Colorectal Cancer Gene Mutations Research Articles

STEAP3 promotes colon cancer cell proliferation and migration via regulating histone acetylation.

Colorectal cancer (CRC) is the third most prevalent diagnosed cancer in men and second most prevalent cancer in women. H3K27ac alterations are more commonly than gene mutations in colorectal cancer. Most colorectal cancer genes have significant H3K27ac changes, which leads to an over-expression disorder in gene transcription. Over-expression of STEAP3 is involved in a variety of tumors, participating in the regulation of cancer cell proliferation and migration. The purpose of this work is to investigate the role of STEAP3 in the regulation of histone modification (H3K27ac) expression in colon cancer. Bioinformatic ChIP-seq, ChIP-qPCR and ATAC-seq were used to analyze the histone modification properties and gene accessibility of STEAP3. Western blot and qRT-PCR were used to evaluate relative protein and gene expression, respectively. CRISPR/Cas9 technology was used to knockout STEAP3 on colon cancer cells to analyze the effect of ATF3 on STEAP3. STEAP3 was over-expressed in colon cancer and associated with higher metastases and more invasive and worse stage of colon cancer. ChIP-seq and ChIP-qPCR analyses revealed significant enrichment of H3K27ac in the STEAP3 gene. In addition, knocking down STEAP3 significantly inhibits colon cancer cell proliferation and migration and down-regulates H3K27ac expression. ChIP-seq found that ATF3 is enriched in the STEAP3 gene and CRISPR/Cas9 technology used for the deletion of the ATF3 binding site suppresses the expression of STEAP3. Over-expression of STEAP3 promotes colon cancer cell proliferation and migration. Mechanical studies have indicated that H3K27ac and ATF3 are significantly enriched in the STEAP3 gene and regulate the over-expression of STEAP3.

Exploration of the application of gene editing technology in the treatment of colorectal cancer, taking CRISPR technology as an example for editing FAT4 genes

Colorectal cancer, the fourth most common cancer in the world, is the most common digestive tract tumor. At present, conservative treatment is still the main treatment method for colorectal cancer in clinical practice. Among the pathogenic factors of colorectal cancer, genetic factors need to be emphasized. Currently, many studies have focused on pathological studies of related gene mutations, mostly focusing on genes such as p53 and APC, and their molecular biological reactions have been thoroughly studied. However, research on other pathogenic genes is still lacking, and there is limited research on specific treatment methods. With the birth of CRISPR-Cas9 technology, applying of gene editing technology in the field of cancer has made breakthrough progress. This article will consider combining gene editing technology with related gene mutations in colorectal cancer, attempting to explore the possibility of applying gene editing technology in the field of cancer treatment, proposing feasibility for new treatment plans for colorectal cancer, and also providing new treatment ideas for future cancer treatment research.

Ceramide synthase CERS4 gene downregulation is associated with KRAS mutation in colorectal cancer

Ceramide, the central molecule in sphingolipid synthesis, is a bioactive lipid that serves as a regulatory molecule in the anti-inflammatory responses, apoptosis, programmed necrosis, autophagy, and cell motility of cancer cells. In particular, the authors have reported differences in sphingolipid content in colorectal cancer tissues. The associations among genetic mutations, clinicopathological factors, and sphingolipid metabolism in colorectal cancer (CRC) have not been investigated. The objective of this study is to investigate the association between genes associated with sphingolipid metabolism, genetic variations in colorectal cancer (CRC), and clinicopathological factors in CRC patients. We enrolled 82 consecutive patients with stage I–IV CRC who underwent tumor resection at a single institution in 2019–2021. We measured the expression levels of genes related to sphingolipid metabolism and examined the relationships between CRC gene mutations and the clinicopathological data of each individual patient. The relationship between CRC gene mutations and expression levels of ceramide synthase (CERS), N-acylsphingosine amidohydrolase (ASAH), and alkaline ceramidase (ACER) genes involved in sphingolipid metabolism was examined CRES4 expression was significantly lower in the CRC KRAS gene mutation group (p = 0.004); vascular invasion was more common in colorectal cancer patients with high CERS4 expression (p = 0.0057). By examining the correlation between sphingolipid gene expression and clinical factors, we were able to identify cancer types in which sphingolipid metabolism is particularly relevant. CERS4 expression was significantly reduced in KRAS mutant CRC. Moreover, CRC with decreased CERS4 showed significantly more frequent venous invasion.

