAS) gene is approximately seen 35.0-45.0% of the CRC cases [4]. KRAS is known to be a poor prognostic marker in CRC, which is still debatable based on the type of mutation and location [5]. KRAS is found be usually seen in Microsatellite Stable Tumors (MSS) which is responsive to the 5Fluoro-Uracil in stage II-III cancers [6,7]. Three distinct molecular pathways have been defined in CRC: Microsatellite Instable (MSI) phenotypic tumors; Chromosomal Instability (CIN) phenotype; CpG Island Methylator Phenotype (CIMP) [8]. The significance of the Cancer Genome Atlas (TCGA) integrated molecular classification have been clearly laid out. [9] KRAS is known to be associated with CIN molecular pathway and is also biological marker of resistance to targeted therapy [10].

Somatic KRAS mutations in codons 12, 13 (exon 2), 59, 61 (exon 3), and 146 (exon 4) are common hotspots. Other non-hotspot mutations in codons 2, 3, 4, 63 and 154 occur less frequently in CRC. Most of the reported mutations are single nucleotide point mutations, particularly G > A transitions and G > T transversions [11]. According to published western study by Tan C, among the codon 12 mutations, p.Gly12Asp, and p.Gly12Val are the most frequent, while in codon 13, the substitution of Glycine by Aspartate (p.Gly13Asp) is the most common [3]

KRAS has been found to be more common on the left sided location along with other genes like APC, PIK3CA, p53 mutations [5]. The mutation usually results in constitutionally active RAS protein, leading to cell proliferation and cell survival. It is essential to investigate the underlying mutations in the KRAS gene in mCRC patients to identify the prevailing patterns based on topographical location of the tumor i.e. either right sided/proximal colon or left sided/distal colon (Figure 1) [12]. Two clinical trials have shown that KRAS-Wild (WT) left sided CRC have survived longer when treated with Cetuximab; whereas KRAS-WT right sided CRC showed longer survival when Bevacizumab was added to the treatment regimen.

Figure 1: Representation of the Right and Left sided colon [13].

(ClinicalTrials.gov identifier: NCT00265850) [13]. In metastatic settings, KRAS-Mutant (MT) patients showed worse overall survival than KRAS-WT type patients [14].

KRAS has been difficult gene to target due to a unique intrinsic structure of the KRAS protein, the surface of which is a smooth and shallow leading to difficulty in binding of drugs. However, Sotorasib and Onvansetinib, new targetable treatments against KRAS p.12C are able to bind GTP binding pocket and has been approved by FDA [15,16].

A retrospective descriptive study was conducted on both in-situ and metastatic colorectal cancers (n = 87) that were registered for KRAS gene mutation testing in the Department of Molecular Pathology and Genomics from January 2016 to January 2022 at Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute, Mumbai. Using patients’ medical records, data was collected regarding variables such as demographic characteristics (age and gender) and pathological tumor features (primary site, and staging).

Around 10 Curls from the patient's Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks were obtained and carried out DNA isolation by QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). The slides were specifically chosen by Molecular Pathologist to include tumor tissue with > 30% tumor cells without significant necrosis, mucin, or inflammation [17]. All the tumor sections were placed in two baths of xylene for deparaffinization and after, for rehydratation, in two baths of ethanol. For a complete lysis, they were placed in the lysis buffer with proteinase K at 56°C overnight. After lysis, DNA was precipitated with ethanol and was fixated on the QIAamp silica membrane by centrifugation. Two washes were performed and the purified DNA was eluted in buffer AE or water. The purified DNA was used for Polymerase Chain Reaction (PCR). Results of the PCR amplification reaction were visualized using gel electrophoresis. The products were subjected to post amplification purification followed by Sanger's sequencing by Capillary electrophoresis on ABI 3500Dx Genetic Analyzer. Data were analyzed with respect to age groups, gender, KRAS genotype, location on right/left side and TNM classification using standard descriptive statistics [15].

The colorectal tissue samples of 87 patients were tested with age between 20 to 80 years (median age, 63 years). Of these, 26 (30%) tested positive for KRAS mutations. The remaining 61 (70%) samples had the wild-type allele. Overall, there were 50 (57%) males and 37 (43%) females. Among the KRAS positive cases, there were 12 (45%) males and 14 (54%) females. The highest percentage of mutant KRAS cases was found in the 50-70 years age group (Figure 2).

