Specifically identifying one-base mismatched gene sequences is of significance for early diagnosis, prognosis, treatment, and evaluation. However, traditional polymerase chain reaction and ...next-generation sequencing methods require relatively long, carefully implemented analytical procedures. Here, we report a nanoconjugate that can synchronously discriminate two types of KRAS gene mutation due to the dual-recognition mechanism of the CRISPR/dCas system with the surface-enhanced Raman scattering (SERS) response output. This method applied two sets of CRISPR/dCas9 units to collect all KRAS genes via a magnetic force and the specific mutant sequence labeled through SERS nanotags, forming a dual-clamping architecture. This sensing conjugate was sufficient to quantify the amount of the two different mutations simultaneously with a low cross-reactivity. Consequently, we deem that this sensing assay satisfies the great characteristics of a genetic analytical tool with high specificity, accuracy, and simplicity, highlighting its potential as an alternative for screening multigene events and relevant genetic biomarkers.
KRAS is the most frequently mutated oncogene in non-small cell lung cancers (NSCLC), with a frequency of around 30%, and encoding a GTPAse that cycles between active form (GTP-bound) to inactive form ...(GDP-bound). The
mutations favor the active form with inhibition of GTPAse activity.
mutations are often with poor response of EGFR targeted therapies.
mutations are good predictive factor for immunotherapy. The lack of success with direct targeting of KRAS proteins, downstream inhibition of KRAS effector pathways, and other strategies contributed to a focus on developing mutation-specific KRAS inhibitors.
p.G12C mutation is one of the most frequent KRAS mutation in NSCLC, especially in current and former smokers (over 40%), which occurs among approximately 12-14% of NSCLC tumors. The mutated cysteine resides next to a pocket (P2) of the switch II region, and P2 is present only in the inactive GDP-bound KRAS. Small molecules such as sotorasib are now the first targeted drugs for
G12C mutation, preventing conversion of the mutant protein to GTP-bound active state. Little is known about primary or acquired resistance. Acquired resistance does occur and may be due to genetic alterations in the nucleotide exchange function or adaptative mechanisms in either downstream pathways or in newly expressed
G12C mutation.
Developing new targeted therapy for pancreatic cancer is one of the major current challenges in cancer research. KRAS mutations and miRNA dysregulation (e.g. miR-21-5p oncomiR) play key roles in ...Pancreatic Ductal Adenocarcinoma (PDAC), leading to rapid progression of the disease. As the KRAS mutation is a main driver of PDAC, anti-KRAS strategies remain a major therapeutic approach for PDAC treatment. Previously, utilization of either siKRAS or small chemically modified single-stranded RNA molecules that specifically disable miR-21 (anti-miR-21) were effective in slowing PDAC tumor growth in various tumor models when packaged in an innovative delivery system (TPN) required for efficient drug delivery to the PDAC tumor site. Here we have tested the utility of targeting the KRAS pathway through multiple mechanisms and via dual targeting of a PDAC oncomiR and oncogene. Initially we found that miR-217, which has been shown to directly regulate KRAS expression, is downregulated in our PDAC samples, thus we tested the benefits of anti-miR-21, miR-217 mimic or siKRAS loaded into the tumor-penetrating nanoparticles (TPN) that we had previously shown to potently target the largely impenetrable PDAC tumors, and found an enhanced anti-tumoral response upon dual treatments in KRAS-mutated PDAC models.
Background
Despite the acknowledged predictive value of KRAS in immune checkpoint inhibitor (ICI) responses, the heterogeneous behavior of its mutations in this sphere remains largely unexplored. As ...of now, no studies have definitively categorized KRAS subtype variations as independent prognostic indicators for ICI responses in lung cancer patients.
Methods
We analyzed a cohort of 103 patients, all harboring different KRAS mutation subtypes, and complemented this data with information from TCGA and GEO databases. Our research focused on delineating the relationships between KRAS mutation subtypes and factors like immunotherapy markers and immune cell composition, in addition to examining survival rates, drug sensitivity, and PD-L1 responses corresponding to distinct KRAS subtypes.
Results
We found that the G12V and G12D subtypes demonstrated elevated expressions of immunotherapy markers, implying a potentially enhanced benefit from immunotherapy. Significant variations were identified in the distribution of naive B cells, activated CD4+ memory T cells, and regulatory T cells (Tregs) across different KRAS mutant subtypes. A notable difference was observed in the Tumor Mutation Burden (TMB) levels across the four KRAS subtypes, with the G12D subtype displaying the lowest TMB level. Furthermore, G12C subtype showcased the worst prognosis in terms of progression-free intervals (PFI), in stark contrast to the more favorable outcomes associated with the G12A subtype.
Conclusion
Our study reveals that KRAS mutations exhibit considerable variability in predicting outcomes for LUAD patients undergoing ICI treatment. Thus, the evaluation of KRAS as a biomarker for ICIs necessitates recognizing the potential diversity inherent in KRAS mutations.