Mathematical modeling the order of driver gene mutations in colorectal cancer.

Tumor heterogeneity is a large obstacle for cancer study and treatment. Different cancer patients may involve different combinations of gene mutations or the distinct regulatory pathways for inducing the progression of tumor. Investigating the pathways of gene mutations which can cause the formation of tumor can provide a basis for the personalized treatment of cancer. Studies suggested that KRAS, APC and TP53 are the most significant driver genes for colorectal cancer. However, it is still an open issue regarding the detailed mutation order of these genes in the development of colorectal cancer. For this purpose, we analyze the mathematical model considering all orders of mutations in oncogene, KRAS and tumor suppressor genes, APC and TP53, and fit it on data describing the incidence rates of colorectal cancer at different age from the Surveillance Epidemiology and End Results registry in the United States for the year 1973-2013. The specific orders that can induce the development of colorectal cancer are identified by the model fitting. The fitting results indicate that the mutation orders with KRAS → APC → TP53, APC → TP53 → KRAS and APC → KRAS → TP53 explain the age-specific risk of colorectal cancer with very well. Furthermore, eleven pathways of gene mutations can be accepted for the mutation order of genes with KRAS → APC → TP53, APC → TP53 → KRAS and APC → KRAS → TP53, and the alternation of APC acts as the initiating or promoting event in the colorectal cancer. The estimated mutation rates of cells in the different pathways demonstrate that genetic instability must exist in colorectal cancer with alterations of genes, KRAS, APC and TP53.

Clinical significance of SPOP and APC gene alterations in colorectal cancer in Indian population.

Speckle-Type Poz Protein (SPOP) involved in the regulation of proteasome-mediated degradation of several oncoproteins, resulting in cancer initiation and progression. Mutations in Adenomatous Polyposis Coli (APC) gene is reported in most sporadic and hereditary colorectal cancer (CRC). Identifying the cellular changes involved in carcinogenesis when APC is mutated is an important issue that needs attention. The tumor suppressive function of SPOP and APC has long been a major focus in the research field of colorectal cancer. However, the clinical significance of SPOP and APC gene alteration in CRC has not been established to date. Mutational analysis was performed by single-strand conformational polymorphism followed by Sanger sequencing, methylation status by methylation-specific PCR, and protein expression by immunohistochemistry on 142 tumor tissues along with their adjacent non-cancerous specimens. The overall survival (OS) and recurrence free survival (RFS) were estimated by Kaplan-Meier Curve. Mutation rates of APC and SPOP gene were 2.8% and 11.9% while that of promoter hypermethylation were 37% and 47%, respectively. The grade of differentiation and Lymph node metastasis were significantly correlated with APC methylation pattern (p ≤ 0.05). The down regulation of APC was more often seen in colonic cancer compared to rectal cancer (p = 0.07) and more commonly in T3-4 depth of invasion (p = 0.07) and in patients without lymphovascular and perineural invasion (p = 0.007, p = 0.08 respectively). The median overall survival and recurrence free survival (RFS) was 67 & 36months while 3-yr and 5-yr OS and RFS were 61.1% & 56.4% and 49.2% & 44.8%, respectively. APC promoter methylation had a better overall survival (p = 0.035) while loss of SPOP expression had a worse survival (p = 0.09). Our findings reveal high percentage of SPOP gene mutations in CRC. A significant link is found between promoter hyper methylationand protein expression in all mutant cases of APC and SPOP, suggesting that both genes may be associated in the development of colorectal cancer in people of Indian decent. Hypermethylation of APC gene and loss of SPOP expression have shown an association with disease prognosis and could be further studied looking at its potential role in planning adjuvant treatment in CRC patients.

Homologous recombination repair gene mutations in colorectal cancer favors treatment of immune checkpoint inhibitors.