Figure 2: Distribution of mutations within different codons.

Out of these 26 positive cases, the main point mutations were as follows: codon 12-18 (69%), codon 13-4 (15%), codon 61-2 (8%), codon 146-1(4%), and codon 10-1(4%) (Figure 3).

Figure 3: Distribution of KRAS Codon 12 mutations.

The codon 12 mutations showed the following mutations: G12D-9 (50%), G12V-3 (17%), G12A-3 (17%), G12S-2 (11%), G12C-1 (5%), (Figure 2) and codon 13 mutation observed was G13D-4. The p.Gly12Asp (c.35G > A) mutation had the highest frequency in both females -7 (27%) and males -2 (8%). Multiple mutations in the same individual were not detected in any of the patients in this cohort.

KRAS is located on Chromosome 12p12.1, is a member of RAS family of genes and it is responsible for oncogenesis in various malignancies and the sustained signalling makes it vulnerable to high profile therapeutic target [19]. Activated KRAS binds to GTPase protein, produces a conformational change in the KRAS protein which affects its interaction with the downstream transducer GTPase Activating Proteins (GAPs) [19]. KRAS is the one of the most common gene mutations occurring in 17-25% of all cancers [20]. Approximately, 30-40% of colorectal cancer carries KRAS mutation which are associated with poor prognosis and advanced disease [21,22]. The overall frequency of positive KRAS mutations observed in this study was 30% which is slightly lesser than the reported studies. There are significant differences between the frequencies of KRAS gene mutation in patients with CRC from different continents: Asia -24%, Europe -36% and Latin America - 40% as reported by Yamane L, et al. [23].

The present study cohort is widely spread across age groups from 21 to 80 years, with most mutations were found in age group 61-70 (38%) followed by 71-80 (27%) and 51 to 60 (19%) and less commonly in younger patients (Figure 4). An Indian study by Veldore, et al. [24] showed similar findings with respect to age. Conversely, Ozer M, et al. [25] in their study cohort reported 42% KRAS mutations in of young patients, which is higher than previously reported studies. They also inferred that prognosis was better in patients with less than 50 years, and in between 50-70 years. The prognostic impact is lost above 70 years due to underutilization of targeted therapy and more vulnerability to chemotherapy. Further studies might elucidate the clinical characteristics of young-onset colorectal cancer. The prevalence of KRAS positive mutations was higher in females (54%) as compared to males (45%) in the present study. This was contrary to the Srilankan study by Sirisena ND, et al. [26] where they reported KRAS mutations in 56% of males and 44% in females.

Figure 4: Incidence of KRAS mutation across different age groups.

The distribution of mutations in different codons in KRAS was studied and the majority of KRAS mutation are mainly present in codon 12, 13 and codon 64 together corresponding to 92% positivity. The mutations on minor (146, 10) codons were concluded to 8%. Knijn N, et al. [27] reported 95% KRAS mutations in codon 12 and 13; other mutations in codons 61, 146 in a rare scenario, accounting for 5% of all the mutations. Specific mutations at different codons are known modify the biologic behaviour of the tumor. KRAS mutational subtypes in the present study did not show any differentiation into mucinous/nonmucinous morphology. Further studies will help to understand further associations with morphogenomics.

In the study, the most common mutation detected on codon 12 was G12D (c.35G > A; p.Gly12Asp) which accounted for 50% of the mutations; followed by 17% each of G12V (c.35G > T; p.Gly12Val) and G12A (c.34G > A; p.Gly12Ser) mutations. The most common KRAS mutation types observed are p.Gly12Asp (Codon 12), p.Gly12Val (Codon 12)and p.Gly13Asp (Codon 13), accounting for almost 62% of all mutations. Overall studies have shown that these three mutations account for around 70% of the codon 12 mutations [28,29].

All 26 cases of KRAS were located to different anatomical sites and vastly grouped into left sided/distal and right sided/proximal location. The tumor site with higher incidence is left sided colon composed of 1/3 transverse colon, descending colon, sigmoid colon and rectum constituting 58% of total mutations. G12D was found to be the most common mutation (33%) on left side colon. This was similar to the study reported by Indian Study, Turkish and Chinese Study [24,30,31]. But Watanabe, et al. [32] found a higher frequency of KRAS mutation in right sided colon cancers rather than left sided colonic cancers. In an interesting finding, 1 case of codon 13 (G13D) mutations was located on jejunum which is a rare finding.

