KRAS Gene In Colorectal Cancer Research Articles

HUBUNGAN MUTASI GEN K-RAS TERHADAP RESPON TERAPI PADA PASIEN KANKER KOLOREKTAL DI RSUP PROF. DR. IGNG. NGOERAH TAHUN 2017-2019

K-RAS is one of the most frequently mutated genes found in colorectal cancer cases. The K-RAS gene has about 30%-40% contribution to the formation of colorectal cancer. This research is an analytic observational study with a cross-sectional design to explain the characteristics of patients with colorectal cancer at Prof. dr. IGNG. Ngoerah 2017-2019 and assessed the relationship between K-RAS gene mutations and response to therapy. The results of the chi-square test showing a significant relationship between the type of KRAS gene mutation and the response to therapy where p=0.035 (p<0.05). This shows that there is a significant relationship between mutations in the K-RAS gene in colorectal cancer and the response to therapy in colorectal cancer patients at Prof. Dr. IGNG. Ngoerah General Hospital in 2017-2019.

Single-Nucleotide-Specific Lipidic Nanoflares for Precise and Visible Detection of KRAS Mutations via Toehold-Initiated Self-Priming DNA Polymerization.

Accurate identification of single-nucleotide mutations in circulating tumor DNA (ctDNA) is critical for cancer surveillance and cell biology research. However, achieving precise and sensitive detection of ctDNAs in complex physiological environments remains challenging due to their low expression and interference from numerous homologous species. This study introduces single-nucleotide-specific lipidic nanoflares designed for the precise and visible detection of ctDNA via toehold-initiated self-priming DNA polymerization (TPP). This system can be assembled from only a single cholesterol-conjugated multifunctional molecular beacon (MMB) via hydrophobicity-mediated aggregation. This results in a compact, high-density, and nick-hidden arrangement of MMBs on the surface of lipidic micelles, thereby enhancing their biostability and localized concentrations. The assay commences with the binding of frequently mutated regions of ctDNA to the MMB toehold domain. This domain is the proximal holding point for initiating the TPP-based strand-displacement reaction, which is the key step in enabling the discrimination of single-base mutations. We successfully detected a single-base mutation in ctDNA (KRAS G12D) in its wild-type gene (KRAS WT), which is one of the most frequently mutated ctDNAs. Notably, coexisting homologous species did not interfere with signal transduction, and small differences in these variations can be visualized by fluorescence imaging. The limit of detection was as low as 10 amol, with the system functioning well in physiological media. In particular, this system allowed us to resolve genetic mutations in the KRAS gene in colorectal cancer, suggesting its high potential in clinical diagnosis and personalized medicine.

KRAS Promoter Methylation Status and miR-18a-3p and miR-143 Expression in Patients With Wild-type KRAS Gene in Colorectal Cancer.

Although some mutations of KRAS proto-oncogene, GTPase (KRAS) have been associated with the prognosis and therapeutic management of colorectal cancer (CRC), the epigenetic mechanisms (DNA methylation and microRNA expression) that regulate wild-type KRAS expression in patients with CRC are poorly known. The aim of this study was to establish whether there is a relationship between the expression of the wild-type KRAS gene, the methylation status of its distal promoter, and miR-143 and miR-18a-3p levels in samples of sporadic CRC. A total of 51 cases of sporadic CRC with wild-type KRAS were analyzed. The expression levels of KRAS mRNA, miR-18a-3p, miR-143, and KRAS protein, as well as methylation in the distal promoter of the KRAS gene were evaluated. In the analyzed cases, KRAS mRNA expression was detected in 51.1%; wild-type KRAS protein was found in the membrane in 31.4% and in the cytoplasm in 98% of cases. An inverse relationship of marginal significance was observed between miR-18a-3p and KRAS protein expression in the cytoplasm (odds ratio=0.14, 95% confidence interval=0.012-1.092; p=0.08). The methylation status of the distal promoter of KRAS at four CpG islands was analyzed in 30 cases (58.8%): partial methylation of the four CpG islands evaluated was observed in two cases (6.7%). In these cases, KRAS protein expression was not evidenced at the membrane level; miR-18a-3p expression was not detected either but high expression of miR-143 was observed. No association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.