Immune checkpoint inhibitor (ICI) therapy is insensitive for Colorectal cancer (CRC)patients with microsatellite stable (MSS). Genomic data of three CRC cohort, n = 35), and the Cancer Genome Atlas (TCGA CRC cohort, n = 377), were analyzed. A cohort treated with ICIs from Memorial Sloan Kettering Cancer Center (MSKCC CRC cohort, n = 110) and two cases from the local hospital were characterized the impact of the HRR mutation on prognosis of CRC. Homologous recombination repair(HRR) gene mutations were more common in CN and HL cohorts (27.85%; 48.57%) than in TCGA CRC cohort (15.92%), especially in the MSS populations, the frequencies of HRR mutation were higher in CN and HL cohort (27.45%, 51.72%) than in TCGA cohort (6.85%). HRR mutations were associated with high tumor mutational burden (TMB-H). Although HRR mutation uncorrelated with an improved overall survival in the MSKCC CRC cohort (p = 0.97), HRR mutated patients had a significantly improved OS compared to the HRR wildtype population particularly in MSS subgroups (p = 0.0407) under ICI treatment. It probably contributed by a higher neoantigen and increased CD4+ T cell infiltration which found in the TCGA MSS HRR mutated CRC cohort. The similar phenomenon on cases was observed that MSS metastatic CRC patient with HRR mutation seemed more sensitive to ICI after multi-line chemotherapy in clinical practice than HRR wildtype. This finding suggests the feasibility of HRR mutation as an immunotherapy response predictor in MSS CRC, which highlights a potential therapeutic approach for these patients.

Genetic compensation response could exist in colorectal cancer: UPF3A upregulates the oncogenic homologue gene SRSF3 expression corresponding to SRSF6 to promote colorectal cancer metastasis.

Genetic compensation response (GCR) is a mechanism that maintains the robustness of functional genes, which has been recently identified. Whether GCR exists in tumors and its effects on tumor progression remains unknown. Whole exome sequencing was performed to identify premature termination codon (PTC) gene mutations in colorectal cancer (CRC) tissues. RNA sequencing, Cancer Cell Line Encyclopedia database analysis, and high-throughput output of homologous genes using the Ensemble genome database were performed to further identify homologous genes of target PTC gene mutations. Serine and arginine-rich splicing factor 3 (SRSF3) increased the invasion ability in CRC cells and could be the target gene of up-frameshift 3A (UPF3A). The deletion of the 660th base A in the coding sequence region of SRSF6 caused a frameshift mutation of serine at position 220 (s220fs), which contributed to a PTC UAA termination of translation in HCT116 cells. We further found that SRSF3 was the only homologue of SRSF6 with a frameshift mutation. The transfection of s220fs of SRSF6 into HCT116 cells led to upregulation of its corresponding oncogenic homologue gene SRSF3 expression to promote CRC metastasis. SRSF3 was highly expressed in CRC liver metastases and was positively correlated with UPF3A expression and contributed to poor prognosis. GCR may exist in CRC and exert effects on the progression of CRC. Targeted inhibition of UPF3A could reduce the GCR effects and suppress the expression of oncogenic homologue genes corresponding to PTC mutations, indicating a novel therapeutic strategy for treatment of CRC metastasis.

Relationship between PET/CT images and KRAS gene mutations in colorectal cancer in Vietnamese patients.

We conducted this study to determine the relationship among standardized uptake value (SUV), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) indexes of Flourine-18 fluorodeoxyglucose positron emission tomography/computed tomography18 (FDG-PET/CT) imaging and Kirsten rat sarcoma (KRAS) gene mutations in colorectal cancer (CRC). This cross-sectional study was conducted in Bach Mai Hospital from 2020 to 2022. It included newly diagnosed CRC patients who underwent PET/CT examination prior to primary tumor resection. The maximum SUV (SUVmax - SUVmean), MTV, and TLG were considered. All pathologically confirmed CRC patients were accepted with further KRAS mutation status analysis. We enrolled 63 newly diagnosed CRC patients who underwent PET/CT examination prior to primary tumor resection. Among them, 31 (49.2%) patients had KRAS gene mutation. Patients with KRAS mutation status showed significantly different and higher SUVmax (p-value = 0.025), SUVmax t/b (p-value = 0.013), SUVmax t-b (p-value = 0.014), MTV (p-value = 0.023), and TLG (p-value = 0.011) than patients with WT KRAS. Other characteristics, including age, gender, tumor location, SUVb, SUVmean, SUVmax of lymph nodes, and SUVmax of liver metastasis, were insignificantly different between the two groups of patients with KRAS mutation status. Receiver operating curve analysis showed that the area under the curve was 0.672 for SUVmax (p-value = 0.019), SUVt/b (p-value = 0.045), and SUVt-b (p-value = 0.020). We observed a relationship, considering the quantitative parameters (SUVmax, SUVmax, SUVmax t-b, MTV, and TLG), between 18FDG-PET/CT images and the KRAS gene mutation in CRC by analyzing 63 patients prior to treatment.

