Search Results (15)

Aberrant activation of the RAS signaling pathway is a halmark of various cancers. This activation, is often caused by mutations in RAS genes or other pathway components and, drivesi uncontrolled cell growth and proliferation. Studies have demonstrated that certain codon mutations can significantly influence the clinical outcomes of cancer patients. Historically, KRAS was considered “undruggable”; however, recent advancements in drug discovery have led to the development of promising KRAS inhibitors. Accurately identifying the specific type of KRAS mutation in a patient is essential for making optimal treatment decisions. Several methods have been developed for detecting KRAS mutations to address this need, focusing on creating robust, rapid, sensitive, accurate, and cost-effective approaches, particularly for point-of-care applications. Starting with the Ras family and RASopathies, this review provides a comprehensive overview of KRAS mutation detection methods, ranging from research-use-only techniques to in vitro diagnostic-certified tests. Published results are critically evaluated in terms of accuracy, sensitivity, cost, throughput, and suitability for various sample types and clinical settings. This, offers researchers and clinicians an up-to-date resource for. Full article

KRAS mutations occur in over 90% of pancreatic ductal adenocarcinomas (PDACs), most commonly at codon 12, but the clinical implications of codon-specific subtypes in advanced disease remain unclear. We retrospectively analyzed 269 patients with advanced PDAC who underwent next-generation sequencing between 2020 and 2024 at a single tertiary cancer center. Clinical features, co-mutations, treatment outcomes, and survival were evaluated. KRAS mutations were detected in 92% of patients, most frequently G12V (36%) and G12D (34%), followed by G12R (10%) and non-G12 variants (9%). TP53 was the most frequent co-mutation (67%) and was significantly enriched in G12D tumors compared with wild type (74.2% vs. 31.8%). Mutations in homologous recombination and DNA damage response genes were more common in non-G12 and wild-type tumors, although not statistically significant. Serum CA 19-9 was elevated in most G12-mutant tumors, whereas approximately 40% of non-G12 and wild-type patients showed normal levels despite advanced disease. No significant survival differences were observed among KRAS subtypes in the overall or treated cohorts. However, patients with G12V mutations achieved significantly longer survival with fluorouracil-based than gemcitabine-based chemotherapy. These findings suggest that, while KRAS subtyping alone lacks prognostic value, the G12V subtype may inform chemotherapy selection and warrants further prospective validation. Full article

Background and Objectives: KRAS genes are among the most prominent oncogenes that trigger tumor formation in colorectal cancer (CRC) and serve as predictive biomarkers for resistance to anti-EGFR therapies in metastatic colorectal cancer (mCRC) patients. However, the prevalence and mutation spectrum of the KRAS gene family in mCRC patients in Turkey have not been sufficiently analyzed. This study investigates the frequency and distribution of mutations in the KRAS gene family across different regions of Turkey and examines gender-related variations. Materials and Methods: This multicenter observational study included 2458 histologically confirmed mCRC patients collected from 52 centers across Turkey. In a central laboratory, KRAS mutations in codons 12 and 13 were analyzed using polymerase chain reaction (PCR). Statistical analyses were performed using chi-square tests and Monte Carlo simulations, with a significance threshold set at p < 0.05. Results: Depending on the region, KRAS mutations were detected in 45% of patients, ranging from 39.6% to 47.5%. The mutation rate was significantly higher in female patients (48.8%) compared to male patients (42.6%) (p = 0.002). Codon 12 mutations were more frequent than codon 13 mutations. G12D, G12V, and G13D mutations accounted for 80% of all detected mutations. The G12V mutation was prevalent in female patients (p = 0.007). Based on region, mutation diversity was similar, and no statistically significant difference was found (p > 0.05). Conclusions: This large-scale, multicenter study provides the most comprehensive dataset of KRAS mutations in mCRC patients in Turkey. This study revealed regional trends, as well as gender differences. The findings highlight the importance of routine KRAS genotyping in guiding personalized treatment strategies, especially regarding candidate selection for anti-EGFR therapies. Further research is required to elucidate the prognostic and therapeutic implications of specific KRAS mutations. Full article