CT Radiomics in Colorectal Cancer: Detection of KRAS Mutation Using Texture Analysis and Machine Learning

In this work, by using descriptive techniques, the characteristics of the texture of the CT (computed tomography) image of patients with colorectal cancer were extracted and, subsequently, classified in KRAS+ or KRAS-. This was accomplished by using different classifiers, such as Support Vector Machine (SVM), Grading Boosting Machine (GBM), Neural Networks (NNET), and Random Forest (RF). Texture analysis can provide a quantitative assessment of tumour heterogeneity by analysing both the distribution and relationship between the pixels in the image. The objective of this research is to demonstrate that CT-based Radiomics can predict the presence of mutation in the KRAS gene in colorectal cancer. This is a retrospective study, with 47 patients from the University Hospital, with a confirmatory pathological analysis of KRAS mutation. The highest accuracy and kappa achieved were 83% and 64.7%, respectively, with a sensitivity of 88.9% and a specificity of 75.0%, achieved by the NNET classifier using the texture feature vectors combining wavelet transform and Haralick coefficients. The fact of being able to identify the genetic expression of a tumour without having to perform either a biopsy or a genetic test is a great advantage, because it prevents invasive procedures that involve complications and may present biases in the sample. As well, it leads towards a more personalized and effective treatment.

Investigation of FIH-1 and SOCS3 expression in KRAS mutant and wild-type patients with colorectal cancer.

Colorectal cancer (CRC) is a multistep process based on the accumulation of somatic mutations in genes such as APC and KRAS. Data on the presence of mutations in KRAS gene in CRC and its relationship with clinicopathological parameters and expression of genes involved in tumor progression are scarce. We unbiasedly examined the KRAS status in samples from 99 patients and its correlation with clinicopathological parameters such as age, sex, tumor location, lymph node metastasis, tumor stage, tumor grade, and vascular invasion. Consistent with reports of other researchers, 38.4% of our samples harbored KRAS mutation in their genomes with preferential mutation in codon 12 (89.4%). Nevertheless, unlike previous reports, we were not able to correlate KRAS status with clinicopathological parameters (P > 0.05) except for vascular invasion. Patients with KRAS mutation have more vascular invasion compared with patient having wild-type KRAS. Next, we investigated the expression of two tumor suppressor genes, factor-inhibiting hypoxia-inducible factor 1 (FIH-1) and suppressor of cytokine signaling (SOCS3), in both KRAS mutant and wild-type groups and looked for any correlation between their expression and clinicopathological parameters. Although the expression of both genes was not regular, none of the clinicopathological parameters were associated with the expressions of FIH-1 and SOCS3 at mRNA level (P > 0.05). However, decline in FIH-1 expression at protein level in KRAS mutant group was correlated with stage IV and grade 2 of tumor (P ≤ 0.05). Our results demonstrated that there is no or low correlation between KRAS status, FIH-1, and SOCS3 expression with epidemiologic and clinicpathological characteristics in CRC.

P65: Development of allele-specific PCR assays for detection of mutations in KRAS gene in colorectal cancer in Russian patients