Mathematical modeling the gene mechanism of colorectal cancer and the effect of radiation exposure.

Cancer is the result of continuous accumulation of gene mutations in normal cells. The number of mutations is different in different types of cancer and even in different patients with the same type of cancer. Therefore, studying all possible numbers of gene mutations in malignant cells is of great value for the understanding of tumorigenesis and the treatment of cancer. To this end, we applied a stochastic mathematical model considering the clonal expansion of any premalignant cells with different mutations to analyze the number of gene mutations in colorectal cancer. The age-specific colorectal cancer incidence rates from the Surveillance, Epidemiology and End Results (SEER) registry in the United States and the Life Span Study (LSS) in Nagasaki and Hiroshima, Japan are chosen to test the reasonableness of the model. Our fitting results indicate that the transformation from normal cells to malignant cells may undergo two to five driver mutations for colorectal cancer patients without radiation-exposed environment, two to four driver mutations for colorectal cancer patients with low level radiation-exposure, and two to three driver mutations for colorectal cancer patients with high level radiation-exposure. Furthermore, the net growth rate of the mutated cells with radiation-exposure was is higher than that of the mutated cells without radiation-exposure for the models with two to five driver mutations. These results suggest that radiation environment may affect the clonal expansion of cells and significantly affect the development of tumors.