This study aimed to validate the utility of the new histological classification proposed by the International Association for the Study of Lung Cancer (IASLC), American Thoracic Society (ATS), and ...European Respiratory Society (ERS) for identifying the prognostic subtypes of adenocarcinomas in Japanese patients; correlations between the classification and the presence of EGFR or KRAS mutation status were also investigated.
We retrospectively reviewed 440 patients with lung adenocarcinoma, who underwent resection. The tumors were classified according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were detected using the established methods.
Five-year disease-free survival rates were: 100% for adenocarcinoma in situ (n = 20) and minimally invasive adenocarcinoma (n = 33), 93.8% for lepidic-predominant adenocarcinoma (n = 36), 88.8% for invasive mucinous adenocarcinoma (n = 10), 66.7% for papillary-predominant adenocarcinoma (n = 179), 69.7% for acinar-predominant adenocarcinoma (n = 61), 43.3% for solid-predominant adencoarcinoma (n = 78), and 0% for micropapillary-predominant adenocarcinoma (n = 19). Multivariate analysis revealed that the new classification was an independent predictor of disease-free survival. EGFR and KRAS mutations were detected in 90 cases (53.9%) and 21 cases (13.3%), respectively; EGFR mutations were significantly associated with adenocarcinoma in situ, minimally invasive adenocarcinoma, lepidic- and papillary-predominant adenocarcinoma, and KRAS mutations adenocarcinomas with mucinous tumor subtypes.
We found that the IASLC/ATS/ERS classification identified prognostic histologic subtypes of lung adenocarcinomas among Japanese patients. Histologic subtyping and molecular testing for EGFR and KRAS mutations can help predict patient prognosis and select those who require adjuvant chemotherapy.
Background
Although immune checkpoint inhibitors (ICIs) are one of the most important treatments for advanced-stage non-small-cell lung cancer (NSCLC), NSCLC patients with ALK-rearranged usually ...don’t obtain a clinical benefit. The reason may be related to the unique tumor microenvironment (TME). We evaluated the characteristics of immune biomarkers of the TME and their prognostic value in ALK-rearranged NSCLC.
Methods
Tumor samples from patients with ALK-rearranged (N = 39) and EGFR- (N = 40)/KRAS- (N = 30) mutated NSCLC were collected. Immunohistochemistry (IHC) was used to assess the expression of 9 tumor immune markers as well as 6 immune markers of tumor-infiltrating cells. To research the TME of ALK-rearranged NSCLC, EGFR/KRAS-positive patients were used as controls. Furthermore, the correlation between the efficacy and prognosis of patients with advanced-stage (IIIC-IV) ALK rearrangements treated with targeted drugs was analyzed in terms of the TME.
Results
The proportion of PD-L1+ tumors was lower in ALK-positive NSCLC than in KRAS-positive NSCLC. Besides, the proportion of T cells expressing TIM-3-CD8+ (15.38%), CTLA4-CD8+ (12.82%), LAG3-CD8+ (33.33%) and PD-1-CD8+ (2.56%) in ALK-positive NSCLC was lower than that in EGFR/KRAS-positive NSCLC. The expression of CD3, CD8 T cells and CD20 B cells was lower in ALK-positive NSCLC than in KRAS-positive NSCLC (p < 0.0001, < 0.005, and < 0.001, respectively). Nevertheless, the level of CD4 helper T cells was higher in ALK-positive NSCLC than in EGFR/KRAS-positive NSCLC (p < 0.0001 and p < 0.05, respectively). The repression of TIM3 was higher in ALK-positive NSCLC than in KRAS-positive NSCLC (p < 0.001). In addition, our data showed that high expression of PD-L1 (HR = 0.177, 95% CI 0.038–0.852, p = 0.027) and CTLA4 (HR = 0.196, 95% CI 0.041–0.947, p = 0.043) was related to lower OS in advanced-stage ALK- rearranged NSCLC patients treated with ALK tyrosine kinase inhibitors (TKIs).
Conclusions
Immunosuppressive status was characteristic of the TME in patients with ALK-positive NSCLC compared with EGFR/KRAS-positive NSCLC. High expression of PD-L1 and CTLA4 was an adverse prognostic factor in advanced-stage ALK-rearranged NSCLC patients treated with ALK-TKIs. Immunotherapy for ALK-rearranged patients requires further exploration and validation by clinical trials.
Objectives
To assess the association between CT features and EGFR, ALK, KRAS mutations in non-small cell lung cancer.
Methods
Patients undergoing chest CT and testing for the above gene mutations ...were included. Qualitative evaluation of CTs included: lobe; lesion diameter; shape; margins; ground-glass opacity; density; cavitation; air bronchogram; pleural thickening; intratumoral necrosis; nodules in tumour lobe; nodules in non-tumour lobes; pleural retraction; location; calcifications; emphysema; fibrosis; pleural contact; pleural effusion. Statistical analysis was performed to assess association of features with each gene mutation. ROC curves for gene mutations were drawn; the corresponding area under the curve was calculated.