The clinical correlation also elucidated that KRAS mutations were found in late stage of presentation of the colorectal cancer. The results showed 20% of cases in stage 2, 20% in stage III, and 50% in stage IV (Table 1). Inoue, et al. [2] also reported similar findings with the frequency of KRAS mutation was 20% in stage I, 30% in stage II, 40% in stage III and 58% in stage IV.

Novel Mutation: A rare case of codon 10 insertion was found in our study, also known as codon 10 duplication. Codon 10 mutation is a rare case of insertion where GGA is inserted that leads to two peaks from that codon in the electropherogram. The patient has a metastatic sigmoid cancer. The metastasis has reached to the abdominal skeletal muscles. Patient has been treated with FOLFIRI (Leucovorin + 5FU + Irinotecan) regimen of chemotherapy along with Bevacizumab, an anti-angiogenic immunotherapy drug. This rare mutation 10G11, which suggested the insertion of an additional Glycine residue between Glycine (amino acid 10) and Alanine (amino acid 11). Only 2 cases have been reported of which the first was reported in the patient with myeloid leukemia and the other in colorectal cancer [33,34].

Another rare mutation of codon 146 was found on the right sided colon and required further investigation.

KRAS is a negative prognostic factor with the mutational diversity is potentially oncogenic by conveying selective growth advantage to the cells and potentially contributes to primary resistance for anti-EGFR mAb targeted therapy [2,10]. The KRAS mutation can still respond to 5-Fluorouracil (5-FU) based regimens but doesn’t predict response to 5-FU [5]. Historically, KRAS has been known to be untargetable, probably due to high affinity to cytoplasmic GTP, RAS protein with no pocket or groove for binding the drug and multiple resistance mechanism due to activation of bypass upstream and downstream signals. [35] Sotorasib, a small molecule inhibitor, is the first KRAS targeting drug approved by FDA in KRASp.G12C positive lung cancers which covalently binds novel surface groove and locks the KRAS 12C protein irreversibly [15][36] Onvansertinib, a pole like kinase inhibitor (PLK-1) has been approved with KRAS mutant metastatic CRC in combination with FOLFIRI (5-Fluorocil, Leucovorin, and Irinotecan) and Bevacizumab. [16] The resistance mechanism include reactivaton of receptor tyrosine kinase (RTK) pathway by an alternate mechanism, where the combination therapy of targeted pathways with drugs targeting RTKs can be a promising strategy.

Adagrasib, a new drug approved in United States in December 2022, developed for treating non-small cell lung cancer for KRAS G12C mutated patients, can be a promising discovery also in relation with CRC after further research and development [37].These characteristics of the study cohort can help pathologists and oncologists to thoroughly understand the behaviour of mutations and will increase efficiency in the approach towards managing the disease.

In the present study, 29% of colorectal cancer tissue samples were positive for KRAS mutations. KRAS 12D was found to be the most common mutational site. The majority of mutations were detected on left sided/distal colon, in elderly population, more common in females and presented at an advanced TNM stage. Clinicopathological significance due to histomorphology was not observed in the given study.

Using KRAS testing to restrict the use of EGFR inhibitor therapy would help to select the appropriate patients. Understanding drug development through binding of development drugs at different mutational sites, might give a comprehensive understanding of KRAS inhibition. Hence, KRAS is a promising biomarkers and shows both prognostic and predictive value.

We express our gratitude towards Mrs. Varsha Vadera for always supporting us.

Author contributions

Conceptualization: Amrit Kaler; Methodology: Omkar D; Validation: Smita K; Formal analysis: Sweta L; Investigation: Yash T; Data curation: Samrudhi, Ankita; Writing-original draft preparation: Amrit Kaler; Writing-review and editing: Bijal K, Meenal H, Nivetha A; Supervision: Sandeep G, Imran Shaikh; Project administration: Rajesh Mistry; Funding Acquisition, Not applicable; All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute (Protocol code: KDAH/PUB/2022/38) and date of approval: 7 September 2022).

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