RAS Pathway Alterations in Pediatric AML Patients

RAS pathway alterations are highly prevalent in hematologic malignancies1,2, often involving gain of function KRAS, NRAS or HRAS mutations and have been linked to tumorigenesis and associated with poorer prognosis in different cancer entities3-5. In adult myeloid leukemia, RAS mutations are one of the most common mutations, yet show no clear prognostic value6. In a study of the Japanese pediatric AML cohort, NRAS mutations were associated with a favorable outcome, the effect of KRAS mutations could not be analyzed due to the limited patient size7. Here we report the outcome of 372 pediatric AML patients of the German AML-BFM study between 2016 and 2021 that were characterized by morphology, multicolor flow cytometry, classical cytogenetics and mutation analysis via panel sequencing. In total, 27% (n=102) of the patients showed a RAS pathway alteration, involving n=32 patients (9%) with a KRAS mutation and n=75 (20%) with a NRAS mutation. Notably, no HRAS mutation was detected in our cohort. Age distribution did not differ between the groups (Fig. A). Interestingly, the KRAS cohort consisted mostly of male patients (72%, p=0,0087), while the overall cohort was balanced (48,7% male patients). NRAS and KRAS mutations were strong predictors of therapy response and early death. Only 1/75 (1,3%) of all patients harboring a NRAS mutations showed poor response to induction therapy compared to patients without a NRAS mutation (10,10%, p=0,01). KRAS mutations on the opposite were strongly associated with non-response (21,9%; p<0,01) and subsequent early deaths (n=6/32; p=0,001) compared to patients without KRAS mutations (3,2%). Of note, only 1/75 (1,3%) of patients with NRAS mutation and 17/372 (4,6%) of all AML patients suffered early deaths. Risk stratification taking into account re-stratification after poor therapy response showed a significant higher proportion of high-risk patients in the KRAS-mutated cohort (43,8 %) compared to 20% in the patients with NRAS mutations (p=0,0022). KRAS mutations correlated with KMT2A-MLLT1 (15,6%; p=0,01) translocations, which were significantly rarer in patients without KRAS mutations (3,5%). NRAS mutations occurred frequently with CBFA2T3-RUNX1 (14,7%; p=0,0002) and CBFB-MYH11 translocations (12%; p=0,0068%) as well as KIT mutations (12%; p=0,026). Existence of RAS mutations showed overall no significant impact on event-free survival (EFS=69,6% ±4,8%) and overall survival (OS=82,8% ±4,2%) compared to RAS wildtype (EFS=66,4% ±3,1% ; OS=76,5% ±2,9%) (Fig. B). Multivariate analysis comparing outcome for several subgroups also showed no significant differences. In summary, we show worse therapy response and increased early death rates in patients with KRAS mutations, while NRAS mutations are associated with better therapy response. These findings warrant further analyses and longer follow-up on the cohort to determine long-term significances of RAS pathway alterations in pediatric AML patients. 1.de Luca, A., Maiello, M. R., D'Alessio, A., Pergameno, M. & Normanno, N. The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets16 Suppl 2, (2012). 2.Hobbs, G. A., Der, C. J. & Rossman, K. L. RAS isoforms and mutations in cancer at a glance. Journal of Cell Science129, 1287-1292 (2016). 3.Hsu, H. C. et al. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients. Oncotarget7, 22257 (2016). 4.Vauthey, J., Zimmitti, G., Kopetz, S., ... J. S.-A. of & 2013, undefined. RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases. ncbi.nlm.nih.gov. 5.Dinu, D. et al. Prognostic significance of KRAS gene mutations in colorectal cancer - preliminary study. Journal of Medicine and Life7, 581 (2014). 6.Neubauer, A. et al. Patients With Acute Myeloid Leukemia and RAS Mutations Benefit Most From Postremission High-Dose Cytarabine: A Cancer and Leukemia Group B Study. Journal of Clinical Oncology26, 4603 (2008). 7.Kaburagi, T. et al. Clinical significance of RAS pathway alterations in pediatric acute myeloid leukemia. Haematologica107, 583-592 (2022). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histomorphological and molecular genetic characterization of different intratumoral regions and matched metastatic lymph nodes of colorectal cancer with heterogenous mismatch repair protein expression.

To better understand the clinicopathological characteristics and molecular alterations in different intratumoral components of colorectal cancer (CRC) with heterogeneity of mismatch repair (MMR) protein expression and microsatellite instability (MSI) status. The histopathological features, MSI status, and other molecular alterations were analyzed in separately microdissected intratumoral regions and matched metastatic lymph nodes in four cases with intratumoral heterogenous MMR expression screened from 500 CRC patients, using PCR-based MSI testing, MLH1 promoter methylation, and targeted next-generation sequencing (NGS). High microsatellite instability (MSI-H) was identified in MLH1/PMS2-deficient regions in Cases 1 to 3 and in MSH2/MSH6-deficient regions in Case 4, while microsatellite stability (MSS) was detected in all the intratumoral regions and metastatic lymph nodes with proficient MMR expression (pMMR). Intratumoral heterogeneity of MLH1 promoter methylation and/or other common driving gene mutations of CRC, such as KRAS and PIK3CA mutations, was identified in all four CRCs. Further, three cases (75%) showed heterogeneous histomorphological features in intratumoral components and metastatic lymph nodes (Cases 1, 2, and 4), and the corresponding metastatic lymph nodes showed moderate differentiation with MSS/pMMR (Cases 2 and 3). Intratumoral heterogeneous MSI status is highly correlated with intratumoral histomorphological heterogeneity, which is also an important clue for the intratumoral heterogeneity of drive gene mutations in CRC. Thus, it is essential to detect MMR protein expression and other gene mutations in metastases before treatment, especially for CRCs with intratumoral heterogenous MMR protein expression or heterogenous histomorphological features.