NRAS belongs to the RAS family of GTPases. In colorectal cancer (CRC), NRAS mutations are rare compared to KRAS, but may lead to worse outcomes. We report the functional characterization of the novel NRAS mutants—G48C, Q43K, and E37K—identified in Filipino young-onset CRC patients. Unlike previously characterized NRAS mutants with no apparent effects on cell proliferation, these mutants enhanced proliferation of both HCT116 and NIH3T3 cells. This was confirmed in 3D spheroid assays to mimic the spatial organization of cells. G48C and E37K showed apoptosis resistance in both cell lines, and Q43K showed resistance in HCT116 cells. All three showed no effect on cellular migration in NIH3T3, but G48C enhanced the migration rate of HCT116 cells. Actin staining of NIH3T3 cells expressing the mutants showed a shrunken cytoplasm and transient structures associated with motility and invasiveness. Docking simulations show that GDP is only able to bind fully within the binding pocket of wild-type NRAS, but not in the mutants. Further, G48C, Q43K, and E37K all have less negative ΔG values, indicating a weaker GDP-binding affinity compared to wild-type NRAS. Taken together, the results suggest that oncogenic readouts of NRAS mutants are codon- and mutation-specific, with potential repercussions on the aggressiveness, resistance, and therapeutic response. Full article

Background/Objectives: Although the overall survival prognosis of patients in advanced stages of pancreatic ductal adenocarcinoma (PDAC) is poor, typically ranging from days to months from diagnosis, there are rare cases of patients remaining in therapy for longer periods of time. Early estimations of survival prognosis would allow rational decisions on complex therapy interventions, including radical surgery and robust systemic therapy regimens. Understandably, there is great interest in finding prognostic markers that can be used for patient stratification. We determined the role of various KRAS mutations in the prognosis of PDAC patients using biopsy samples and circulating tumor DNA. Methods: A total of 118 patients with PDAC, clinically confirmed by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNB), were included in the study. DNA was extracted from cytological slides following a standard cytology evaluation to ensure adequacy (viability and quantity) and to mark the tumor cell fraction. Circulating tumor DNA (ctDNA) was extracted from plasma samples of 45 patients in stage IV of the disease. KRAS mutations in exons 12 and 13 were detected by denaturing capillary electrophoresis (DCE), revealing a minute presence of mutation-specific heteroduplexes. Kaplan–Meier survival curves were calculated for individual KRAS mutation types. Results:KRAS mutations were detected in 90% of tissue (106/118) and 44% of plasma (20/45) samples. All mutations were localized at exon 2, codon 12, with G12D (GGT > GAT) being the most frequent at 44% (47/106) and 65% (13/20), followed by other types including G12V (GGT > GTT) at 31% (33/106) and 10% (2/20), G12R (GGT > CGT) at 17% (18/106) and 10% (2/20), G12C (GGT/TGT) at 5% (5/106) and 0% (0/20) and G12S (GGT/AGT) at 1% (1/106) and 5% (1/20) in tissue and plasma samples, respectively. Two patients had two mutations simultaneously (G12V + G12S and G12D + G12S) in both types of samples (2%, 2/106 and 10%, 2/20 in tissue and plasma samples, respectively). The median survival of patients with the G12D mutation in tissues was less than half that of other patients (median survival 101 days, 95% CI: 80–600 vs. 228 days, 95% CI: 184–602), with a statistically significant overall difference in survival (p = 0.0080, log-rank test), and furthermore it was less than that of all combined patients with other mutation types (101 days, 95% CI: 80–600 vs. 210 days, 95% CI: 161–602, p = 0.0166). For plasma samples, the survival of patients with this mutation was six times shorter than that of patients without the G12D mutation (27 days, 95% CI: 8–334 vs. 161 days, 95% CI: 107–536, p = 0.0200). In contrast, patients with detected KRAS G12R in the tissue survived nearly twice as long as other patients in the aggregate (286 days, 95% CI: 70–602 vs. 162 days, 95% CI: 122–600, p = 0.0374) or patients with other KRAS mutations (286 days, 95% CI: 70–602 vs. 137 days, 95% CI: 107–600, p = 0.0257). Conclusions: Differentiation of specific KRAS mutations in EUS-FNB and ctDNA (above all, the crucial G12D and G12R) is feasible in routine management of PDAC patients and imperative for assessment of prognosis. Full article