P
-values <0.05 were considered significant.
Results
Of 285 patients, 60/280 (21.43 %) were positive for EGFR mutation; 31/270 (11.48 %) for ALK rearrangement; 64/240 (26.67 %) for KRAS mutation. EGFR mutation was associated with air bronchogram, pleural retraction, females, non-smokers, small lesion size, and absence of fibrosis. ALK rearrangements were associated with age and pleural effusion. KRAS mutation was associated with round shape, nodules in non-tumour lobes, and smoking.
Conclusions
This study disclosed associations between CT features and alterations of EGFR (air bronchogram, pleural retraction, small lesion size, absence of fibrosis), ALK (pleural effusion) and KRAS (round lesion shape, nodules in non-tumour lobes).
Key Points
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Air bronchogram
,
pleural retraction
,
small size relate to EGFR mutation in NSCLC
.
•
Pleural effusion and younger age relate to ALK mutation
.
•
Round lesion shape
,
nodules in non
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tumour lobes relate to KRAS mutation
.
RAS (H-ras, K-ras, and N-ras), as the second largest mutated gene driver in various human cancers, has long been a vital research target for cancer. Its function is to transform the extracellular ...environment into a cascade of intracellular signal transduction. RAS mutant protein regulates tumor cell proliferation, apoptosis, metabolism and angiogenesis through downstream MAPK, PI3K and other signaling pathways. In KRAS or other RAS-driven cancers, current treatments include direct inhibitors and upstream/downstream signaling pathway inhibitors. However, the research on these inhibitors has been largely restricted due to their escape inhibition and off-target toxicity. In this paper, we started with the role of normal and mutant RAS genes in cancer, elucidated the relevant RAS regulating pathways, and highlighted the important research advancements in RAS inhibitor research. We concluded that for the crosstalk between RAS pathways, the effect of single regulation may be limited, and the multi-target drug combined compensation mechanism is becoming a research hotspot.
Abstract
Quantifying mutant or variable allele frequencies (VAFs) of ≤10−3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for ...quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10−8 VAF, it is not a quick, easy, or inexpensive method. We report a method for quantifying VAFs that are ≥10−4 that is as easy and quick for processing samples as standard sequencing kits, yet less expensive than the kits. The method was developed using PCR fragment-based VAFs of Kras codon 12 in log10 increments from 10−5 to 10−1, then applied and tested on native genomic DNA. For both sources of DNA, there is a proportional increase in the observed VAF to input VAF from 10−4 to 100% mutant samples. Variability of quantitation was evaluated within experimental replicates and shown to be consistent across sample preparations. The error at each successive base read was evaluated to determine if there is a limit of read length for quantitation of ≥10−4, and it was determined that read lengths up to 70 bases are reliable for quantitation. The method described here is adaptable to various oncogene or tumor suppressor gene targets, with the potential to implement multiplexing at the initial tagging step. While easy to perform manually, it is also suited for robotic handling and batch processing of samples, facilitating detection and quantitation of genetic carcinogenic biomarkers before tumor formation or in normal-appearing tissue.
Non-invasive molecular analysis of circulating tumor DNA (ctDNA) is a promising application in personalized cancer management, although there is still much to learn about the biological ...characteristics of ctDNA. The present study compared absolute amounts of
mutated ctDNA and total circulating cell-free DNA (cfDNA) in colorectal cancer (CRC) patients (n=50) from various stages and healthy controls (n=8) by Intplex allele-specific and digital droplet PCR. In addition, the impact of two prominent extraction techniques (silica-based membrane vs. magnetic beads) on cfDNA and ctDNA recovery was analyzed in 38 paired samples from CRC patients and specific spike-in DNA controls. CfDNA fragment size was assessed using the Agilent 2100 Bioanalyzer. Relative quantities of total cfDNA quantities were measured using the Qubit fluorometer. Statistical analysis on total cfDNA yield revealed a strong correlation (r=0.976) between Qubit and absolute Intplex allele-specific PCR measurements in cancer patients and healthy controls. Total cfDNA was significantly increased in cancer patients compared to healthy controls, with the highest yield in distant metastatic disease. In line, the highest amount of ctDNA (1.35 ng/μL) was found in patients with distant organ metastasis. Of great interest, the silica-based membrane method significantly promoted extraction of long cfDNA fragments. In contrast, the magnetic bead system more efficiently recovered short cfDNA fragments in serum of cancer patients. Further, a decreased
allele frequency was observed in serum compared to plasma. This study suggests that the source of cfDNA and choice of pre-analytical extraction systems needs to be more carefully validated in routine clinical practice.
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