A comprehensive analysis of the microbiota composition and host driver gene mutations in colorectal cancer

Studies of both, microbiota and target therapy associated with gene mutations in colorectal cancer, (CRC) have attracted increasing attention. However, only a few of them analyzed the combined effects on CRC. we analyzed differences in intestinal microbiota of 44 colorectal cancer patients and 20 healthy controls (HC) using 16S rRNA gene sequencing of fecal samples. For 39 of the CRC patients, targeted Next Generation Sequencing (NGS) was carried out at formalin fixed paraffin embedded (FFPE) samples to identify somatic mutation profiles. Compared to the HC group, the microbial diversity of CRC patients was significantly lower. In the CRC group, we found a microbiome that was significantly enriched for strains of Bifidobacterium, Bacteroides, and Megasphaera whereas in the HC group the abundance of Collinsella, Faecalibacterium, and Agathobacter strains was higher. Among the mutations detected in the CRC group, the APC gene had the highest mutation rate (77%, 30/39). We found that the KRAS mutant type was closely associated with Faecalibacterium, Roseburia, Megamonas, Lachnoclostridium, and Harryflintia. Notably, Spearman correlation analysis showed that KRAS mutations were negatively correlated with the existence of Bifidobacterium and positively correlated with Faecalibacterium. By employing 16S rRNA gene sequencing, we identified more unique features of microbiota profiles in CRC patients. For the first time, our study showed that gene mutations could directly be linked to the microbiota composition of CRC patients. We hypothesize that the effect of a targeted colorectal cancer therapy is also closely related to the colorectal flora, however, this requires further investigation.

High sensitivity and rapid detection of KRAS and BRAF gene mutations in colorectal cancer using YbTixOy electrolyte-insulator-semiconductor biosensors

The KRAS and BRAF genes are attractive for a predictive biomarker to determine whether the colorectal cancer (CRC) patient predicts response to anti-epidermal growth factor receptor treatment. The detection of KRAS and BRAF mutation statuses has to be considered as a diagnostic and prognostic factor for the treatment of metastatic CRC patients because the targeted therapy may be expensive as well as toxic. In this work, we develop an YbTixOy electrolyte-insulator-semiconductor (EIS) biosensor to detect the KRAS and BRAF gene mutations in CRC. X-ray diffraction, atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry were used to explore the structural features of YbTixOy films prepared under three Ti plasma power conditions. We found that the YbTixOy-based EIS sensor fabricated at the 80 W condition exhibited a higher sensitivity of 69.54 mV/pH (in pH range 2–12), a lower hysteresis voltage of 1 mV (in pH loop of 7→4→7→10→7), and a smaller drift rate of 0.15 mV/h (in pH 7) than those of the other Ti plasma power conditions. Furthermore, we successfully demonstrated that the single strain DNA probe-immobilized YbTixOy EIS biosensors functionalized with 3-aminopropyl triethoxysilane followed by glutaraldehyde were screened for the KRAS and BRAF gene mutations in CRC. High sensitivity and rapid detection of the YbTixOy-based EIS biosensor are expected to serve as the diagnostic tool for clinical examination and screening with CRC.

Combination of artesunate and WNT974 induces KRAS protein degradation by upregulating E3 ligase ANACP2 and \u03b2-TrCP in the ubiquitin\u2013proteasome pathway

BackgroundKRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin.MethodsThe synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or β-TrCP. Western blot assay was utilized for the protein levels of NRAS, HRAS, KRAS, ANAPC2, β-TrCP, GSK-3β, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, β-TrCP, GSK-3β or ANAPC2 in tumor tissues.ResultsOur study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3β and E3 ligase β-TrCP that is known to degrade GSK-3β-phosphorylated KRAS protein. Knockdown of β-TrCP- and inhibition of GSK-3β abolish the combination treatment-induce KRAS ubiquitination and reduction in expression. Last but not least, combination treatment suppresses PI3K/Akt/m-TOR signaling pathway.ConclusionsOur data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin–proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment.Bh4nRY9p6vgUp6geXci3gHVideo

Prevalence of KRAS, PIK3CA, BRAF and AXIN2 gene mutations in colorectal cancer and its relationship with dental agenesis: a systematic review