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS. Full article

The KRAS proto-oncogene is a major driver of pancreatic tumorigenesis and is nearly ubiquitously mutated in pancreatic ductal adenocarcinoma (PDAC). KRAS point mutations are detected in over 90% of PDAC cases, and these mutations have been shown to be associated with worse therapy response and overall survival. Pathogenic KRAS mutations are mostly limited to codons 12, 13 and 61, with G12D, G12V, G12R, Q61H, and G13D accounting for approximately 95% of the mutant cases. Emerging data have shown the importance of specific mutant subtypes, as well as KRAS variant allele frequency on clinical prognosis. Furthermore, novel technologies and therapies are being developed to target specific mutant subtypes, with encouraging early results. In this paper, we aim to review the recent studies regarding the relative impact of specific mutant KRAS subtypes on oncologic outcomes, the application of variant allele frequency in next generation sequencing analyses, and the ongoing research into therapies targeting specific mutant KRAS subtypes. Full article

Oncogenic K-RAS mutations occur in approximately 25% of human lung cancers and are most frequently found in codon 12 (G12C, G12V, and G12D). Mutated K-RAS inhibitors have shown beneficial results in many patients; however, the inhibitors specifically target K-RAS^G12C and acquired resistance is a common occurrence. Therefore, new treatments targeting all kinds of oncogenic K-RAS mutations with a durable response are needed. RUNX3 acts as a pioneer factor of the restriction (R)-point, which is critical for the life and death of cells. RUNX3 is inactivated in most K-RAS-activated mouse and human lung cancers. Deletion of mouse lung Runx3 induces adenomas (ADs) and facilitates the development of K-Ras-activated adenocarcinomas (ADCs). In this study, conditional restoration of Runx3 in an established K-Ras-activated mouse lung cancer model regressed both ADs and ADCs and suppressed cancer recurrence, markedly increasing mouse survival. Runx3 restoration suppressed K-Ras-activated lung cancer mainly through Arf-p53 pathway-mediated apoptosis and partly through p53-independent inhibition of proliferation. This study provides in vivo evidence supporting RUNX3 as a therapeutic tool for the treatment of K-RAS-activated lung cancers with a durable response. Full article

KRAS is frequently mutated in tumors. It is mutated in approximately 30% of all cancer cases and in nearly 50% of cases of metastatic colorectal cancer (CRC), which is the third leading cause of cancer-related deaths worldwide. Recent advancements in understanding CRC biology and genetics have highlighted the significance of KRAS mutations in the progression of CRC. The KRAS gene encodes a small GTPase (Guanosine TriPhosphatases) that plays a key role in signaling pathways associated with important proteins involved in amplifying growth factor and receptor signals. Mutations in KRAS are frequently observed in codons 12 and 13, and these mutations have oncogenic properties. Abnormal activation of KRAS proteins strongly stimulates signals associated with various cancer-related processes in CRC, including cell proliferation, migration and neoangiogenesis. In this review, we explore the distinct prognostic implications of KRAS mutations. Specifically, the KRAS p.G12C mutation is associated with a worse prognosis in metastatic CRC. The correlation between structure, conformation and mutations is visually presented to emphasize how alterations in individual amino acids at the same position in a single protein can unexpectedly exhibit complex involvement in cancer. Last, KRAS p.G12C is discussed as an emerging and promising therapeutic target in metastatic CRC, providing a concise overview of available clinical data regarding the use of new inhibitors. Full article

Activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS), in particular, a point mutation leading to a glycine-to-cysteine substitution at codon 12 (G12C), are among the most frequent genomic alterations in non-small cell lung cancer (NSCLC). Several agents targeting KRAS G12C have recently entered clinical development. Sotorasib, a first-in-class specific small molecule that irreversibly inhibits KRAS G12C, has since obtained Health Canada approval. The emergence of novel KRAS-targeted therapies warrants the development of evidence-based consensus recommendations to help clinicians better understand and contextualize the available data. A Canadian expert panel was convened to define the key clinical questions, review recent evidence, and discuss and agree on recommendations for the treatment of advanced KRAS G12C-mutated NSCLC. The panel agreed that testing for KRAS G12C should be performed as part of a comprehensive panel that includes current standard-of-care biomarkers. Sotorasib, the only approved KRAS G12C inhibitor in Canada, is recommended for patients with advanced KRAS G12C-mutated NSCLC who progressed on guideline-recommended first-line standard of care for advanced NSCLC without driver alterations (immune-checkpoint inhibitor(s) [ICIs] +/− chemotherapy). Sotorasib could also be offered as second-line therapy to patients who progressed on ICI monotherapy that are not candidates for a platinum doublet and those that received first-line chemotherapy with a contraindication to ICIs. Preliminary data indicate the activity of KRAS G12C inhibitors in brain metastases; however, the evidence is insufficient to make specific recommendations. Regular liver function monitoring is recommended when patients are prescribed KRAS G12C inhibitors due to risk of hepatotoxicity. Full article