KRAS is component of Ras/MAPK signaling cascade that regulates cell proliferation and cell survival. Somatic mutations in KRAS gene are often found in tumors and affect the sensitivity of tumors to target therapy. Mutations in codons 12 or 13 of KRAS gene in colorectal cancer (CRC) are associated with resistance to anti-EGFR antibodies Cetuximab and Panitumumab. The objectives of this work were to develop PCR tests for detection mutations in KRAS gene and analyze the frequency of mutations in KRAS gene in CRC in Russia. DNA sequencing by Sanger is the most common method for mutation analysis. However, the method has a sensitivity of 20% mutant allele, which is often not sufficient for the analysis of somatic mutations in tumors. One of the most sensitive mutation analysis methods is allele-specific real-time PCR. This method allows to detect 1% of mutated DNA in the sample. This sensitivity is sufficient for analysis of mutations in tumor samples containing 2–5% or more of tumor cells in normal tissue. In this study we developed and compared 3 new KRAS assays (1) real-time PCR with allele-specific primers; (2) real-time wild-type blocking PCR with LNA (locked nucleic acid) blocker; and (3) Sanger sequencing with LNA-blocker. First assay is a PCR test with seven reactions using allele-specific primers for detection and genotyping 7 mutations in 12 and 13 codons of KRAS gene. Second assay is real-time PCR with only a single pair of primers and LNA oligonucleotide blocker. LNA-blocker is an oligonucleotide which has a wild-type sequence of codons 12 and 13 of KRAS gene. LNA-blocker binds strongly to wild-type KRAS DNA and suppresses its amplification, but does not block amplification of mutant DNA. The real-time PCR with LNA -blocker can detect mutant DNA but does not genotype mutation. Such assay can be used as a simple and sensitive screening test for mutant KRAS cases if exact genotyping of mutation is not required. We also used LNA-blocker to increase sensitivity of Sanger sequencing. To evaluate sensitivity and specificity of new tests DNA standards were prepared with different ratios of normal and mutant alleles using normal human DNA without mutation and recombinant plasmids with mutations in KRAS (G12C, G12S, G12R, G12V, G12D, G12A, G13D). After optimization all three assays had sensitivity 5% of mutant alleles for the detection of mutations in KRAS gene, using 2.5–40 ng of human DNA. Performance of new assays for KRAS mutations was compared using 81 colorectal tumor samples. Before analysis relative content of tumor cells in the samples was evaluated by pathologist. If tumor content was less than 20% in the sample then regions with a maximum number of tumor cells were manually macrodissected before the DNA extraction. DNA was purified from formalin fixed paraffin embedded (FFPE) tissue using “FFPE-DNA Kit” (Biolink). All three assays had high sensitivity (95–100%) and specificity (100%) for detection of KRAS mutations in clinical tumor samples. Mutations of the KRAS gene were found in 37 of 81 cases (46%) of CRC including 12 cases of mutation G13D, 11 cases of G12D, 5 cases of mutation G12V, 4 cases G12C, 3 cases of mutation G12A, 2 cases G12S, 1 case G13R. A single case with mutation G13R was missed by allele-specific PCR but was detected by real-time PCR with LNA-blocker and confirmed by sequencing. New assays have high sensitivity and specificity and are suitable for detection of KRAS mutations in clinical FFPE tumor samples.

Coexistence of two different mutations in codon 12 of the Kras gene in colorectal cancer: Report of a case supporting the concept of tumoral heterogeneity

Evaluation of the mutational status of KRAS is a crucial step for the correct therapeutic approach in treating advanced colorectal cancer as the identification of wild-type KRAS tumors leads to more specific and less toxic treatments for patients. Although several studies have highlighted the differences between primary and metastatic tumors, the possibility of two or more mutations in the same codon has seldom been reported. The present study reports an additional case of an advanced adenocarcinoma of the colon showing two somatic mutations (p.G12D and p.G12V) in the same codon (codon 12) of exon 2 of the KRAS gene, thus supporting the possibility of two differing clonal origins of the tumor. Although the clinical significance of multiple mutations remains unknown at present, based on the limited data available in the literature, this rare event appears to be associated with a more aggressive disease, as in the present case. This case report demonstrates the existence of intratumoral heterogeneity and the coexistence of distinct clones within a tumor that may have profound clinical implications for disease progression and therapeutic responses.