Introduction: The study of allelic and genotypic frequencies contributes to determining the distribution of genetic variants in different populations and their possible association with biomarkers. This knowledge could improve the decision-making process regarding the management of some diseases such as colorectal cancer (CRC), in which the detection of clinical biomarkers such as dental agenesis could be crucial in clinical practice. Objective: To evaluate the available scientific evidence on the prevalence of KRAS, PIK3CA, BRAF and AXIN2 mutations and their possible association with dental agenesis in people with CRC. Materials and methods: A systematic search was conducted in PubMed, EMBASE and Cochrane Library databases using the following search strategy: type of studies: observational studies reporting the prevalence of KRAS, PIK3CA, BRAF and AXIN2 mutations in people diagnosed with CRC and their possible association with dental agenesis; publication language: English and Spanish; publication period: 2010-2020; search terms: “Genes”, “RAS”, “Kras”, “PIK3CA”, “BRAF”, “AXIN2”, “Mutation”, “Polymorphism”, “Colorectal Neoplasms”, “Colorectal Cancer”, used in different combinations (“AND” and “OR”). Results: The initial search yielded 403 records, but only 30 studies met the eligibility criteria. Of these, 11, 5, 5 and 1 only reported the prevalence of PIK3CA, KRAS, BRAF and AXIN2 mutations, respectively; while 8 reported the prevalence of more than one of these mutations in patients with CRC. The prevalence of KRAS (p.Gly12Asp), PIK3CA (p.Glu545Lys), and BRAF (p.Val600Glu) mutations ranged from 20.5% to 54%, 3.5% to 20.2%, and 2.5% to 12.1%, respectively. There were no findings regarding the association between the occurrence of these mutations and dental agenesis. Conclusions: KRAS mutations were the most prevalent; however, there is no evidence on the association between dental agenesis and the occurrence of KRAS, PIK3CA and BRAF germline mutations in individuals with CRC.

Crosstalk Between DNA Methylation and Gene Mutations in Colorectal Cancer.

Colorectal cancer (CRC) is often characterized by mutations and aberrant DNA methylation within the promoters of tumor suppressor genes and proto-oncogenes. The most frequent somatic mutations occur within KRAS and BRAF genes. Mutations of the KRAS gene have been detected in approximately 40% of patients, while mutations in BRAF have been detected less frequently at a rate of 10%. In this study, the DNA methylation levels of 22 candidate genes were evaluated in three types of tissue: mucosal tumoral tissue from 18 CRC patients, normal adjacent tissues from 10 CRC patients who underwent surgical resection, and tissue from a control group of six individuals with normal colonoscopies. A differential methylation profile of nine genes (RUNX3, SFRP1, WIF1, PCDH10, DKK2, DKK3, TMEFF2, OPCML, and SFRP2) presenting high methylation levels in tumoral compared to normal tissues was identified. KRAS mutations (codons 12 or 13) were detected in eight CRC cases, and BRAF mutations (codon 600) in four cases. One of the CRC patients presented concomitant mutations in KRAS codon 12 and BRAF, whereas seven patients did not present these mutations (WT). When comparing the methylation profile according to mutation status, we found that six genes (SFRP2, DKK2, PCDH10, TMEFF2, SFRP1, HS3ST2) showed a methylation level higher in BRAF positive cases than BRAF negative cases. The molecular sub-classification of CRC according to mutations and epigenetic modifications may help to identify epigenetic biomarkers useful in designing personalized strategies to improve patient outcomes.

Comparative analysis of KRAS and NRAS gene mutations in colorectal cancer

Comparative analysis of KRAS and NRAS gene mutations in colorectal cancer

Noninvasive KRAS mutation estimation in colorectal cancer using a deep learning method based on CT imaging