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future. Full article

In NSCLC, KRAS mutations occur in up to 30% of all cases, most frequently at codon 12 and 13. KRAS mutations have been linked to adenocarcinoma histology, positive smoking history, and Caucasian ethnicity, although differences have been described across KRAS mutational variants subtypes. KRAS mutations often concur with other molecular alterations, notably TP53, STK11, and KEAP1, which could play an important role in treatment efficacy and patient outcomes. For many years, KRAS mutations have been considered undruggable mainly due to a high toxicity profile and low specificity of compounds. Sotorasib and adagrasib are novel KRAS inhibitors that recently gained FDA approval for pre-treated KRAS mutant NSCLC patients, and other molecules such as GDC-6036 are currently being investigated with promising results. Despite their approval, the efficacy of these drugs is lower than expected and progression among responders has been reported. Mechanisms of acquired resistance to anti-KRAS molecules typically involves either on target secondary mutations (e.g., G12, G13, Q61H, R68S, H95, Y96C, V8L) or off-target alterations. Ongoing trials are currently evaluating strategies for implementing efficacy and overcoming acquired resistance to these compounds. Finally, the efficacy of immune-checkpoint inhibitors still needs to be completely assessed and responses to anti-PD-1/PD-L1 agents may strongly depend on concomitant mutations. Full article

Increased insulin-like growth factor (IGF) axis activity is associated with the development and progression of different types of malignancies, including colorectal cancer (CRC). MicroRNAs (miRNAs) belonging to the let-7 family have been reported to target genes involved in this axis and are known as tumor suppressors. In this study, in silico bioinformatic analysis was performed to assess miRNA–mRNA interactions between eight miRNAs belonging to the let-7 family and genes involved in the IGF signaling pathway, coding for receptors and substrates. miRNAs’ expression analysis revealed that hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let- 97 7d-5p, hsa-let-7e-5p, hsa-let-7f-5p, and hsa-let-7g-5p were significantly down-regulated in 25 CRC tumoral tissues (T) compared to the corresponding adjacent peritumoral tissues (PT). Moreover, our results showed an upregulation of miR-let-7e-5p in CRC tissues with mutations in KRAS codon 12 or 13, and, for the first time, found a specific dysregulation of let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, and let-7i-5p in CRC with perineural invasion. Our results sustain the relationship between the IGF axis, let-7 miRNAs, and CRC and suggest an association between the expression of these miRNAs and perineural invasion. Full article

The KRAS oncogene is involved in the pathogenesis of several types of cancer, particularly colorectal cancer (CRC). The most frequent mutations in this gene are associated with poor survival, increased tumor aggressiveness and resistance to therapy with anti-epidermal growth factor receptor (EGFR) antibodies. For this reason, KRAS mutation testing has become increasingly common in clinical practice for personalized cancer treatments of CRC patients. Detection methods for KRAS mutations are currently expensive, laborious, time-consuming and often lack of diagnostic sensitivity and specificity. In this study, we describe the development of a Lab-on-Chip assay for genotyping of KRAS mutational status. This assay, based on the In-Check platform, integrates microfluidic handling, a multiplex polymerase chain reaction (PCR) and a low-density microarray. This integrated sample-to-result system enables the detection of KRAS point mutations, including those occurring in codons 12 and 13 of exon 2, 59 and 61 of exon 3, 117 and 146 of exon 4. Thanks to its miniaturization, automation, rapid analysis, minimal risk of sample contamination, increased accuracy and reproducibility of results, this Lab-on-Chip platform may offer immediate opportunities to simplify KRAS genotyping into clinical routine. Full article

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples. Full article