KRAS mutation profile in Puerto Rican patients with colorectal cancer: Trends in expression of KRAS mutation in PR between April 2009 to January 2011.

e14102 Background: The prevalence of a mutated KRAS gene in colorectal cancer shows ethnic variation with a range between 30-48% among the Caucasian, Asian, and European populations. The purpose of this study was to define the profile of KRAS mutation in colorectal cancer patients in Puerto Rico. Methods: Between April 2009 and January 2011, we collected the expression of KRAS gene among colorectal cancer patients in Puerto Rico. The assay was performed by a central laboratory which began to offer the test in the island in April 2009 (GENOPTIX). Data for the presence and type of mutation, age, sex, cancer histology, site of tumor, and cancer stage were collected. The site of the mutation in codon 12 and 13 were identified by the real time polymerase chain reaction (PCR). The study was approved by IRB. A total of 501 cases were available for study. SPPS version 14 was used for statistical analysis. Results: A mutated KRAS was present in 39% (n=195) of the cohort, more commonly in the 51-69 age group (55.4%; p=0.004), in females (55.4%; p=0.095), in the proximal area of colon (43.8%; p=0.017), in the codon 12 (91.2%), and 8.8% in the codon 13. The majority of mutated genes were in codon 12 (91.2%) with 12 ASP (39.5%) and 12 VAL (25.4%) most often seen. Mucinous histology was associated with 46.7% mutated KRAS, poorly differentiated tumor with 40.0% of KRAS mutation. The mutant expression of KRAS in cases with rectum carcinoma was 39.5%, where the most common mutation was 12 VAL. Conclusions: The prevalence of KRAS mutation in Puerto Rican population is similar to other reported studies. A higher prevalence of 12 VAL mutation in rectal carcinoma and a trend towards a higher mutation rate in females are different in this cohort as compared with published studies. This study was supported by NIH grant numbers G12RR-03035 & 8U54MD007587-03.

Multiple mutations in the Kras gene in colorectal cancer: review of the literature with two case reports

Kras mutations are negative predictors of anti-EGFR therapy, occurring in 40% of colorectal carcinomas (CRCs). Point substitutions in codon 12 or 13 are the most frequent mutations in Kras, but multiple mutations (MMs) in other codons can also develop. Few data exist on MMs with regard to their frequency and the codons and amino acids that are affected. We report two cases of Kras double mutations in codons 12 and 13 and review Kras MMs in primary CRC in PubMed databases. A 53-year-old woman and a 70-year-old man presented with deep, invasive, moderately differentiated CRC at an advanced clinical stage. The former had regional lymph node involvement and vaginal wall neoplastic implantation, and the latter had liver metastasis. Primary tumors were examined for Kras mutations by pyrosequencing, which were confirmed by direct sequencing. Both tumors had a mutation in codons 12 and 13, wherein codon 12 was mutated to GAT, and codon 13 became GAC. We identified 69 reported cases of Kras MMs and reported two other cases, representing 2.1% of all mutated tumors; the incidence of such mutations is 1.0% in CRC patients. In most cases (59%), MMs develop in a single codon, usually codon 12. Codons 12 and 13 are affected simultaneously in only 27% of cases. These findings add information about the impact of specific amino acid changes in the Kras gene.

Significance of K-ras detection in colorectal cancer

To determine the mutation status of K-ras gene in colorectal cancer and analyze the associations between its mutation status and clinicopathological characteristics in colorectal cancer so as to select the patients likely to benefit from a targeted therapy. A total of 208 colorectal cancer tissue samples were collected from September 2008 to February 2009. DNA was extracted with a genomic DNA miniprep kit. Then PCR was performed with the designed primers and the product directly sequenced by the Sanger method. Then the associations between K-ras mutation status and clinicopathological characteristics in colorectal cancer were analyzed. Of 208 cases, 91 cases of K-ras gene mutation were detected. The 12 or 13 codon had a mutation rate of 43.8%. There were no significant differences in gender, tumor location, histopathological grading and Duke's stage between the wild and mutated groups. Detection of K-ras gene status in colorectal cancer will help to select the patients likely to benefit from the monoclonal antibody therapy of targeting epidermal growth factor receptor.