BackgroundThe detection of Kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutations in colorectal cancer (CRC) is key to the optimal design of individualized therapeutic strategies. The noninvasive prediction of the KRAS status in CRC is challenging. Deep learning (DL) in medical imaging has shown its high performance in diagnosis, classification, and prediction in recent years. In this paper, we investigated predictive performance by using a DL method with a residual neural network (ResNet) to estimate the KRAS mutation status in CRC patients based on pre-treatment contrast-enhanced CT imaging.MethodsWe have collected a dataset consisting of 157 patients with pathology-confirmed CRC who were divided into a training cohort (n = 117) and a testing cohort (n = 40). We developed an ResNet model that used portal venous phase CT images to estimate KRAS mutations in the axial, coronal, and sagittal directions of the training cohort and evaluated the model in the testing cohort. Several groups of expended region of interest (ROI) patches were generated for the ResNet model, to explore whether tissues around the tumor can contribute to cancer assessment. We also explored a radiomics model with the random forest classifier (RFC) to predict KRAS mutations and compared it with the DL model.ResultsThe ResNet model in the axial direction achieved the higher area under the curve (AUC) value (0.90) in the testing cohort and peaked at 0.93 with an input of ’ROI and 20-pixel’ surrounding area. AUC of radiomics model in testing cohorts were 0.818. In comparison, the ResNet model showed better predictive ability.ConclusionsOur experiments reveal that the computerized assessment of the pre-treatment CT images of CRC patients using a DL model has the potential to precisely predict KRAS mutations. This new model has the potential to assist in noninvasive KRAS mutation estimation.

Evaluation and identification of factors related to KRAS and BRAF gene mutations in colorectal cancer: A meta-analysis

Evaluation and identification of factors related to KRAS and BRAF gene mutations in colorectal cancer: A meta-analysis

188P - A clinical validation study of RASKET-B: A multiple detection kit for RAS and BRAF gene mutations in colorectal cancer

Background: The detection of RAS and BRAF gene mutations has been essential to determine the treatment strategy for metastatic colorectal cancer (CRC). A multiplex kit simultaneously detecting both gene mutations is required, although few genetic assays with high-quality assurance are established. We evaluated RAS/BRAF KIT (RASKET-B), a multiplex assay using PCR-reverse sequence specific oligonucleotide (PCR-rSSO) and xMAP technology to concurrently detect 48 types of RAS mutations as well as the BRAF V600E mutation in a short turnaround time (4.5 h/96 specimens). Methods: Formalin-fixed paraffin-embedded tissues were obtained from 309 consenting patients with histologically-confirmed CRC. The primary endpoint was the concordance rate (CR) between RAS mutations identified with RASKET-B and a PCR-rSSO method kit (RASKET) and between BRAF mutations identified with direct sequencing (DS), respectively. As the secondary endpoints, we evaluated the CR between RASKET-B and DS for RAS mutations and between RASKET-B and the pyrosequencing (PYRO) method for BRAF V600E mutation. Results: We registered 302 samples, all of which were tested using the RASKET-B and reference methods. In RAS genes, 142 samples (47.0%) were detected by RASKET-B: 114 KRAS exon 2 and 28 other RAS mutations. The CR between RASKET-B and RASKET or RASKET-B and DS were 100% (95%CI: 99-100%) and 97.4% (95%CI: 95-99%), respectively. In BRAF genes, 18 samples (6.0%) were detected using RASKET-B. The CR between RASKET-B and DS or RASKET-B and PYRO were 100% (95%CI: 99-100%) and 99.7% (95%CI: 98-100%), respectively. The result of RASKET-B was consistent with that of DS. We also analyzed RAS and BRAF mutation ratios for each tumor cell ratio and tumor cell area ratio (0-25%, 25-50%, 50-75%, 75-100% respectively) and report it. Conclusions: RASKET-B provides rapid, precise and simultaneous detections of RAS and BRAF mutations in CRC. Clinical trial indentification: Study ID: UMIN000022742. Legal entity responsible for the study: Medical & Biological Laboratories Co., Ltd. Funding: Medical & Biological Laboratories Co., Ltd. Disclosure: W. Okamoto: MSD, K. Muro: Takeda, Chugai Pharma, Yakult Honsha, Merck Serono, Taiho Pharmaceutical, Lilly., H. Taniguchi: Takeda, Chugai Pharma, Merck Serono, Taiho Pharmaceutical, Bayer, Lilly Japan, Yakult Honsha., H. Hara: Chugai Pharma, Taiho Pharmaceutical, Merck Serono, Yakult Honsha, Lilly, Ono Pharmaceutical, Chugai Pharma., T. Satoh: Bayer Yakuhin, Lilly, Ono Pharmaceutical, Takara Bio, Merck Serono, Chugai Pharma, Bristol-Myers Squibb, Takeda, Yakult Honsha. All other authors have declared no conflicts of interest.